ASBMR 25th Annual Meeting SU001–SU482


Relationship of Peak Lean Tissue Accrual to Peak Bone Mineral Accrual in Boys and Girls.R. A. Faulkner, A. D. G. Baxter-Jones*, R. L. Mirwald*, D. A. Bailey. Kinesiology, University of Saskatchewan, Saskatoon, SK, Canada.

It has been postulated that muscle and bone form an operational unit; that is, factors that affect muscle will also influence bone. It also is thought that muscle action is paramount in affecting bone adaptation; if so, then it would be expected that muscle development (or lean tissue as a surrogate of muscle) during the growing years should precede bone mineral accrual. We have shown previously that this is the case for total body mass; however, in theory this relationship should be even stronger at the extremities where muscle action is more isolated. The purpose of this study was to investigate the relationship of the timing of bone-free peak lean mass accrual (PLM) to peak bone mineral mass accrual (PBM) at the arms and legs. Subjects (70 boys and 67 girls) were measured annually (DXA: Hologic 2000 QDR in array mode). Whole year velocity values were calculated for bone-free lean tissue and bone mineral content. The mean age of peak tissue accrual was then calculated as the mean of the peak. Dependent t-tests were done to test for differences between the age of PLM and PBM (p<.05). As shown in the following table, PLM occurred prior to PBM at both sites in both boys and girls; however, this difference was not statistically significant at the legs in girls. These preliminary data support the hypotheses that PLM (a surrogate measure for muscle mass) precedes PBM and that muscle and bone development are closely related. 

Table  . Comparison of age at peak velocity (Mean + S.D.)
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Disclosures: R.A. Faulkner, None.


IGF-1 and IGFBP-3 Influence Bone Mass Acquisition in Children via Independent Mechanisms.J. H. Tobias1, C. D. Steer*2, P. M. Emmett*2, A. R. Ness*2, D. J. Gunnell*3, J. M. P. Holly*4. 1Rheumatology Unit, University of Bristol, Bristol, United Kingdom, 2Alspac, University of Bristol, Bristol, United Kingdom, 3Social Medicine, University of Bristol, Bristol, United Kingdom, 4Surgery, University of Bristol, Bristol, United Kingdom.

Previous studies suggest that IGFBP-3 augments the stimulation of bone formation by IGF-1, which has been attributed to the ability of IGFBP-3 to prolong IGF-1 half-life. However, since IGFBP-3 is known to exert cellular actions independently of IGF-1, separate effects of IGFBP-3 on bone formation may also be involved. To explore the respective roles of IGF-1 and IGFBP-3 in regulating bone formation, we examined the relationship between serum levels of these proteins and bone mass acquisition in childhood. Serum concentrations of IGF-1 and IGFBP-3 were analysed in samples from 751 children randomly selected from the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort at age 7.9, and whole body bone mineral content (BMC) subsequently measured in 652 of these children at age 9.9, using a Lunar Prodigy DXA scanner. Serum levels of IGF-1 and IGFBP-3 were found to be significantly associated with BMC (standardised regression coefficient = 0.299 and 0.297 for IGF-1 and IGFBP-3 respectively, p < 0.001). Subsequent analysis suggested that these associations reflected influences on bone growth, since equivalent relationships of IGF-1 and IGFBP-3 were observed to height, weight and DXA-derived skeletal area. Moreover, when BMC was adjusted for DXA-derived skeletal area, a significant association with serum IGF-1 and IGFBP-3 was no longer observed. Multi-variable regression analysis was then carried out to determine whether the relationships of serum IGF-1 and IGFBP-3 to bone growth which we found are inter-dependent. Highly significant associations with parameters related to skeletal growth were still observed after adjusting IGF-1 levels for IGFBP-3, and IGFBP-3 levels for IGF-1 (data shows standardised regression coefficients):- 

Table  .  
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Moreover, no significant association was observed between these parameters and serum IGF-I/IGFBP-3 ratio. We conclude that serum levels of IGF-1 and IGFBP-3 are independent determinants of bone mass acquisition in childhood, suggesting that these two proteins regulate bone growth in children via distinct mechanisms.

Disclosures: J.H. Tobias, None.


Relationship Between Body Composition and Hip Bone Mass in Women of African Ancestry: Tobago Women's Health Study.D. D. Hill1, J. A. Cauley1, V. Wheeler*2, J. M. Zmuda1, A. Patrick*2, P. Joseph*2, C. Baker*1, G. Beckles*3, C. Bunker*1. 1U of Pitt, Pittsburgh, PA, USA, 2Tobago Regional Hospital, Scarborough, Trinidad and Tobago, 3CDC, Atlanta, GA, USA.

Aging is often accompanied by loss of lean mass and bone mass and an increase in fat mass. Studies examining the interrelationships between body composition and bone mass have primarily on Caucasian women and limited to areal bone measures. To test the hypothesis that lean mass is related to total hip and femoral neck bone mineral density (BMD), independent of fat mass in women of African ancestry, we recruited 355 postmenopausal women, aged 50+ on the Caribbean Island of Tobago.

Body composition and BMD were measured by DXA (Hologic QDR 4500W). We estimated volumetric femoral neck BMD by calculating bone mineral apparent density (BMAD). The mean age, BMI, weight, and height of the women were 63.3 ± 8.2 years, 30.5 ± 5.7 kg/m2, 81.1 ± 16.1 kg, and 163.1 ± 6.4 cm, respectively. In models considering soft tissue lean mass or fat mass separately, both were related to total hip and femoral neck BMD. We compared mean bone mass across tertiles of lean and fat mass, and tested for an interaction, adjusting for age and height using ANCOVA. Femoral neck BMD and BMAD increased across tertiles of lean mass, but not across fat mass. (Table 1.) We observed similar results for total hip BMD.

Our results suggest that fat mass is not significantly associated with bone mass in this population, after controlling for the effects of covariates. In conclusion, lean mass is independently related to bone mass in postmenopausal Tobagonian women of African ancestry. The stronger association between lean mass and BMD could reflect a multifactorial relationship encompassing bone strength, culture, lifestyle, genetics, hormonal, and growth factors.

Table Table 1.. Age and Height Adjusted Mean Femoral Neck BMD (g/cm2) and BMAD (g/cm3) across Tertiles of Lean Mass and Fat Mass
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Disclosures: D.D. Hill, None.


Growth “Tracking” of Femoral and Humeral Strength from Childhood to Late Adolescence.C. B. Ruff. Functional Anatomy and Evolution, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

The degree to which skeletal parameters “track”, or remain at the same level relative to other individuals during growth and development, has important implications for both early detection and future prediction of increased fracture risk. Several recent studies have indicated an apparently high level of tracking of bone mineral content (BMC), width (BW), area and/or density in children measured over periods ranging from 2 to 6 years. In this study growth tracking of femoral and humeral diaphyseal strength was assessed in a sample of children measured radiographically from infancy to late adolescence. Radiographs were obtained from the archives of the Denver Child Research Study, a longitudinal study carried out in the 1940's-1960's. 20 individuals, 10 males and 10 females, with almost complete radiographic records (average, 34.5 time points at 6 month or yearly intervals) were selected and measured. Periosteal and cortical breadths at midshaft of the femur and 40% of bone length from the distal end of the humerus were measured with sharp-tipped digital calipers, and used to calculate bone section moduli, measures of bending and torsional strength. Comparisons are presented here for a 10 year age interval, from 7 years of age to 17 years of age, representing early school-age to late adolescence. Spearman rank-order correlations of bone strength, within sex, between these two age endpoints are moderate (r=.45--.79), except for the femur in males (r=-.10). Correlations are higher for the humerus (r =.66--.79, p <.05) than for the femur (r=-.10--.45, n.s.). In 30% of the comparisons (12/40), individuals cross between bottom and top halves of the strength distributions between 7 and 17 years. These results argue for caution in accepting a high level of growth tracking in mechanically relevant measures of bone strength over extended time periods during pre-adult development. Previous studies of growth tracking that have included different time intervals have found lower correlations for more extended periods and for periods that include early adolescence or childhood in addition to later adolescence. The results presented here are consistent with these findings, and suggest that extrapolations from more limited time intervals to longer periods may be unwarranted. Thus, prediction of bone strength in late adolescence or adulthood from measures taken in earlier in development may be more problematic than previously indicated.

Disclosures: C.B. Ruff, None.


Bone Mineral Accrual During Childhood and Adolescence: An Analysis of Size-Correction Techniques.K. S. Davison*1, R. A. Faulkner2, D. Drinkwater*2, D. Bailey2. 1Medicine, McMaster University & University of Laval, Hamilton/Quebec City, ON, Canada, 2Kinesiology, University of Saskatchewan, Saskatoon, SK, Canada.

The true pattern of bone mineral density changes during childhood and adolescence is unclear owing to a lack of longitudinal investigations, a lack of adequate control for maturational differences, and size-mediated errors in the measurement of areal bone mineral density (aBMD). This investigation incorporated mixed-longitudinal (distance data) and longitudinal (velocity data) designs to describe pediatric changes in bone density at the total body (TB), femoral neck (FN) and lumbar spine (LS). Maturational differences were controlled by aligning participants on their age at peak height velocity (PHV). Changes in areal BMD (aBMD) were compared with densities obtained from two methods of size correction: bone mineral apparent density (BMAD) based on geometric assumptions, and statistically-corrected BMD (sBMD), which utilizes linear regression. Correlations between bone projected area (PA) and density were strongest with aBMD, intermediate with BMAD, and generally insignificant with sBMD, supporting sBMD's size independence. With the distance data aBMD increased over the entire growth period at all sites. Contrastingly, TB BMAD declined initially and then stabilized after PHV, and FN and LS BMAD were generally stable until PHV, increasing afterward. Similarly, TB and LS sBMD decreased until PHV, increasing afterward, and FN sBMD was stable until PHV, increasing thereafter. aBMD velocity was positive at all sites and all ages. In contrast, TB and FN BMAD had a negative velocity until after PHV, and LS BMAD velocity was generally stable until near PHV, with a positive velocity afterwards. sBMD velocity was negative at the TB and LS until PHV and there was a stabilization (males) or decrease in velocity (females) in FN sBMD until PHV. Velocity curves for PA and bone mineral content displayed a consistent dissociation at all sites with bones increasing in area first and later consolidating when rapid growth ceased or slowed. The point of minimal density suggested from the corrected data coincided with PHV and is supported by epidemiological data that reports the highest rates of fracture in adolescents during this time. These results highlight the size-dependence of aBMD and cautions against its use in the pediatric population. Physiologically, sBMD appeared the more appropriate size-correction technique.

Disclosures: K.S. Davison, None.


Vitamin D Status and Bone Accretion Profiles in Young Females from Childhood to Young Adulthood.N. E. Badenhop-Stevens*1, J. D. Landoll*1, E. Ha*1, P. Goel*2, B. Li*2, B. Hollis*3, L. Nagode*4, A. Clairmont*5, V. Matkovic1. 1Bone and Mineral Metabolism Laboratory, OSU, Columbus, OH, USA, 2Statistics, OSU, Columbus, OH, USA, 3University of South Carolina, Charleston, SC, USA, 4Veterinary Biosciences, OSU, Columbus, OH, USA, 5OSU, Columbus, OH, USA.

Effect of vitamin D insufficiency on bone mass accumulation during growth is currently unknown. To evaluate this relationship calcidiol blood levels and bone mineral areal density (BMD) of the proximal forearm (33% site) were measured in a cohort of young females (N=236), participants of a 7-year long randomized study with calcium supplementation from childhood to young adulthood. Blood samples were collected annually while bone measurements were done every 6 months. Serum 25(OH)D3 was measured by a radioimmunoassay (RIA) (excluding baseline) with 125I labeled tracer (Hollis et al. Clin Chem 39/3,529, 1993). BMD was measured by DXA, GE-Lunar DPX-L. Bone accretion profiles (linear mixed effect model-LME based on => 7 measuring points) versus yearssince-menarche (YSM) were used to assess the effects of vitamin D status (below and above the vitamin D threshold of 31.5 ng/ml) in subjects with cumulative calcium intake above (1494±292) and below (748±161) the median (1006 mg/day). There was no difference in BMD measurements between subjects with cumulative average vitamin D levels below and above the threshold in the high calcium intake group (p=0.88, difference in BMD at YSM 4=-0.70%). However, in the low calcium intake group, the difference between BMD profiles of subjects with cumulative calcidiol levels below and above the threshold was of borderline statistical significance (p=0.08). Furthermore, the subjects above the threshold had much denser bones at YSM 4 (2.7%). Given that the sample size for this subgroups comparison is small, the results of this research indicate that vitamin D status may play an important role with regard to bone mass accumulation in young females accustomed to a relatively low dietary calcium intake.

Disclosures: N.E. Badenhop-Stevens, None.


Forearm pQCT Measurements in Young Adult Women Accustomed to Different Calcium Intakes during Adolescence.J. D. Landoll*1, N. E. Badenhop-Stevens*1, E. Ha*1, S. L. Mobley*1, A. Clairmont*2, V. Matkovic1. 1Bone and Mineral Metabolism Laboratory, OSU, Columbus, OH, USA, 2OSU, Columbus, OH, USA.

We previously showed that calcium is an important determinant of bone mineral areal density during growth. In this study, we examined the influence of calcium on volumetric bone mineral density in the same cohort of young women (N=175; average age ∼18 y) at the end of a 7-year intervention study with calcium supplementation. Volumetric bone mineral density measurements of the non-dominant radius at the distal (4%) and proximal sites (33%) were performed using a pQCT (Norland-Stratec XCT 2000) densitometer with contour, peel, and separation modes of 2,7,2 (distal site) and 2,2,2 (proximal site), respectively. Calcium-supplemented individuals had higher volumetric density at both the distal radius (394±6 vs 383±6 mg/cm3), and proximal radius (1002±7 vs 986±6 mg/cm3), and higher cortical/total area (CA/TA) ratio (0.789±0.005 vs 0.782±0.005) than placebo subjects, however, none of the differences were statistically significant. Dividing the subjects into subgroups according to cumulative calcium intake over time (upper and lower tercile) revealed a significant influence of calcium on volumetric bone mineral density at the distal (404±6 vs 378±8 mg/cm3, p=0.013) and proximal (1009±7 vs 980 mg/cm3, p=006) sites, and CA/TA (0.794±0.006 vs 0.774±0.005, p=0.018). There were minimal differences in total cross sectional areas at the distal site (251±4 vs 265±5 mm2, p=0.03) between the upper and lower tercile subgroups, and no difference at the proximal site (92±1 vs 93±2 mm2, p=0.5). The above analysis confirmed previous findings, suggesting that calcium exerts its action on bone accretion primarily by influencing volumetric bone mineral density.

Disclosures: J.D. Landoll, None.


Bone Mineral Density by Age, Gender and Pubertal Stages in Healthy Lebanese Children and Adolescents.A. Arabi1, M. Choucair*2, M. Nabulsi*3, J. Maalouf*1, R. Vieth4, G. El-Hajj Fuleihan1. 1Calcium Metabolism and Osteoporosis Program, American University of Beirut, Beirut, Lebanon, 2Endocrinology, American University of Beirut, Beirut, Lebanon, 3Pediatrics, American University of Beirut, Beirut, Lebanon, 4Mt Sinai Hospital, Toronto University, Toronto, ON, Canada.

Gender, ethnicity and lifestyle factors affect bone mass acquisition during childhood, thus there is a need to have age and sex adjusted Z-scores using ethnic specific data for BMD measurement with DEXA. We have previously demonstrated that peak BMD is lower in healthy Lebanese compared to western standards. This cross-sectional study aims at obtaining normative data of BMD in healthy Lebanese children and adolescents.

363 children aged 10 to 17 years were enrolled in a randomized controlled trial evaluating the impact of vitamin D on musculoskeletal parameters. Data obtained at baseline is used in this study. Each child underwent physical examination including height, weight and Tanner staging of breast and genitalia. BMD and BMC were measured by DEXA using a Hologic 4500 A device. Apparent volumetric BMD (BMAD) of the lumbar spine and the femoral neck were calculated using the following formula: spine BMAD=BMC/A3/2 and femoral neck BMAD= BMC/A2, where BMC is the bone mineral content and A is the projected area. Low density software was applied to all analyses and subtotal body measurements were used. Children were subdivided into 8 subgroups of one year interval, and mean±SD of BMD, BMC and BMAD were given for each age group and Tanner stage, for boys and girls separately.

The mean age was 13.13±2.04 years. There was a significant effect of age and puberty on all bone parameters by one way ANOVA, except at the femoral neck BMAD in boys. At cortical sites, all bone parameters were higher in boys than in girls, whereas at the lumbar spine girls had higher values, including BMAD. Children who completed their puberty (Tanner V) had mean BMD about 40% higher at the lumbar spine and 20% higher at cortical sites than those who started their pubertal development (Tanner II). At age 17 years, the mean BMD of the lumbar spine were 9.8% lower in boys and 9.5% lower in girls than the peak BMD of the lebanese population. Except at the trochanter, the mean BMD were low compared to western normative values. Z-score ranged between -0.25 and -1.1 (p<10--4) in both sexes.

Data obtained from this study confirms previous reports on the powerful effect of puberty and gender on bone acquisition, and provide valuable reference enabling calculation of ethnic, gender, age, and Tanner adjusted Z- scores in our pediatric population

Disclosures: A. Arabi, None.


Leptin Negatively Predicts Total Body Bone Mineral Density in Young Healthy Women.C. W. Gunther*1, R. M. Lyle*2, G. P. McCabe*3, D. Teegarden1. 1Foods and Nutrition, Purdue University, West Lafayette, IN, USA, 2Health and Kinesiology, Purdue University, West Lafayette, IN, USA, 3Statistics, Purdue University, West Lafayette, IN, USA.

The role of leptin has been widely studied in relationship to body fat and obesity. However, more recently, leptin has been shown to have a significant impact on other biological endpoints, such as bone. For example, leptin has been implicated in the inhibition of bone mass accumulation. In this prospective analysis, the relationship of leptin with total body bone mineral density (TBBMD) was assessed in 132 healthy women, aged 18--30 years old, who were participating in a one year dairy calcium intervention trial. Fasting serum leptin levels (ELISA) and TBBMD (Lunar DXA) were measured at baseline and 12 months. Baseline (B0) means ± SD were: age=20.1±2.4 yrs, weight=62.4±10.3 kg, BMI=22.6±3.3 kg/m2, fat mass=18.5±7.4 kg, lean mass=39.8±4.4 kg, TBBMD=1.2±0.1 g/cm2, ln leptin=2.4±0.7 (ng/ml). Potential confounders (age, oral contraceptive usage, assignment to the dairy calcium intervention, and B0 and 1 year changes in weight, fat mass, physical activity, dietary calcium, and calories) were assessed. As expected, the 1 year change in fat mass was significantly correlated with the 1 year change in ln leptin (r=0.34, p<0.0001). The 1 year change in ln leptin negatively predicted the 1 year % change TBBMD in a regression model (R2=0.04, p=0.03) and remained significant when the 1 year change in fat mass was included in the model. Thus, increased serum leptin levels may contribute to a decrease in the accumulation of bone mass.

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Disclosures: D. Teegarden, None.


Cheese as a Source of Calcium and its Effects on Body Composition in Pubertal Finnish Girls.A. Lyytikäinen1, M. Narva*2, R. Korpela*2, C. Lamberg-Allardt3, M. Suuriniemi4, Q. Wang*1, S. Cheng1. 1Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland, 2Valio LTD, Helsinki, Finland, 3Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland, 4Department of Cell Biology, University of Jyväskylä, Jyväskylä, Finland.

The purpose of this study was to compare cheese (16% fat, 100 g/day) and calcium-tablets (calciumcarbonate, 500 mg Ca x 2/d) as a source of supplementation for calcium (1000 mg/d), and their effects on body composition in pre- and peripubertal Finnish girls. Altogether 149 girls aged 10--12 years at Tanner stage I-II were randomized into the tablet (n=75), cheese (n=38), and placebo group (n=36). The total bone mass (TBM), lean tissue mass (LTM), and fat mass (FM) were measured by DXA (Prodigy, GE Lunar). The food consumption and the intake of nutrients were evaluated with a three-day food record. At baseline, the mean intake of energy (1480/1480/1420 kcal/day), calcium (663/694/653 mg/day), protein (14/15/15 E%), and other nutrients, as well as the food consumption were similar between the groups. Neither body composition nor maturation status differed between the groups. No significant differences were found in the compliance between the groups (70/63/72 %). After the two-year intervention, the total calcium intake was 1623 in the tablet, 1335 in the cheese, and 915 mg/day in placebo group. The energy intake increased by 210/260/250 kcal/day in the groups. The tablet group was found to consume more cereals than the cheese and placebo group (p< .05). The energy intake was higher from protein (15/17/15 E%) in cheese group compared to the tablet and placebo group (p< .01), whereas the intake of saturated fatty acids (14/16/15 E%) was higher and intake of carbohydrates (53/49/51 E%) was lower in the cheese group compared to the tablet group (p< .01). There were no significant differences in the percentage increase of the TBM, LTM, and FM between groups. However, when taking into account the speed of maturation (Tanner stage at 24 months) and the compliance, the TBM of the girls at Tanner stage I at the baseline increased significantly in the cheese group compared to the tablet and placebo group (40 vs 36/35 %, p= 0.008). No differences were found in TBM between the girls at Tanner stage II. Our results indicate that adequate calcium intake by high cheese consumption (> 60 g/day) is more beneficial for the total bone mass accrual than the calcium from tablets with similar amount of calcium. This might be due to the high protein content in cheese or increased absorption of calcium induced by caseinphosphopeptides. Our results suggest that components in cheese protein has a vital role in bone mass accrual in growing girls transiting from pre- to peripuberty.

Disclosures: A. Lyytikäinen, None.


Comparison of Areal and Volumetric Vertebral Bone Densities Measured by DXA and CT in Pediatric Patients.A. Kovanlikaya*1, T. A. L. Wren2, X. D. Liu*1, P. D. Pitukcheewanont*3, V. Gilsanz1. 1Radiology, Childrens Hospital Los Angeles, Los Angeles, CA, USA, 2Orthopaedic Surgery, Childrens Hospital Los Angeles, Los Angeles, CA, USA, 3Endocrinology and Metabolism, Childrens Hospital Los Angeles, Los Angeles, CA, USA.

Dual-energy x-ray absorptiometry (DXA) values of areal bone mineral density (BMD) in children are influenced by bone size and soft tissue composition, while computed tomography (CT) measures of volumetric bone density (BD) are not. The purpose of this study was to determine the extent to which the assessment of osteoporosis differs when it is performed using DXA versus CT. DXA areal BMD and Z-score (ZDXA) and CT volumetric BD and Z-score (ZCT) were compared in 45 girls and 43 boys ages 3--20 yrs who were referred for assessment of bone density and osteoporosis. Z-score represents the number of standard deviations (SD) the BMD or BD is above or below the mean for sex- and age-matched controls, and patients were considered osteoporotic if they had a Z-score below - 2.5 (2.5 SD below normal). Plain radiographs of the spine were blindly and independently reviewed by two experienced pediatric radiologists. Of the 5 patients whose radiographs depicted osteoporosis, 5 had ZCT <=-2.5, and 4 had ZDXA <=-2.5. There was only a moderate correlation between the DXA and CT densities (r=.459, p=.0013 for girls; r=.439, p=.0029 for boys; r=.448, p<.0001 for all subjects) and between the DXA and CT Z-scores (r=.490, p=.0005 for girls; r=.537, p=.0001 for boys; r=.513, p <.0001 for all subjects). Paired t-tests indicated that the DXA Z-scores were systematically lower than the CT Z-scores (p=.0003 for girls; p=.0001 for boys; p<.0001 for all subjects). Nine of the 16 children with ZDXA <=-2.5 had ZCT between +1.0 and -1.0. Consequently, a substantial number of children (6/45 girls and 9/43 boys) who would not be considered osteoporotic based on CT measurements were classified as osteoporotic based on DXA results (see Table). Caution should therefore be exercised in using DXA to assess osteoporosis. Treatment for osteoporosis may not be warranted for many children who have DXA BMD values more than 2.5 SD below normal. 

Table  .  
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Disclosures: A. Kovanlikaya, None.


Micro-Architectural Strut Analysis Study on Paediatric Bone.S. Picard1, D. Brown*1, F. Rauch2, R. Travers2, F. H. Glorieux2, J. P. Brown1. 1Groupe de recherche en maladies osseuses, Centre de recherche du CHUQ - Pavillon CHUL, Ste-Foy, PQ, Canada, 2Shriner's Hospital for Children - Genetics Unit, McGill University, Montreal, PQ, Canada.

Bone quality is defined by a sufficient mineralization of tissue and also by an adequate micro-architecture organization. Quantitative trabecular bone micro-architecture studies from aged skeleton have already been reported but none on pediatric bones. The aim of this study was to perform strut analysis histomorphometric measurements on iliac crest biopsies from children aged 1,5 to 22,9 years. Bone biopsies from 54 healthy Caucasian subjects were obtained during surgery for various orthopaedic conditions. All subjects were ambulatory and no evidence of any metabolic bone disease was found. At least two Masson Goldner's stained slides were selected from each biopsy and JPEG images of the whole section were captured for quantification and measurement. Strut analyses were performed with Nova Prime Bioquant's (R&M biometrics, Nashville) image-analysis software. Trabecular node, free-end and node-to-free ratios have been quantified and calculated for each biopsy. Strut length has also been measured and defined as node-to-node, node-to-free, free-to-free, cortical-to-free, cortical-to-node and node-to-loop struts. The progressive increase in node number per tissue area and node-to-node strut percentage (p = 0.0013) associated with a significant decrease in free-node and free-to-free strut percentages (p = < 0.0001) show an architectural organizational change during skeleton growth. The results may suggest an architecture quality improvement which could contribute in the development of bone quality in growing children.

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Disclosures: S. Picard, None.


Fecal Calcium Density, Serum PTH, and 24-hr Urinary Calcium as Biological Indicators of Compliance with Calcium and Calcium Efficacy in a 7-year Clinical Trial.E. Ha*1, J. D. Landoll*1, L. Nagode*2, P. Goel*3, N. E. Badenhop-Stevens*1, B. Li*3, S. L. Mobley*1, A. Clairmont*4, V. Matkovic1. 1Bone and Mineral Metabolism Laboratory, OSU, Columbus, OH, USA, 2Veterinary Biosciences, OSU, Columbus, OH, USA, 3Statistics, OSU, Columbus, OH, USA, 4OSU, Columbus, OH, USA.

Effectiveness of biological indicators of calcium (Ca) nutritional status in predicting bone accretion at the proximal radius versus years-since-menarche (YSM; linear mixed effect models) was evaluated in a 7-year double blind placebo-controlled clinical trial with Ca supplementation (1000 mg/day) in addition to ∼830 mg habitual dietary Ca intake. The data from 236 subjects were analyzed as they had =>7 out of 15 measuring points. Subjects were divided into subgroups based on each cumulative biological indicator above and below the median. The biological indicators of compliance with Ca were: fecal Ca density (FCD, Ca mg/g dry stool weight), serum PTH, and 24 hour urinary Ca excretion. Bone mass measurement by DXA (GE-Lunar DPX-L) were conducted semiannually while fecal (excluding baseline), blood (drawn between 8AM and 5PM), and urine samples were collected annually. FCD was measured according to the method previously described (Heaney RP, 1991). Ca was measured by AAS. Serum PTH was measured by Allegro immunoassay kits (Nichols Institute). The difference in bone accretion profiles between FCD subgroups was highly significant (p<0.0001, difference in BMD at YSM 4=2.8%). Average cumulative FCD for the subgroup above the median was 47.3 mg/g while below the median 26.1 mg/g. The difference in bone accretion profiles between PTH subgroups was significant (p=0.035, difference in BMD at YSM 4=-1.3%). Average cumulative PTH concentration for the subgroup above the median was 31 pg/ml while below the median 19 pg/ml. Urinary Ca excretion was a poor predictor of Ca efficacy (p=0.564; difference in BMD at YSM 4=0.9%). Average urinary Ca excretion in the high urinary calcium subgroup was 147 mg/day and for the low urinary calcium subgroup 65 mg/day. The above research indicate that FCD is a superior marker of Ca nutritional status and compliance with Ca intervention in a clinical trial and serum PTH is a moderately good predictor.

Disclosures: E. Ha, None.


Compromised Skeletal Growth and Mineralization following a Period of Dietary Calcium Deficiency Are not Recovered in Prepuberal Pigs.D. K. Schneider*, C. E. Pardo*, K. L. Saddoris*, T. D. Crenshaw. Animal Sciences, University of Wisconsin, Madison, WI, USA.

Dietary Ca deficiency inhibits skeletal mineralization. The potential for recovery of skeletal growth and mineralization following a period of deficiency is not established. Failure to regain skeletal growth and mineralization during early growth stages may compromise peak bone mass. Twenty-four young pigs (6 kg, 3 wk old) were fed diets that differed in Ca (100 vs 70% of Ca requirements) for 4 wk. Both groups were then fed the same diet (108% of Ca requirements) for an additional 5 wk. Pigs were scanned using a Prodigy (GE Lunar) bone densitometer at 0, 2, 4, 6, and 9 wk of the trial. Skeletal mineralization was assessed by bone mineral density (BMD, g/cm2) and skeletal growth was assessed by bone mineral content (BMC, g). At 4 wk, mineralization (BMD) was reduced (P<0.001) by 12% (0.625 vs 0.550 g/cm2) and skeletal growth (BMC) was reduced (P<0.001) by >35% (256 vs 163 g). During recovery no differences were detected (P>0.50) in the rate of skeletal growth (BMC, g/d) during wk 4 to 6 (16.9 vs 16.6 g/d) or wk 6 to 9 (21.4 vs 21.2 g/d). The approximately 100 g differences in skeletal growth induced by dietary Ca during the first 4 wk was maintained until wk 9 (BMC, 942 vs 840 g). Likewise, no evidence was detected for recovery of skeletal mineralization. BMD at wk 9 (0.889 vs 0.838 g/cm2) was lower (P<0.02) for pigs previously fed Ca deficient diets. In conclusion, a 35% reduction in BMC of rapidly growing pigs was not compensated during a recovery period that allowed a fourto five-fold increase in total skeletal growth. These results do not support an improvement in efficiency of Ca use following a period of Ca depletion.

Disclosures: T.D. Crenshaw, None.


Comparison of TGF-beta/BMP Pathways Signaled by BMP-2 and Demineralized Bone Powder in Human Dermal Fibroblasts.J. Glowacki, S. Zhou, K. E. Yates. Orthopedic Surgery, Brigham and Women's Hospital, Boston, MA, USA.

Both BMP-2 and Demineralized Bone Powder (DBP) stimulate endochondral osteogenesis in vivo. When human dermal fibroblasts (hDFs) are cultured with DBP in a porous collagen sponge for 7 days, cartilage-specific genes, aggrecan and collagen II, are upregulated and cartilage matrix accumulates around the cells in proximity to the DBP. We reported that this chondroinduction is preceded by shifts in some signaling genes. This study tests the hypothesis that DBP and BMP-2 affect similar signal pathways.

One million hDFs were cultured for 3 days in each porous 3D collagen sponge containing 0 or 3 mg DBP, or 6 μg/4 μL rhBMP-2 or PBS/BSA solution (manufacturer's recommended conc.) The rhBMP was delivered in a 4×4×2 mm coupon of the absorbable collagen and inserted into the porous collagen sponges. The absorbable collagen and rhBMP-2 were generously provided by Wyeth, Cambridge, MA. After 3 days, specimens were prepared for histology, for cDNA macroarrays for BMP/TGFb Signaling Pathways (SuperArray HS-023), RT-PCR, and Northern hybridization.

Histology showed homogeneous cellularity through each of the sponges at day 3. Similarities and differences in gene expression were observed (Figure), confirmed by RT-PCR. First, there were very similar responses of some genes, such as IGFBP3, ID2, and ID3, to DBP and rhBMP-2. This finding suggested that the dosing and timepoint were apt. In contrast, some of the genes that were most dramatically increased by DBP, such as TGFβ-Induced protein (1260%) and Collagen 3A1 (1770%), were barely affected by rhBMP-2. Although there was concordance with ID2 and ID3, ID4 was decreased by DBP by 60% and by rhBMP-2 by only 10%. Both PAI-1 and TIMP1 were greatly increased by DBP, but PAI-1 was decreased by rhBMP-2 (30%) and TIMP was not at all changed. DBP increased cyclin-dependent kinase inhibitor P21/Waf1/Cip1 by 260%, whereas it was decreased (30%) by rhBMP-2. It is possible that DBP's inhibition of proliferation may contribute to its effects to promote differentiation. Cbfa1 was highly expressed in target hDFs but was moderately decreased by both DBP (10%) and rhBMP-2 (20%). Smad 6 and 7 were increased by only rhBMP-2, but smad 2--5 and 9 were low in hDFs and were not modulated by either BMP-2 or DBP. In sum, although BMP was originally isolated as the active factor in DBP, rhBMP-2 alone and DBP do not affect all the same genes.

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Disclosures: J. Glowacki, None.


Intraosseous Delivery of rhBMP-2 in HYAFF-11/P65 Paste Rapidly Increases Trabecular Bone Mass in Nonhuman Primates.E. A. Smith-Adaline, H. J. Seeherman, X. J. Li, H. Kim*, R. Li*, M. L. Bouxsein, J. M. Wozney. Women's Health and Bone Division, Wyeth Research, Cambridge, MA, USA.

Recombinant BMP-2 (rhBMP-2) is a potent osteoinductive factor with the potential to lower the risk of hip and wrist fractures in osteopenic patients by inducing dramatic anabolic bone formation following local injection. However, previous research has shown that local administration of rhBMP-2 can result in transient bone resorption that precedes bone formation (Seeherman HJ, et al., ASBMR 2001). This study evaluated the efficacy of intraosseous (IO) delivery of rhBMP-2 to increase bone mass locally when co-administered with bisphosphonates (BP) to block the initial resorption phase. One distal radius of ten adult male cynomolgus monkeys received a single 0.25ml IO injection of 2mg/ml rhBMP-2 delivered in a hyaluronan paste (HYAFF-11/p65, Fidia Advanced Biopolymers) (BMP/p65). The contralateral radius was not treated. Animals were divided into groups receiving 1) no BP (n=4), 2) oral alendronate (2.7mg daily for 4 weeks, n=3), or 3) IV zoledronate (0.067mg/kg weekly for 4 weeks, n=3). Four weeks after BMP/p65 injection, trabecular BMD decreased 28.3±13.2% in animals not receiving BP. While oral alendronate was not effective (δBMD = -22.9±8.3%), IV zoledronate eliminated bone resorption at 4 weeks (δBMD = +1.6±7.5%) (Fig. 1). Trabecular BMD (pQCT) increased 42.3±15.1% and 45.8±17.8% by 4--6 months respectively in all groups after BMP/p65 treatment compared to baseline values (Fig. 1). In contrast, BP had no effect on BMD of the contralateral untreated radius (6 mo δBMD = -0.5±6.4%). Histological evaluation demonstrated both de novo and appositional bone formation in response to BMP/p65. Abundant osteoid present on bone surfaces 6 months after treatment indicated an ongoing anabolic effect of BMP at this time. Additional studies in OVX female cynomolgus monkeys demonstrated that a single injection of zoledronate (0.067mg/kg, IV) administered 3 days after IO injection also minimized BMP-induced bone resorption. In summary, local administration of BMP/p65 resulted in a rapid 46% increase in trabecular BMD, and coadministration with zoledronate eliminated the transient resorption phase. This combination treatment represents a promising anabolic therapy for prevention of osteoporosis-related hip, wrist and spine fractures.

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Disclosures: E.A. Smith-Adaline, Wyeth 1, 3.


Osteoinduction by Genetically Modified Ligamentum Flavum With Human BMP-2 Gene.K. Yang*, S. Moon*, H. Kim*, K. Kim*, U. Kwon*, H. Kim*, J. Jahng, H. Lee*. Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Republic of Korea.

Ossification of spinal ligament can be found in certain pathologic conditions. Recently spontaneous expression of osteogenic phenotype from degenerated ligamentum flavum (LF) was demonstrated in vitro study. Furthermore in vivo application of recombinant human bone morphogenetic protein-2 (BMP-2) to spinal ligament resulted in ossification of ligament. Biologically modified LF with human BMP-2 gene can be an agent for osteoinduction. Therefore, we implanted genetically modified LF to nude mice to test feasibility of LF as bone graft substitution and a carrier for ex vivo gene therapy.

LF tissue was harvested and cultured. Type 5 adenovirus constructs with lacZ and BMP-2 cDNA (Ad/lacZ, Ad/BMP-2) were prepared. LF cell cultures were exposed to Ad/lacZ and stained with X-gal. Also cultures were exposed to Ad/BMP-2 and stained with alkaline phosphatase, Alizarin Red-S, Von Kossa method. Transgene expression and expression osteocalcin were assessed by immunocytochemistry and Western blot with BMP-2 and osteocalcin antibody. RT-PCRs for mRNA expression of collagen type I, II and osteocalcin were also performed. For in vivo assessment of osteoinductivity by Ad/BMP-2, control group was injected saline and osteoinductive group was injected 1×1011 particle to human donor LF tissue, which were implanted to immune-deficient mice. At 1 and 2 month postimplantation, all animals were sacrificed for histological evaluation. Implant tissue were isolated and immunohistochemical staining with osteogenic marker antibody.

Ad/lacZ efficiently transduced LF cells. Culture with Ad/BMP-2 demonstrated transgene expression (BMP-2), expression of osteocalcin in immunocytochemisty and Western blot. Culture with Ad/BMP-2 showed strong reactivity in alkaline phosphatase, Alizarin Red-S, Von Kossa stain. In RT-PCR, cultures with Ad/BMP-2 showed upregulation of mRNA expression for collagen type I and osteocalcin. Newly bone formation was observed in the section from the Ad/BMP-2/hLF tissue implanted side while no bone formation was detected on the control side.

This study demonstrated feasibility of gene transfer to LF and also osteoinductive gene transfer to LF clearly upregulated osteogenic phenotype and induced osteogenesis. In the surgery of spinal stenosis, large amount of LF is removed and destabilized spinal segment is often restabilized with instrumented fusion with autogenous bone graft. The results of this study imply that removed LF can be a carrier for ex-vivo gene therapy and an osteoinductive agent to augment autogenous bone graft through genetic modification.

Disclosures: S. Moon, None.


Prostaglandin E EP4 Receptor Agonist Enhances Ectopic Bone Formation Induced by rhBMP-2.H. Terai, R. Sasaoka*, H. Toyoda*, Y. Imai*, R. Sugama*, K. Takaoka. Orthopedic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan.

For clinical use, it is important to establish methods to maximize the effects of recombinat human bone morphogenetic protein- 2 (rhBMP- 2). In the present study, we investigated the synergic effects of Prostaglandin E EP4 receptor agonist (ONO-4819) in ectopic bone formation induced by rhBMP-2 in mice.

Forty lyophilized disks (6mm diameter) of bovine type I tendon- derived collagen containing 5 μg of rhBMP-2 were implanted respectively on the paravertebral muscle of 4 weeks old ICR mice. These mice were divided into four groups (n=10 per each group) according to the single administrated concentration of Prostaglandin E EP4 receptor agonist; (1) 0 μg/ kg, as controls (2) 10 μg/kg (3) 30 μg/kg (4) 100 μg/kg. Prostaglandin E EP4 receptor agonist was injected subcutaneously every 8 hours after implantation until sacrifice. After 3 weeks of implantation, all mice were sacrificed and disks and left tibias were harvested. Soft X- ray photographs were taken for all specimens. All harvested disks and tibias were received histological analysis after measurements of bone mineral density (BMD) and bone mineral content (BMC) by dual energy X-ray absorption (DEXA). In another experimental protocol, serum osteocalcin, phosphate, calcium and ALP were measured under the administration of Prostaglandin E EP4 receptor agonist without rhBMP- 2. Forty- five mice were divided into 3 groups (n=15 per each) according to the administrated concentration and the interval; (I) 0 μg/kg, every 8 hours, as controls (II) 30 μg/kg, every 8 hours (III) 90 μg/kg, every 24 hours. Five mice were sacrificed every week in each group to collect blood samples.

BMD values (mg/cm2) of group 4 (15.7±4.1) and group 5 (16.8±4.5) were significantly higher than in the group 1 (8.1±2.2) (p< 0.01). BMC value (mg) of group 4 (7.9±2.8) was significantly higher than in the group 1 (4.4±3.6) and group 2 (3.9±2.1) (p< 0.01). No difference was observed in BMD or BMC values of tibiae among any groups. Serum osteocalcin and ALP were significantly higher in group II at 2 weeks (p< 0.05). We concluded that Prostaglandin E EP4 receptor agonist enhanced the ectopic bone formation induced by rhBMP- 2 in mice. The optimal administrative dose was 30 μg/kg when injected subcutaneously every 8 hours, in which dose there was no effect to BMD or BMC of tibiae. 

Table  . BMC and BMD of implanted collagen disks
  1. (* : p< 0.01)

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Disclosures: H. Terai, None.


Regulation of BMP Signaling by Smurfs.T. Imamura*, S. Maeda*. Biochemistry, The JFCR Cancer Institute, Tokyo, Japan.

Smurf1, a HECT type E3 ubiquitin ligase, has been reported to interact with receptor-regulated Smads (R-Smads) for bone morphogenetic proteins (BMPs), Smads 1 and 5, and promotes their degradation. Here we show that, in addition to direct binding to BMP-R-Smads, Smurf1 also interacts with inhibitory Smads (I-Smads), Smads 6 and 7, to form E3 ubiquitin ligase complexes to target BMP type I receptors (BMPR-Is) and activated BMP-R-Smads for degradation. Smurf1 interacts with nuclear I-Smads and induces their translocation to the cytoplasm in a nuclear export receptor (CRM1)-dependent fashion. Smurf1 then associates with BMPR-Is, and enhances turnover of the receptors. Furthermore, Smad6-Smurf1 complex binds to activated BMP-R-Smads, and induces their ubiquitination and degradation. Consistence with these data, Smurf1 synergies with Smad6 to inhibit BMP signaling in Xenopus. These results elucidate novel mechanisms whereby I-Smads-Smurf1 complexes inhibit BMP signaling both in vitro and in vivo, by mediating downregulation of activated BMPR-Is and activated BR-Smads.

Disclosures: T. Imamura, None.


Use of LMP-1 Fusion Protein to Induce Bone Formation without Risks of Gene Therapy.S. Sangadala*, L. Titus, M. Viggeswarapu, Y. Liu, G. A. Hair*, E. Gibson*, C. Oliver*, S. D. Boden. Orthopaedic Surgery, Emory University, Decatur, GA, USA.

LMP-1, an intracellular osteoinductive protein, can induce multiple BMPs and facilitate spine fusion in vivo. Induction of bone formation by LMP-1 has required gene therapy techniques to deliver its cDNA. The goal of this study was to determine the feasibility of delivering a recombinant LMP-1 fusion protein directly into cells. The fusion protein includes an 11 amino acid cationic transduction domain from the HIV TAT protein that facilitates entry into cells without the need for chemicals or electrical stimulus. Phase I: Athymic rats (N=58) received 3--4 subcutaneous (SQ) implants consisting of a collagen disc loaded with 1--1.5 million buffy coat cells from rabbit, human or non-human primate peripheral blood. The cells were treated with TAT-LMP-1 fusion protein (0.08 to 200 nM) for 30 min before implantation. Phase 2: 46 adult New Zealand white rabbits underwent a posterolateral lumbar spine fusion with a collagen sponge loaded with 6 million autologous buffy coat cells per side after 30 min treatment with TAT-LMP-1 protein (0.25 to 60 nM). Spines and SQ implants were harvested after 4 weeks and assessed by radiographs for bone formation. In rats bone nodules were formed in 2 experiments with the non-lyophilized TAT-LMP-1 protein at doses of 0.6 nM (4/4 implants), 1.2 nM (4/4), or 1.6 nM (2/3). The lyophilized protein appeared more stable and made bone in 3 experiments at 2.5 nM (11/11) when rabbit buffy coat cells were used. In addition human and non-human primate cells transduced with TAT-LMP-1 induced bone formation at 20 nM (6/6, human; 4/6, non-human primate). The challenging rabbit posterolateral spine fusion model also demonstrated the successful use of recombinant TAT-LMP-1 protein. Palpable fusion masses were formed in 2 experiments with the non-lyophylized TAT-LMP-1 protein at 5.0 nM (3/3) or 5.5 nM (2/2). The lyophilized TAT-LMP-1 protein was successful in 3 experiments at 5.0 nM (7/8) and in 2 experiments at 5.5 nM (5/5). In all of the above experiments higher and lower doses of TAT-LMP-1 had a lower rate of success or failed to induce bone formation. This study shows that a short peptide sequence (TAT) added to recombinant LMP-1 protein will facilitate the entry of the protein itself directly into buffy coat cells and initiate ectopic bone formation in rats and spine fusions in adult rabbits. Induction of bone formation by rabbit, human, or non-human primate buffy coat cells transduced with TAT-LMP-1 allows the ectopic rat model to be used to optimize delivery of TAT-LMP-1 prior to spine fusion studies in these species. This approach can avoid the challenges of gene therapy for LMP-1 delivery to induce bone formation.

Disclosures: L. Titus, Medtronic Sofamor Danek 2, 5.


Bone Formation Induced by Bone Morphogenetic Protein Is Stimulated by Prostaglandin E EP4 Receptor Agonist in its Early Phase.T. Hiromitsu*, H. Terai, R. Sasaoka*, Y. Imai*, R. Sugama*, K. Takaoka. Orthopedic Surgery, Osaka City Univerity Graduate School of Medicine, Osaka, Japan.

Administration of Prostaglandin E EP4 agonist (EP4, ONO-4819) enhances ectopic bone formation induced by recombinant human bone morphogenetic protein (rhBMP-2) in mice. It is important to investigate the mechanism by which Prostagrandin E EP4 receptor agonist regulates bone formation induced by rhBMP-2. In this study, we examined the effects of Prostaglandin E EP4 agonist in vivo and in vitro to determine the phase of its action in BMP inducing bone formation.

Mouse MC3T3-E1 cell line and ST-2 cell line were grown in 24- well plates with alpha MEM containing 10% fetal bovine serum. Cells were exposed to various concentration of Prostaglandin E EP4 agonist with or without BMP when they were reached to 60% of confluence. After 3 days of incubation, Alkaline phosphatase (ALP) activity was measured in each well. ALP activity values were analyzed statistically. In another protocol, 15 lyophilized disks (6 mm diameter) of bovine type I tendon- derived collagen containing 5 μg of rhBMP- 2 were implanted respectively on paravertebral muscle of 4 weeks old ICR mice. All mice were received 30 μg/kg of EP4 subcutaneously every 8 hours until sacrifice (3 weeks after implantation). These mice were divided into 3 groups (n=5 in each group) by administrated period of EP4; Group (I) for 3 weeks after implantation, Group (II) for 2 weeks (from 1 week after implantation), Group (III) for 1 week just prior to sacrifice (2weeks after implantation). After 3 weeks of implantation, all disks were harvested and examined bone mineral content (BMC) and bone mineral density (BMD) by dual energy X- ray absorption (DEXA).

Administration of EP4 to ST2 cell lines increased ALP activity levels in dose- dependent manner in the presence and absence of BMP. However, ALP activity levels in 3T3E1 cell lines were not increased in dose- dependent manner in the absence of BMP. In vivo study, the values of BMC and BMD of specimens in Group (I) were significantly higher than in other groups (See table). We concluded that Prostaglandin E EP4 receptor agonist enhanced ectopic bone formation induced by BMP by stimulating osteogenic cells in the early stage of bone formation. 

Table  . BMC and BMD of implanted collagen disks
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Disclosures: T. Hiromitsu, None.


Durable Adeno-Associated Virus Delivery of a Systemic Noggin Mutein Prevents BMP Induced Heterotopic Ossification.D. L. Glaser*1, A. N. Economides*2, L. Wang*3, K. V. Sachs*1, N. Stahl*2, J. M. Wilson*3, F. S. Kaplan1, E. M. Shore1. 1Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA, USA, 2Regeneron Pharmaceuticals, Tarrytown, NY, USA, 3Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Heterotopic ossification (HO) occurs in a wide variety of diseases, and prevention regimens have had varying success. In patients with fibrodysplasia ossificans progressiva (FOP), a catastrophic genetic disorder of HO with no available treatment, evidence supports an imbalance in the BMP4 pathway with inductive signals overwhelming antagonist signals. We previously demonstrated that a circulating mutein of Noggin (hNOGδB2), a BMP antagonist, delivered by an adenovirus gene therapy approach was effective in preventing BMP4 induced HO, although transient viral expression with immunogenicity was observed. To develop an effective gene therapy for HO, sustained and controllable expression is required. This study investigated AAV-mediated gene transfer of systemic Noggin mutein for durable expression that is sufficient to prevent induced HO.

A mouse model of BMP-induced HO was used. C57/B16 mice (5 groups of 10) were treated with control virus, or one of two serotypes of AAV (2/5 or 2/8TBGhNOGδB2) at either 1×1011 or 1×1010 GC/mouse. Serum Noggins level were monitored by ELISA at 1, 7, 14, 21 and 28 days. On day 28, the abdominal musculature was injected on one side with carrier alone, and on the other with carrier + BMP4. Serum Noggin levels and implants were studied at 7 and 14 days after implant. Histologic stages of bone formation were evaluated by standard methods.

Implants containing BMP4 induced an aggressive, fibroproliferative lesion with cartilage formation at 7d and HO at 14d. In animals treated with AAV 2/8TBGhNOGδB2 at a viral titer of 1×1011, implants with BMP4 demonstrated a minimal, mixed inflammatory cell infiltrate at 7d and a thin pseudocapsule several cell layers thick surrounding the unresorbed plug at 14d, indistinguishable from carrier implants with no BMP. HO was evident in low titer AAV 2/8, high and low titer AAV 2/5, and control groups. Serum Noggin levels were detected as early as 7 days after viral injection. By 24d, Noggin levels of <1ug/ml were detectable in the 3 AAV groups that formed HO, while, in the therapeutic group, Noggin levels ranged from 9 ug/ml to 21 ug/ml.

This study demonstrates that delivery of a systemic Noggin mutein through AAV gene therapy provides durable gene delivery at therapeutic levels, suggesting that gene transfer of a circulating BMP4 antagonist may offer a solution to catastrophic disorders of progressive HO when all other modalities have failed.

Disclosures: D.L. Glaser, None.


Deficiency of Caf1, a Novel Type Inhibitor of BMP, Promotes Nodule Formation in vitro, Enhances Cancellous Bone Mass and Promotes OrthotopicBbone Formation in Response to BMP Injection in vivo.K. Oikawa1, T. Nakamura*2, M. Usui1, K. Tsuji1, I. Ishikawa*3, A. Nifuji1, T. Noda*4, T. Yamamoto*2. 1Dept of Molecular Pharmacology, Tokyo Medical and Dental University, Tokyo, Japan, 2University of Tokyo, Tokyo, Japan, 3Periodontology, Tokyo Medical and Dental University, Tokyo, Japan, 4Dept of Cell Biology, Cancer Institute, Tokyo, Japan.

Caf1 (CCR4-associated factor 1), which is a component of CCR4-NOT complex, is involved in gene transcription and mRNA modulation in yeast. In mammal, Caf1 associates with Tob, which belongs to Tob/BTG antiproliferative protein family. Since Tob gene product could modulate BMP signaling, Caf1 may also be involved in the regulation of BMP. This paper examined whether Caf1 deficiency would modulate BMP signaling in vitro and bone mass levels in vivo. Skeletal patterns in Caf1-deficient (-/-) mice and heterozygous littermates (Caf1+/−) were similar to those in wild type mice. Bone mineral density (BMD) of the whole femur and tibia was similar among Caf1-/-, Caf1+/− and wild type mice. However, microCT analysis of the cancellous bone volume revealed enhancement of bone volume per tissue volume (BV/TV) in Caf1-deficient mice compared to heterozygous mice. The bone volume levels in heterozygous mice were similar to those in wild type mice and therefore heterozygous littermate mice were used as control for further analyses. In order to examine the osteoblastic properties of the cells, bone marrow cells were cultured in the presence or absence of betaglycerophospate and ascorbate. Caf1 deficiency enhancement of the alizarin red positive nodule formation compared to wild type. In contrast, TRAP-positive osteoclast like cell development in the bone marrow cells was similar regardless of the genotypes suggesting that Caf1 deficiency mainly affects the cells in osteoblastic lineage. Further examination indicated that Caf1 deficiency enhanced BMP-stimulated alkaline phosphatase activities in the calvaria-derived osteoblast enriched cells compared to wild type. Caf1 deficiency did not alter the actions of TGF-beta. To test whether BMP signaling could be enhanced by Caf1 deficiency in vivo, BMP was injected onto the calvaria of the mice. BMP injection induced new bone formation on the calvaria in wild type. Caf1 deficiency increased the amount of BMP-induced new bone formation compared to wild type. These observations indicated that Caf1 is a novel type of inhibitor of BMP actions in osteoblasts and suppresses the levels of cancellous bone volume in vivo.

Disclosures: K. Oikawa, None.


Necessity of Immune Recognition and Control of a Human Adult Stem Cell, Circulating in Blood as a Monocyte, in order to Avoid some Proliferating Diseases.M. Labat. Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France.

Damage to stem cells has been reported to contribute to neoplasia. Organ stem cells present in blood might represent one single population of pluripotent stem cells in homeostatic equilibrium with the ‘reserve’ stem cells present in the organs. These circulating stem cells are normally almost quiescent. Under precise circumstances such as wound healing they may proliferate and migrate in order to participate in the regeneration of the damaged tissue. Indeed, such a process has to be tightly controlled. Time-lapse videomicroscopy shows how a subpopulation of CD4+ T lymphocytes, called phagic T lymphocytes, destroy the stem cells when they differentiate in vitro. These stem cells that express constitutively HLA-DR molecules are both the activators and the targets of phagic T lymphocytes that penetrate and circulate inside them until the stem cells ‘explode’. It is a beneficial exception to self-tolerance, restricted to normal stem cells, in order to avoid their accumulation out of a repair purpose. In disorders such as fibrosis and chondrosarcoma, these circulating stem cells proliferate, escape destruction by phagic T lymphocytes and accumulate, giving rise in vitro to a ‘tissue’ evoking the lesion the patient. This process observed in vitro may mimic what happens in vivo at the blood-tissue interface. Hence, failure in the regulation of circulating organ stem cells might be involved in the common early steps leading to fibrosis and some malignancies. On the opposite, excessive activation of phagic T lymphocytes might be involved in autoimmune diseases.

The spontaneous expression by normal circulating stem cells of neural markers, including nestin, and of a specific neural crest marker, suggest a neural crest origin.

Disclosures: M. Labat, None.


Role of Twisted Gastrulation (Tsg) in Osteoblast Differentiation and Mineralization.R. Gopalakrishnan1, N. P. Lowe*1, A. Petryk*2. 1Oral Sciences, University of Minnesota, Minneapolis, MN, USA, 2Pediatrics, University of Minnesota, Minneapolis, MN, USA.

Bone morphogenetic proteins (BMPs) play crucial roles in osteoblast differentiation and function. The activity of BMPs in the extracellular matrix of osteoblasts has previously been shown to be regulated by their antagonist, Noggin. Twisted Gastrulation (TSG) is a recently identified protein that also interacts with BMPs, although the nature of the interaction is not completely understood. TSG initially forms a complex with BMP and full-length Chordin to antagonize BMP signaling. However, following cleavage of full-length Chordin by Xolloid, TSG acts as a BMP agonist by dislodging BMP from Chordin and destabilizing the Chordin fragments, thereby facilitating BMP binding to its receptor. The specific role of TSG in osteoblast differentiation and function is not known. In this study, we investigate the role of TSG during osteoblast differentiation and mineralization in vitro. Using a strongly mineralizing subclone of MC3T3-E1 cells, we examined the effect of TSG on mineralization. MC3T3-E1 cells were allowed to differentiate (with ascorbic acid) for 10 days in the presence or absence of TSG. TSG treatment during differentiation of osteoblasts completely inhibited mineralization. To determine if the inhibitory effect of TSG on mineralization is related to differentiation, we will examine the mRNA expression of osteoblast differentiation markers such as bone sialoprotein, osteocalcin, alkaline phosphatase, type I collagen and Runx2/Cbfa1 following TSG treatment. Further, we will also examine the effect of TSG treatment on the expression of BMP-2, -4, -7, Noggin, Chordin, Xolloid and TSG in differentiating MC3T3-E1 cells in order to understand the interactions between BMPs, TSG, and known BMP antagonists. To conclude, we have demonstrated that TSG blocks mineralization in MC3T3-E1 cells. Following completion of our proposed experiments, we will begin to understand the role of TSG during osteoblast differentiation and explain the mechanisms involved in its ability to inhibit mineralization.

Disclosures: R. Gopalakrishnan, None.


A Conditional Null Allele for the BMP Antagonist Noggin Using a Novel Method for Generating Conditional Knock-Out Mice.E. Gazzerro*1, E. Canalis1, X. Wang*2, R. Raz*2, W. Auerbach*2, L. Brunet*3, M. Boucher*2, D. Frendeway*2, A. J. Murphy*2, T. M. DeChiara*2, D. M. Valenzuela*2, R. M. Harland*3, G. D. Yancopoulos*2, A. N. Economides*2. 1Department of Research, St. Francis Hospital and Medical Center, Hartford, CT, USA, 2Regeneron Pharmaceuticals, Inc, Tarrytown, NY, USA, 3Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.

Noggin is a secreted protein that acts as an extracellular antagonist of bone morphogenetic proteins (BMPs). Noggin binds to its cognate BMPs and as a result inhibits binding to their receptors. The importance of these interactions is exemplified by the following evidence: (a) osteogenic BMPs induce expression of their antagonists, presumably to maintain a balance between instructive and inhibitory signals; (b) overexpression or lack of expression of the antagonists lead to profound phenotypic effects: overexpression of noggin causes severe osteopenia whereas noggin null mice display abnormal skeletogenesis and embryonic lethality. Since BMPs and noggin have multiple biological roles pre- and postnatally, it is not surprising that noggin null mice display multiple phenotypic defects and die before birth. Thus it is not possible to dissect the role of BMP inhibition by noggin postnatally. This would be particularly important for understanding the role of BMPs in adult organisms. To this purpose, we generated a Cre-dependent conditional null allele for noggin, using VelocigeneTM, a novel method for rapidly generating genetically modified alleles, ES cells, and mice. LoxP sites were placed within the single exon noggin gene so that noggin expression would not be affected before Cre-induced recombination. After the noggin gene polyadenylation site, but within the floxed region, a PGK/Neo cassette was placed, followed by an open reading frame (ORF) encoding enhanced green fluorescent protein (eGFP) cloned after the 2nd LoxP site. After exposure to Cre recombinase, the ORF of noggin will be deleted and replaced by eGFP, expression of which will indicate deletion of the floxed sequence. The resulting floxed noggin allele was tested in culture. Cos7 cells were transiently transfected with a pcDNA/floxed noggin construct and were shown to secrete noggin protein by Western Immunoblot analysis -- they did not express eGFP. Cotransfection of pcDNA/floxed noggin with a Cre vector resulted in expression of eGFP, indicating appropriate recombination and deletion of the gene of interest. Targeted ES cells carrying the floxed allele were generated and after similar testing were used to generate floxed noggin mice. In conclusion, we present a novel methodology to engineer conditional single exon gene knock-out mouse models.

Disclosures: E. Gazzerro, None.


BMP-2 Controls Cardiomyocyte Contractility by Activating Phosphatidylinositol 3 Kinase Signaling Pathway.N. Ghosh-Choudhury1, B. Chandrasekar*2, S. L. Abboud1, G. Ghosh Choudhury*2. 1Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, 2Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

Cardiac development during vertebrate embryogenesis is critically dependent on the activity of bone morphogenetic protein-2 (BMP-2). We have previously reported that BMP-2 activates phosphatidylinositol 3 kinase (PI 3 K) in cardiomyocyte precursor cell line P19 CL6 (CL6). To determine the role of PI 3 K in BMP-2-induced cardiomyocyte differentiation, we examined the expression of sarcomeric myosin heavy chain (MHC), a marker of mature cardiomyocyte, in CL6 cells. BMP-2 significantly increased expression of MHC, as determined by anti-MHC immuofluorescence, suggesting formation of mature cardiomyocytes. Ly294002, a pharmacological inhibitor of PI 3 K, blocked BMP-2-induced PI 3 K activity, resulting in inhibition of MHC expression. To confirm the involvement of PI 3 K in BMP-2-induced cardiomyocyte differentiation, we used an adenovirus vector containing the constitutively active p110 catalytic subunit of PI 3 K (Ad-Myr-p110). Immunoblot analysis of lysates of CL6 cells infected with Ad-Myr-p110 showed increased expression of constitutively active PI 3 K catalytic subunit, resulting in significant expression of sarcomeric MHC in the absence of BMP-2. These data indicate that PI 3 K positively regulates differentiation of cardiac precursor cells into MHC-expressing mature cardiomyocyte in response to BMP-2. To further understand the functional implication of our finding, we studied the role of BMP-2 in cardiomyocyte contractility, loss of which in many heart diseases results in cardiac dysfunction. BMP-2 significantly increased PI 3 K activity in freshly prepared rat ventricular myocytes with concomitant increase in fractional shortening, a measure of contractility of these cells. Treatment of these primary cells with Ly294002 prior to BMP-2 addition completely inhibited BMP-2-induced PI 3 K activity and contractility. Expression of constitutively active PI 3 K, using adenovirusmediated gene transfer, resulted in significant increase in cardiomyocyte contractility in the absence of BMP-2. These data for the first time show the role of BMP-2 in cardiomyocyte contractility. Furthermore our data provide the first evidence that PI 3 K regulates cardiomyocyte differentiation and function. Together our data demonstrate a plausible use of BMP-2 in diseases, where cardiomyocyte differentiation and contraction are impaired.

Disclosures: N. Ghosh-Choudhury, None.


The Relations between Lumbar Spine, Hip, Distal Femur and Proximal Tibia Bone Mineral Density in Healthy Individuals.K. Beattie*1, P. Boulos*2, C. Webber3, C. Gordon4, D. Inglis*1, A. Papaioannou5, E. Jurriaans*6, J. D. Adachi2. 1McMaster University, Hamilton, ON, Canada, 2Dept. of Rheumatology, McMaster University, Hamilton, ON, Canada, 3Dept. of Nuclear Medicine, McMaster University, Hamilton, ON, Canada, 4Dept. of Radiology, McMaster University, Hamilton, ON, Canada, 5Dept. of Geriatrics, McMaster University, Hamilton, ON, Canada, 6Dept. of Radiology, St. Joseph's Healthcare, Hamilton, ON, Canada.

Objective: To investigate the relation between hip, lumbar spine, distal femur and proximal tibia bone densities in healthy men and women.

Methods: Healthy individuals (no knee pain, history of knee injury or diagnosis of a bone or joint disease) aged 20--69 yrs. were invited to participate. Volunteers underwent a fixed flexion knee X-ray and bone mineral density (BMD) scans of the lumbar spine, hip, distal femur and proximal tibia. Femur and tibia scans were acquired using the lumbar spine protocol. X-ray films were graded according to the Kellgren-Lawrence (K-L) scale. BMDs were evaluated by X-ray technicians and a research assistant.

Results: Of 46 participants, 4 cases were excluded (3 scored ⩾2 on K-L scale, 1 had T<−2.5 at lumbar spine). The remaining 42 individuals included 32 females and 10 males, mean age (SD) 41.9 (13.6) years and body mass index (BMI) 25.5 (3.9) kg/m2. Total proximal tibia and total distal femur BMDs were significantly positively correlated (r=0.81, p<0.01) as were subchondral femoral and subchondral tibial BMDs (r=0.642, p<0.01). Linear regression analyses revealed that the femoral neck was a significant predictor of total distal femur BMD (β coeff. 0.745, p<0.01) and subchondral femoral BMD (β coeff. 0.617, p<0.01). Lumbar spine and total hip BMDs also significantly predicted total distal femur BMD (β coeffs. 0.589 and 0.716, p<0.01) and subchondral femoral BMD (β coeffs. 0.321 and 0.522, p<0.01). In a multivariable regression analysis with all regions entered as independent variables, femoral neck BMD consistently and significantly predicted total distal femur BMD and subchondral femoral BMD (β coeffs. 0.604 and 0.617, respectively, p<0.01). A regression analysis including these same independent variables showed that lumbar spine, femoral neck and trochanter were significant predictors of total proximal tibial BMD at the p<0.01 level (β coeffs. 0.251, 0.514 and 0.339 respectively). They also significantly predicted subchondral tibial BMD (β coeffs. 0.321, 0.347 and 0.46, respectively).

Conclusion: Femoral neck and lumbar spine BMD significantly predict both distal femur and proximal tibia BMD in healthy men and women. The trochanter also significantly predicts proximal tibial BMD.

Disclosures: A. Papaioannou, None.


Evaluation of Human Trabecular Bone Properties by Scanning Acoustic Microscopy.I. Leguerney*1, K. Raum*2, A. Saied1, H. Follet*3, G. Boivin4, P. Laugier1. 1Laboratoire Imagerie Parametrique, CNRS - Paris 6 - UMR 7623, Paris, France, 2Q-BAM group, Martin Luther University of Halle, Halle, Germany, 3Laboratoire de Mecanique des Solides, INSA, Lyon, France, 4Laboratoire Physiopathologie des Osteopathies fragilisantes, INSERM Unity 403, Lyon, France.

The study aims at evaluating local changes of trabecular bone material properties using a 50 MHz - 200 MHz reflection scanning acoustic microscope (SAM). A multi-layer analysis (MLA) method was used to estimate local variations of acoustic impedance of bone that is related to changes in both bone density and elasticity, and to reconstruct acoustic impedance images. This analysis method allows measurements that are independent of the system transfer function, sample topography and inclination.

Measurements were performed on calcaneus samples taken from 13 male and 6 female cadavers between 61 and 91 years of age. After dehydration, embedding in methylmethacrylate and polishing, the bone samples were explored with SAM. For each bone sample, the acoustic impedance (Z) was estimated from the entire sample section of at 50 MHz (30 μm lateral resolution) and from 5 selected regions of interest at 200 MHz (7.5 μm lateral resolution). The mean degree of mineralization of bone (MDMB) was measured over the entire section of bone samples by quantitative microradiography.

No correlation was found between age and acoustic impedance. At 50 MHz, a least square linear fit between Z and MDMB showed a moderate but significant correlation (R=0.56, p<0.05) between acoustic impedance and mean degree of mineralization for all the samples. No correlation was found between local mean Z-values obtained at 200 MHz and mean degree of mineralization of bone.

These findings indicate that mean degree of mineralization of bone contribute to changes in acoustic impedance. Further analysis are under way and complementary techniques are necessary to establish the influence of bone micromechanical properties on acoustic impedance.

Nevertheless, quantitative acoustic microscopy using MLA method has potential to be a relevant tool for the evaluation of bone properties and follow-up of the therapeutic effects at the tissue level.

Disclosures: P. Laugier, None.


Geometrical Changes in Micro-architecture Related to Bone Loss on a Mice Model.F. C. Peyrin*1, A. Bonnassie*1, E. Martin-Badosa*2, D. Amblard*3, L. Vico*3. 1CREATIS, Villeurbanne, France, 2Dept. de FAO, Lab. d'Optica, Barcelone, Spain, 3LBTO INSERM, Saint-Etienne, France.

A tail suspension model of bone loss was investigated to characterise the changes occurring in micro-architecture on C57BL/6J (B6) inbred mice (male, 4-months old). Previous studies using histomorphometry revealed micro-architectural changes, and especially a decrease of trabecular thickness. In B6 mice, two groups were submitted to hind limb unloading through tail suspension (S, n=8) or had the tail suspension device but were not suspended (ANS, n=8). We investigated the distal metaphysis in the left femur using 3D synchrotron radiation microtomography. The voxel size was 6.65 μm, and a ROI encompassing the femoral bone with a total height of 2.35mm was selected in each image. 3D Model independent architecture parameters were computed, in particular bone volume fraction (BV/TV*) and trabecular thickness (Tb.Th*).

We developed a new technique for analysing the geometry's of bone micro-architecture. The 3D original volume is decomposed in three sub-volumes, each of them corresponding to the set of voxels in tube, plate or branching structures respectively. This geometrical approach allows to define new architectural parameters : plate volume to total volume (PV/TV), rod volume to total volume (RV/TV), as well as the direct thickness of each structures respectively denoted as (P.Th*) and (R.Th*). The parameters on the two groups are reported in table 1. 

Table  .  
  1. Table 1: Geometrical architecture parameters in two groups of mice

  2. ( significantly different (p<0.05). NS, non-significantly different)

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The computation of morphological parameters shows a significant thinning of the bone trabeculae with suspension. The geometrical analysis shows that the two groups have a similar dominant rod structure and that rods are significantly thicker than the plates (p=0.012 in B6 ANS group and p=0.025 in B6 S group). In addition it shows that suspension induces a statistical significant thinning of both of plate-like and rod-like structures.

Disclosures: F.C. Peyrin, None.


Monoclonal Antibodies Production and its Application to Study Possible New Markers in Osteoblastic Cells.L. M. Dos Reis*1, R. C. Monteiro*2, M. Benhamou*2, V. Jorgetti1. 1Nephrology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 2Nephrologie, INSERM E0225 - Immunopathologie Renale, Recepteurs et Signalisation, Faculté de Médecine Xavier Bichat, France.

Monoclonal antibodies have been used to study the cellular differentiation pathway in osteoblast lineage. However, the majority of the antibodies recognizes antigens present in cells from the mature stage of differentiation and none of them is lineage and cell stage specific. In the present study we obtained monoclonal antibodies against human osteosarcoma cells and osteoblastic like cells by immunizing mice with MG-63. After several steps of tests we sub cloned three monoclonal antibodies: PSP 4--5, PSP 42--22 and PSP 85--9. These antibodies recognizes antigens which size are 100, 20 and 16 kDa, respectively. We evaluated the antigen expression in cryostat sections of undecalcified normal bone, cartilage, adipocyte and cardiac muscle from fetus. Also, in normal osteoblastic like cells and from patients with metabolic bone diseases. Finally, we tested these antibodies in normal fibroblasts like cells from human skin. The three antibodies showed antigen expression in periosteum of undecalcified bone sections. The pattern of expression of PSP 42--22 in this tissue was restricted in some regions. The antibodies were positive also in osteoblasts, osteocytes and in condrocytes. In cardiac muscle the antibody PSP 85--9 showed high antigen expression in some cells. PSP 4--5 detected its antigen in some cells of this tissue and PSP 42--22 in blood vessels. In adipocytes and fibroblasts like cells the antigen expression for PSP 4--5 and 85--9 was also detected. There was a similar pattern of antigen expression with PSP 4--5 e 85--9 in normal osteoblasts like cells and in cells obtained from patients with osteoporosis. In cells obtained from patients with primary and secondary hyperparathyroidism PSP 85--9 antigen expression was higher than PSP 4--5. In contrast, PSP 4--5 antigen expression in osteoblastic like cells from patients with osteomalacia was higher than PSP 85--9. PSP 42--22 was negative in all cells tested by immunocytochemistry. These monoclonal antibodies recognize antigens from undifferentiated to differentiated bone cells and they are not bone specific. We concluded that they may be useful to study the relations between the different cells lineage and in the comprehension of mechanisms involved in metabolic bone diseases.

Disclosures: L.M. Dos Reis, None.


Micro-Computed Tomographic Analysis of Endosseous Titanium Implant Integration: A Novel Approach for Assessing the Anchorage of Titanium Implants in Bone.Y. Gabet*1, D. Kohavi*2, T. Kohler*3, R. Müller3, I. Bab1. 1Bone Laboratory, Hebrew University of Jerusalem, Jerusalem, Israel, 2Dental Implantology Center, Hebrew University of Jerusalem, Jerusalem, Israel, 3Institute for Biomedical Engineering, Swiss Federal Institute of Technology (ETH) & University of Zürich, Zürich, Switzerland.

Uncemented endosseous titanium implants constitute the standard of care in restorative dentistry and orthopaedic surgery. However, the mechanisms involved in this unique biological interaction with a foreign substance are poorly understood, mainly due to the absence of robust animal models and tools to analyze the implant-bone system. Here, we report a novel method to quantitatively evaluate endosseous implant anchorage by micro-computed tomography (μCT). A 5 mm long, 1 mm diameter titanium screw was inserted horizontally into the proximal tibial metaphysis of 4 months old male rats. The implantation site was examined 2--8 weeks thereafter by a desktop μCT system at 15 μm resolution. To better penetrate the highly radio-opaque titanium and improve the signal-to-noise ratio the system was operated at 70 KeV and 350 ms integration time, respectively, vs. the standard 50 KeV and 100 ms. The titanium and mineralized tissue were individually segmented by a multi-level thresholding procedure using Image Processing Language and topological operators developed with software devised specifically for this purpose. Unlike the traditional assessment of implant anchorage which is limited to the determination of the implant surface fraction in direct contact with bone (percent osseointegration, %OI) in a few histological sections, the present method analyses the %OI as well as the peri-implant trabecular bone (PIB) bone volume (BV/TV) and connectivity (Conn.D) densities directly in three-dimensional μCT images. After the μCT analysis the same specimens were subjected to biomechanical testing by a pullout test. All the μCT parameters showed high correlations with the ultimate force and toughness revealed by the implant pullout test. Surprisingly, the correlation coefficients between the biomechanical and PIB parameters were higher than those with the %OI, approaching the ultimate value (1.00), thus highlighting the critical role of PIB in connecting the implant to the surrounding cortex. The presently developed μCT operational definitions and software for analyzing the implant-bone system comprise an efficient, automatic, non-destructive and highly robust tool proposed as the “gold standard” for the experimental evaluation of endosseous implantation.

Disclosures: Y. Gabet, R&D Pharmaceuticals, Inc. 2, 7.


NMR Spectroscopy Shows a Structure Stabilizing Role for Water Found in Bone Mineral.L. W. Beck*1, E. Wilson*1, M. D. Morris*1, D. H. Kohn*2, M. M. J. Tecklenburg*3. 1Department of Chemistry, University of Michigan, Ann Arbor, MI, USA, 2Department of Biologic and Materials Science & Department of Biomedial Engineering, University of Michigan, Ann Arbor, MI, USA, 3Department of Chemistry, University of Central Michigan, Mt. Pleasant, MI, USA.

We propose that water in bone mineral functions to occupy vacancies in the apatitic mineral lattice caused by incorporation of carbonate and other ions in the crystallites. The water content of bone mineral thereby stabilizes the mineral against mechanical weakening caused by these substitutions. Here, proton solid state NMR with magic-angle spinning (MAS) has been used to investigate the water and hydroxide content of deproteinated rat cortical bone tissue and a reference mineral, synthetic carbonated hydroxyapatite (CAP). The reference mineral was obtained by the standard room temperature precipitation resulting in a mineral that is 6-wt% (B-type) carbonate. At least two distinct types of structural water were found in the bone mineral, whereas three distinct types of water were found in the CAP material. The major type of water found in the deproteinated bone mineral sample was observed at 5.6 ppm in the 1H NMR after the sample was heated to 120°C to remove non-structural surface water. This water occupies calcium vacancies in the apatite lattice and is strongly hydrogen-bonded to phosphate or carbonate anions. A second type of structural water was observed in higher abundance in the synthetic carbonated apatite mineral, at 4.3 ppm 1H NMR. This water is assumed to also occupy calcium lattice vacancies. A third type of water, ca. 2--2.5 ppm, is most likely located in the hydrophobic environment of the calcium lined hydroxide channel (apatite lattice). This third type water is present at significantly higher levels in the deproteinated bone mineral as compared to the synthetic CAP material. There is a concomitant decrease in the fraction of hydroxide observed in the bone mineral, which suggests hydroxide is replaced by the structure water (third type).

Our results suggest that fat mass is not significantly associated with bone mass in this population, after controlling for the effects of covariates. In conclusion, lean mass is independently related to bone mass in postmenopausal Tobagonian women of African ancestry. The stronger association between lean mass and BMD could reflect a multifactorial relationship encompassing bone strength, culture, lifestyle, genetics, hormonal, and growth factors.

Table Table 1.. 1H MAS-NMR spectral analysis of deproteinated bone and B-type carbonated hydroxyapatite at 220°C.
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Disclosures: L.W. Beck, None.


Visualizing GFP Positive Cells in Frozen Decalcified and Nondecalcified Histological Sections of Bone.X. Jiang*, D. W. Rowe. Genetics & Dev Biology, University of Connecticut Health Center, Farmington, CT, USA.

The ability to image specific population of cells within the osteoblast lineage in transgenic mice bearing reporter-GFP constructs can reveal new levels of tissue physiology that cannot be assessed by standard histological techniques. Last year we reported that frozen sections preserved a significantly stronger GFP signal than paraffin sections and allowed GFPcyan, GFPsaph and the widely used eGFP to be visualized. However great skill in preparing the frozen sections was required and the method could not be performed with calcified tissues. The CryoJane adaptation to cryostat sectioning has to potential to overcome these technical problems of tissue sectioning. It uses a piece of adhesive tape to transfer the cut section to a temperature controlled slide thereby preserving tissue integrity. The slide comes precoated with an adhesive that crosslinks the tissue to the slide after a brief flash of UV light. Using this approach we have been able to cut 5--7 μm decalcified sections from mice of any age with a disposable blade and calcified section from mice < 8 months of age with a tungsten blade. The histological sections are imaged with a computer controlled Zeiss Axioplan 200 microscope workstation that records a series of image files that are concatenated into a single file of the majority of the bone section. We will present composite images showing the markedly improved signal of GFP of a frozen versus a paraffin section and the remarkable quality of the frozen section prepared with the CryoJane system. The approach has a very low endogenous fluorescence background and allows weaker GFP signals from other elements within the bone marrow to be appreciated (endothelial cells expressing Tie2eGFP), and to co-localize immunostained and enzymatic stained sections with GFP. Immunostaining intracellular targets that require denaturation of the DNA (BrdU) destroys the GFP signal. Nondecalcified sections can be prepared that show dynamic labeling with calcein and xylenol orange that also preserve the endogenous GFP signal. The step distinguishes those areas expressing GFP that are and are not producing a bone matrix. While we are still learning how to use this approach to histology to a greater advantage, it can reveal information that is not possible with standard histological methods and significantly increases sample throughput for transgene analysis in murine bone. An additional advantage of the tape method is the relative ease such that an inexperienced user can produce their own histological sections.

Disclosures: D.W. Rowe, None.


Induction of Mesenchymal Progenitor Cell Differentiation into Chondrocytes by BMP 2 and Promotion of Hypertrophy by Thyroxine.Y. Okubo*1, K. Bessho*1, T. Iizuka*1, H. Reddi2. 1Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto university, Kyoto, Japan, 2Department of Orthopaedic Surgery, School of Medicine, UC Davis, Sacramento, CA, USA.

Mesenchymal stem cells have the potential to differentiate into adipocytes, myoblasts, chondrocytes and osteoblasts. The chondrogenesis of mesenchymal cells is followed by maturation and terminal differentiation and hypertrophy. The faithful mimicry of the sequence in vitro will be useful. The induction of chondrogenesis of two mesenchymal progenitor/ stem cell lines, C2C12 and C3H10T1/2 in three-dimensional micromass and pellet cultures was investigated. An adenoviral gene transfer system with bone morphogenetic protein 2 (BMP 2) converted C3H10T1/2 cells to chondrocytes in three-dimensional cultures. However, C2C12 cells were not converted to chondrocytes, but expressed alkaline phosphatase activity, as osteoblast marker. Treatment of C3H10T1/2 with 5-azacytidine prior to BMP2 treatment increased the intensity of Alcian blue staining and alkaline phosphatase (AP) activity on days 14 (Fig) and 21. Next, we examined the effects of thyroxine and growth hormone on the terminal differentiation and hypertrophy of chondrocytes. Thyroxine treatment resulted in an increase in markers of chondrocyte hypertrophy such as AP activity and type X collagen. However, growth hormone alone did not affect the terminal differentiation of chondrocytes. These experiments demonstrated that BMP 2 and thyroxine are sufficient to initiate chondrogenesis and promote hypertrophy of mesenchymal progenitor/ stem cells.

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Disclosures: Y. Okubo, None.


Study of Growth Plate Homeostasis Markers in Rats with Growth Retardation or Hypophosphatemic Rickets.V. Lascau-Coman*, N. Dion, G. Mailhot*, M. Gascon-Barré, L. G. Ste-Marie. Hǒpital Saint-Luc, CHUM Research Centre, Montréal, PQ, Canada.

Normal longitudinal growth depends on a well-defined sequence of chondrocyte maturational stages occurring within the epiphyseal growth plate (GP). During this endochondral ossification, a population of stem cells in the resting zone (RZ) proceeds through proliferation (PZ), differentiation and hypertrophy (HZ) which finally gives way to bone. Several proteins have been involved in chondrocyte proliferation and differentiation such as PTH-related peptide (PTHrP), Indian hedgehog (Ihh) and its receptor Patched (Ptc), fibroblast growth factor 18 (FGF18) and its receptor FGFR3. To ensure normal endochondral ossification and growth, adequate dietary intake of calcium (Ca) and vitamin (D) are essential. It was previously observed that young rats (9 week old) fed a low Ca diet depleted in D (Ca-D-) showed growth retardation whereas young rats fed a D depleted diet but high in Ca (Ca+D-) developed hypophosphatemic rickets. Immunohistochemistry (IHC) performed on rat tibial GP showed that the expression of PTHrP, Ihh, Ptc, FGF18 and FGFR3 was modulated by the diet. When compared to rats fed a normal diet (N), PTHrP and Ihh proteins were decreased in Ca-D- but were abundant in Ca+D-. In Ca+D-, Ptc was present in all GP zones in contrast to Ca-D- and N where Ptc was only localized in PZ. There were no differences among groups in the localization of FGF18 except that its expression was higher in Ca+D- RZ and PZ. IHC of FGFR3 showed a similar expression pattern in all groups but the intensity of staining was lowered in Ca+D- HZ. One of the mechanisms proposed to be part of the fate of hypertrophic chondrocytes is apoptosis. To further characterized growth abnormalities in these rats, we studied chondrocyte apoptosis by performing TUNEL assay and IHC for Caspase 3, the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2. Ca-D- were found to have TUNEL and IHC stainings comparable to N. However, there was a marked decrease of apoptosis as indicated by TUNEL and Caspase 3 IHC in Ca+D- HZ compared to N. In addition, Bax IHC staining was lower and conversely, Bcl-2 was higher in Ca+D- than in HZ of N. Taken together, these results suggest that growth retardation observed in Ca-D- is mainly due to a diminution of proliferation and differentiation of GP chondrocytes. In Ca+D- the development of rickets characterized by a widening HZ is partly attributable to the increment of chondrocyte proliferation and differentiation but further amplified by decreased apoptosis. The role of hypophosphatemia and the mechanism by which chondrocyte apoptosis is lowered in this group remain to be elucidated.

Disclosures: V. Lascau-Coman, None.


ACTH Enhances Chondrogenesis In Vitro.J. F. Evans*1, D. Sima*1, C. Shen*2, J. F. Aloia1, J. K. Yeh1. 1Medicine, Winthrop University Hospital, Mineola, NY, USA, 2Pathology, Texas Tech Health Sciences Center, Lubbock, TX, USA.

Childhood obesity has been linked to increased growth and most forms of obesity are associated with hyperphagia. Recently overfeeding has been linked to increased production of pro-opiomelanocortin (POMC), the melanocortin peptide pre-cursor. Familial Glucocorticoid Deficiency (FGD), a non-obesity syndrome, is also associated with both increased production of melanocortin peptide and increased growth. The association of melanocortin peptide overproduction with enhanced linear growth prompted the current investigation of ACTH's effects on resting zone chondrocytes and bone marrow stromal cells in culture. Resting chondrocytes isolated from the rib of young rats were cultured in differentiation medium plus or minus 10--7M ACTH. Secondary stromal cells derived from long bones of the same rats were grown in basal medium (α-MEM plus 10% charcoal/dextran treated FBS and 50αg/ml ascorbic acid) plus or minus 10--7M ACTH. 3H-thymidine incorporation measurements in Day 3 cultures indicate that ACTH significantly decreases proliferation of chondrocytes (CON 318.06±11.14×103 vs. ACTH 244.14±11.35 × 103 dpm, P=0.001) but significantly increases the proliferation of stromal cells (CON 1563.23±86.20 vs. ACTH 2674.97±136.64 dpm, P<0.001). ACTH significantly increased protein content of both chondrocytes (CON 7.3±0.4 vs. ACTH 8.2±0.4 αg/well, P<0.05) and stromal cells (CON 4.66±0.10 vs. ACTH 7.69±0.46 αg/well, P<0.001). Total collagen was also significantly elevated in ACTH treated chondrocyte cultures throughout the culture period (day 7--28), as measured by 3H-proline incorporation. The increase in protein content and total collagen in the chondrocyte cultures indicates an increased matrix formation while the increase in protein content of the stromal cell cultures may reflect increased cell numbers. Examination of chondrogenic cell and molecular markers in both chondrocyte and stromal cultures indicates an increase in differentiation. Peak AP-activity is significantly greater in ACTH treated chondrocyte (CON 82.36±5.77 vs. ACTH 131.83±11.85 nmol pnp/min/ well, P<0.001) and stromal cell cultures (CON 5.59±0.83 vs. ACTH 11.80±0.38 nmol pnp/ min/104 cells, P<0.001). mRNA expression of COLL II and AGR is also elevated in ACTH treated chondrocyte and stromal cell cultures. These data show that ACTH increases both chondrocyte differentiation and stromal cell chondrogenesis which implicates ACTH as a regulator of chondrocyte differentiation and a promoter of chondrocyte phenotype development from bone marrow stromal cells. These findings provide a link between enhanced linear growth and the overproduction of melanocortin peptides.

Disclosures: J.F. Evans, None.


Hypoxia-Inducible Factor-1α and Effects of Hypoxia on Chondrocytes and Induced Human Chondrocytes.J. Glowacki, C. C. Wykoff*, S. Mizuno. Orthopedic Research, Brigham and Women's Hospital, Boston, MA, USA.

The heterodimeric transcriptional complex hypoxia-inducible factor 1 (HIF-1) is a key mediator of cellular response to hypoxia and directs expression of a large family of hypoxia-inducible genes. HIF-1 comprises an oxygen-sensitive subunit, HIF-1α, and a constitutively expressed subunit, HIF-1 β. Hypoxia and hypoxia-mimetics (e.g. deferoxamine, DFO, 150 microM) are capable of inducing the accumulation of HIF-1α protein in normal and cancer cells. Cartilage is an avascular and relatively oxygen-deficient tissue. Hypoxia maintains chondrogenesis in monolayer culture [Dev Bio 19:52, 1969]. HIF-1α was recently identified in mouse fetal cartilage and was required for normal growth plate viability and development [Genes Dev 15:2865, 2001]. Little is known about HIF function in normal adult chondrocytes or neochondrocytes. We tested the hypotheses 1) that HIF-1α regulation in chondrocytes differs from other cell types and 2) that hypoxia enhances chondrogenesis in 3D culture. Freshly isolated bovine articular chondrocytes (bACs), human dermal fibroblasts (hDFs), and control HeLa cells were cultured in normoxia (19%) and in hypoxia (1--5%) in a custom-made incubator by autoregulating air, N2, and CO2. The hDFs were also cultured in 3D collagen sponges +/− chondroinductive demineralized bone powder (DBP). Expression of HIF-1α was examined by Western blot. Chondrogenesis was measured by ELISA in bACs, hDFs, and chondroinduced hDFs.

First, hypoxia-inducibility and DFO-inducibility of HIF-1α were confirmed in HeLa and hDFs. Second, HIF-1α was expressed at a relatively high level in the normoxic bACs, followed for 6 days. Third, HIF-1α was not greater in hypoxic bACs; rather, it appeared that HIF-1α was constitutively expressed in normoxia and hypoxia. Fourth, DFO treatment of bACs further increased HIF-1α expression. Fifth, hDFs that were cultured with DBP for 7 days showed histochemical, immunochemical, and molecular evidence of chondroinduction. Those induced neochondrocytes expressed HIF-1α under all conditions. Sixth, hypoxia (2%) enhanced accumulation of cartilage-specific chondroitin 4-sulfate by both bACs (14%, p<0.05) and hDFs with DBP (18%, p<0.05), but not by control hDFs.

In conclusion, we show that 3D chondrogenesis is enhanced in hypoxia and that HIF-1α is expressed in bACs and in induced human chondrocytes. In contrast to HeLa cells and hDFs, bACs demonstrated an impressive normoxic level of HIF-1α expression. Furthermore, the expression of HIF-1α in bACs was constitutive, unresponsive to hypoxia, and increased with exposure to DFO. Enhanced chondrogenesis with hypoxia may be related to viability and other HIF target genes that are unique in chondrocytes.

Disclosures: J. Glowacki, None.


Overexpression of p57Kip2 Is Not Sufficient for, but Augments, Collagen Type X Induction in Differentiating Chondrocytes.M. Stewart1, R. M. Kadlcek*2. 1Veterinary Clinical Medicine, University of Illinois at UrbanaChampaign, Urbana, IL, USA, 2Orthopaedics, Case Western Reserve University, Cleveland, OH, USA.

Introduction: The CDK inhibitor p57Kip2 has been implicated in regulating collagen type X (Coll X) expression in differentiating chondrocytes (1). This study was carried out to investigate the link between p57 expression and the induction of Coll X in chondrocytes and to determine whether p57 overexpression is sufficient for the induction of hypertrophic differentiation.

Methods: Neonatal rat epiphyseal and growth plate chondrocytes were maintained as nonadherent aggregates in serum-free medium. Hypertrophic differentiation was induced by BMP-2 administration. p57 mRNA and protein levels were assessed by Northern and Western blot analyses. Associated changes in proliferative activity were monitored by total DNA and 3H thymidine incorporation rates. An adenoviral vector was constructed to assess the phenotypic consequences of p57 overexpression. Vectors expressing LacZ and the related CDK inhibitors p21Cip1 and p27Kip1 were used as controls and in co-expression studies.

Results: Steady-state levels of p57 mRNA and protein were not affected by BMP-2 treatment, despite transient effects on chondrocyte proliferative activity and the induction of ALP and Coll X. Adenoviral p57 overexpression induced growth arrest in epiphyseal chondrocytes in a dose-dependent manner, as did vectors expressing p21 and p27. p57 overexpression did not induce Coll X, either alone or when co-expressed with p21. Further, pretreatment of epiphyseal chondrocytes with BMP-2 for 4 days to initiate differentiation did not induce “p57-responsiveness” in these cells. Similar results were obtained with more mature tibial growth plate chondrocytes that were actively expressing Coll X at the time of isolation. p57 overexpression did significantly augment Coll X induction by sub maximal BMP-2 doses. This effect was specific to Coll X since ALP was not affected, and was also specific to p57, since parallel experiments using p21 or p27 had no effect on Coll X levels.

Conclusions: These results indicate that growth arrest is not sufficient for expression of the hypertrophic phenotype, but rather occurs in parallel with other aspects of the differentiation pathway, analogous to results derived from keratinocyte and skeletal myoblast differentiation studies(2,3). The study also suggests a specific activity for p57 in controlling Coll X expression in differentiating chondrocytes, independent of its direct effect on cell cycle progression in these cells.

References. 1. Zhang P et al (1997) Nature 387:151--158 2. Harvat BL et al (1998) J Cell Sci 111:1185--1196. 3. Zhang P et al (1999) Genes & Dev 13:213--224

Disclosures: M. Stewart, None.


Expression of Mouse Receptor of NF-kappaB Ligand (RANKL) in Growth Plate and Articular Cartilage.K. Kishimoto*, R. Kitazawa, A. Darwanto*, T. Kondo*, S. Maeda*, S. Kitazawa. Division of Molecular Pathology, Kobe University Graduate School of Medicine, Kobe, Japan.

RANKL (receptor activator of NF-kappaB ligand) is a membrane-associated osteoblastic molecule that, along with the macrophage-colony-stimulating factor, is crucial for osteoclast formation. In addition to the osteoblasts that actively support osteoclasts, we and others have reported that RANKL expression is observed in hypertrophic chondrocytes during endochondral ossification in bone tissue. To investigate the molecular mechanism controlling RANKL gene expression in hypertrophic chondrocytes, we examined the expression of RANKL and Runx2/Cbfa1, a key transcription factor of RANKL gene expression, and the methylation status of the RANKL gene promoter. The articular cartilage, a cartilage that does not undergo endochondral ossification, in the joint was used as an internal control for the entire study. Knee joints of male BALB/c mice aged 1--24 weeks were dissected and fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH7.4) at 4 C for 3 days, then decalcified with 20% EDTA in 0.1M PB for 4 days. Tartrateresistant acid phosphatase (TRAP) staining was used to detect osteoclasts. To characterize hypertrophic chondrocytes, type X-collagen mRNA was detected by in situ hybridization (ISH). Immunostaining was carried out using anti-RANKL and Runx2/Cbfa1 antibodies (Santa Cruz). The methylation status of the mouse RANKL gene promoter in both hyper-trophic and articular cartilage was analyzed by sodium bisulfite mapping using microdissected mouse bone tissue. By immunostaining and ISH, besides osteoblasts lining the trabecular bone surface, RANKL expression was observed on hypertrophic chondrocytes expressing type-X collagen in the growth plate, but not on chondrocytes in the articular cartilage. A similar localization pattern of Runx2/Cbfa1 was demonstrated by immunostaining. Although the overall methylation in the RANKL promoter region was low, chondrocytes in the articular cartilage showed a higher methylation rate than hypertrophic chondrocytes in the growth plate. These results indicate that RANKL expression in hyper-trophic chondrocytes is regulated as in osteoblasts and contributes to the formation of osteo(chondro)clasts during endochondral ossification.

Disclosures: K. Kishimoto, None.


Regulation of Chondrogenic Differentiation of ATDC5 Cells: A Role for Estrogens?J. Hoogendam1, C. Lowik2, J. Wit*1, M. Karperien3. 1Pediatrics, LUMC, Leiden, Netherlands, 2Endocrinology, LUMC, Leiden, Netherlands, 3Pediatrics and Endocrinology, LUMC, Leiden, Netherlands.

Sex-steroids have profound effects on longitudinal bone growth. They can exert direct effects on growth plate chondrocytes, due to the presence of ERa and ß mRNA and protein. The mechanism of action by which these receptors regulate growth and differentiation of chondrocytes are largely elusive. One possibility is that they influence the activity of the growth restraining PTHrP/Ihh-feedback loop which is operational in the prenatal and postnatal growth plate.

To study the interactions between sex-steroids and the PTHrP/Ihh-feedback loop in more detail, we have started to use the mouse chondrogenic ATDC5 cell line. This cell line differentiates into mineralising hypertrophic chondrocytes in monolayer in the presence of insulin. Hypertrophic differentiation is enhanced by the addition of BMP4 and can be inhibited by PTHrP. RT-PCR analysis identified the presence of ERa, but not ERß mRNA. Hypertrophic differentiation of ATDC5 cells was dramatically accelerated by inducing cell aggregation. Interestingly, under these culture conditions, insulin was not required for terminal chondrocyte differentiation. However, in the presence of insulin aggregates were larger and their integrity was better maintained. Culturing of ATDC5 cells as aggregates in the presence of estrogen reduced terminal chondrocyte differentiation.

In order to study the effect of estrogens on chondrocyte differentiation in more detail, we have generated stable cell lines overexpressing wild type hERa (WT), constitutive active hERa (CA) and dominant negative hERa (DN) using FLP-mediated recombination. For this purpose ATDC5 cells are stable transfected with an FRT-recombination site. One clone, which behaved comparable to the parental cells was selected, and used for generating overexpressing variants of hERa (WT, CA and DN) isogenic cell lines.

In all cell lines overexpression of hERa variants could be detected. Using transient transfection with EREluc, parental and overexpressing cell lines responded differentially to estrogen (E2). The WT was highly sensitive and gave almost a ten fold induction in luciferase activity. The CA overexpressing cell line increased basal activity and did not react to E2. Like the CA, the DN did not react to E2, but had low basal levels.

In conclusion, hypertrophic differentiation of ATDC5 cells can be accelerated by culturing the cells in aggregates. In addition, cell aggregation appears to be more important for hypertrophic differentiation than treatment with insulin. The flp-in stable ATDC5 cell line appears to be a suitable model to study interactions between sex-steroids and the PTHrP/ Ihh-feedback loop.

Disclosures: J. Hoogendam, None.


Effects of Pyrrolidine Dithiocarbamate on Chicken Chondrocytes.N. C. Rath. USDA, Agricultural Research Service, Fayetteville, AR, USA.

Thiocarbamates are widely used as fungicides, seed fumigants, and accelerators in the vulcanization of rubber. Pyrrolidine dithiocarbamate (PDTC) is used as an antioxidant and a metabolic inhibitor of transcription factor NF-kB. PDTC, and a few other thiocarbamates including, thiram and disulfiram induce tibial dyschondroplasia (TD), a common cartilage defect in meat-type poultry. TD is characterized by the presence of a thickened plug of cartilage in the proximal end of tibial growth plates which impedes bone formation and causes bone distortion, fracture, and lameness. To understand how PDTC affects chondrocyte metabolism to induce TD, we studied its effects on the expression of several chondrocyteassociated genes using avian-leukosis virus transformed chicken epiphyseal growth plate chondrocytes (CEGC) in culture. CEGC were grown at a concentration of 105 cells /ml/ well in 24 well plate for14 days before they were treated with 2- and 4μM concentrations of PDTC for 24h. The choice of the concentrations of PDTC was based on studies which showed these to be nontoxic to cells. Total RNA extracted from control and PDTC treated cells, were reverse transcribed and cDNA was amplified using PCR and chicken gene specific primers for glyceraldehyde phosphate dehydrogenase (GAPDH), aggrecan, bone morphogenetic protein (BMP)-4 and 7, collagen types II and X, matrix metalloproteinase-2 (MMP-2), and transforming growth factor-β (TGF-β). The PCR products were analyzed using capillary electrophoresis and laser-induced fluorescence detection which allows both size determination and quantification of amplicons. The expression of different genes was normalized against GAPDH and the results (n=3/ treatment) were analyzed using Duncan's GLM procedure. PDTC at the concentration of both 2 and 4μM down regulated the expression of aggrecan and type II collagen genes in a dose dependent manner but had no effect on other genes. However, at 4μM concentration PDTC also down regulated the expression of BMP-4, collagen X genes without significant effect on MMP-2, or TGF-β. These results suggest that PDTC negatively affects chondrocyte development most likely affecting the maturation of prehypertrophic population of chondrocytes. On the other hand, the hyper-trophic population of chondrocytes appear to be less drastically affected since other studies under similar conditions showed that PDTC up to 16μM was not inhibitory to alkaline phosphatase that is responsible for calcification. Extrapolating these in vitro observations to an in vivo situation of growth plate, it is likely that PDTC prevents maturation of chondrocytes eventually leading to their premature death and accumulation as an unresolved plug of dead cartilage, which characterizes tibial dyschondroplasia.

Disclosures: N.C. Rath, None.


Perlecan Domain I Synergizes with BMP-2 to Promote Maturation of Cartilage Condensations.R. R. Gomes*, W. Yang*, M. C. Farach-Carson, D. D. Carson*. Biological Sciences, University of Delaware, Newark, DE, USA.

C3H10T1/2 cells differentiate along a chondrogenic pathway when plated onto the extracellular matrix (ECM) protein perlecan (Pln) or recombinant domain I of Pln (PlnDI). Pln-stimulated condensations undergo chondrogenic maturation in response to exogenous recombinant human bone morphogenic protein 2 (rhBMP-2) treatments. In this investigation, we tested the hypothesis that exogenous rhBMP-2 treatments of PlnDI-stimulated condensations promote chondrogenic maturation in vitro. To test this hypothesis PlnDI was first purified from conditioned media of EBNA-293 cells stably transfected with a vector constitutively expressing PlnDI. Purified PlnDI is decorated with both heparan-sulfate and chondroitin-sulfate side chains, as determined by SDS-PAGE analysis of heparitinase I-III, and chondroitinase ABC treated samples. In addition, dot-blot analysis demonstrated that PlnDI binds basic fibroblast growth factor, and that this binding is heparan sulfate dependent. To test the ability of rhBMP-2 to synergize with PlnDI-stimulated cartilage-like condensations, C3H10T1/2 fibroblasts were seeded in chambered “Permanox” slides uncoated or coated with PlnDI. The cells were maintained in CMRL-1066 media supplemented with ascorbic acid, citrate, and pyruvate. C3H10T1/2 fibroblasts seeded on PlnDI coated wells attach and began to aggregate, forming cartilage-like condensations within minutes of plating. On day 3 post plating, the media was replaced and supplemented with or without rhBMP-2 (100 ng/ml). On day 6 post plating, phase contrast microscopy suggested that cells in rhBMP-2 treated cultures had proliferated relative to untreated cultures. Subsequent observations of cells in rhBMP-2 treated cultures suggested that differentiation also occurs, as changes in cell shape (from fibroblastic to round) and the appearance of increased matrix deposition were noted. By day 12 of culture, indirect immunofluoresence visualized by confocal microscopy revealed that cells within PlnDI-stimulated condensations treated with rhBMP-2 express a late stage marker of chondrogenesis, collagen type X. Morphologically, collagen type X expressing cells appear larger in diameter, hyper-trophic, with large nuclei, relative to cells within PlnDI-stimulated condensation not treated with rhBMP-2. Thus, PlnDI-stimulated cartilage-like condensations respond to rhBMP-2 treatment similar to intact Pln. This in vitro model mimics key events that comprise the early cartilaginous model of bone development.

Disclosures: R.R. Gomes, None.


Analysis of Gene Expression Profiles Among Calvarial Bones, Sutures, and Osteogenic Fronts Composing of Cranial Vaults.H. J. Kim1, W. B. Lee*1, H. J. Kim*2, H. M. Ryoo1. 1Biochemistry, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea, 2Pediatric Dentistry, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea.

Enlargement of the skull vault occurs by appositional growth at the fibrous joints between the bones, termed cranial sutures. However, little is known about patterns of gene expression from cells populating mouse cranial vaults. This study investigated the possible variances of gene expression profile among calvarial bones, sutures, and osteogneic fronts of mouse cranial vaults at embryonic day (E) 15 -- E16. Expression was analyzed on a mouse gene array panel of 7,400 mouse cDNAs representing tightly regulated genes with key roles in various biological processes. Of these genes, total 455 were found to have at least 2-fold or higher expression changes in calvarial bones (269 genes), sutures (174 genes), and osteogenic fronts (12 genes). A large number of these genes have never been previously recognized in the context of calvarial development and most genes belong to the following protein families: growth factors and receptors; signal transduction pathway proteins; transcription factors; structural and matrix proteins; immune response-related proteins; transporter, etc. For example, cDNAs for Col1a1, Col3a1, Col5a2, osteopontin, Pkd1, Hoxa5, PDGF receptor beta, Gli, and Ncor1 had higher normalized hybridization intensities in calvarial bones than both in sutures and osteogenic fronts. In suture area, Ftl1, Collla1, Abl-1, and insulin-like growth factor 2 were higher expressed than other both tissues. Osteogenic fronts had higher expression levels of FGF3, Gfi1, Hoxc4, interleukin 1 alpha, and Sn. The establishment of the expression profiles of these and other genes from each tissue of cranial vault will allow us to distinguish the molecular events for cranial vault development.

Disclosures: H.J. Kim, None.


Hypoxia Promotes Chondrocytic Differentiation of C3H10T1/2 Cells induced by Bone Morphogenetic Protein-2 via p38 Kinase Pathway.M. Hirao*, N. Tamai*, A. Myoui, H. Yoshikawa. Department of Orthopaedics, Osaka University Graduate School of Medicine, Suita, Japan.

Cartilage is an avascular tissue that functions at a lower oxygen tension than do most other tissues. On the other hand, ossification is always accompanied by vascularization, suggesting high oxygen requirement. Recently, it was reported that hypoxia-inducible factor 1 (HIF-1), one of the major regulators of the hypoxic response, is essential for chondrocyte growth arrest and survival in in vivo experiment. These observations lead us to hypothesize that hypoxia plays a critical role in the chondrocytic differentiation of the cells in mesenchymal lineage. In this study, we investigated the role of oxygen tension on chondrocytic differentiation induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) using pluripotent mesenchymal cell line C3H10T1/2. C3H10T1/2 cells were cultured in normoxia (O2 20%) and hypoxia (O2 5%) conditions in the presence of rhBMP-2 (500ng/ml). Alcian blue staining revealed that more abundant proteoglycan synthesis in the cells cultured in hypoxia than in normoxia. In parallel, type II collagen (Col2a1) gene expression by Northern blotting was elevated in hypoxic condition. However, hypoxia did not alter gene expression of sry-Y-box-9 (Sox9), a transcription factor that has been shown to be involved in chondrocyte differentiation. To determine the signaling pathway involved here, activation of Smad 1/5 and p38 mitogen activated protein kinase (MAPK) were evaluated using antibodies specific for phosphorylated form. We found hypoxia upregulated phosphorylation of p38 MAPK but downregulated that of Smad 1/5. Since p38 MAPK has been suggested to be one of the regulators of chondrocyte differentioation, we next determined the effect of p38 MAPK inhibitor (FR167653) on chondrocytic differentiation induced by hypoxia. Interestingly, FR167653 abolished the upregulation of Alcian blue staining and Col2a1 gene expression induced by hypoxia but did not alter Sox9 expression. Taken together, these results suggest that hypoxia promotes chondrocytic differentiation of C3H10T1/2 cells induced by BMP-2 via p38 MAPK pathway. Oxygen tension may play an important role in regulating BMP's various physiological function.

Disclosures: M. Hirao, None.


Heparanase Expression in Developing Mouse Limb and its Relationship with Heparin Binding Growth Factor Delivery System(s).A. J. Brown*, C. Kirn-Safran*, D. D. Carson, M. C. Farach-Carson. Biological Sciences, University of Delaware, Newark, DE, USA.

Heparanase, an endoglucuronidase that cleaves heparan sulfate (HS) chains from various proteoglycans including perlecan (Pln), has been identified in a wide variety of tissues. Pln is a large multi-domain HSPG with a diverse tissue distribution including high expression in the pericellular matrix of cartilage that supports pre-chondrocyte aggregation and early chondrogenic differentiation. We have demonstrated that Pln domain I, when decorated with three glycosaminoglycan (GAG) chains, can trigger aggregate formation, early chondrogenesis, and facilitate heparin binding growth factor (HBGF) delivery. Despite the ability of heparanase to release active HBGFs in various tissues, the importance of heparanase in the chondrogenic pathway has not been determined. To investigate this role, we employed an ATDC5 cell line, a clonal mouse embryonal carcinoma cell line, to study the changes in heparanase expression and function(s) as these cells progress through the chondrogenic pathway. ATDC5 cells serve as good models for endochondral bone formation because under the appropriate conditions they display both early and late stage markers of cartilage differentiation. We propose that heparanase expression during endochondral bone formation is essential for the delivery of HBGFs that regulate cartilage tissue growth and differentiation. Conventional RT PCR was performed to analyze expression of heparanase transcripts in relation to other markers of chondrogenesis. Real time PCR showed that as these cells progress through the chondrogenic pathway, heparanase mRNA expression is transiently increased by 60 fold in maturing chondrocytes. In future studies, Laser Capture Microdissection (LCM) will allow us to obtain homogeneous populations of cells from the long bone of developing mouse limb to visualize heparanase mRNA expression in vivo. In addition to investigating the mRNA expression, enzymatic and immunodetection assays will be employed to monitor changes in heparanase protein expression and activity during cartilage development in vivo and in the ATDC5 model. Our results support the notion that heparanase is a highly regulated enzyme in the growth plate likely to be involved in HBGF delivery and functions by releasing active HBGFs from HSPGs such as Pln.

Disclosures: A.J. Brown, None.


Hepatocyte Growth Factor (HGF) Is Responsible of a High Degree of Bone Remodeling and some Cartilage Matrix Modifications.C. Boileau*1, J. Martel-Pelletier*1, M. Guévremont*1, J. Pelletier*1, G. Merlino*2, P. Reboul*1. 1Arthritis Unit, University of Montreal, Montreal, PQ, Canada, 2Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD, USA.

Our recent in vitro findings suggest that HGF found in human osteoarthritic (OA) cartilage is produced by osteoblasts and could seep from the subchondral bone. Therefore, we investigated whether some modifications of bone and cartilage matrix could be found in knee joints of HGF transgenic mice.

Transgenic mice were compared to an OA model developed in the knee joint of non-transgenic mice of the same strain. OA was induced by an intra-articular injection of mono iodoacetate (MIA, 0.5%) and animals were sacrified 7, 14 and 21 days post-injection. Joints were processed for histological analyses (O-Safranin and toluidine bleu). Using stain intensity, cellularity, structural modifications and remodelling, a histological score was established for bone and cartilage.

Both HGF transgenic mice and MIA induced a loss in both O-Safranin and toluidine bleu staining of cartilage compared to control, indicating a disruption of the proteoglycans. This effect is weaker in HGF tansgenic mice than in MIA model. In MIA model, the proteoglycan loss was maximal at day 14 while a beginning of tissue regeneration was observed at day 21. MIA induced subchondral bone remodeling consisting of formation of large lacunae with osteoclast presence and also irregularities close to the cartilage border. HGF transgenic mouse joints showed almost similar results however with some particularities. Subchondral bone was more remodeled than OA (ie: presented larger lacunae and smaller trabeculae). On the other hand, although some loss of proteoglycans and hypercellularity were found in cartilage, the effect was weaker than in MIA model.

Results obtained with transgenic mice confirmed our in vitro data indicating that HGF source found in cartilage was bone dependent. Moreover, these new data showed that bone was also the main HGF target since high degrees of remodeling were found in HGF transgenic mice. The impact of HGF on the cartilage matrix seemed to be a remodeling effect rather than a complete destruction. Therefore, we may hypothesize that HGF found in human OA cartilage is an attempt to repair the matrix.

Disclosures: C. Boileau, None.


Runx1/Cbfa2 Contributes to Proper Endochondral Ossification During Fracture Healing.K. E. McDougall1, R. M. Belflower*1, Y. Dong*1, A. J. van Wijnen2, J. L. Stein2, J. B. Lian2, G. S. Stein2, E. M. Schwarz1, R. J. O'Keefe1, H. Drissi1. 1Department of Orthopaedics, University of Rochester, Rochester, NY, USA, 2Department of Cell Biology, University of Massachusetts, Worcester, MA, USA.

Runx family members are key regulators of organogenesis and pathogenesis. The leukemia-associated transcription factor Runx1 is required for definitive hematopoiesis, such that mice lacking functional Runx1 protein die during mid-gestation, prior to skeletal formation. We investigated Runx1expression patterns in mouse embryos between E13.5 and E18.5 by in situ hybridization. Runx1 transcript was detected in the sclerotome, concomitant with Sox9 and Collagen type II, and was maintained throughout cartilage development along with Collagen type X in the axial skeleton. Real time RT-PCR revealed that Runx1 mRNA levels are up-regulated between E9.5 and E11.5 and down-regulated thereafter, while Runx2 mRNA continues to be enhanced throughout embryonic development. Since Runx1 is present in these pluripotent mesenchymal condensations, we assessed its expression during chondrocyte differentiation using micromass from mouse limb buds collected at E11.5. While the MASDS isoform of Runx1 is up-regulated throughout chondrocyte hypertrophy, as evidenced by expression levels of cartilage phenotypic genes, the MRIPV isoform is down-regulated between 4 and 10 days of culture. Our results show that Runx1 expression is regulated during embryonic development and chondrocyte differentiation. Both isoforms of Runx1 are expressed and differentially regulated in maturing mesenchymal condensations, suggesting an interplay between Runx1 isoforms in mediating cartilage formation.

We further investigated the function of Runx1 in cartilage, by performing tibial fractures in heterozygous mice (HZ) for the Runx1 gene. Wild-type (WT) littermates were used as controls. X-ray analysis shows that while WT tibia achieved bony union by day 14, radiolucent lines at the fracture site were still evident in the HZ mice. Furthermore, although HZ mice show callus formation as early as day 7, the bone at the fracture site remains uncalcified after 14 days. Histological staining reveals presence of fibrous non-union as well as persistence of uncalcified cartilage in the HZ fractures, while the WT fractures contained bridging bone with little cartilage. Taken together, our results indicate that haploinsufficiency of Runx1 delays chondrocyte maturation thus suggesting Runx1 may be required for endochondral ossification at the fracture sites.

Disclosures: K.E. McDougall, None.


Bonnin Stimulates Osteoblast Differentiation.L. X. Bi, E. G. Mainous, W. L. Buford*, R. R. Throndson*. Departments of Surgery and Orthopaedics, University of Texas Medical Branch, Galveston, TX, USA.

Early studies showed that bone formation is influenced by small electric currents. When an electrode was implanted in bone or bone defects, the new bone formation was observed in the vicinity of the cathode. And our previous studies have also shown that negatively charged resins increase bone formation and accelerate bone defect healing in vivo. In order to investigate potential effects of bonnin, negatively charged peptide, on human preosteoblast, we examined expression of bone morphogenetic protein-7 [BMP-7] and type I collagen, alkaline phosphatase activity (ALP) and mineralization after treatment of cells with bonnin. Human preosteoblast cells were cultured in a-minimum essential medium [a-MEM] and 10% fetal bovine serum with or without bonnin (5ug/ml) for 7, 10, 14 days, respectively. To determine mineralization, the cells were cultured in mineralizing-growth medium. The levels of ALP were assayed using a commercial kit (Sigma Chemical Co., St. Luis, MO). Expression of BMP-7 and type I collagen (polyclonal antibody, Santa Cruz Biotechnology, Inc. CA) was examined using immunoquantitative assay. The mineralization was assessed by Von Kossa technique. ALP activities were significantly elevated (38--47%, P<0.01) in bonnin treated group, compared to control group. BMP-7 expression was increased (21--35% P<0.01) after bonnin treatment compared to control. Expression of type I collagen was increased (23% at day 10 and 38% at day 14, respectively, P<0.001). There was significant increase in the levels of mineralization (2--3 fold, P<0.001) within 14 days of culture. We conclude that bonnin significantly stimulates osteoblast differentiation in vitro. It might be an important regulator of bone formation by accelerating fracture and bone defect healing, and controlling bone diseases.

Disclosures: L.X. Bi, None.


Genes that Influence Cell Differentiation Are Altered by Demineralized Bone In Vitro.K. E. Yates, S. Zhou, J. Glowacki. Orthopedic Surgery, Brigham and Women's Hospital, Boston, MA, USA.

Demineralized bone powder (DBP) induces endochondral bone formation in vivo. An early event in this process (differentiation of chondrocytes) can be modeled in vitro with a porous, 3D collagen sponge system [Exp Cell Res 227:89,1996]. We previously showed some of the gene expression shifts on day 3, prior to expression of the chondroblast phenotype [Exp Cell Res 265:203,2001]. In addition, changes in extracellular matrix genes suggested that the DBP-altered environment supports cell differentiation [Conn Tiss Res, in press]. Altogether, those gene expression shifts begin to define a “determined neochondroblast” phenotype at 3 days in the DBP/collagen sponge.

In this study, the hypothesis was that DBP alters expression of differentiation factors (i.e., peptide factors and extracellular matrix components) in determined neochondroblasts. Human dermal fibroblasts were used as targets cells for chondroinduction by DBP. Cells were cultured in DBP/collagen or control collagen sponges for 3 days. RNA was isolated from pooled sponges (n=10 per group) and used for cDNA macroarray and RT-PCR analyses. cDNA synthesis, labeling, hybridization, and data analysis were performed as per the manufacturer's instructions (Human Signal Transduction PathwayFinder and TGFB-BMP Signaling Pathway GE Arrays, Q Series; SuperArray Inc., Bethesda MA).

We found that DBP increased expression of several peptide differentiation factors, receptors, and matrix proteins (Table). Increases in other factors (TGFb1; BMP1; BMP antagonist-1; anti-mullerian hormone) and receptors (activin receptors I, IB, II, and II-like) was detected but could not be quantified due to low levels in control cells. Decreases in some differentiation factor receptors (TGFbR II and III; Patched 1) were detected. It is notable that no changes in expression were found for nodal (a differentiation factor) or BMP receptors IA, IB, or II. Several TGF-b/BMP signaling genes (RUNX1/AML1, RUNX2/cbfa1; Smad 2, 3, 4, 5, and 9) were also not changed by DBP. Of note, many TGFb superfamily proteins (BMP2--11, -15; GDF1--3, 5, 8--11; TGFb2, b3) were below the limits of detection in this system. Overall, the changes reflect coactivation of a unique and specific set of TGFb superfamily signaling and target genes in determined neochondroblasts that may be necessary for initiating post-natal differentiation. 

Table Table.. DBP-regulated “differentiation genes.”
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Disclosures: K.E. Yates, None.


Effects of RGD-Peptide(224) on Bone Metabolism and Mechanical Strength in Ovariectomized Mice.X. Yan*. Department of Biochemistry, BeiJing JI Shui Tan Hospital, Beijing, China.

The purpose of this investigation is to compare the effects of a synthtic RGD peptide (224) with 17β- Estrodiol on bone metabolism and mechanical strength in ovariectomized (OVX) mice. Methods: Bone mineral density (BMD), bone mineral content (BMC), bone strength, bone histomorphometry and biochemistry indicators were measured in all mice. Results: 36 female Kunming mice, weighing an average of 35g, were randomly divided into 4 groups. After 6 weeks treatment RGD peptide(224) at 4.41 and 17β-Estradiol at 30αg.Kg-1d-1, in OVX mice, femal BMD and BMC increased respectively 0.7%, 4% and 5.9%, 5.7%. Maximun load of bone increased respectively 12.9% and 16% and serum AKP decreased 33% and 37% respectively. Conclusion:RGD(Arg-Gly-Asp)-containing sequence into the mutant proinsulin by replaceing C-peptide and four basic amino acid residues with designed CRGDSC hexa-peptide.RGD peptide may maintain BMD, improve BMC and bone mechanical strength and decrease bone turnover in OVX mice.

Disclosures: X. Yan, None.


Bisphosphonate Bone Affinity, Differences Predicted By In Vitro Carbonated Apatite Model.Z. J. Henneman*1, R. Tang*1, S. Gulde*1, G. H. Nancollas*1, R. J. Phipps2, R. G. G. Russell3, F. H. Ebetino2. 1University of Buffalo, Buffalo, NY, USA, 2Procter & Gamble Pharmaceuticals, Mason, OH, USA, 3University of Oxford, Oxford, United Kingdom.

Bisphosphonates (BPs) are effective inhibitors of bone resorption. Important components in their ability to inhibit bone resorption are their affinity for calcium phosphate surfaces and their ability to target to bone mineral. This can be predicted in vitro through studies of their effects on the dissolution and growth of calcium phosphates. The effects of BPs on the dissolution of a bone-like mineral, hydroxyapatite (HAP), has been previously reported. We now also report data on BP effects on carbonated apatite (CAP), which is thought to be more relevant to human bone. These effects have been studied by a constant composition method at both physiological ionic strength and temperature, 0.15M and 37°C. Adsorption affinity constants, K, were calculated from the kinetics data. For HAP growth at pH=7.4, differences were observed between BPs in the order of zoledronate > alendronate> risedronate. For HAP dissolution at pH = 5.50, the rank order of inhibition was similar but the difference between the BPs was less marked. Adsorption affinity constants for CAP dissolution rank ordered as observed with HAP, but with a greater discrimination between zoledronate, alendronate, and risedronate at pH = 5.50. The degree of inhibition was also markedly dependent on the relative undersaturation, a, with respect to CAP. When compared at a more physiologically relevant undersaturation, a = 0.3, K values for risedronate (4.93×104 M-1) and alendronate (1.10×105 M-1) were only 25% and 50% of that for zoledronate (2.19×105 M-1), respectively. The inhibition of CAP dissolution by the BPs was also related to the carbonate content of the crystallites. Lower dissolution rates were obtained with crystallites containing more carbonate. Thus, at a = 0.3 and pH = 5.50, K values for zoledronate inhibited dissolution of CAP containing 3.1±0.1% and 8.0±0.1% carbonate were 1.47×105 and 2.19×105 M-1, respectively. These results point to the importance of carbonate in more accurately distinguishing between the ability of BPs to target and affect bone mineral, and further substantiate the significantly lower bone affinity of risedronate compared to alendronate and zoledronate. These differences in bone affinity may contribute to the observed differences in pharmacokinetics among these three BPs in the clinic. Partial support by NIH/NIDCR grant # DE03223.

Disclosures: Z.J. Henneman, None.


The Relationship of Antiresorptive Drug Use to Osteoarthritis.L. D. Carbone1, S. B. Kritchevsky*1, K. Wildy*2, K. D. Barrow*1, M. Visser*3, F. Harris*4, T. B. Harris*5, B. W. E. Wang*1, M. C. Nevitt*4. 1UTHSC, Memphis, TN, USA, 2Univ. of Pittsburgh, Pittsburgh, PA, USA, 3EMGO Institute, Amsterdam, Netherlands, 4UCSF, San Francisco, CA, USA, 5NIA, Bethesda, MD, USA.

Subchondral bone changes accompany cartilage degradation in knee OA. Antiresorptive treatments (ARTx) could be beneficial in OA, although there is limited data. We analyzed 1360 women ages 69--81 (44% Black) in the Health ABC study to examine the relationship of current use of ARTx to symptomatic knee OA (Sx) (knee pain plus xray OA in tibiofemoral/patellofemoral area), bone and cartilage changes in the knee by MRI and knee pain severity. Women were interviewed for knee pain and medication use. Those with knee pain, and a random sample without, had PA fixed flexion knee x-rays and bilateral MRI of the knee done on 1.5 T GE Signa scanners (axial/coronal T2 FSE, sagittal T2 FSE with fat saturation). MRIs were read using the Peterfy Whole Organ MRI Scoring (WORMS) method. Logistic and linear regression, with GEE to adjust for inter-knee correlations, were used to examine the association of ARTx use with outcomes. Models were adjusted for age, race, BMI, NSAIDS, thiazides, calcium supplements, smoking, quads strength, whole body BMD and study site. 313 (23%) women had Sx knee OA. In adjusted analyses, there was no association of Sx knee OA with use of any ARTx (OR: 1.11; 95% CI: 0.64--1.90) nor with use of individual ARTx. WORMS data were available for 898 knees. Results were similar for knees with and without pain, so these were combined. Bisphosphonate, but not other ARTx use, was associated with lower bone attrition (BA) and bone marrow edema (BME) scores compared with nonuse (p<0.05). There were no differences in osteophyte (OP), total cartilage (TC) or WOMAC scores by use of any ARTx or by use of individual ARTx (p>0.11). In women in the Health ABC study, BA and BME were significantly reduced with bisphosphonate use, supporting a potential effect of ARTx in knee OA. 

Table  . MRI and WOMAC Scores by Use of ARTx (Mean (SD))
  1. *p<0.05 Compared to nonusers

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Disclosures: L.D. Carbone, Proctor and Gamble 5, 8; Aventis 2, 5, S; Merck 5, 8; Eli Lilly 5, 8.


Serotonin Affects Bone Metabolism in Vitro and Leads to Increased Bone Mineral Density (BMD) in Female Rats.B. Gustafsson*, L. Thommesen*, R. Fossmark*, H. L. Waldum*, U. Syversen. Institute of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Faculty of Medicine, Trondheim, Norway.

Serotonin (5-hydroxytryptamine or 5-HT) is a peptide neurotransmitter. Outside the central nervous system it is mainly produced by the enterochromaffin cells of the gut where it participates in the regulation of intestinal motility, fluid secretion and regional blood flow. Cell culture studies have shown that serotonin has proliferative effects on fibroblasts, myofibroblasts, smooth muscle cells and endothelial cells. Serotonin is also known as a regulator of craniofacial morphogenesis. Recent studies have shown the expression of several serotonin receptors in chicken osteocytes and osteoblasts, mouse and rat osteoblasts, as well as the serotonin transporter 5-HTT in rat osteoblasts. In the present study we have studied the effect of serotonin on proliferation of preosteoblasts, the effect on osteoclast differentiation and finally the long-term effects of serotonin on BMD in female rats.

The murine preosteoblast cell line MC3T3-E1 was incubated with serotonin at different concentrations (0.01--50uM), and the proliferation rate was determined by using a ELISA BrdU proliferation kit. Human peripheral blood mononuclear cells were differentiated into osteoclasts by stimulation with RANK-L, MCSF and dexametazone, in the presence or absence of serotonin (0.1--10uM). Tartrate resistant acid phosphatase positive multinucleate giant cells were considered as genuine osteoclasts. Female Sprague-Dawley rats were given daily s.c serotonin injections (5 mg/kg) for 3 months and BMD was thereafter measured by DXA.

Serotonin induced a 10--15 % increase in osteoblast proliferation. This increase was totally abolished in the presence of the PKC inhibitor, GF109203. Serotonin increased the total number of differentiated human osteoclasts by 90--300%, in a dosage-dependent manner. After 3 months total body BMD was significantly higher in the serotonin treated rats, (mean±SEM: 0,1976 ±0,0012 g/cm2) compared to controls, (0,1913 ±0,0015 g/cm2, p=0,004). There was no significant difference in femoral BMD between the 2 groups, which might be explained by the fact that the serotonin treated rats were less active and weighed 5% less than the controls.

Conclusion: Serotonin induces murine osteoblast proliferation and human osteoclast differentiation in vitro. Treatment with serotonin increases total body BMD in female rats. These results indicate that serotonin might have an important regulating role in bone metabolism.

Disclosures: U. Syversen, None.


Light-Activated Gene Transduction of Recombinant Adeno-Associated Virus in Human Mesenchymal Stem Cells.H. Ito, J. Goater*, P. Tiyapatanaputi*, P. T. Rubery*, R. J. O'Keefe, E. M. Schwarz. Department of Orthopaedics, University of Rochester Medical Center, Rochester, NY, USA.

Deficiencies in skeletal tissue repair and regeneration lead to conditions like osteoarthritis, osteoporosis and degenerative disc disease. While no cure for these conditions is available, the use of human bone marrow derived-mesenchymal stem cells (HuMSCs) has been shown to have potential for cell-based therapy. Furthermore, recombinant adenoassociated viruses (rAAV) could be used together with HuMSCs for in vivo or ex vivo gene therapy. Unfortunately, the poor transduction efficiency of these cells remains a significant obstacle. Here, we describe the properties of ultraviolet (UV) light activated gene transduction (LAGT) with rAAV in HuMSCs, an advance toward overcoming this limitation. Using direct fluorescent image analysis and real-time quantitative PCR to evaluate enhanced green fluorescent protein (eGFP) gene expression, we found that the optimal effects of LAGT with limited cytotoxicity occurred at a UV dose of 200J/m2. Furthermore, this UV irradiation had no effect on either the chondrogenic or osteogenic potential of HuMSCs. Significant effects of LAGT in HuMSCs could be detected as early as 12 hours after exposure and persisted over 21 days, in a time and energy-dependent manner. This LAGT effect was maintained for more than 8 hours after irradiation and required only a 10-minute exposure to rAAV after UV irradiation. Finally, we show that the production of secreted TGFβ1 protein from rAAV-TGFβ1-IRES-eGFP infected HuMSCs is highly inducible by UV irradiation. These results demonstrate that LAGT combined with rAAV is a promising procedure to facilitate gene induction in HuMSCs for human gene therapy.

Disclosures: H. Ito, None.


Environmental Effects on Osteogenesis.Z. Valkusz MD*1, O. Vetró*2, A. Juhász*3, A. Petri*4, M. Gálfi*2. 1Department of Endocrinology, Medical Faculty, University of Szeged, Szeged, Hungary, 2Environmental Protection Group, University of Szeged, Szeged, Hungary, 3Department of Psychiatry, Medical Faculty, University of Szeged, Szeged, Hungary, 4Department of Surgery, Medical Faculty, University of Szeged, Szeged, Hungary.

Bone and cartilage cells differentiate from fibroblasts. These processes are influenced by the extracellular space, and during the development and differentiation of fibrocytes they are determined by the receptors on/in the cells, and genetic factors in nucleus and mitochondria. The modifications of the extracellular enviroment therefore determine the formation and eventually the functionability of cells. The chemical and physical enviromental effects play a role in inducing osteoporosis. We studied the chronic effects of sub-toxic doses of chlorobenzene- given to pregnant rats- on embrionic and newborn osteogenesis changes. Model experiments were applied to investigate the causes of observed bone (femur) destruction. During investigation, Wistar rats were treated through gastric tubes with hexachlorobenzene:trichlorobenzene=1:1, and the dosage was 3, 6, 12.5 μg/kg body weight for 30 or 60 days. After treatment, we studied the pregnant, embrionic and newborn animals. The samples were taken from the bone tissues of the treated, untreated control, and solvent-treated control animals. Tissue structure and Ca2+ content were examined. Liver function (γGT, SGOT, SGPT) and sturture were also investigated. Monolayer cultures were generated from the fibroblasts of treated and control animals, and membrane fluidity (fluorescent anisotropy), collagen synthesis (protein content in the presence and absence of Collagenase-E) and structure (morphometry) were examined. As a result of the chronic hexa-and trichlorobenzene treatment, femur tissue destruction was observed in all the animals. This general structural damage of the bone tissues is in correlation with a significant change in the Ca2+ content of the femur matrices. Although there was found no fundamental change in the amount of procollagen I, destruction was detected in the protein structure.

Disclosures: Z. Valkusz MD, None.


From Marrow to Bone and Adipose Tissues: Isolation, Cultivation, and Induction of Differentiation in Adult Rat.C. Shih, H. W. Wu*. Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan Republic of China.

Mesenchymal stem cells(MSCs) are present in a variety of tissues during development, and its potential roles in tissue engineering and cellular and gene therapy are promising in the near future. Bone marrow MSCs contribute to the regeneration of tissues such as bone, cartilage, muscle, ligament, tendon, and adipose tissues. Despite extensive experimentation has defined conditions for their isolation and characterization both in vivo and in vitro, there is still no well-defined protocol for isolation and expansion of these cells. MSCs isoltated primarily by tight adherence to plastic Petri dishes in most experiments shows heterogenerous. More recently, sorting by size differences and surface markers has been developed to obtain more homogenerous population. The purpose of this study was to isolate, characterize, and induce cell differentiation into different lineage cells by density gradient separation method, cell culture, CD marker selection, alkaline phosphatase activity(ALP), type I collagen synthesis, bone colony formation, light and phase contrast inverted microscopy, H&E stain, oil red O stain and flow cytometry. Rat bone marrow cells were harvested from both femur and tibia and MSCs isolated by density gradient separation, cultivation and CD markers (e.g. CD 90 and etc.) selection. Isolated bone marrow MSCs were cultured to semiconfluent and treated with osteogenic differentiation medium(DMEM-LG supplemented with 10% FBS, ascorbic acid, 100 nM dexamethasone, with or without 10 mM beta-GP) or adipogenic differentiation medium (100 nM dexamethasone, indomethacin and insulin). Appearance of cuboid-shaped osteoblastic cells, unmineralized or mineralized colony was noted approximately 5, 10 and 14 days after osteogenic induction. Histological sections showed mineralized matrix with lining osteoblasts and entrapped osteocytes. With adipogenic induction, small sized lipid droplets began to appear within cytoplasm 2--4 days. More and larger lipid droplets accumulation were noted 5--10 days adter induction. We also noted that lipid droplets-containing adipocytes terminally differentiated into round shaped adipocyte and finally became unadherent and floating within the medium. The results of this study indicated that bone marrow MSCs could be isolated, cultured, and induced to become osteogenic and adipogenic cells/ tissues.

Disclosures: C. Shih, None.


Regeneration of Critical-sized Bone Defect after Transplantation of Rat Marrow Mesenchymal Stem Cells.C. Shih1, H. W. Wu*1, J. Shyu1, P. Chu2. 1Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan Republic of China, 2Nephrology, Tri-Service General Hospital, Taipei, Taiwan Republic of China.

Bone marrow has been shown to contain a population of mesenchymal stem cells (MSCs) that are capable of forming bone, tendon, cartilage, muscle, and other connective tissues. The repair/regeneration of bone tissue requires 1) MSCs capable of differentiating into osteoblasts, 2) a scaffold to support the attachment and migration of MSCs or osteogenic cells and 3) growth factors that direct these cells to migrate into bone defects, to proliferate, to differentiate into osteoblasts, and form new bone. The purpose of this study was to evaluate the ability of cultured marrow MSCs to elicit repair/regeneration at the site of a critical-sized bone defect in the rat femur. The strategy is based on the hypothesis that such an approach decreases or eliminates the need for chemotaxis of osteoblast progenitor cells into the defect and their massive proliferation. MSCs has been proved to have the osteogenic potential with addition of differentiation inducing factors in vitro before implantation in this study. Forty 12-month old male Sprague-Dawley male rats were used in this study. A model for the resection of an osteoperiosteal segment and stabilization of the limb was developed, and the defect was left untreated, filled with osteoset that had been loaded with MSCs, MSCs alone, osteoset alone, and isolated adherent light density marrow cells. Bone formation and healing at the site of the defect were evaluated by microradiography, histological, and bone histomorphometric analysis. Microradiographic analysis was performed at 2 week intervals. Eight weeks after surgery, new bone formed throughout the body of the implant by direct conversion of MSCs into osteoblsts, without progression through a cartilaginous intermediate. Microradiographs showed radiopaque area(area of mineralization) began to appear eight weeks (in osteoset with MSCs group) or 10 weeks (in MSCs alone group) after implantation. It is also noted that MSCs were superior to isolated adherent light density marrow cells. The results of this study indicating that osteoset loaded with marrow MSCs effectively repair or regenerate the critical-sized bone defect.

Disclosures: C. Shih, None.


Platelet-Derived Growth Factor Stimulates Osteoblastic Migration and Proliferation via Independent Signaling Pathways.M. Mehrotra*, S. Mehrotra*, C. C. Pilbeam. Medicine, University of Connecticut Heath Center, Farmington, CT, USA.

Osteoblastic migration is important for skeletal development as well as bone remodeling and fracture repair. Platelet-derived growth factor (PDGF) is a potent stimulator of both osteoblastic migration and proliferation. We used a wounding model to study migration of osteoblastic MC3T3-E1 cells on a collagen substrate in response to PDGF. Non-tissue-culture-treated Petri dishes were coated with 5 μg/cm2 of rat-tail collagen type I and cells were plated at 100,000 /cm2 in DMEM with 10% FCS. 24 h later half the cell layer was removed by gentle scraping and the cultures were treated with PDGF-BB (10 ng/ml) in serum-free DMEM. The average distance of cell migration from the wound line was measured in arbitrary units (pixels) using NIH Image software. PDGF stimulated a 1.8 and 2.4-fold increase in the distance that cells moved out from the wound line over 24 and 48 h, respectively. Treatment with PDGF for 24 h also stimulated a 10-16-fold increase in cell proliferation as measured by 3[H]-thymidine incorporation into replicating DNA ([3H]-TdR) over the last 2 h of culture, normalized to total DNA content. Aphidicolin, 30 μM and 90 μM, inhibited the PDGF stimulated increase in [3H]-TdR by 90% and 98%, respectively, but did not reduce the distance of migration from the wound line. [3H]-TdR results were confirmed by BrdU staining of cells. Previous studies have shown mitogen activated protein (MAP) kinase pathways to be involved in PDGF stimulated fibroblast migration and proliferation. Using flow cytometry, we analyzed the carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled cells to measure the % of cells that were replicating in cultures treated with PDGF, with and without specific MAP kinase inhibitors. An inhibitor of ERK activation, U-0126 (20 μM), reduced the number of replicating cells in both vehicle and PDGF treated cultures by 50%, while a p38 MAP kinase inhibitor, SB-203580 (10 μM), had little effect. Conversely, migration in PDGF treated cultures was reduced 80--95% by the p38 inhibitor and only 25% by the ERK inhibitor. We conclude that PDGF stimulated both proliferation and migration of MC3T3-E1 osteoblasts. Proliferation occured via an ERK pathway. Migration was independent of proliferation and occured via a p38 MAP kinase pathway.

Disclosures: M. Mehrotra, None.


Identification of a Biflavone Found in Ginkgo Biloba that Stimulates Bone Formation In Vitro and In Vivo.W. E. Gallwitz, G. Gutierrez, I. R. Garrett, M. Jalomo*, J. Esparza*, A. Escobedo*, B. McCluskey*, P. Rivas*, D. Horn*, G. R. Mundy. OsteoScreen, San Antonio, TX, USA.

There is considerable interest in the effects of various herbal remedies on bone, since many have been proposed to have beneficial consequences. To identify and characterize constituents of herbal preparations that may have stimulatory effects on bone formation, we examined hundreds of natural product compounds frequently found in herbal preparations. We discovered that the biflavone isoginkgetin, one of five biflavones found in Ginkgo biloba leaf extracts, stimulated bone formation in organ cultures of neonatal murine calvaria at concentrations of 5--20 μM. This compound was also tested for its capacity to stimulate bone formation in vivo, using the murine local calvarial injection model. Isoginkgetin at doses from 2--20 mg/kg per day caused a dose-dependent increase up to 40% in local appositional bone growth after 5 days of treatment. To determine the mechanism by which isoginkgetin stimulated bone formation, we examined its capacity to inhibit the 20S proteasome, since we have recently found compounds that are inhibitors of the chymotryptic catalytic activity of the proteasome enhance osteoblast differentiation. We found that isoginkgetin in concentrations of 0.1--3 μM inhibited proteasome activity. Since proteasome inhibitors stimulate osteoblast differentiation in part by their capacity to stimulate BMP-2 expression, we tested the effects of isoginkgetin in concentrations from 0.075--40 μM on BMP-2 promoter activity, using osteoblasts stably transfected with the murine BMP-2 promoter linked to the firefly luciferase gene. We found that isoginkgetin increased BMP-2 gene transcription 3 fold at 5 μM. In conclusion, our results show that the biflavone isoginkgetin, which is a constituent of herbal remedies, stimulates bone formation in vitro and in vivo. Isoginkgetin is a powerful inhibitor of the osteoblast proteasome, and our data is consistent with the notion that isoginkgetin stimulates osteoblast differentiation and bone formation by its effects to enhance BMP-2 transcription.

Disclosures: W.E. Gallwitz, None.


Alterations in Bone Formation in a Glucose-Dependent Insulinotropic Peptide Functional Receptor Knockout Mouse on a Low Calcium Diet.D. Xie1, Q. Zhong1, K. Ding1, R. J. Bollag*1, C. M. Isales2. 1Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA, 2Medicine, Medical College of Georgia and the Augusta VA Hospital, Augusta, GA, USA.

Glucose-dependent insulinotropic peptide (GIP) is a 42 amino acid peptide secreted by endocrine cells in the small intestine. Our laboratory has previously shown that GIP has anabolic effects on osteoblastic-like cells and that daily injection of GIP in rats results in higher bone mass. To further evaluate the role of GIP in bone metabolism we generated a GIP-overexpressing transgenic mouse with a metallothionein inducible promoter. Even at baseline the transgenic mice were found to have a greater than two-fold higher level of GIP than controls (Control: 197.1+/−26.3 vs. MT-GIP: 561+/−130 pM+/−SEM p<0.001), which rose even higher with zinc (25mM): 1,160+/−189 pM/ml+/−SEM. We have previously shown that continuous high levels of GIP lead to GIP receptor downregulation. MT-GIP transgenic mice and controls were started on zinc feeding at one month of age, resulting in receptor downregulation and thus generating a GIP receptor knockout mouse. Twenty mice were then divided into four groups: (1) control (2) control + low calcium diet (0.1% calcium) (3) MT-GIP and (4) MT-GIP on a low calcium diet. Several parameters of bone turnover were measured including osteocalcin and bone mineral density (PIXImus, Lunar Corp.). Osteocalcin (a marker of bone formation) levels were: Group 1: 18.89+/−5.491 and Group 3: 10.89+/−1.74 (ng/ml+SEM). Thus, the MT-GIP have significantly lower osteocalcin levels than control (p<0.01) suggesting that the MT-GIP mice were more prone to lose bone. Similarly, at the end of five months MT-GIP had a lower bone mineral density than controls (0.0503+/−0.0021 vs. 0.0526+/−0.0028 g/cm2+/−SEM). As expected a low calcium diet led to a drop in BMD in both controls and MT-GIP but this was greater in the MT-GIP (0.0400+/−0.003vs 0.0429+/−0033g/cm2+/−SEM). Taken together this data is consistent with an anabolic role for GIP and when the GIP effect is lost (via receptor downregulation) the animals are more prone to lose bone.

Disclosures: D. Xie, None.


N-cadherin-mediated Distribution of Stabilized β-catenin Alters the Effect of MAP Kinase and BMP-2 Signaling on Gene Expression during Chondrogenesis.R. Modarresi*1, J. Roman-Blas*2, T. Lafond*1, K. G. Danielson1, R. S. Tuan3, M. R. Seghatoleslami*2. 1Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA, USA, 2Medicine/ Rheumatology, Thomas Jefferson University, Philadelphia, PA, USA, 3Cartilage Biology and Orthopaedics Branch, NIH, NIAMS, Bethesda, MD, USA.

We have examined the effect of calcium dependent adhesion, mediated by N-cadherin, on cell signaling involved in chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan and Sox 9 was examined in monolayer (nonchondrogenic), or micromass (chondrogenic), cultures of C3H10T1/2, or C3H10T1/2 cell lines expressing a dominant negative form of N-cadherin that is missing the extracellular calcium binding domain (δ390--10T1/2) and also the MNCD2--10T1/2 subline that expresses N-cadherin at twice the level compared to the endogenous gene. Our findings show that overexpression or inhibition of N-cadherin in C3H10T1/2 cells result in changes in chondrogenic gene expression. In 2.5 day micromass cultures, reduced N-cadherin function in the δ390--10T1/2 cell line resulted in increased cytoplasmic levels and nuclear localization of ß-catenin which is accompanied by increased ratio of type IIB /type IIA collagen splice forms but no significant effect on aggrecan gene expression. On the other hand, over-expression of normal N-cadherin in the MNCD2--10T1/2 cell line resulted in reduced type II collagen expression as well as total inhibition of aggrecan gene expression. In addition, levels of nuclear and cytoplasmic ß-catenin were markedly reduced but an increase in cell membrane localization of this protein was observed. Therefore, these findings suggest that adherens junctions involving N-cadherin can control the distribution of stabilized ß-catenin (possibly by Wnt signaling), in regulating gene expression. We have further analyzed the SRF activity, a nuclear target of MAP kinase signaling implicated in chondrogenesis. In general, in semi-confluent mono-layer cultures (minimum cell-cell contact) of C3H10T1/2, NCD2--10T1/2 or δ390--10T1/2 cells, there was not a significant change in MAP kinase or BMP-2 regulation of a construct concatamer of SRF element fused with luciferase reporter gene. In micromass cultures, a culture condition required for upregulation of N-cadherin expression and initiation of chondrogenesis, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of ß-catenin. Specifically, the SRF promoter construct was more responsive to the above signaling in δ390--10T1/2 micromass cultures compared to C3H10T1/2 or NCD2--10T1/2 cells. Support: NIH grant DE12864.

Disclosures: M.R. Seghatoleslami, None.


Developmental Patterning of Fracture Callus Formation: Three-Dimensional Image Analysis and Collagen Gene Expression.L. C. Gerstenfeld, Y. M. Alkhiary, D. M. Cullinane*, F. Nicholls*, D. T. Graves*, T. A. Einhorn. Boston University Medical Center, Boston, MA, USA.

While a standardized set of histological criteria have been developed to assess the metabolic status of bone, identical measurements are not fully applicable to bone repair because of the unique attributes of the endochondral process. Standardized methodologies were developed to assess fracture healing which allowed for the quantitative measure of cartilage and new bone formation, as well as overall fracture callus tissue development and geometry. Standard mid-diaphyseal transverse closed fractures were generated in the femurs of mature male rats or mice. Fracture calluses from both species were collected at 1, 2, 3, and 5 weeks. Serial transverse sections were taken at 100 micron intervals over a total length of 7500 microns of the callus. The quantities of cartilage, bone and marrow space were determined by differential staining with Safranin-O/Fast Green, and TRAP staining was used to assess osteoclast numbers. Statistically reproducible measurements were obtained from sections taken at 500 micron increments from a minimum of three animals. Using 3D reconstruction software, the developmental patterning of the tissue formation of the fracture calluses were determined. These data showed two morphogenetic fields of endochondral development around the fracture line. The endochondral tissue that forms was not symmetrical. Rather, ∼70% of the volume of tissue that forms is on the distal posterior/medial axis, and as it develops proximally the endochondral tissue rotates in an anterior/lateral direction. The confines that defined the volume of original endochondral tissue dictated the pattern of the primary remodeling of the tissue. Serial reconstruction of the pattern of collegen types I and II in situ hybridization were used to establish the developmental patterning of the tissue. Active areas of Type II collagen gene expression were localized on the surface of the callus and centrally around the fracture line. Type I collagen expression was uniformly expressed around the cortical surfaces and was greater proximally and distally away from the fracture line. Both patterns of gene expression and callus formation were identical in both species of bone that were examined. These results demonstrate that traditional sagittal views of fracture callus histology have provided a biased and incomplete description of callus formation. They further suggest that as of yet unidentified aspects of anatomical, biomechanical, and/or developmental principles affect the developmental mechanisms that regulate fracture callus morphogenesis.

Disclosures: L.C. Gerstenfeld, None.


The Collagenase, Matrix Metalloproteinase-13 (MMP-13), Is Required for Osteoclast Formation and Function.M. Inada1, Y. Wang1, M. H. Byrne*1, C. Miyaura2, S. M. Krane1. 1Rheumatology CIID, Mass General Hospital, Boston, MA, USA, 2Department of Biochemistry, Tokyo University of Pharmacy and Life Science, Tokyo, Japan.

To determine potential roles of collagenases in bone remodeling, we targeted a null mutation in mice in the MMP-13 gene splicing out Exon 5 which encodes the critical catalytic, zinc-binding domain. MMP-13 is normally highly expressed in the skeleton during embryonic development and adult bone remodeling. Previously, we presented preliminary results indicating that homozygous mutant mice (MMP-13 -/-) had increased trabecular bone mass (DEXA, microCT), decreased trabecular separation (histomorphometry, microCT) and decreased bone resorption in organ cultures of newborn calvariae in response to parathyroid hormone (PTH) or interleukin-1 alpha, suggesting altered osteoclast formation/function. Here we provide further evidence for altered osteoclast formation/function. In metaphyses of distal femurs from mice 6 weeks of age, osteoclast numbers (TRAP staining) were decreased in MMP-13 -/- vs. MMP-13 +/+ mice by ∼ > 40 % (p< 0.01). Bone resorption area was also decreased (p 0.1--0.4 mm2 was strikingly decreased (p< 0.001) and collagen deposition increased (Sirius red staining) in BMC from MMP-13 -/- vs. MMP-13 +/+ mice. This reduction was associated with reduced levels of osteoclast marker mRNAs, measured by Realtime PCR. The functional capacity of the osteoclasts generated in BMC was measured by formation of pits on dentin. The pit area (mm2) /dentin slice area (mm2) formed by osteoclasts generated by PTH was reduced from ∼ 8 to ∼2 (p<0.01) and that by osteoclasts generated by 1,25(OH)2 vitamin D3 from ∼11 to ∼2 (p<0.01) in BMC from MMP-13 -/- vs. MMP-13 +/+ mice. Osteoblasts normally support osteoclast formation/function. In wild type BMC, MMP-13 is produced by osteoblasts/stromal cells but not by osteoclasts. We propose therefore that absence of osteoblast MMP-13 and increased uncleaved extracellular collagen are primarily responsible for the decreased osteoclast formation/function we observed in the MMP-13 -/- mice.

Disclosures: M. Inada, None.


Differential Effects of Selenium and Iodine on Micro-architecture of Bone in Male and Female Rats.B. J. Stoecker, F. Toure*, E. A. Lucas*, J. B. King*, B. H. Arjmandi. Nutritional Sciences, Oklahoma State University, Stillwater, OK, USA.

Epidemiological studies suggest a relationship between a combined deficiency of selenium (Se) and iodine (I) and impaired bone metabolism. This project investigated the effects of experimental Se and/or I depletion on bone structure in young rats. Dams were fed experimental diets beginning at week 1 of lactation. Pups were weaned at 3 wks of age and a subsample of males and females were fed the experimental diet of their mother for an additional 7 wks. Weight gain was significantly reduced by I deficiency in all rats and by Se deficiency in males. Thyroid weight was increased and serum thyroxine (T4) was reduced by I deficiency. Selenium depletion was confirmed by significantly lower hepatic glutathione peroxidase activity and Se depletion tended (p<0.06) to increase T4. Tri-iodothyronine (T3) was increased in female rats. In male rats selenium depletion reduced T3 levels. Both iodine and selenium depletion decreased bone mineral area and content as measured by DEXA (Hologic QDR4500A Elite with small animal software). Three-dimensional analysis of proximal tibia by micro-computed tomography (μCT) showed a signficantly lower bone volume/total volume (BV/TV) in males than in females. In Se depleted animals, BV/TV was higher when I was adequate. The structure model index (SMI) was lower in females meaning that the structure of the bone was more plate-like. Females also had higher trabecular thickness and lower trabecular separation. In Se-depleted animals, trabecular separation was signficantly higher when I was also depleted. Modes of action of Se and I on bone require further investigation.

Disclosures: B.J. Stoecker, None.


Collagen Crosslinking Modifies the Mechanical Properties of Cortical Bone.P. Garnero1, Y. Proust*2, O. Borel*2, E. Gineyts*2, F. Duboeuf*2, H. Solberg*3, P. D. Delmas2. 1Synarc, Inserm Unit 403, Lyon, France, 2Inserm Unit 403, Lyon, France, 3Nordic Bioscience, Herlev, Denmark.

Bone mineral density (BMD) is the main determinant of bone strength but the contribution of bone's material properties, particularly the extent of crosslinking (XL), is poorly understood. In vitro studies suggest that enzymatic XL including pyridinoline (PYD) and deoxypyridinoline (DPD) and non-enzymatic glycation end products such as pentosine (PEN) may play a role. We also showed that alterations of the isomerisation of C-telopeptide of type I collagen (CTX) - that can be detected in vivo by the urinary ratio of isomerized (β) to native (α) CTX -are associated with fracture risk independently of BMD and bone turnover. We analyzed the role of increasing XL on mechanical properties of bone specimens of constant size and mineral content.

Calibrated fetal bovine cortical bone specimens (11 different animals, 41 samples in total) characterized by a low degree of postranslational modifications were incubated for 0, 60, 90 and 120 days at 37°C to induce collagen crosslinking in vitro. At each time point, 3 point binding mechanical tests were performed to determine the Young modulus (an index of stiffness) and yield stress (an index of elasticity). We also measured on each sample the concentration of PYD, DPD and PEN by HPLC, α and β CTX by ELISA on trypsin bone digests, and BMD by DXA.

After 60 days of incubation, elasticity was decreased by 40% (p=0.01) -with no significant further change up to 120 days- whereas no significant effect was observed on stiffness and BMD. The decline in elasticity at 60 days was associated with increases of PYD (+98%, p=0.005), DPD (+42%, p=0.013), PEN (+ 55 fold, p=0.005) and β/α CTX ratio (+4.9 fold, p=0.005). In univariate analyses, PYD was significantly associated with stiffness (r= -0.37, p=0.003) and elasticity (r=-0.64, p<0.001). PEN was a significant predictor of both elasticity (r=-0.63, p<0.001) and of its 60 days changes (r=-0.64, p=0.04), higher PEN concentration in bone being associated with lower elasticity. The ratio β/α CTX correlated with elasticity (r= +0.46, p=0.002). In multivariate analyses, the prediction of elasticity was significantly improved by combining BMD data (r2=0.36) with PYD (r2 increasing to 0.58, p=0.0003), PEN (r2 increasing to 0.58, p<0.0001) or β/α CTX (r2 increasing to 0.49, p=0.003).

These data indicate that the degree of posttranslational modifications of collagen matrix plays an independent role in determining the mechanical competence of cortical bone. Further studies should explore potential alterations of these biochemical parameters of bone matrix in osteoporosis, as they may represent a key component of bone quality.


Detection by Immunoassay of C-terminal Telopeptides of Bone Collagen Containing Unidentified, Non-fluorescent Cross-links.K. S. Puukka*1, M. K. Koivula*1, S. P. Robins2, K. Savolainen*3, J. P. Risteli1. 1Clinical Chemistry, University of Oulu, Oulu, Finland, 2Rowett Reseach Institute, Aberdeen, United Kingdom, 3Clinical Chemistry, Kuopio University Hospital, Kuopio, Finland.

Two immunoassays, ICTP and CrossLaps®, measure degradation of type I collagen through interaction with carboxyterminal telopeptide structures. ICTP antigen is derived from collagenase or trypsin digestion of bone type I collagen whereas CrossLaps is based on a shorter, synthetic peptide from the same region. The CrossLaps assay is sensitive for physiological bone resorption, but ICTP measures mainly pathological bone destruction. ICTP antigen contains two alfa-1 telopeptides and one helical peptide joined by mature, trivalent cross-links, e.g. hydroxylysylpyridinoline (Pyd) and lysylpyridinoline (Dpd). However, about 60 % of the trivalent cross-links are uncharacterised. To investigate the nature of these unknown cross-links ICTP peptides linking helical and alpha 1 sites were isolated from chymotrypsin digests then treated with cathepsin K. Cross-linked peptide fragments were purified using two successive RP-HPLC fractionations, monitoring both pyridinium fluorescence and CrossLaps reactivity (automatic Elecsys-instrument). The CrossLaps assay used measures only those C-telopeptides where both alfa-1-telopeptides have beta-isomerization of aspartyl residues. Cathepsin K produced five major fragments from the alfa1-alfa1-alfa1-telopeptide, only two of which contained pyridium cross-link fluorescence. The CrossLaps concentration was highest in those fractions where pyridinium cross-links were undetectable. MALTI-TOF analysis revealed differences in the molecular masses of these cross-linked fractions, with CrossLaps detected fragments having m/z values of about 1730 whereas those containing pyridinium cross-links had m/z about 2536. There was also a minor fragment between the main species (m/z about 2180). It is known that ICTP and CrossLaps can be produced from the same type I collagen molecule with different enzymes. However, our results indicate that the ICTP and CrossLaps antigens differ also in the fine structure of collagen molecules. CrossLaps mainly detects only those ICTP antigens where the cross-link is currently uncharacterised. This study provides procedures that will assist in the identification of the unknown cross-links.

Disclosures: J.P. Risteli, None.


Bone Acidic Glycoprotein-75 and Bone Sialoprotein Co-Localize in Primary Bone Prior to Mineralization.J. P. Gorski1, A. Wang2, D. Law*1, K. Powell*2, R. J. Midura*2. 1Molecular Biology and Biochemistry, Univ. of Missouri-Kansas City, Kansas City, MO, USA, 2Biomedical Engineering, Cleveland Clinic and Foundation, Cleveland, OH, USA.

Recent studies demonstrate that bone acidic glycoprotein-75 (BAG-75) is a biomarker for biomineralization foci (BMF) in UMR-106--01 osteoblastic cultures, sites of initial bone sialoprotein (BSP) and hydroxyapatite deposition. Our goals were 1) to identify BMF in primary bone and 2) to define relationships among BAG-75, BSP and mineral during primary bone mineralization in vivo. Primary bone was obtained from the rat tibial marrow ablation model. Our findings can be summarized as follows. First, BMF were identified which are similar in structure to those characterized in UMR cultures, e.g., spherical, punctate, acellular, matrix regions ringed by alkaline phosphatase positive cells. Second, hematoxylin and eosin stained intramedullary tissue five days after ablation is devoid of bone morphology; only cellular mesenchyme and residual fibrin clot were visible. This region contained prominent filaments containing BAG-75 and a network of thinner BAG-75 strands interspersed within the clot. We conclude that BAG-75 is expressed both by condensing mesenchymal cells replacing the fibrin clot, as well as by osteoprogenitor cells derived from pre-existing ossicles of trabecular bone. Third, using confocal microscopy, BAG-75 or BSP immunostaining was compared with nascent mineral detected by Alizarin red-S fluorescence. At the peak of osteogenesis, BSP and BAG-75 co-localized with each other in over 80% of decalcified primary bone at low magnification and this trend persisted at higher magnification yielding 75% co-localization. Both matrix proteins also exhibited a high level of co-localization with the mineral phase in undecalcified sections; there was 3-4 times more detectable protein epitope staining associated with the mineral phase than that identified separately. These values likely underestimate BAG-75 because the mineral phase appears to block antibody access. Fourth, ultrastructural immunoelectron microscopy does not support a direct association with banded fibrils. BAG-75 instead comprises a separate amorphous layer located in close proximity to the fibrillar collagenous network. Within such regions, BAG-75 immunogold particles seem to be preferentially clustered in groups of 2 to 6. Importantly, some of the BSP immunogold signal after double-labeling co-distributes within 50 nm of anti-BAG-75 particles. Our spatial analysis suggests that BAG-75 deposition in primary bone precedes that for BSP, that BSP co-localizes in part with BAG-75 at sub-micron resolution, and that mineral deposition follows BAG-75/BSP co-localization.

Disclosures: J.P. Gorski, None.


Evaluation of RANKL and OPG Gene Expression in Human and Murine Fibroblasts in Response to Orthopaedic Particulate Debris.J. T. Ninomiya, B. C. W. Law*, J. A. Struve. Department of Orthopaedic Surgery, Medical College of Wisconsin, Milwaukee, WI, USA.

Implant loosening remains the primary cause of failure in total joint arthroplasty, and this is largely felt to be due to the generation of particulate wear debris, resulting in osteolysis. Histologic examination of osteolytic membranes has revealed a predominance of macrophages and fibroblasts, and these cells are felt to be critical in the process of implant loosening. Although they are capable of producing large amounts of cytokines, prostaglandins, and proteases in response to particulate debris, little is known about their role in the regulation of the proteins RANKL and OPG. Therefore, we chose to assess the effects of orthopaedic particulate debris on the gene expression and secretion of these proteins in both human and murine fibroblasts.

Both human fibroblasts and murine BALB fibroblasts were grown in tissue culture and titanium particles were added to the cells. Controls consisted of cells grown in the absence of particulate debris. For the murine cells, vitamin D-3 was also included in the culture media. Alterations in the gene expression of RANKL and OPG were assessed using RT-PCR, while the effects on protein secretion were determined by ELISA. The functional results of the addition of the particulate debris were evaluated using a murine bone marrow osteoclast maturation assay.

Following the addition of titanium particles to the human fibroblasts, the gene expression of RANKL increased, resulting in a net increase in the RANKL/OPG ratio. This activity peaked between 6 and 12 hours after the particles were added. No detectable changes in RANKL protein levels were observed in conditioned media using ELISA for the secreted form of RANKL.

In contrast, the murine cells increased RANKL gene expression in response to particles in the presence of vitamin D-3, but not to particles or vitamin D-3 alone. Like the human cells, alterations in RANKL gene expression peaked between 6 and 12 hours following addition of the particles.

Taken together, these studies suggest that fibroblasts may alter the generation of osteolysis through modulation of the gene expression of RANKL. Both human and murine fibroblasts produced similar responses to particulate debris, suggesting that either or both of these may be useful models for the evaluation of the role of fibroblasts in the process of implant loosening.

Disclosures: J.T. Ninomiya, None.


Mechanisms of Assembly of Latent TGFβ Binding Protein-1 into Bone Extracellular Matrix.Q. Chen*1, P. Sivakumar*1, C. Barley*2, D. M. Peters*3, S. L. Dallas1. 1Univ. Missouri, Kansas City, MO, USA, 2Univ. Manchester, Manchester, United Kingdom, 3Univ. Wisconsin, Madison, WI, USA.

Latent TGFβ binding proteins (LTBPs) are members of the fibrillin superfamily of extracellular matrix (ECM) proteins that bind and regulate transforming growth factor betas (TGFβs). Mutations in this superfamily are associated with heritable disorders that often show skeletal abnormalities. LTBP1 regulates secretion of latent TGFβ as well as its storage and release from the ECM. Given these critical functions, and the implication of TGFβ in fibrotic and other diseases, our goal is to understand the structural arrangement of LTBPs in bone ECM and the molecular mechanisms by which they regulate TGFβ.

We have previously identified fibronectin as a key molecule for assembly of LTBP1 into the ECM of osteoblasts. To further define the relationship between LTBP1 and fibronectin, a series of recombinant LTBP1 fragments were expressed and purified in a mammalian expression system. Transient transfection studies showed that an N-terminal LTBP1 fragment (a.a. 21--487) was sufficient for co-localization with fibronectin in 2T3 osteoblasts. A C-terminal fragment (a.a.1008--1394) also showed a weak association with the ECM. Surprisingly, solid phase binding assays demonstrated no direct interaction of any of the purified LTBP1 fragments with fibronectin or fibronectin fragments. However, when conditioned media containing the recombinant LTBP1 fragments was used, the N-terminal (21--487) and C-terminal (1008--1394) fragments as well as a fragment (526--1014) consisting of a central stretch of 11 tandem EGF-like repeats, bound to immobilized fibronectin, suggesting an indirect association. A heparin binding site was identified in the N-terminal LTBP1 fragment by solid phase binding assays and by heparin sepharose chromatography. Since fibronectin also contains at least two heparin binding sites, we explored the possibility that heparan sulphate containing proteoglycans (HSPGs) may mediate binding between the LTBP1 N-terminus and fibronectin. To mimic the clustered glycosaminoglycan pattern on proteoglycans, heparin coupled to bovine serum albumin was used. Treatment of osteoblast cultures with both heparin and heparin-BSA inhibited incorporation of LTBP1 into the ECM, while chondroitin-6-sulphate had no effect. Solid phase binding assays showed that heparin-BSA could mediate binding of the N-terminal LTBP1 fragment with fibronectin.

These studies suggest that heparin binding sites in fibronectin and LTBP1 may be important for assembly of LTBP1 into the ECM and suggest that HSPGs may mediate binding interactions between these two ECM proteins.

Disclosures: Q. Chen, None.


Matrix Metalloproteinase (MMP)-2-deficient Mice Exhibit Hypertelorisms, Cortical Bone Thinnings, and Short Statures, Similar to Human Osteolysis Syndrome.K. Inoue1, Y. Mikuni-Takagaki2, O. Kaoru3, T. Itoh*4, T. Noguchi*1, J. S. Park*1, M. Noda3, S. Itohara*1. 1Behavioral Genetics, Brain Science Institute, RIKEN, Wako, Japan, 2Oral Biochemistry, Kanagawa Dental College, Yokosuka, Japan, 3Molecular Pharmacology, Tokyo Medical & Dental University, Tokyo, Japan, 4Discovery Research Laboratories, Shionogi & Co. Ltd, Osaka, Japan.

In bone tissues, various extracellular matrix (ECM) proteins are metabolized in concert and the degradation of ECM proteins in bone by matrix metalloproteinases (MMPs) is required for normal turnover. MMP-2 degrades type IV collagen as its major substrate, while it also cleaves type I collagen. However, no functional aspects of MMP-2 have been described in terms of skeletal tissue homeostasis. In the present study, we examined the effects of MMP-2-deficiency on the skeletal systems in mice. MMP-2-deficient mice developed osteoporotic phenotype (reduction in BMD) in long bones after weaning. They showed hypertelorisms, cortical bone thinnings, and short statures, which are similar to those observed in human osteolysis syndrome. It was recently reported that human familial osteolysis syndrome was linked with MMP-2-deficiency. We further analyzed cellular mechanism underlying osteoporotic phenotypes in MMP-2-deficient mice based on histomorphometry. MMP-2-deficiency did not affect bone formation parameters such as mineral apposition rate (MAR) and bone formation rate (BFR) in aged (55 wk) cancellus bones. Similarly, bone resorption parameters such as osteoclast surface and eroded surface were not affected in cancellus bones of aged MMP-2-deficient mice. In contrast, MMP-2-deficiency enhanced endosteal BFR and suppressed periosteal BFR in aged cortical bones. In addition, bone resorption (eroded surface/BS) was enhanced in cortical bones in aged MMP-2-deficient mice. Interestingly, osteoblastic and osteoclastic properties were similar between MMP-2-deficient and wild-type mice when they were examined in bone marrow-derived cell culture experiments. These findings suggest that MMP-2 is an important modulator of bone mass in vivo.

Disclosures: K. Inoue, None.


The Inhibition of Calcium Phosphate Precipitation by Fetuin is Accompanied by the Formation of a Fetuin-Mineral Complex.P. A. Price, J. E. Lim*. Division of Biology, University of California San Diego, La Jolla, CA, USA.

Previous studies have shown that the fetuin-mineral complex (FMC) is a complex of a calcium phosphate mineral phase and the proteins fetuin and matrix Gla protein that appears in the blood of rats with on-going, vitamin D-induced artery calcification and in the blood of rats treated with doses of etidronate that acutely inhibit bone mineralization [JBC (2002) 277:3926--34]. The present in vitro studies were carried out in order to better understand the mechanisms responsible for the generation of the FMC in the rat.

Our working hypothesis is that the FMC is formed in vivo under conditions in which fetuin inhibits the precipitation of a calcium phosphate mineral phase, a precipitation that arguably occurs in bone when resorption exceeds formation [JBMR (2002) 17:1171--79]. The first test was carried out in rat serum, since rat serum is known to contain high levels of fetuin (1.5mg/ml), and fetuin is known to be an important serum inhibitor of calcium phosphate precipitation. In this test, the concentration of calcium and phosphate in serum were each increased by 10mM and the modified serum was incubated at 37°C. The serum remained clear with no evidence of calcium phosphate precipitation over 9 days of observation, while precipitation occurred in seconds in the absence of serum under the same ionic conditions. Centrifugation and gel filtration procedures revealed the formation of large amounts of a FMC in the first 3h of this incubation, and the calcium content of the FMC accounted for over 90% of the calcium added to serum. FMC levels remained constant throughout the 9 days of incubation at 37°C.

The second test showed that the addition of purified bovine fetuin to a solution containing 5mM calcium and phosphate at pH 7.4 at 22°C inhibited the precipitation of mineral for over 14 days, while precipitation occurred in minutes without fetuin. There was a biphasic drop in ionic calcium in the fetuin solution, however, from 5 to 3mM in the first hour and from 3 to 0.9mM between 20 and 24h; these changes in ionic calcium proved to be due to the formation of complexes of calcium, phosphate, and fetuin. The complex found after 24h appears to be identical to the fetuin-mineral complex found in the serum of etidronatetreated rats, while the complex found between 1 and 20h is less stable.

These experiments demonstrate that a fetuin-mineral complex apparently identical to that found in rat blood in vivo is indeed generated in vitro during the course of the inhibition of calcium phosphate precipitation in serum containing supersaturating amounts of calcium and phosphate, and during the course of inhibition of calcium phosphate precipitation from supersaturated solutions containing purified bovine fetuin.

Disclosures: P.A. Price, None.


Bone Repair Is Dramatically Delayed in Dentin Matrix Protein-1 (Dmp1) Deficient Mice.H. Huang1, L. Ye1, Y. Xie1, S. Ko1, S. E. Harris1, L. Bonewald1, Y. Mishina2, J. Q. Feng1. 1Oral Biology, School of Dentistry, University of Missouri-Kansas City, Kansas City, MO, USA, 2NIEHS/NIH, Research Triangle Park, NC, USA.

Fracture healing is a complex process that involves both endochondral and intramembranous ossification. Dmp1, an acidic matrix protein, is expressed in both cartilage and bone cells. To address whether Dmp1 is critical for bone repair, rib fractures were generated in wild-type and Dmp1 deficient mice in both sexes and samples collected on days 7, 14 and 21 after fracture. In control animals, Dmp1 expression was highly increased in hypertrophic chondrocytes followed by lower expression in bone cells within the fracture callus, suggesting that Dmp1 is involved in this healing process. In the Dmp1 deficient animals, formation of callus and union was dramatically delayed (see Table). In a search for potential mechanisms for delay of bone healing, we found that Dmp1 null mice display the following: 1) a profound defect in mineralization, not due to a systemic defect in calcium/ phosphate metabolism as serum levels of calcium and phosphate were similar to those in the control mice; 2) a severe delay in blood vessel invasion which could be responsible for the reduction in osteoclast numbers in fracture areas as spleen cells from Dmp1 null mice produce similar numbers of osteoclasts as control animals in vitro; and 3) delayed expression of Cbfa1, Col I, and Col II suggesting a delay in osteogenesis and chondrogenesis. Based on these observations, defects in vascular invasion in this model could be due to defective matrix produced by chondrocytes and osteoblasts that is incapable of supporting blood vessel invasion. 

Table Table. Radiographic Assessments of Fracture Healing
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Disclosures: H. Huang, None.


Smad3 Interacts with JunB and Cbfa1/Runx2 for Transforming Growth Factor-beta1-Stimulated Collagenase-3 Expression in Human Breast Cancer Cells.N. Selvamurugan, S. Kwok*, N. C. Partridge. Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA.

The specific characteristics of breast cancer cells and the unique properties of the bone/ bone marrow microenvironment are largely responsible for the oberved high frequency of skeletal metastases from breast cancer. A series of recent studies have indicated that collagenase-3 (matrix metalloproteinase-13) is overexpressed in a variety of tumors from diverse sources. Collagenase-3 is characterized by its ability to degrade the extracellular matrix (ECM) and is stimulated by TGF-β1 (enriched in bone matrix) in the human breast cancer cell line, MDA-MB231. Collagenase-3-driven ECM proteolysis may support cancer cell growth by exposing the cells to growth factors and cytokines, resulting in bone degradation (osteolysis). A greater understanding of the regulatory mechanisms that control collagenase-3 expression may provide several new avenues for therapeutic intervention. Hence, our interest is to identify the signaling and molecular mechanisms responsible for TGF-β1-stimulated collagenase-3 expression in human breast cancer cells. We have recently shown that transcriptional activation of collagenase-3 by TGF-β1 is via MAPK and Smad pathways in these cells (N. Selvamurugan, Z. Fung, and N.C. Partridge (2002): FEBS Letters 532, 31--35). To understand the molecular mechanisms responsible for this TGF-β1-response, a functional analysis of the promoter region of the collagenase-3 gene was carried out and we identified the distal RD (runt domain) and proximal RD/AP-1 (activator protein-1) sites as necessary for full TGF-β1-stimulated collagenase-3 promoter activity. Gel shift, real time RT-PCR and Western blot analyses showed increased levels of c-Jun, JunB, and Cbfa1/Runx2 upon TGF-β1 treatment in MDA-MB231 cells. Functional importance of the AP-1 factors and Cbfa1/Runx2 for TGF-β1-stimulated collagenase-3 promoter activity was identified by overexpression of antisense-Fra1 and dominant repressor, Cbfa/ETO expression plasmids, respectively. Further analysis of the proximal RD/AP-1 site by biotinylated DNA precipitation experiments showed no direct binding of Cbfa1/ Runx2 to this site. Co-immunoprecipitation in vitro studies identified no physical interaction between JunB and Cbfa1/Runx2; whereas Smad3 interacted with both. Taken together, our results suggest that TGF-β1 stimulated JunB and Cbfa1/Runx2 proteins bind to their respective DNA consensus sites and Smad3 stabilizes their interaction to confer functional TGF-β1-stimulation of collagenase-3 expression in MDA-MB231 cells.

Disclosures: N. Selvamurugan, None.


Development of an “All-Human” Animal Model of Breast Cancer Metastasis to Bone.C. Kupperwasser*1, D. Garnet*2, K. Sperandio*2, R. A. Weinberg*1, M. Rosenblatt2. 1Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA, USA, 2Dept. of Medicine/Division of Bone & Mineral Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA.

A common and serious complication of breast cancer is the development of bone metastasis. Fractures, pain, hypercalcemia, and other disorders often occur as a result of bone metastasis, and the appearance of a skeletal metastasis signals that the disease has entered an incurable phase. We are interested in identifying the mechanisms underlying breast cancer osteotropism, i.e. the molecular basis by which cancer cells adhere to and subsequently establish secondary colonies within bone. In an effort to better understand the biology of cancer osteotropism, we developed a mouse model that replicates the multi-step process by which breast cancer metastasize to bone.

In this model, human bone fragments are implanted subcutaneously in non-obese diabetic/ severe combined immunodeficient (NOD/SCID) mice. These bone grafts are viable and the marrow functional post-engraftment as evidenced by: 1) histologically normal bone cells and architecture; 2) the presence of human immunoglobulin (IgG) in mouse circulation; and 3) the presence of human B-cells in the spleen of mice. In addition, striking neovasculature arises to supply the bone graft. We tested a panel of cell lines for their ability to metastasize to bone after I.V., I.P. or orthotopic (mammary pad) injections. Of the several lines tested, one metastasized to human bone by all three routes of administration, albeit with low frequency. Despite the ectopic extra-skeletal location of the human bone implant, SUM-1315 cells (derived from a metastatic nodule of a patient with breast cancer and bone metastases; generously provided by Dr. S. Ethier) appeared to metastasize preferentially to human bone over the mouse skeleton.

We plan to harvest SUM-1315 metastatic tissue from human bone implants in order to perform gene expression profiling using DNA-microarray analysis. In this manner, we will attempt to identify genes specific for metastases and/or osteotropism. We also plan to refine the model into an assay which can be used to evaluate genes for their role in the osteotropic phenotype or potential skeletal metastasis-specific drugs.

Disclosures: M. Rosenblatt, None.


Paracrine Activation of PDGFR Tyrosine Kinase in Osteoblasts Is Critical to Osteosclerotic Bone Metastases in Breast Cancer Producing PDGF-BB.T. Tsutsumimoto*, B. Yi*, P. Williams*, B. Story*, T. Yoneda. Div Endocrinol, Univ TX Hlth Sci Ctr, San Antonio, TX, USA.

Metastatic cancer cell colonization in bone is significantly influenced by the interactions with bone microenvironment. In osteolytic bone metastases, establishment of a vicious cycle between tumor-produced PTH-rP and osteoclastic bone resorption is shown to be critical. On the other hand, tumor-bone partnership in osteosclerotic bone metastases is still poorly understood. Since osteoblasts play a central role and a recent study has suggested the importance of tumor-produced PDGF-BB in osteosclerotic bone metastases, we studied the interactions between tumor-produced PDGF-BB and osteoblasts using a model of the MCF-7 human breast cancer cells transfected with the proto-oncogene Neu (MCF-7/Neu). MCF-7/Neu cells produced hPDGF-BB and caused osteosclerotic bone metastases. MCF-7/Neu cells showed little expression of PDGF receptor-alpha and -beta (PDGFRa and PDGFRb) by RT-PCR, indicating that PDGF-BB is a paracrine rather than an autocrine factor in MCF-7/Neu cells. On the other hand, we found PDGFRa and PDGFRb expression in cells of osteoblast lineage including C3H10T1/2, C2C12, ST2, and MC3T3-E1 cells. PDGFR expressed in these cells were functional, because hPDGF-BB induced tyrosine autophosphorylation of PDGFR and stimulated cell proliferation. Of interest, western analysis showed that hPDGF-BB also increased osteopontin (OPN) production. Furthermore, a selective PDGFR tyrosine kinase (PDGFR TK) inhibitor AG1296 inhibited PDGFR tyrosine autophosphorylation, cell proliferation and OPN production induced by hPDGF-BB but not EGF and FGF-2 in these osteoblastic cells. In contrast, MCF-7/Neu cell growth was not inhibited by AG1296. AG1296 inhibited hPDGF-BB-stimulated bone formation in mouse neonatal calvariae in organ culture, suggesting a critical role of PDGFR TK in bone formation. We then examined the effects of AG1296 on osteosclerotic bone metastases in vivo. AG1296 (1.25mg/mouse, ip, every 4 days for 6 weeks, total 11.25mg/mouse) significantly decreased MCF-7/Neu tumor burden in bone metastases. Furthermore, AG1296 (1mg/mouse, ip, 3 times/week for 3 weeks, total 9 mg/mouse) also significantly decreased bone metastases in the MDA-MB-231 human breast cancer cells transfected with hPDGF-B cDNA. In conclusion, our results suggest that activation of PDGFR TK signaling pathways in osteoblasts by breast cancer-produced PDGF-BB contributes to the promotion of osteosclerotic bone metastases. The results also suggest that PDGFR TK may be a potential molecular target in the treatment of osteosclerotic bone metastases in PDGF-producing breast cancers.

Disclosures: T. Tsutsumimoto, None.


Opposite Effects of Clodronate and Ibandronate on the Growth of Estrogen Receptor-Positive Breast Cancer Cells in Steroid-Free Medium.F. Journe*1, C. Chaboteaux*1, G. Laurent*2, F. Place*1, J. C. Dumon*1, J. J. Body1. 1Bone Diseases Unit, Institut Bordet, Université Libre de Bruxelles, Brussels, Belgium, 2Laboratory of Histology, Université de Mons-Hainaut, Mons, Belgium.

The skeleton is the most common site colonized by metastatic breast cancer cells, especially those expressing estrogen receptor (ER). Neoplastic cells stimulate osteoclast-mediated bone resorption and bisphosphonates have emerged as a logical approach for the treatment of bone metastasis. Besides their powerful anti-osteoclast activity, we and others have recently demonstrated that bisphosphonates induce growth inhibition and apoptosis of breast cancer cells in vitro (e.g. Fromigué et al, JBMR, 2000). In the present study, we have investigated the effects of two structurally distinct bisphosphonates on MCF-7 breast cancer cells cultured in steroid free medium, with particular emphasis on ER expression and activity. Tested compounds were clodronate (Clod) and ibandronate (Iban). MCF-7 cells were cultured in RPMI 1640 medium supplemented with charcoal-stripped fetal bovine serum to obtain a steroid-free medium (SFM). Cell growth was estimated by photometry after crystal violet staining. In SFM, Clod stimulated cell growth in dose- and time-dependent manners (mean 46% increase after 3 days of exposure to 10−4 M Clod). This mitogenic effect of Clod was not detected when cancer cells were cultured in medium with complete serum. As could be expected, 17β-estradiol (E2) also produced a growth stimulatory effect in steroid-free conditions (mean 77% increase after 3 days of culture with 10−8 M E2), but combination of E2 and Clod did not significantly enhance the effect of the latter. Moreover, partial (4-OH-tamoxifen) and pure (RU 58 668) antiestrogens (10−7 M) completely suppressed the proliferative activity of Clod, suggesting that it is mediated by an activation of ER. In accordance with this view, Clod induced ER downregulation, weakly increased progesterone receptor expression and stimulated transcription of an estrogen-responsive reporter gene. In contrast, in the same steroid-free conditions, Iban still inhibited cancer cell proliferation (mean 39% decrease after 3 days of incubation with 10−4 M Iban in SFM). Furthermore, Iban completely abolished the mitogenic effect of E2 and reinforced the growth inhibitory action of pure antiestrogen in an additive fashion. In conclusion, we report a previously unknown stimulating effect of Clod on MCF-7 breast cancer cells growth, occurring through direct or indirect ER activation in absence of cognate ligand. In the same conditions, Iban inhibited MCF-7 cell growth, prevented E2 stimulatory effects and exerted additive effects with pure antiestrogens.

Disclosures: F. Journe, Hoffmann - La Roche 2.


Influence of Chemotherapy (AC) on Bone Mineral Density (BMD) and Bone Ultrasonometry (QUS) in Women with Breast Cancer.P. Hadji, C. Maskow*, M. Gottschalk*, V. Ziller*, F. Fischer*. Dept. of Menopause and Osteoporosis, Philipps-University of Marburg, Marburg, Germany.

Introduction: The aim of this prospective, case-control pilot study was to investigate the influence of chemotherapy (Adriamycin/Cyclophosphamid) on BMD and QUS in preand postmenopausal women with breast cancer.

Material and Methods: We included 16 patients (12 pre- and 4 postmenopausal), mean age 47,1 ± 8,3 years with an incident diagnose of breast cancer who received a chemotherapy (4 cycles of AC) and 16 age- and BMI-matched controls. Women with metastases, a history of osteoporosis with or without fracture, diseases or treatments known to effect bone metabolism were excluded from the study. BMD was measured by DXA (DPX-L, GE/Lunar) at spine and hip. QUS was performed at the os calcaneus using the Achilles device (GE/Lunar) and at the phalanges using the Bone-Profiler (IGEA). Measurements were performed at baseline (before chemotherapy), after 6 and 12 months and were compared with measurement results of the age- and BMI-matched control group.

Results: DXA results of the spine and hip showed a linear decrease of T- and Z-score in patients with chemotherapy compared controls, but the decrease did not reach statistical significance. In accordance to QUS results, measurement at the os calcaneus showed a similar linear decrease of T- and Z-score without reaching statistical significance. In contrast, QUS results at the phalanges showed a significant decrease of AD-SOS, for T- and Z-Score (p ⩽ 0,008), with the largest difference between baseline and 12 months T-score (p⩽0,001).

Conclusion: The result of our prospective, case controlled pilot study confirms the deleterious influence of chemotherapy on BMD in women with breast cancer. This effect could be observed by DXA and additionally by QUS for the first time in this regard. Further, large scale longitudinal studies are need to improve our understanding of the mechanism of bone changes during chemotherapy.

Disclosures: P. Hadji, None.


Pamidronate Reduces the Severity of Bone Lesions in an Osseous Prostate Cancer Mouse Model by Decreasing Osteoprotegrin and PTHrP.D. W. Burton1, J. Geller*2, M. Yang*2, I. Barken*2, R. M. Hoffman*2, L. J. Deftos1. 1Medicine, University of California and SDVAMC, San Diego, CA, USA, 2AntiCancer, Inc., San Diego, CA, USA.

Prostate cancer is the most prevalent tumor in men and bone metastases are responsible for most of the morbidity associated with this tumor. Bisphosphonates have been shown to attenuate prostate tumor progression in bone, but the mechanism of this action is not clearly understood. This study evaluated the effects of the bisphosphonate, Pamidronate, on the growth of PTHrP-expressing PC-3 prostate cancer cells in a mouse model for prostate cancer in bone. The PC-3 cells were genetically engineered to express green fluorescent protein (GFP) in order to enable convenient and rapid visualization and quantitation of the tumor mass. Immunocompromised mice were injected with PC-3-GFP cells into the bone marrow of the right tibia. The left tibia served as the negative control leg. One month prior to inta-tibial implantation of the prostate cancer cells, the animals received 1.44 mg (low dose) or 7.2 mg (high dose) of Pamidronate i.v./kg body weight. Following the injection of PC-3-GFP cells into the tibias, both groups of mice received monthly doses of Pamidronate i.v. at the same concentrations respectively for 2 months. The control animals received PBS using the same injection protocol. At the end of the study, the mice were imaged for GFP expression, the skeletons analyzed for abnormalities by radiography, and the sera assayed for calcium, PTHrP and selected cytokines and osteokines. The mice that were treated with the high dose of Pamidronate demonstrated a significant reduction in the severity of the bone lesions compared to the control mice as assessed by X-ray and GFP imaging. The low dose group did not demonstrate any significant changes from the control group. The GFP and X-ray score results from the skeletons demonstrated a very high correlation (r = 0.783, P < 0.01). Once the tumor cells starting growing outside the bone, Pamidronate had little effect on the tumor growth. Serum osteoprotegrin (OPG) and PTHrP levels were significantly decreased in the high dose Pamidronate group compared to the control group (76.5%, P < 0.001 and 33.3%, P < 0.05, respectively). No significant changes were noted in serum calcium or interleukin-6 or -8 levels between the groups. Our studies demonstrate that GFP imaging can be used to assess the progression of prostate cancer. Pamidronate's effect on decreasing prostate cancer growth in bone was associated with a decrease in serum OPG and PTHrP. Further studies are need to elucidate the signaling cascade of bisphosphonates' effects on the skeletal progression of cancer.

Disclosures: D.W. Burton, None.


Regulation of Prostate Cancer Progression in Bone by PTHrP.D. W. Burton1, I. Barken*2, J. Geller*2, R. M. Hoffman*2, L. J. Deftos1. 1Medicine, University of California and SDVAMC, San Diego, CA, USA, 2AntiCancer, Inc., San Diego, CA, USA.

We and other investigators have previously reported that PTHrP is widely expressed by human prostate cancer tissue, suggesting that PTHrP might be involved in the progression of this tumor. In order to evaluate the effect of PTHrP on tumor progression in bone, we studied the DU 145 cell line in a mouse model for prostate cancer. The DU 145 cell line was selected because it has a low constitutive PTHrP expression and does not grow well in mouse tumor models. We studied four types of DU 145 cells: 1. Wild type cells, 2. Vector (pCi-neo) transformed cells 3. PTHrP 1--87 transformed cells and 4. PTHrP 1--173 transformed cells. The PC-3 cell line (group 5) was also used as a known prostate cancer cell line that produces extensive bone lesions in immunocompromised mice. The cells were directly injected into the femurs of SCID mice and the mice were evaluated 60 days later for biochemical changes in the sera (see Table 1) and skeletal abnormalities by X-ray (see Figure). Quantitation of the radiographic images of the mouse femurs showed progression of tumor in bone that correlated with PTHrP production by the tumor. The DU 145-PTHrP transformed groups demonstrated increased bone lesions, serum calcium and PTHrP. The PTHrP produced by the DU 145-PTHrP1--173 cells was less than the DU 145-PTHrP1--87, but the femur radiographs nevertheless showed more tumor damage in the DU 145-PTHrP1--173 mice. Our results provide more evidence that PTHrP expression by prostate cancer cells promotes the development of skeletal lesions by the prostate cancer. Furthermore, PTHrP secreted into the blood of tumor-bearing animals served as a tumor biomarker by correlating with the primary prostate tumor volume and the degree of tumor burden in the bone. These animal models can generate clinical hypotheses designed to elucidate the role of PTHrP in human prostate cancer pathogenesis and to identify specific PTHrP species as diagnostic and therapeutic targets for this malignancy.

Disclosures: D.W. Burton, None.


Apoptotic Effects of Parathyroid Hormone Related Protein in Prostate Cancer Cells.V. V. Maheshwari1, D. W. Burton1, R. H. Hastings2, J. A. Aguilera*3, F. S. Pardo*3, L. J. Deftos1. 1Medicine, University of California and SDVAMC, San Diego, CA, USA, 2Anesthesiology, University of California and SDVAMC, San Diego, CA, USA, 3Radiology, University of California, San Diego, CA, USA.

Parathyroid hormone-related peptide (PTHrP) is an autocoid regulator cell of growth, acting via autocrine, paracrine and intracrine pathways. Studies by us and our colleagues have demonstrated that PTHrP regulates prostate tumor development, growth, progression and metastasis to bone, but little is known about its role in apoptosis in prostate cancer. We performed apoptosis studies using wild-type DU 145 cells prostate carcinoma cells and DU 145 cells stably transformed to express various PTHrP peptides. We evaluated PTHrP's effect on survival with clonogenic-cell survival assays. Suspended cells were exposed to gamma irradiation (4 Gy) and replated in triplicate into 60-mm dishes. colonies (>50 cells) were counted after 14 days in culture. Multiple clones of DU 145 PTHrP 1--173 expressing cells demonstrated a 2-fold increase in colony formation compared to the vector control transformed cells (P < 0.01). Conversely, DU 145 PTHrP 1--87 and 1--141 expressing cells demonstrated a 50% and 30% decrease respectively in colony formation after gamma irradiation compared to the vector control cells. We also evaluated PTHrP's effect on staurosporine-induced apoptosis as measured by nuclear condensation and caspases-3 and -9 activities. The DU 145 PTHrP 1--173 expressing cells showed a reduction in nuclear condensation and caspases-3 and -9 activities (P <0.05, P < 0.01 and P < 0.05, respectively) compared to the vector-control cells. DU 145 PTHrP 1--87 and 1--141 expressing cells increased caspases-3 and -9 activities slightly compared to vector control cells. The effects of PTHrP peptides on staurosporine-induced apoptosis were studied in wild-type DU 145 cells. PTHrP140--173 peptide treatment decreased caspases-3 and -9 activities and nuclear condensation compared to vehicle treated cells. No significant effects on nuclear condensation and caspases-3 or -9 were observed with treatment with PTHrP 1--34 or scrambled PTHrP 140--173 peptide. Since protective effects on apoptosis were not observed for PTHrP 1--34 peptide or PTHrP 1--87 and 1--141 gene transfer, the human-specific 140--173 region appears responsible for the anti-apoptotic properties of PTHrP in prostate cancer cells through a paracrine mechanism. The pro-apoptotic effects of PTHrP 1--87 and 1--141 may result from an intracrine, nuclear targeting pathway. Further studies are necessary to understand PTHrP's role in prostate cancer apoptosis.

Disclosures: D.W. Burton, None.


Osteoblastic Properties of Prostate Cancer Bone Metastases are Dependent on Notch Signaling and ERK Activation.M. Zayzafoon, S. A. Abdulkadir*, J. M. McDonald. Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

Prostate cancer bone metastases are characterized by their ability to induce osteoblastic lesions and local bone formation. The pathophysiology of this skeletal response is not currently known. Recently, many reports have indicated that bone metastatic prostate cancer cells are osteomimetic and capable of expressing genes and proteins typically expressed by osteoblasts. The ability of these cells to express osteoblastic genes and to modify their phenotype requires the activation of many pathways, most importantly, the Notch's. Notch1 activation by Dll1 (a Notch ligand) has been recently shown to play a role in inducing osteoblast differentiation. To determine the pattern of Notch expression in skeletal and non-skeletal prostate cancer metastases, we examined gene and protein expression in different metastatic prostate cancer cell lines and in human prostate cancer bone metastases. Our results demonstrate that Notch1 expression is increased 4--5 times in bone derived osteoblastic metastatic cancer cell lines (C4--2B and PCa 2b) compared with lymph node and brain derived cell lines (LNCaP and DU145). Notch1 ligand, Dll1, is expressed only in C4--2B cells. In addition, we demonstrate by immunohistochemistry that Notch1 is present and active in both human prostate cancer bone metastases as well as C4--2B cells. To determine if prostate cancer bone metastases respond to osteogenic induction in a similar way to osteoblasts, C4--2B cells were cultured in osteogenic media that promotes mineralization. C4--2B cells mineralize, like osteoblasts, an effect that is completely inhibited by L-685,458, a Notch activity inhibitor. Interestingly, osteogenic media increases ERK activation independent of Notch signaling. HES-1 expression (a downstream target of Notch activation) is 20-fold increased in C4--2B cells when compared to non-skeletal prostate cancer metastases. This expression is inhibited by 10 uM L-685,458 but not altered by osteogenic media or ERK inhibition by 10 uM U0126. Runt-related transcription factor 2 (Runx2) DNA binding activity is increased in response to osteogenic induction and is inhibited by both L-685,458 and U0126. Supershift analyses indicate that both HES1 and Runx2 are involved in this response. These studies demonstrate that prostate cancer bone metastases acquire osteoblastic properties through independent activation of ERK and of Notch signaling, presumably both pathways being activated in the bone microenvironment.

Disclosures: M. Zayzafoon, None.


Pamidronate in the Prevention of Chemotherapy Induced Bone Loss in Premenopausal Women with Breast Cancer.M. Salamoun*1, A. Shamseddine*2, A. Chehal*2, Y. Abou Mourad*2, Z. Salem*2, Y. Arslanian*2, G. El-Hajj Fuleihan1. 1Calcium Metabolism and Osteoporosis Program, American University of Beirut, Beirut, Lebanon, 2Hematology Oncology Division, American University of Beirut, Beirut, Lebanon.

Overall mortality from breast cancer has decreased worldwide. Improved survival in such patients is partially due to the successful use of adjuvant chemotherapy, a therapy that is not without cost however. Indeed, a common side effect of adjuvant chemotherapy is ovarian failure and ensuing bone loss, bone loss that would be projected to increase these patients' risk of fracture with aging. Limited however is the information on therapies to prevent such anticipated bone loss. This issue is of particular concern in our country, where we observed an increased prevalence of pre-menopausal breast cancer and a lower peak bone mass compared to Western populations.

We describe below the results of a randomized, double-blind controlled trial using pamidronate (APD) 60 mg intravenously versus placebo (PBO) every three months in 38 premenopausal women with non-metastatic breast cancer. Chemotherapy in the APD group included: 16 FAC (5-FU, Adriamycin, Cyclophosphamide) or FAC like therapies, 2 CMF (Cyclophosphamide, Methotrexate, 5-FU); 2 Cisplatinum-vinorelbine followed by FAC; in the PBO 15 FAC/FAC like, 2 CMF and 1 Cisplatinum-vinorelbine followed by FAC. 26 women received tamoxifen: 13 were randomized to APD and 13 to PBO. All subjects were advised to take a calcium/D supplement if their diet was insufficient in these nutrients. Bone mineral density (BMD) was measured using DXA and % change in BMD was compared between the two groups using t-test. Numbers expressed as means (±SD).

The mean age of the patients was 40 (6) yrs, BMI 27 (5) kg/m2, 21 (60%) patients became amenorrheic within the first year of chemotherapy, 11 (61%) in the APD and 10 (59%) in the PBO group. The mean age at study entry of women who became amenorrheic was 42 (5) years, and was 38(5) years in the women who did not become amenorrheic, p=0.02. At 12 months, BMD increased significantly in the APD group by 2.2 (4)% at the spine and was stable at the total hip 0.6 (5) %, whereas BMD decreased significantly in the PBO group by -2.8(5)% at the spine, and by -2.3 (5)% at the total hip. There was a significant difference in BMD % change at 12 months between the two treatment groups at the lumbar spine only, p=0.002. Treatment was well tolerated, only 1 subject reported a flu-like syndrome with her first injection that did not recur with following ones. Cyclical therapy with APD 60 mg every 3 months is well tolerated, can be conveniently administered at the time of the chemotherapy, and prevents chemotherapy-induced bone loss in pre-menopausal women with breast cancer.

Disclosures: G. El-Hajj Fuleihan, Novartis 2.


Breast Cancer Cells Promote Osteoclast Survival by Activating PI3 kinase/ Akt Pathways : Possible Involvement of M-CSF.M. Gallet*, N. Sévennet*, I. El Hajj Dib*, R. Mentaverri*, C. Prouillet*, A. Wattel*, M. Brazier, S. Kamel. Laboratoire de Biologie et Pharmacie Cliniques, UMRO, Amiens, France.

Osteolytic bone metastases are the major complication occurring in breast cancer patients. The osteolytic lesions during bone invasion are believed to be caused by osteoclast-activating factors, which are released by tumor cells in the bone environment. Some of these factors are well known to stimulate osteoclast differentiation and thus bone resorption. Herein, we investigated whether breast cancer cells can directly stimulate osteoclast survival, a mechanism, which contributes to the osteolytic potential of breast cancer cells. Mature osteoclasts were isolated and purified from long bones of 10-day-old New Zealand rabbits. Conditioned media were prepared from MDA-MB-231 cells. Osteoclast apoptosis was evaluated by Hoechst staining and DNA ladder formation.

MDA-MB-231 conditioned media inhibited significantly and dose-dependently osteoclast apoptosis. When osteoclasts are cultured in 20 % conditioned media harvested from breast cancer cells, apoptosis was decreased by approximately 60%. We then investigated the signaling pathways involved in the anti apoptotic effect of conditioned media. By using PD98059, an ERK1--2 inhibitor and LY294002, a PI3 kinase/Akt inhibitor, we demonstrate that both pathways are involved. LY294002 dramatically inhibited the conditioned media-induced survival suggesting that PI3 kinase/Akt constitutes the major pathway. PI3 kinase/ Akt and ERK pathway are known to be survival pathway triggered by many growth factors and cytokines, such as TNFa, IGFI, IGFII and M-CSF. These factors are all secreted by breast cancer cells and could interact with mature osteoclasts, which express their receptors. In order to identify the possible factors secreted by MDA-MB-231 cancer cells, responsible for the anti apoptotic effect, we used neutralizing antibodies anti-TNFa, anti-IGFI, anti-IGFII and anti-M-CSF. Anti-M-CSF strongly prevented the anti apoptotic effect. Blockade by anti-IGFs was much less pronounced and anti-TNFa was without effect. Finally, using rh-TNFa, rh IGFI, rh IGFII and rh M-CSF ranged from 0.1 to 100 ng/ml, we checked the direct effects of this components on osteoclast apoptosis. We demonstrate that among these factors, only M-CSF increased significantly and dramatically osteoclast survival.

Taken together, these results demonstrate that breast cancer cell-derived factors promote osteoclast survival by activating PI3 kinase/Akt pathway. M-CSF was identified as the major factor responsible for the anti-apoptotic effect suggesting that M-CSF may be a potential therapeutic target in bone metastasis treatment.

Disclosures: M. Gallet, None.


Bone Morphogenetic Protein Signaling in Prostate Cancer Cell Lines.K. D. Brubaker, R. L. Vessella*, L. G. Brown*, E. Corey. Urology, University of Washington, Seattle, WA, USA.

Prostate cancer (CaP) cells express bone morphogenetic proteins (BMPs), which stimulate bone formation, implying a role in the development of osteoblastic lesions associated with CaP. Furthermore, CaP cells express BMP receptors (BMPRs) suggesting the possibility of direct effects of BMPs on CaP cells. The objective of this study was to evaluate the effects of BMP-2 and BMP-4 on CaP cells.

BMPs -2 and -4 exhibited growth inhibitory effects on androgen-sensitive LNCaP cells, while eliciting no effect on proliferation of androgen-independent PC-3 cells. Flow cytometric analysis revealed that BMP-2 treatment caused G1 arrest in LNCaP cells. Also, p21CIP1/WAF1, a cyclin-dependent kinase inhibitor involved in G1 cell cycle arrest, levels increased in LNCaP cells after BMP-2 treatment. Previously, we reported that both LNCaP and PC-3 cells express BMPR II, but that only LNCaP cells express BMPR IB. It is possible that the inhibitory response of BMPs on CaP cells requires BMPR IB receptor. To determine whether both LNCaP and PC-3 cells posses intact BMP signaling, we evaluated activation of SMAD-1, a signal transducer downstream of BMPRs. Interestingly, the treatment with BMP-2 and BMP-4 increased phosphorylation of SMAD-1 in both LNCaP and PC-3 cells. To further increase our understanding of the effects of BMP treatment on CaP cells, we evaluated the levels of osteoprotegerin (OPG), a factor involved in bone remodeling, in the media of LNCaP and PC-3 cells after BMP treatment. BMP-2 treatment decreased OPG production in LNCaP, but significantly stimulated the levels in PC-3 cells. In summary, our results demonstrate that BMPs exhibit direct effects on CaP cells, and that these effects are different in androgen-sensitive LNCaP and androgen-insensitive PC-3 cells. Detailed understanding of the effects of BMPs on prostate cancer and its bone metastases will require further studies.

Disclosures: E. Corey, None.


Export of TR3 Orphan Nuclear Receptor Mediates Rapid Mitochondrial Effects of Vitamin D Receptor Agonists and Antagonists.J. Barsony, K. Prufer. Laboratory of Cell Biochemistry and Biology, NIH/NIDDK/DHHS, Bethesda, MD, USA.

We have recently discovered that our vitamin D receptor (VDR) antagonist, BCA11, induces growth inhibition in various cancer cells, similarly to VDR agonists such as calcitriol. To understand the mechanisms of this growth inhibition, we explored both the effect of BCA11 on VDR coregulator binding and on TR3 orphan nuclear receptor export. TR3 export has recently been reported to induce mitochondrial permeabilization, cytochrome c release, and growth inhibition. We used GST-pull-down experiments to characterize BCA11 effect on corepressors and coactivators binding to VDR and TR3. To visualize subcellular trafficking and protein interactions in real-time, we used microscopy on cells expressing fluorescent protein chimeras of VDR, RXR, TR3, and histone deacetylase 3 (HDAC3). GST pull-down assays showed that both VDR agonists and VDR antagonists induce corepressor release, although only the agonists induce coactivator binding. Microscopy showed that TR3 translocates from the nucleus into the cytoplasm within one hour after treatment with calcitriol or BCA11 in MCF7 breast cancer and LNCaP prostate cancer cells. One-hour treatment with calcitriol and BCA11 also induced cytochrome c release from mitochondria as shown by Western blot analysis of MCF7 cell extracts. Experiments in COS7 cells demonstrated VDR-BFP expression was necessary for both calcitriol and BCA11 induced GFP-TR3 export, but VDR was not necessary for TPA induced GFP-TR3 export. Analysis of VDR and TR3 export mechanisms revealed that both calcitriol and BCA11 treatment results in the loss of Crm-1 dependent export of VDR and the gain of Crm-1 mediated export of GFP-TR3. Additional experiments indicated that the swap of corepressor-associated HDAC3 might be responsible for the hormone-induced switch of Crm-1 dependent export from VDR to TR3. Our results reveal a novel mechanism for the rapid hormone-induced TR3 export from the nucleus and suggest that this TR3-mediated mitochondrial pathway plays a role in the growth inhibitory effects of calcitriol agonists and antagonists.

Disclosures: J. Barsony, None.


Osteoporosis Screening in Primary Care with Heel Bone Densitometry for Women Aged 65 and Over.J. D. McCrea1, T. Hewer*2, D. G. Hayes*3. 1Rheumatology, Cumberland Infirmary, Carlisle, Cumbria, United Kingdom, 2Bone Densitometry, West Cumberland Hospital, Whitehaven, Cumbria, United Kingdom, 3Brunswick Medical Group, Carlisle, Cumbria, United Kingdom.

Access to bone mineral densitometry (BMD) in the UK is usually restricted to selective case finding of individuals considered to be at high risk for osteoporotic fracture. This pilot study was designed to examine the feasibility of measuring heel bone BMD in all women over 65 in a primary care setting.

All female patients aged 65 or over (n = 1013) registered with the group were eligible for inclusion in the study. Seventy eight were excluded (previous dxa scan in 49, frailty in 29). Those remaining were invited for a heel BMD scan using a PIXI® (GE Lunar) densitometer. A clinical risk factor questionnaire was also sent to those who agreed to participate. Patients had height and weight measurements performed at the time of scanning. Modified Study of Osteoporotic Fractures risk factor score, Fracture Index score, Osteoporosis Screening Tool score and Femur T score equivalent were calculated for all patients. Manufacturer's T score recommendations were adopted for classification of results: i.e. normal (T ⩾ - 0.5), osteopenia (- 1.6 > T ⩽ - 0.6), osteoporosis (T ⩽ - 1.6). Treatment was advised for osteoporotic patients and osteopenic patients on oral steroids or with fractures. Patients with T scores between - 1.3 and - 1.5 without fractures or steroids were referred for central dxa along with patients with normal density and fractures.

One hundred and seventy eight patients declined to participate, 279 failed to reply, 49 did not attend and 4 patients were excluded (previous scan in 3, frailty in 1). Four hundred and thirty two patients entered the study but 3 could not be scanned for technical reasons. Fractures occurring after age 50 were found in 25 of 195 normals (12.8%), in 28 of 106 osteopenics (26.4%) and 34 of 128 osteoporotics (26.6%). A treatment decision was made in 378 patients at the time of scanning (88.1%).

Heel BMD in primary care is a cheap, simple effective means of assessing osteoporosis risk in the elderly female population. Less than 12% of patients needed referral for central dxa scanning. Large numbers of untreated patients with osteoporosis and low trauma fractures were discovered in this study despite the provision of a local central dxa service for 10 years. This study suggests that a selective case finding strategy may fail to identify many individuaIs at high risk for osteoporotic fracture. If, however, the full potential of heel BMD measurement is to be realised in this population, additional efforts may be needed to raise the patient participation rate.

Disclosures: J.D. McCrea, MSD 2, 8.


Performance of Computer Assisted Densitometry (CAD): Comparison with Visual Assessment by Experienced Densitometrists.E. N. Schwartz, D. M. Steinberg*. Foundation for Osteoporosis Research and Education, Oakland, CA, USA.

Modern dual-energy x-ray absorptiometry (DXA) systems provide semi-automated analyses of lumbar spine and femur bone mineral density (BMD). However, visual assessment by the user is necessary to a) assure proper acquisition mode and patient positioning, b) assure accurate identification of regions of interest (ROI), and c) identify scans with abnormal conditions that could artificially elevate BMD. Recently, automated software known as Computer Assisted Densitometry (CAD) (GE Medical Systems Lunar) was introduced to assist in identifying scans with common acquisition and analysis irregularities. The aim of this study was to determine how well CAD agreed with the assessment of two experienced DXA users, and to determine if CAD could identify abnormal scans which might be missed by visual assessment.

A total of 71 spine and 70 femur scans were analyzed, with 67% of scans exhibiting some abnormal condition flagged by CAD. Two experienced users (an ISCD-certified densitometrist and an ISCD-certified technologist) together evaluated the scans for abnormalities, unaware of the CAD results. The consensus assessment by the densitometry team was compared to the CAD assessment. In addition, scans where CAD and the visual assessment differed were reviewed to determine if a potential abnormality might have been missed by the densitometry team.

Results showed a strong agreement between CAD classification and visual assessment. Experienced users agreed with the CAD assessment in 76% to 86% of scans prior to knowing the CAD result. Review of scans where disagreement was present, after revealing the CAD recommendations, resulted in an assessment change by the densitometry team in 20% to 40% of cases. Agreement increased to 83% to 92% after CAD assessment was known. Femur results were similar. We conclude that use of CAD can 1) provide valuable information for inexperienced users regarding DXA scan quality and 2) assist experienced densitometrists in identifying potentially abnormal scans which might be missed by visual assessment alone. 

Table  . Agreement between CAD and Expert Evaluation of Normal and Abnormal Spine Scans
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Disclosures: E.N. Schwartz, GE Medical Systems Lunar 2.


Dual Energy X Ray Absorptiometry of Ex Vivo Mouse Long Bones: Site and Side Comparisons.G. E. Lopez-Franco*1, S. Litscher*1, T. K. O'Neil*1, M. Urban-Piette*1, P. Demant*2, R. D. Blank1. 1Endocrinology/Medicine, University of Wisconsin Medical School, Madison, WI, USA, 2Molecular Genetics, Netherlands Cancer Institute, Amsterdam, Netherlands.

Dual energy X ray absorptiometry (DXA) has become a popular analytical technique in mice and other small animals, either as a substitute for more demanding techniques, such as biomechanical testing, histomorphometry, and gravimetry, or as an adjunct to them. As small animal skeletal research continues, more detailed questions regarding differences in the behavior of cortical and trabecular bone and at different anatomical sites are becoming ever more prominent. Such experiments require that site-specific data be generated and interpreted. The existing rodent site-specificity data are therefore insufficient to help guide interpretation of comparative densitometry of left and right bones. Similarly, there are no published data addressing the degree to which contralateral bones are similar in the absence of an experimental intervention. In order to address these gaps in our knowledge, we undertook a comparison of excised mouse femora, humeri, and radii from 383 mice using DXA. At all 3 sites studied, we found that left bones were slightly but significantly (P < 0.00001) denser than right bones. Correlation coefficients between left and right sides ranged from 0.53 ius to 0.78 for bone mineral density (BMD), between 0.56 and 0.86 for bone mineral content (BMC), and between 0.45 and 0.66 for bone area. Correlation coefficients between side-averaged femoral and humeral, femoral and radial, and humeral and radial BMD were 0.81, 0.51, and 0.61, respectively. Correlations were lower when limited to ipsilateral or contralateral pairs, but these were equal to each other. Our findings demonstrate the impact of region of interest analyses on measurement precision relative to whole-body analyses and entail the following recommendations for future DXA studies in mice. To the extent that the scientific questions being addressed are amenable to whole body BMD rather than region of interest BMD measurement, the whole body measurement should be preferred. If site-specific differences are being sought, it is desirable to use the largest practical regions of interest and to adjust sample sizes in accordance with the loss of precision entailed by region of interest analysis. In longitudinal studies assessing regions of interest, care should be taken to compare the same side at each measurement. In comparisons of paired bones, it is important either to have a suitable method for randomizing sides for treatment or adequate reporting of assignment of sides to treatment or control.

Disclosures: G.E. Lopez-Franco, None.


DXA Bone Mineral Density (BMD) Measurements within the Femoral Neck Are Critically and Clinically-dependent on the Placement of the Region of Interest (ROI).M. T. DiMuzio. The North Shore Osteoporosis Center, Deerfield, IL, USA.

Hip fracture is the most severe clinical outcome related to osteoporosis and its evaluation is critical for establishing the true bone health status of patients. Bone health status, in part, requires the measurement of the BMD of key skeletal sites and is the most sensitive factor in the evaluation of fracture risk. Dual Energy X-ray Absorptiometry (DXA) is the gold standard for determining BMD, however it is sensitive to the specific region of interest (ROI) under evaluation. It has been determined that the most sensitive measure of hip fracture risk is the evaluation of the femoral neck ROI within the proximal femur, since most hip fractures occur at the femoral neck without trauma or are independent of the fall characteristics in patients suffering loss of balance.

In order to evaluate patients at highest risk for hip fracture, it is critical to measure the site where fractures occur within the proximal femur: the femoral neck. The purpose of this study was to determine which ROI along the femoral neck is of most clinical relevance, since a significant disparity exists in the choice of such an ROI between the manufacturers of the most common instruments used for measuring BMD in the world (Hologic and GE-Lunar).

This prospective study uses an Hologic QDR-4500A bone densitometer to determine the BMD along the length of the femoral neck in 250 patients aged 60 to 85 years of age referred for a bone density evaluation. Patients' scans were chosen at random with no preference for WHO category, sex, weight or body size. A femoral neck ROI (a rectangle of constant width) for each patient was used to measure BMD. Initial measurements were taken at the edge of the greater trochanter and at equal increments along the neck, up to the edge of the head of the femur. The equal increments consisted of a pixel (one cursor stroke). After each movement up along the neck, the BMD was determined, and for each patient, the BMD along the distance between the edge of the greater trochanter and head of the femur was plotted.

Conclusions: In all cases, the BMD increased as one moved up along the length of the femoral neck from the greater trochanter to the head of the femur. The average increase in BMD was 7.8%. Moreover, for all patients, the lowest BMD was measured at the edge of the greater trochanter. Since fracture risk increases with decreasing BMD, it is critical in evaluating a patients hip fracture risk, that BMD measurements of the neck of the femur are made at the edge of the greater trochanter where the ROI has the lowest BMD and highest risk for fracture. Also, in comparing BMD measurements of the proximal femur between manufacturers, close attention must be paid to specific ROIs in order to determine true rates of change.

Disclosures: M.T. DiMuzio, None.


Site of Bone Densitometry in Middle-Aged Women: Comparison of Bone Mineral Density of 3 Anatomic Regions.L. W. Turner1, L. Wallace2, B. Perry*3, J. Ballard4. 1Health Science, University of Arkansas, Fayetteville, AR, USA, 2Medical Sciences, University of Tennessee, Knoxville, TN, USA, 3Medical Sciences, University of Arkansas, Little Rock, AR, USA, 4Health and Exercise Science, University of Texas, Tyler, TX, USA.

Because the bone mineral density (BMD) in different anatomic regions is heterogenous the number of women who meet the World Health Organization definition of osteopenia or osteporosis increases with the number of sites examined. The purpose of this study was to compare bone mineral density measures of the left femur, spine (L2--L4) and whole body among middle-aged women. Three hundred and forty two (342) healthy middle-aged women (mean age 49.5 years) were studied using dual-energy x-ray absorptiometry (DXA Lunar, Prodigy, software version 2.5). Subjects lay supine on the scanner table and were wearing no metal when measurements were assessed by a single certified densitometry technologist. Mean t-scores were: -0.267, SD=1.17 for hip; -0.101, SD=1.38 for spine, and 0.269, SD=1.14 for whole body. Correlation coefficients between hip and spine were .73, between hip and total body were .77, and between spine and total body were .78. All correlations were significant (p<0.0001). Using WHO definitions, the diagnoses of osteopenia based on a single measurement varied from 28% using the hip region to 22% using the spine area and 13% using the whole body site. Diagnoses of osteoporosis based on a single measurement varied from 2% using the hip area, 4% using the spine site, and 1% using the whole body region. Using a single measurement of the whole body, 3% of osteoporotic patients would have been misclassified as osteopenic and 15% of osteopenic patients would have been misclassified as normal. Six percent more women were diagnosed with osteopenia based on hip measurement compared with spine. Two percent more subjects were diagnosed with osteoporosis based on spine measurement when compared with hip measurement. The choice of anatomic region has considerable impact on diagnoses of osteoporosis and osteopenia. While correlations between anatomic regions are high and statistically significant, considerable variability exists in the classifications of osteoporosis and osteopenia with potential for misdiagnosis, especially when the whole body method is used alone. A combination of densitometry examination using skeletal sites of spine and femur should be used in diagnosing osteopenia and osteoporosis among middle-aged women.

Disclosures: L.W. Turner, None.


High Fracture Risk Does Not Increase Bone Mass Measurement in Older Women.J. M. Neuner*1, A. B. Nattinger*1, N. C. Binkley2. 1Medicine, Medical College of Wisconsin, Milwaukee, WI, USA, 2Medicine, University of Wisconsin-Madison, Madison, WI, USA.

BACKGROUND: Use of many preventive strategies decreases with increasing patient age. Little is known about current bone mass measurement in patients at high fracture risk, including older women. Thus, we examined the effect of age on this measurement in female Medicare patients before and after Medicare reimbursement of postmenopausal osteoporosis screening began in 7/98.

METHODS: We studied 43,250 women aged 67 or over from three random population-based 1% samples of those eligible for Medicare parts A/B in several regionally diverse cities and states. After excluding those with prior testing, we determined the percent who underwent bone mass measurement (central DEXA, peripheral DEXA or QUS) in the three independent cohorts each studied over an 18-month period (7/95--12/96, 1/97--6/98, or 7/98--12/99). We examined the effect of age upon testing in the total sample in a logistic regression model with adjustment for time period (cohort), race, history of hip fracture, Medicaid insurance, urban residence, and Charlson-Deyo comorbidity categories (including diagnoses often treated with glucocorticoids). We repeated the analysis with an interaction term between age and time period.

RESULTS: Of subjects in all three cohorts combined, 85% were white and 11% had a prior hip fracture. Bone mass measurement increased over the three time periods for all age groups, but remained low overall even after Medicare payment began (table). Older women underwent less testing during each period. When compared with women aged 85 or older, those 67--74 were 3.2 times (CI 2.7, 3.9) and those 75--84 were 2.4 times (CI 2.0, 2.8) more likely to undergo testing. Hip fracture had no effect upon testing, and the effect of age upon testing did not differ over time (interaction term p >.1).

CONCLUSION: Despite the known increase in fracture risk with age, older female Medicare patients were much less likely than younger women to receive bone mass measurement. Women with prior hip fracture were no more likely to be tested. As the effect of age upon testing did not improve soon after Medicare reimbursement began, efforts to improve adherence with recent osteoporosis screening guidelines should particularly target older women and those with hip fracture. 

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Disclosures: J.M. Neuner, None.


Precision and Accuracy of DXA and pQCT For Densitometry of the Rat Femur.J. A. Horton*, G. M. Murray*, J. A. Spadaro, B. S. Margulies*, M. J. Allen*, T. A. Damron*. Orthopedic Surgery, SUNY Upstate Medical University, Syracuse, NY, USA.

Background: Bone mineral density (BMD) and bone mineral content (BMC) are key data in the study of osteoporosis and other pathologic skeletal diseases. Dual-energy X-ray Absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT) are used in human and small animal studies of bone density. The purpose of this study was to compare the precision and accuracy of three techniques of measurement of the rat femur.

Method: Validation of relative and absolute accuracy was assessed by scanning the intact and excised femora of 11-week old rats using DXA (Lunar DPX-IQ and PIXImus2) and pQCT (Stratec XCT-2000). The study compared machine measured parameters with standard, non-radiographic measurements using regression analysis and calculated percent difference from standard values to determine the accuracy of each densitometry technique. Machine-specific and subject-specific precision was determined by calculating coefficient of variation of repeated scans, with the subject being repositioned between scans.


Table Table 1.. Accuracy- Regression Analysis (R2).
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Table Table 2.. Accuracy-Mean Percent Difference from Standard Measurement.
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Table Table 3.. Results of Machine- and Subject-specific Precision Studies.
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Discussion: Each of the methods of densitometry examined in this study produced comparable results and was sensitive to small differences. Further, our assessment of the precision and accuracy observed between methods of scanning excised rat femurs supports use of these methods in studies using small animals and may facilitate comparisons of similar data with other laboratories.

Disclosures: J.A. Horton, None.


Bony Densitometry and Prediction of Risk of Fracture of the Hip.L. J. A. Lunar*. Endocrinology, Endocrinologycal Foundation, Caracas, Venezuela.

This paper is the result of a multicenter investigation that served as a clinical assay for the validation of a bone densitometer (Degos 7032). The study was based on the comparison of two groups of persons: one of the 50 apparently sound volonteers and the other of 39 patients with hip fracture. To compare both groups it was used for the evaluation the F Fischer's test if the difference between the bone density variances (CMO-width) was significant in the distal extreme of the ratio by using the method of simple photon absorptiometry. Then, another comparison was made by the t of Student test (for unmatched values). The difference between the means was significant. It was concluded that the bone density magnitude is an index that allow to differentiate both groups and that it may be used to monitor the hip fracture risk and to anticipate to the trauma caused by fracture.

Disclosures: L.J.A. Lunar, None.


Interviews on Osteoporosis in an Area of Health.L. J. A. Lunar*. Endocrinology, Endocrinologycal Foundation, Caracas, Venezuela.

With the aim of knowing the repercussion of osteoporosis among the elderly in a health area, 88 outpatients of both sexes were studied. They were selected by using a simple randomized method, and they stood for 1/6 of all persons 65 years old or over from the health area of the “Endocrinologycal Foundations. This figure represents 91.6 % of the cases that were programmed. It was attained an expectancy of the problem of 50 % with a reliability of 10 %. Every individual underwent conventional radiological examinations of the following segments: dorsal spine, lumbar spine, pelvic bones, thorax, and right hand, in order to analyze the behaviour of senile osteoporosis in an open health area. 100 % of the persons studied showed some radiological sign of osteoporosis. All the skeletal segments were affected, with the exception of the pelvic bones in one patient and the lumbar spine in another. 19 % of these patients had bone fractures. It is concluded that the severity of osteoporosis differs just a little between both sexes and that there is a clear trend to increase with age.

Disclosures: L.J.A. Lunar, Eli Lilly and Company 2.


Predictive Value of DXA Measured at Different Sites for Non Vertebral Fracture Risk in a Sample of 586 Elderly Swiss Women Aged > 70 years.K. Lippuner1, A. W. E. Popp1, M. Kaufmann*1, R. Perrelet*1, M. A. Krieg2. 1Osteoporosis Unit, University Hospital, Berne, Switzerland, 2CHUV, Lausanne, Switzerland.

Bone density measurements at various skeletal sites are predictive of fracture risk. Whereas prospective studies have been conducted using DXA at the spine, hip, forearm and calcaneus, it is not known whether BMD at the tibia predicts the risk of future fracture. We have previously shown that DXA performed at the tibia allows assessment of cortical (diaphysis) and trabecular (distal epiphysis) bone density in a single weight-bearing bone (J Bone Miner Res 1994 9: 1851--7).

To study the predictive value of tibial BMD for non vertebral fracture risk in comparison to BMD at other skeletal sites, 586 Swiss women from the center of Berne of the SEMOF study (age 76.2 ± 3.0 yrs (mean ± SD) were measured using DXA (QDR 4500, Hologic TM) at the following sites: hip (total hip, femoral neck, trochanter, Wards), forearm (1/3 distal radius, ultradistal radius), and distal tibia (epiphysis, diaphysis). Follow-up lasted 2.5 years and non vertebral fractures were reported by the participants and confirmed by their family physicians or by the hospitals in charge of the patients. To assess the predictive value of BMD at the various sites, COX proportional hazard regression analysis was performed. Results were expressed as relative risks (RR and 95% CI) per decrease in BMD of 1 standard deviation (SD) at each site. Comparison between individual predictive values was performed by comparison of the areas under the ROC curves (AUC).

A total of 60 non vertebral fractures occurred during the follow-up. The respective age-adjusted RRs and AUCs for the various sites are presented in the table. 

Table  .  
  1. *p<0.05 compared to tibia diaphysis and trochanter.

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We conclude that in our randomly selected cohort of 70 to 80 yrs old women, the predictive value for non vertebral fracture risk was comparable for BMD measured at tibial diaphysis and at standard sites. BMD measured at tibial epiphysis had a significantly better predictive value than BMD measured at the trochanter or at the tibial diaphysis.

Disclosures: K. Lippuner, None.


Cost-Saving Classifications for Hip Fracture Prediction.H. Jin*1, Y. Lu1, S. T. Harris1, D. M. Black2, K. Stone2, M. C. Hochberg3, H. K. Genant1. 1Radiology & OARG, University of California, San Francisco, CA, USA, 2Epidemiology and Biostatistics, University of California, San Francisco, CA, USA, 3Medicine, University of Maryland, Baltimore, MD, USA.

This paper presents an extension of the Classification And Regression Tree (CART) methods (Brieman, et al, 1984) and its application to cost-saving identification of subjects at high risk of osteoporotic hip fracture within 5 years. Two major modifications were made to the splitting procedures of the standard CART methods: the tree was made more robust, and an algorithm was developed to generate a cost-saving classification rule with classification efficiency not inferior to the optimum rule. We applied the new algorithm to the data from the Study of Osteoporotic Fracture (SOF). Forty-three previously documented predictive variables for osteoporotic hip fracture were examined. The SOF data were randomly separated into two data sets: two thirds were used to generate classification rules (generating data) and the remaining one third was used to compare the rules (validation data). First, using the new algorithm, we generated a robust optimum classification rule for subjects at high risk of 5-year hip fracture without consideration of the cost of the predictive variables. The variables included age, weight, body mass index, height and height loss, bone mineral density (BMD) by dual x-ray absorptiometry (DXA), functional status assessment, visual examination, and prevalent fracture determined by X-ray. This optimum algorithm had a sensitivity and specificity of 76.0% and 74.2% respectively for the data generating the rule, and 65.8% and 73.8% for the validation data. We then generated an alternative cost saving rule with equivalent diagnostic utility, but using only age and BMD by hip DXA scan. This rule had a sensitivity and specificity of 71.4% and 73.6% respectively for the data generating the rule, and 76.3% and 74.6% for the validation data. The two rules were statistically equivalent. We further compared them with the use of a single BMD measurement and results of the standard CART. These comparisons demonstrated the superior performance of the rules generated by our modified algorithm. This paper suggests that our modified CART algorithm can be a useful too to assist clinical decisions and, more importantly, that a DXA hip scan and age can identify subjects at high risk of osteoporotic hip fracture within 5 years as efficiently as more costly and complicated algorithms.

Disclosures: Y. Lu, None.


Correcting Phalanx Bone Mass Measurement for Clinical Risk Factors Increases its Validity in the Diagnosis of Osteoporosis.D. Picard, J. P. Brown*, M. Couturier*, L. Rosenthall*, J. Lévesque*, M. Dumont*, L. Ste-Marie*, A. Tenenhouse*, S. Dodin*. Quebec Osteoporosis Study Group, Montreal, PQ, Canada.

Peripheral bone mass measurement by dual-energy X-ray absorptiometry (DXA) is an appealing method for the diagnosis of osteoporosis, especially in distant areas, where central DXA, the gold standard, is not available. On the other hand, clinical risk factors can provide information on bone mineral density (BMD). The aim of this study is to evaluate the ability of phalanx DXA to predict central BMD after being corrected for two important clinical factors that can affect bone mass, age and weight.

We determined BMD of the spine (s), femoral neck (fn) (1 Hologic, 3 Lunar), as well as phalanx (p) with Schick AccuDXA in 831 women aged 20 to 85 years recruited in four centers. The sBMD and fnBMD were converted to a Hologic base using the method of Genant et al. and T-scores were then derived using data from the Canadian Multicentre Osteoporosis Study (CaMos). There was a good correlation between BMD of the different anatomic regions (r = 0.599--0.671, p<0.0001). After applying regression analyses on the gold T-score (lowest between s and fn) where age, weight and phalanx T-score were retained as the main predictors, we corrected phalanx T-score according to the regression formula obtained. To examine the performance of DXA and corrected phalanx DXA as a diagnostic test for osteoporosis, we performed ROC curves where a positive case was defined as a T-score <=-2.5 either on s or fn. The performance was evaluated at optimal cutoff T-scores selected on the basis of highest sensitivity and specificity. Results are reported in the table below. The main improvement of correcting phalanx T-score for age and weight is observed in an increase in sensitivity by 7% translated through a reduction of the false negatives by 32%. Furthermore, among the false negatives, most of the subjects are near the osteoporosis threshold as only 5 present a central T-score < -3. The mean central T-score among the false negatives is -2.91 (+/− 0.39). Hence, this study suggests that correcting phalanx bone mass measurement for age and weight, increases its validity in the diagnosis of osteoporosis as reflected by a greater number of osteoporotic subjects identified without affecting the proportion of correctly identified normal subjects. 

Table  .  
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Disclosures: D. Picard, None.


Equine Third Metacarpal Bone Mineral Density Assessment with a Mobile Dual Energy X-Ray Absorptiometry Device: An In vivo and Ex Vivo study.M. Donabedian*1, G. Perona*2, C. Delguste*3, P. Lebecque*4, F. Duboeuf*5, O. M. Lepage6, W. Martin-Rosset*7. 1Départements NASA & ENA, INRA, Theix, France, 2Dipartimento di Produzioni Animali, Facoltà di Medicina Veterinaria di Torino, Grugliasco, Italy, 3Département des Sciences Cliniques, Université de Liège, Liège, Belgium, 4Département NASA, INRA, Theix, France, 5Inserm U 403, Hǒpital Edouard Herriot, Lyon, France, 6Département Hippique, Ecole Nationale Vétérinaire de Lyon, Lyon, France, 7Département ENA, INRA, Theix, France.

Dual energy X-Ray absorptiometry (DXA) is the most widely used osteodensitometric method in man. Until recently, because of anatomical specificities and lack of specific DXA device, only post-mortem or in vivo studies under general anesthesia were possible in horses. The development of a mobile DXA device (PIXI, Lunar Corp) offers new opportunities for in vivo equine bone mineral density (BMD) measurements. The purpose of this study was to analyze in vivo usability, and ex vivo - in vivo precision of this device. Region of interest (ROI) was located 5-cm above the distal end of MCIV. In order to test ex vivo precision on one equine limb, 20 successive BMD measurements of the ROI were achieved without (repeatability) and then with (reproducibility) bone repositioning between each procedure. In vivo, intra and inter operator reproducibility was tested. Horses were not sedated and their left front foot was placed on a wood positioning block in order to keep MCIII vertically and forward. For in vivo intra operator reproducibility testing, 3 different operators' pairs achieved 10 successive BMD measurements on 2 different horses. For inter operator investigations, 4 operators tested 12 combinations of PIXI and foot positioning. Five successive complete sets of 12 combinations were achieved. Coefficient of variation (CV% = standard deviation/mean) was established for each test. Ex vivo repeatability and reproducibility CVs was respectively 1.47 and 1.69 %. In vivo intra operator reproducibility varied between 2.91and 4.06 % depending on horse and operator. In vivo inter operator reproducibility varied between 3.13 and 5.42 % according to the measurement set. Close results for ex vivo repeatability and reproducibility in one hand, and intra and inter operator in vivo reproducibility in the other hand show that positioning process was well standardized and induced few imprecision. With a sensitivity threshold of 2√2.CV, lowest significant BMD variation would be 8.23 % in an in vivo longitudinal follow up. The present results suggest adequacy of PIXI for metacarpal BMD measurement in the horse in vivo. It would be of interest to test PIXI's accuracy at the high BMD ranges of equine MCIII.

Disclosures: M. Donabedian, None.


Discriminant Power of DXA Parameters in Low Trauma Fractures in IJO Subjects.P. Pludowski*1, M. Lebiedowski*2, H. Matusik*1, R. S. Lorenc*1. 1Dep. of Biochemistry and Exp. Medicine, Children's Memorial Health Institute, Warsaw, Poland, 2Dep. of Rehabilitation, Children's Memorial Health Institute, Warsaw, Poland.

In Idiopathic Juvenile Osteoporosis (IJO) long bone and vertebral fractures are common and etiology is unknown. The aim of study was to test the ability of DXA derived parameters in fracture discrimination. The study population comprised 61 IJO children aged 5--19 yrs (34f, 27m) evaluated during acute and chronic phases of disease as well as 412 healthy children aged 5--19 yrs (187f, 225m). DXA derived total body BMC (TBBMC, an indicator of bone strength), BMD (TBBMD, g/cm2), lean mass (LBM, a surrogate of muscle mass) and body height (BH) were analyzed. BH/LBM and TBBMC/LBM ratios were established (after transformation: X/Y=>ln(X)/ln(Y)) in healthy and diseased children. Logistic regressions (LR) and ROC analyses were performed to evaluate the parameter that most efficiently discriminate healthy and IJO children, with assumption the IJO sustained at least 1 fracture. LR analyses revealed higher chi-square values for TBBMC/LBM ratio (girls 87.25; boys 50.59) than for TBBMD (83.13; 20.62), TBBMC (50.59; 6.99), LBM (11.51; 1.11), BH/LBM (0.87; 4.24) in both genders of IJO children (acute phase). The chi-square values for BH/LBM ratio and for LBM indicated lack or only small relationship between muscle status and probability of fracture condition. Lower significance was shown for the analyses based on results calculated during chronic phase of IJO. In boys chi-square values were very low and not significant (except LBM) indicating no difference with reference data. The ROC showed an analogous decreasing order of significance for the corresponding areas under the curves (AUC). It could be pointed, that during acute phase of IJO, measured TBBMC (AUC girls 80.8%; boys 60.4%), TBBMD (86.7%; 67.6%) and TBBMC/ LBM (87.8%; 81.0%) discriminated markedly better healthy from diseased groups than BH/LBM (48.2%; 59.4%) or LBM (67.7%; 47.3%). During chronic phase of IJO the AUC values were markedly lower indicating lack of significant differences between analyzed groups. The ROC assessed “cut off” values were close to those corresponding to the inflexion points of logistic curves. On the basis of AUC values, we found that TBBMC/LBM ratio was the most efficient discriminator in IJO children (irrespective of the phase of disease) in assayed analyses conditions.

Our study pointed on significant advantage of TBBMC/LBM ratio over typically used TBBMD in general assessment of bone mechanical status in IJO cases what postulate on inclusion of this parameter into standard diagnostic procedures in pediatric study of skeletal status.

Disclosures: P. Pludowski, None.


Resolution and Magnification Error Using a Cone Beam Bensitometer for Femoral Morphometry.V. Boudousq*, E. Thomas, I. Ruiz*, P. O. Kotzki*. Médecine Nucléaire, Hǒpital Carémeau, Nǐmes, France.

The bone mineral density (BMD) is the mean determinant of the hip fracture risk. However other factors like the lifestyle, the propensity for falls and the femoral geometry could influence the hip fracture risk. The femoral geometry could be evaluated using pencil beam densitometers. With the fan beam densitometers the spatial resolution was improve but there was an inherent magnification of scanned structures in the direction of the beam inducing a distortion of the image. This distortion of the image did not allow to evaluate easily the femoral geometry.

The purpose of this study is to evaluate in vitro the potentiality of a new cone beam densitometer -the DMS Lexxos- in order to realise femoral morphometry. This densitometer used a 2D digital radiographic detector that did not require scanning for image build up of the lumbar spine or the hip. The system required only two 0.75 s X-rays exposures to complete an examination of these areas. The detector was 20 cm square, provided 512 × 512 pixels, each pixel was 0.4 mm on a side.

The resolution was assessed using a line pair test pattern include in a water container to simulate soft tissue. The resolution was defined as the last bar section in which a clear distinction could be seen between lines and spaces. The resolution was explored in the two principal perpendicular directions.

Magnification and distortion are assessed using a matrix test object placed on the examination table and at different levels from the table between 25 and 295mm. The analysis was on one hand a visual analysis looking for a line distortion and on the other hand some crossing points were identified, the coordonates of the points in pixels were noted and the Euclidean distance was computed.

The resolution results on both the X and Y axis are the same. The resolution was evaluated between 1.4 and 0.5 line pairs /mm for an attenuation varying for 32 to 316 mm of water. The magnification is about 1.17% cm-1 when increasing the distance of the phantom above the examination table, this magnification is isotropic without distortion.

Disclosures: V. Boudousq, None.


Precision and Accuracy of the GE Lunar DPX-Bravo Bone Densitometer.R. H. Nord, H. S. Barden, S. Krepanith*, K. G. Faulkner. GE Medical Systems Lunar, Madison, WI, USA.

The DPX Bravo (GE Medical Systems Lunar) is a small footprint, compact DXA system designed to measure the spine, femur and forearm. The spine and both hips can be measured using a single acquisition protocol (OneScan) designed to reduce procedure time by eliminating the need to reposition the subject between scans. The purpose of this study was to evaluate the clinical performance (precision and accuracy) of the DPX Bravo compared to existing DXA systems.

Forty-seven volunteer subjects (18 men and 29 women) ranging in age from 23 to 87 were measured on the DPX Bravo. Each subject was measured at the AP spine (L1--L4), and either at the left forearm or at both femora. A total of 45 spine, 30 femur, and 31 forearm measurements were performed. Precision was evaluated by measuring each site 3 times on the DPX Bravo and computing the root mean square coefficient of variation. Accuracy was evaluated by comparing the DPX Bravo BMD values to measurements on the Prodigy and the DPX Pro, bone densitometers currently available from GE Medical Systems Lunar. Precision error results for the DPX-Bravo are given in the first table. Accuracy is shown as regression results in the second table. Overall comparison results are plotted below. 

Table  .  
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We found the clinical precision of the DPX Bravo to be about 1% at the three body sites it is designed to measure. Regression of BMD values with those of currently accepted densitometers gave extremely high correlation coefficients and slopes that did not differ significantly from unity.

We conclude that BMD measurements taken with DPX Bravo will be clinically equivalent, in both precision and accuracy, to those taken with existing GE Lunar densitometers.

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Disclosures: R.H. Nord, None.


Precision Comparison of Two DXA Densitometers -- Prodigy and Delphi.E. M. Lewiecki1, P. D. Miller2. 1New Mexico Clinical Research & Osteoporosis Center, Albuquerque, NM, USA, 2Colorado Center for Bone Research, Lakewood, CO, USA.

When measuring bone mineral density (BMD), precision errors can be caused by many factors: differences in patient positioning, variations in scan analysis, and both short- and long-term fluctuations of the densitometry equipment. Minimization of these errors is essential to providing an accurate assessment of BMD change over time. In this study, we compared the short-term precision error of two DXA devices, the Prodigy (GE Medical Systems Lunar) and the Delphi (Hologic). Both are fan-beam DXA devices predominantly used to measure BMD of the spine and proximal femur. In this study, 41 women age 51--80 years (mean age 61) were measured in duplicate on both Prodigy and Delphi systems at either of two centers. The DXA technologists were ISCD certified and used manufacturer recommended scanning and analysis procedures. All scans were performed using the manufacturer recommended 30-second scan modes. BMD precision error was calculated as the root-mean-square standard deviation (RMS SD) and coefficient of variation (RMS-%CV) for the repeated measurements. Data from right and left femora were evaluated individually (single femur precision) and using the combined value (bilateral femur precision). The precision error (variance) of the Prodigy and Delphi measurements at each measurement region was compared using an F-test to determine the significance of any observed differences. 

Table  . Precision Error for Prodigy and Delphi
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Prodigy precision errors were significantly less than the Delphi at the femoral neck, total femur and bilateral total femur. There was no significant difference in precision error at the spine and trochanter. Using bilateral femur measurements, precision errors were improved for both systems by 40% compared to the single femur results. We conclude that there are skeletal site-specific differences in precision error depending on whether a Prodigy or Delphi is used. In clinical practice, these differences should be considered when determining the time interval for least significant change in BMD to occur.

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Disclosures: E.M. Lewiecki, GE Medical Systems Lunar 2.


Whole Body and Lateral Vertebral Assessment with a Digital 2D Bone Densitometer.C. Robert-Coutant*1, M. Julier*2, L. Gerfault*1, G. Gonon*1, L. Gaucher*2, C. Gagnepain*2, R. Grando*2, J. M. Dinten*1. 1Leti, CEA-GRENOBLE, Grenoble Cedex 9, France, 2Diagnostic Medical Systems, Montpellier, France.

The Lexxos bone densitometer (DMS, France) is the first axial DEXA bone densitometer using a digital 2D radiographic flat panel detector. Previous presentations (SPIE Medical Imaging 20011, ASBMR 20012) detailed how Lexxos realizes, without scanning, in two X-Rays flashes, spine, hip and forearm exams and presented its BMD measurement performance. One major Lexxos advantage is a quasi radiological image quality.

In order to take profit of Lexxos high image quality, its functionalities have been extended to whole body and lateral vertebral examinations. Due to the limited detector size, images are built from a set of acquisitions, composed of overlapping elementary low energy and high energy radiographs. They are combined by a dedicated reconstruction algorithm which includes a breathing artefacts correction process. Reconstructed images are isotropic and free of distortions.

Whole body mode provides with three 1.6mm pixel size images. Whole body bone, fat and lean tissue images are realized in a continuous mode in less than 6 minutes. An automatic ROI detection algorithm provides with BMD, BMC, fat mass, lean tissue mass at the different ROI. Fat and lean tissue spatial distribution can also be analysed on whole body fat and lean tissue images.

Lateral vertebral mode provides with a 0.4mm pixel size vertebral bone image, obtained in a sequential mode in less than 30 seconds. ROI, corresponding to the different lumbar and thoracic vertebra, are automatically detected. A supervised tool enables a semi-quantitative analysis of vertebral deformations on each vertebral region, either on the bone or on the low energy image.

  • “Dual-Energy X-Rays absorptiometry using a 2D digital radiography detector. Application to bone densitometry”, J.M. Dinten et al.; SPIE Medical Imaging 2001

  • “In vitro short term reproducibility evaluation of a Flash Beam X-Rays bone densitometer”

V. Boudousq et al.; ASBMR 2001

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Disclosures: J.M. Dinten, DMS 2.


Real Volumetric Compared to Apparent Volumetric and to Areal Bone Mineral Density for the Evaluation of Bone Mass in Turner's Syndrome.A. Lage*1, I. Verreschi*1, J. Mendes*1, M. Huayllas*2, B. Liberman*2, B. Mendonça*3, E. Costa*3, M. Lazaretti-Castro1. 1Endocrinology, UNIFESP, Sao Paulo, Brazil, 2Brigadeiro Hospital, Sao Paulo, Brazil, 3USP, Sao Paulo, Brazil.

Turner Syndrome (TS) is a chromosome abnormality characterized by short stature and hypogonadism. Extremes statures are a known error factor for the bone mineral density (BMD) determination using the conventional dual energy X-ray absorbtiometry (DEXA). In the present cross-sectional study we evaluated lumbar spine BMD in 62 patients with TS aged from 10 to 45 years old obtained by different methods in order to determine the magnitude of the confounding effect of short stature. Using a Hologic 4500 A on postero-anterior (PA) and lateral projections, we obtained the areal (aBMD) and calculated the apparent volumetric (apBMD) and the real volumetric (volBMD) bone mineral densities. One hundred and two normal females (10 to 45 years old) were used as controls, and their BMD values were used as reference. We considered “Normal” Z score for TS patient if >-1. In TS, the mean of Z-score for aBMD was significantly lower than for volBMD (-2.31+1.42 g/cm2 and -0.64+1.55 g/cm3, respectively; P <0.0001). The mean for apBMD was higher than for aBMD (-1.72 + 1.51 g/cm2 and -2.31+1.42 g/cm2, respectively; P <0.0001), even though it was lower than for volBMD (P <0.0001). Most of the patients (83.8%) had abnormal Z-score for aBMD. On the other hand, most of them (58.1%) had normal Z-score for volBMD. For apBMD, 30.6% of the patients had normal Z-score. When we evaluated the patients with normal Z-score for aBMD and volBMD in the age sub-groups, we noticed that in the sub-group of 10 to 12 years old, the percentage of girls that had normal Z-score for aBMD was similar to those with normal volBMD in the proportion of 1:1.3. In the other sub-groups the proportion was much larger culminating in the sub-group of 16 to 21 years old (1:11). When we compared the volumetric and areal Z-score, when patients were normalized for statural-age, we noticed that the mean for areal Z-score for statural-age was higher than the volumetric Z-score (0.02+1.31 and -0.64+1.55, respectively; P = 0.0008). There was no significant difference in the percentage of previous fractures between the 2 groups (TS and control). In conclusion, the aBMD underestimates the bone mass of the lumbar spine in patients with TS. This error is more evident in girls above 12 years old. Volumetric BMD minimized the confounding effect of short stature and approached the bone mass of control patients, while apBMD just partially minimized that effect. The prevalence of fractures in the 2 groups (TS and control) was not different and, therefore, in the analyzed patients, we did not find indications of increased bone fragility.

Disclosures: M. Lazaretti-Castro, None.


Increased Awareness of Osteoporosis Improves Radiologists' Reporting of Plain Films and DXA Examinations.L. Lenchik1, A. J. Laster2, S. Morgan*3. 1Wake Forest University, Winston-Salem, NC, USA, 2Duke University, Raleigh-Durham, NC, USA, 3University of Alabama, Birmingham, AL, USA.

Purpose: Little is known about how physicians' attitude toward a disease influences their practice pattern. The purpose of this study was to determine how increased awareness of osteoporosis influences bone densitometry reporting among radiologists in the United States.

Methods: A one page survey was faxed to a random sample of 11,400 radiologists. Awareness of osteoporosis was determined based on responses indicating agreement (on a scale of 1 to 7) with 15 general statements about epidemiology, diagnosis, and treatment of osteoporosis. The responses were used to calculate the osteoporosis awareness score (scale of 1--14). Six multiple choice questions determined the approach to reporting of plain films and DXA examinations. Reporting practices were compared based on osteoporosis awareness.

Results: 397 (3.5%) complete surveys were returned and used in this analysis. When reporting central DXA examinations, 85% used the T-score < -2.5 for diagnosis of osteoporosis, 77% included the absolute BMD, 57% included fracture risk, and 27% included treatment recommendation. When reporting plain films in elderly patients, 96% routinely reported vertebral fracture, 87% reported osteopenia, and 43% recommended a DXA scan when either osteopenia or fracture (hip or spine) are present. General awareness of osteoporosis was satisfactory (mean score = 8.7/14, SD = 2.7). Radiologists who recommend DXA scans based on plain film findings and those who recommend treatment based on DXA results had higher osteoporosis awareness scores (9.5 vs 8.1 and 9.9 vs 8.3, p<0.0001).

Conclusion: Increased awareness about the epidemiology, diagnosis, and treatment of osteoporosis improves the quality of plain film and DXA reports among radiologists.

Disclosures: L. Lenchik, Procter and Gamble/Aventis 2.


Impact of Degenerative Spinal Diseases on Bone Mineral Density of the Lumbar Spine in Elderly Women.S. Muraki*1, S. Yamamoto2, H. Ishibashi*2, T. Horiuchi*2, T. Hosoi*2, H. Orimo2, K. Nakamura1. 1Orthopaedics, Tokyo University School of Medicine, Tokyo, Japan, 2Tokyo Metropolitan Geriatric Medical Center, Tokyo, Japan.

Most of the elderly have degenerative diseases of lumbar spine, which can affect the accuracy of bone mineral density (BMD) of lumbar spine. This study serves as the first to determine the relationship between lumbar spine or femoral neck BMD and the degree of several degenerative diseases at the same. The aim of this study is to determine whether BMD of lumbar spine is related to the degree of degenerative diseases. This study included six hundred and thirty women aged 60 or above (mean age: 73.3 plus/minus 6.9 years) visiting the Osteoporosis Outpatient Clinic in Tokyo Metropolitan Geriatric Medical Center. At entry to this study, subjects were undergone anteroposterior and lateral X-ray of the lumbar spine including L1 to L5. Radiographs were read for the presence and severity of osteophyte (Nathan classification), osteoarthritis (Kellgren method), bone sclerosis, joint space narrowing and spondylolisthesis (Meyerding method) involving L1--2 through L4--5 interspaces. Within one month after taking X-ray, BMD of L2--L4 anteroposterior lumbar spine and femoral neck were measured. The relation between BMD and the score of each item was assessed by regression analysis. Among 630 subjects, 619 (98.3%) had degenerative diseases of lumbar spine. BMD of femoral neck was correlated with age, while, BMD of lumbar spine was not correlated with age. However, among subjects with slight degenerative diseases, lumbar spine BMD was significantly correlated with age. The score of osteophyte, osteoarthritis, bone sclerosis, joint space narrowing and spondylolisthesis were correlated positively with BMD of lumbar spine, however, they had no correlation with BMD of femoral neck. In multiple regression analysis including age, BMI and all items of degenerative diseases, only BMI, osteophyte, bone sclerosis and joint space narrowing were independently correlated with BMD of lumbar spine. According to the multiple regression analysis, adjusted lumbar spine BMD 0.122mg/cm2 lower than observed BMD. Unlike observed BMD of lumbar spine, adjusted BMD of lumbar spine was correlated with age. This study suggests that degenerative diseases of lumbar spine are important sources of BMD overestimation at this site, thus leading to clinical errors. We conclude that BMD of lumbar spine was related to the degree of degenerative diseases.

Disclosures: S. Muraki, None.


Comparison between Osteoprotegerin and Bone Markers in Relation to Age Related Decline in Bone Mass.O. S. Indridason*, L. Franzson*, O. Gunnarsson*, S. L. Gudmundsdottir*, G. Sigurdsson. Department of Medicine, University Hospital, Reykjavik, Iceland.

The purpose of this study was to compare age related differences in osteoprotegerin (OPG) and the bone markers osteocalcin (OC), Collagen crosslinks (CX) and tartrate resistant acid phosphatase (TRAP) in relationship with bone mineral density (BMD).

Data were derived from a cross-sectional study on bone health in a random sample of community dwelling adults aged 30 to 85 years in the Reykjavik area in Iceland. All subjects had whole body, hip and lumbar spine BMD measured (DEXA), gave blood and urine samples and answered a thorough questionnaire on medications and medical history. OPG was measured with an ELISA (Biomedica, Austria), OC and CX with an ECLIA (Roche Diagnostics), TRAP with an ELISA (Suomen Bioanalyttica). We used ANOVA to compare age groups and assessed relationships using partial correlation coefficient. For the current analysis we excluded subjects receiving bisfosfonates, HRT, thiazides or glucocorticoids and those with diseases affecting bone and mineral metabolism. Men and women were analysed separately.

Of 2310 subjects invited over 2 years, 1630 participated. After exclusion, 527 women (age 55.7±16.9) and 496 men (age 58.4±14.9) remained for analysis. In women, BMD at the hip declined steadily after age 50. Women in their 50's had higher OC (33%), TRAP (30%) and CX (55%) than women in their fourties (P<0.001) but the levels plateaued above age 50. OPG increased steadily with age and this increase was most marked in the oldest age groups rather than around menopause. In men, hip BMD remained stable until after age 70 after which it declined significantly (P<0.001). Significantly higher OC (14%), TRAP (6%) and CX (14%) were seen in these oldest age groups compared to those younger than 70 (P<0.05). OPG increased steadily with age as in women. After controlling for age and weight, neither TRAP nor OPG were significantly related to hip BMD in either sex, but OC and CX were associated with hip BMD, r=-0.16, P<0.001 and r=-0.10, P=0.02 in women, and r=-0.17, P<0.001 and r=-0.18, P<0.001 in men, respectively.

We conclude that age related bone loss starts in women around age 50 but in men at age 70. Serum levels of markers of bone turnover increase when age related bone loss starts and this increase is greater for markers of bone resorption (CX). OPG shows a gradual increase with advancing age independent of bone mineral density. The significance of OPG in maintenance of bone remains to be elucidated.

Disclosures: O.S. Indridason, None.


Regional Variations in Cancellous BMD within the Distal Metaphysis of the Rat Femur.H. Lemmon*1, E. Johnson*2, D. D. Cody2, C. G. Ambrose*3, H. A. Hogan1. 1Mechanical Engineering, Texas A&M University, College Station, TX, USA, 2M. D. Anderson Cancer Center, University of Texas, Houston, TX, USA, 3Orthopaedic Surgery, University of Texas Medical School, Houston, TX, USA.

This study was motivated by efforts to measure mechanical properties of cancellous bone tissue in the metaphysis regions of rat femur and tibia specimens. The reduced platen compression (RPC) test utilizes specimens that are 2mm thick and located adjacent to the growth plate, but at a distance intended to avoid primary spongiosa. The specific location of individual specimens is routinely determined using high-resolution contact radiographs (coronal view), which inherently leads to some variability in the locations of the specimens relative to the growth plate region. The purpose of this study was to estimate the effects of this variability on mechanical properties by analyzing variations in bone mineral density (BMD), as a surrogate for mechanical properties. Nine distal femur specimens were selected for analysis from animals from another study, with the goal to encompass a wide range of BMD values. Adult (353 gm avg. initial BW) female Sprague-Dawley rats were sacrificed after 8 weeks. MicroCT scans (model RS-9, GE Medical Systems, London Ontario) were made of the distal third of the right femur (excised) at a resolution of 27 microns. 3D volumes were reconstructed using vendor software. BMD was calculated using a cylindrical region of interest (ROI) that was 2mm long in all cases (to correspond to the size of RPC specimens). The diameter of each ROI was based upon the largest circular region that would inscribe the endocortical perimeter at the proximal extent of the ROI (where the endocortical compartment is smallest). A reference location for the ROI was defined such that the distal surface of the ROI was aligned with the most proximal part of the growth plate (i.e., junction between epiphysis and metaphysis). BMD was measured for this reference location plus 10 additional locations (5 proximal & 5 distal to reference, in 0.2mm increments). BMD ranged from 1 to 662 gm/cc. To quantify “variability,” BMD values were averaged for all specimens at each ROI location and then expressed as a percent of this average. The range (over the 11 locations) of this % for each of the 9 specimens was: 1--30, 7--37, 13--48, 36--100, 43--84, 74--82, 150--186, 175--335, 205--222, resp., which indicates only modest variation as a function of ROI location for most specimens. For ROI locations limited to 0.2mm proximal to 1.0mm distal, the variations become much smaller: 11--30, 11--37, 17--48, 83--100, 74--84, 74--78, 150--179, 175--225, 208--222. These results suggest that specimen locations closer to the growth plate should give more consistent results than those more proximal.

Disclosures: H.A. Hogan, None.


Use of Heel Ultrasound to Screen for Osteoporosis: Comparison with Spine and Femur DXA.P. K. Burke. Osteoporosis Diagnostic and Treatment Program, Richmond, VA, USA.

Recently the International Society for Clinical Densitometry (ISCD) recommended the use of peripheral densitometry (including heel ultrasonometry) to identify patients who might have osteoporosis and should therefore undergo bone densitometry at the hip and spine. This recommendation requires the use of a device specific T-score cutpoint on the peripheral device that detects 90% of individuals with osteoporosis (T-score ⩽ -2.5) at either the spine or hip. In this study, we wished to determine the 90% sensitivity cutpoint that could be used with the Achilles InSight bone ultrasonometer.

The Lunar Achilles InSight (GE Medical Systems) is an imaging ultrasonometer that uses inflatable membranes and an alcohol spray to couple the measurement transducers to the heel. The actual ultrasound measurement takes 8 to 10 seconds; the complete examination (including data entry, patient preparation and positioning) takes 3 to 5 minutes. In this study, 85 female patients (mean age 65 ± 12 years) referred for bone densitometry were assessed. Each patient had DXA measurements of the spine and both hips using a Lunar Prodigy (GE Medical Systems), as well as heel ultrasound measurement using the Achilles InSight. The lowest T-score at the L1--L4 spine, femoral neck, trochanter and total femur was determined for each patient. Osteoporosis was diagnosed when the lowest T-score was ⩽ 2.5. Using heel T-scores ranging from 0.0 to -1.6, the sensitivity and specificity for detecting central osteoporosis was calculated. The T-score cutpoint was then determined which correctly identified 90% of the patients with osteoporosis as defined by the spine and hip measurements. 

Table  . Sensitivity and Specificity of Central Osteoporosis Based on Heel T-Score
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From the DXA results, 34 of the 85 patients were classified as osteoporotic. Sensitivity of the heel T-score ranged from 76% to 97%, reaching 90% at a value between -0.6 and -0.8. At a heel T-score of -0.6, sensitivity was 97% and specificity was 41%. A lower cutpoint of -1.0 provides almost 80% sensitivity but with much better specificity (59%). We conclude that the Achilles InSight can be used as a valid screening tool for osteoporosis according to ISCD recommendations. Using a T-score cutpoint of -0.6 identified more than 90% of patients with osteoporosis at the spine or hip, with a specificity of greater than 40%.

Disclosures: P.K. Burke, GE Medical Systems Lunar 5.


Impaired Bone Quality in Bisphosphonate (BISPHOS) Treatment and Renal Transplantation by Ultrasound Critical Angle Reflectometry (UCR).E. Richer*1, M. Lewis*1, C. V. Odvina2, M. A. Vasquez*3, C. Y. C. Pak2, P. P. Antich*1. 1Radiology, UT Southwestern Medical Center, Dallas, TX, USA, 2Center for Mineral Metabolism and Clinical Research, UT Southwestern Medical Center, Dallas, TX, USA, 3Medicine, UT Southwestern Medical Center, Dallas, TX, USA.

This study was undertaken in order to determine whether intrinsic bone quality is altered following long-term BISPHOS treatment. The UCR technique previously described by us (1) has been incorporated in a new device designed to measure cortical and cancellous bone ultrasound velocities (v) at multiple orientations in the calcaneus in vivo. These measurements are consistent with the hypothesis that bone has transverse symmetry, characterized by the relationship v2 = a+bx2+cx4, where x = cos (orientation), and v2 is elasticity normalized to a density of 1 g/cc. By fitting v data to this formula, the minimum (E*min) and maximum (E*max) elasticities of trabecular and cortical bone were calculated as a measure of bone material quality.

We obtained E* in 14 normal postmenopausal women (NPO), 9 with postmenopausal osteoporosis on conventional treatment (PO), 25 on BISPHOS treatment, and 11 with renal transplantation on steroids (RT-St). Values were expressed as percentage of values in 14 normal premenopausal women (Table 1). Mild or no decreases (<5%) were observed in NPO. A mild-moderate reduction in E* (∼10%) was disclosed in untreated PO. In BISPHOS, trabecular E* declined by ∼ 20%, and cortical E* decreased by 26% (mean duration = 3.5 y). Much of the decline in E* occurred during the first 3 y of BISPHOS treatment. In RT-St, the decline in E* was qualitatively similar to that of BISPHOS. In BISPHOS and RT-St, E* was independent of heel bone mineral density (DEXA). In other groups, E* was modestly correlated with bone density. 

Table Table 1.. Trabecular and Cortical Elasticities Measured by UCR
  1. *p < 0.05; ** p < 0.01; + p < 0.001 vs. normal premenopausal women.

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In conclusion, quality of cortical and trabecular bone is significantly impaired following long-term BISPHOS treatment, as in a condition associated with low bone turnover and propensity for fractures (RT-St).

  • Antich PP, Anderson JA, Ashman RB, Dowdey JE, et al.: Measurement of mechanical properties of bone material in vitro by ultrasound reflection: methodology and comparison with ultrasound transmission. J Bone Miner Res, 6:417--426, 1991.

Disclosures: E. Richer, None.


Bone-Density-Independent Association of Quantitative Ultrasound Measurements and Fracture Risk.T. V. Nguyen, J. R. Center, J. A. Eisman. Bone and Mineral Research Program, Garvan Institute of Medical Research, Sydney, Australia.

Quantitative ultrasound measurements (QUS) of bone, that may reflect micro-architectural aspects of bone, have been shown to be associated with fracture. However, it is not clear whether this association is independent of bone mineral density (BMD). This study was designed to examine the contributions of cortical QUS and BMD measurements to the prediction of fracture risk in postmenopausal Caucasian women.

Speed of sound (SOS) at the distal radius, tibia, and phalanx (Sunlight Omnisense) and BMD at the lumbar spine and femoral neck (GE Lunar) were measured in 549 women, aged 63.2 ± 12.3 yr (mean ± SD; range: 49 -- 88 yr), including low trauma 77 fracture cases. Lower SOS at the distal radius, tibia and phalanx, which were correlated with each other, were associated with increased risk of fracture. Independent predictors of fracture risk (in multivariate analysis) were: distal radius SOS (OR: 1.8 per SD; 95% CI: 1.3 -- 2.4), femoral neck BMD (OR per SD: 1.9; 95% CI: 1.4 -- 2.4) and age (OR per 5-yr: 1.2; 95% CI: 1.0 -- 1.5). Approximately 30% of the women had distal radius SOS T-scores < -2.5; however, only 6.6% of women had both BMD and SOS T-scores < -2.5. Among the 77 fracture cases, only 14 (18.2%) had both BMD and QUS T-scores below -2.5.

In these postmenopausal women, speed of sound at the distal radius was associated with fracture risk, independent of BMD and age. The combination of QUS and BMD measurements may improve the accuracy of identification of women who likely sustain a low trauma fracture.

Disclosures: T.V. Nguyen, None.


Calcaneal Quantitative Ultrasound in Japanese Elementary Schoolchildren and Proper Positioning of Heel Using Adapters for CM-100.K. Nakatsuka1, K. Mimura*2, T. Yamamoto3, H. Morii1, T. Arai*4. 1Osaka City University Medical School, Osaka, Japan, 2Osaka University of Education, Osaka, Japan, 3Dept. of Pediatrics, Minoh City Hospital, Osaka, Japan, 4Furuno Electric corpration, Nishinomiya, Japan.

Assessment of bone health is important in growing children. Although change of bone mass measured by using quantitative ultrasound (QUS) at the calcareous have been previously reported, little is shown on details on how to overcome difficulties in applying QUS devices developed for adults to children. The aims of the present study is to measure calcaneal bone mass by using specially designed adapters which can adjust the position of the heel according to the given foot length of children, and to examine the reliability of the values obtained for these purposes. Subjects consisted of 189 Japanese children living in Osaka Prefecture and belonging to elementary school (6--12 years of age). Body heights, body weights and foot lengths were measured at the same time of QUS measurements. Calcaneal bone mass of right feet were measured using 3 different devices CM-100 (Furuno Electric Co. Japan), AOS-100 (Aloka Co. Japan) and UBIS3000 (DMS Co. France). In case of measurements using CM-100 the heel position of the subjects were adjusted by placing 2 specially designed adapters A and B according to foot lengths. Tomographic imaging of the calcaneal region was also obtained by echo effects of UBIS3000, which makes it easier to adjust manually the heel to the proper position exposed with ultrasonic wave. The lower the SOS values (CM-100) the longer the feet length in the subjects with feet length of 22 or less cm. Although there was no significant differences in SOS between UBIS3000 and CM-100 (Adapter A) in boys, significant differences in SOS were found when CM-100 (Adapter B) and AOS-100 were employed. Similar trends were observed in evaluating differences in SOS among devices in girls. In addition, values of SOS measured by CM-100 (Adapter A) had a greater relationship with that measured by UBIS3000 than those measured by other devices in both boys and girls. In conclusion, proper positioning of heel using an automatic adapter should be recommended for the QUS calcaneal bone mass measurement in growing children.

Disclosures: K. Nakatsuka, None.


Quantitative Ultrasound to Evaluate Bone Health in Term and Preterm Infants.H. McDevitt*, C. Tomlinson*, M. White*, S. F. Ahmed. Bone and Endocrine Research Group, Yorkhill Hospital, Glasgow, United Kingdom.

Quantitative ultrasound (QUS) assessment of speed of sound (SOS)has been used extensively in the adult population, and to a lesser extent in paediatrics, as a marker of bone health. It has been used only in limited settings in the neonates. In this study we assessed the ease of use and reliability of the Sunlight Omnisense 7000P in the neonatal population.

Infants were eligible for the study if they were born between 24 and 42 weeks and parents consented to the study. 75 infants (39 male) with gestational age range 25 -- 41 weeks and birthweights 790 -- 4500g had at least one ultrasound scan performed at the right tibia, 20 of these 75 infants also had measurements performed at the left tibia and both radii. Overall the Omnisense scanner was easy to use at the tibia in the neonate with low intraobserver error. 23 infants had 3 or more measurements at the right tibia with a mean SOS of 3068 m/ s and SD of 45 m/s. Radial measurements were found to be technically much more difficult and time-consuming and the results were found to be similar to tibial measurements. The 52 term infants tibial SOS varied between 2850 - 3300 m/s with a mean of 3082 m/s and SD 99 m/s. There was no correlation between birthweight or sex and SOS within the term group. 23 preterm infants gestation 24--36 weeks had an average SOS 2925 m/s and SD of 132 m/s. This difference was statistically significant p<0.0001 from the term infant group. Within the preterm group there was no relation between sex or birth weight and SOS.

In conclusion, we found that the Omnisense 7000P is able to perform QUS measurements in both term and preterm babies with reproducible results. There is no advantage to measuring multiple sites. SOS measurements are significantly lower in preterm babies. We obtained normative data on tibial SOS for term and preterm babies and this may be of use in future studies. Further studies are needed to establish QUS as a tool for assessing bone mineralisation in term and preterm infants.

Disclosures: H. McDevitt, None.


Bone Ultrasonography and Dual X-Ray Absorptiometry in the Assessment of Corticosteroid Induced Osteoporosis.C. Cepollaro*1, S. Gonnelli1, A. Montagnani*1, C. Caffarelli*1, N. Nikiforakis*2, M. Martino*2, P. Rottoli*2, R. Nuti1. 1Department of Internal Medicine, Endocrine-Metabolic Sciences and Biochemistry, University of Siena, Siena, Italy, 2Division of Pulmunary Diseases, University of Siena, Siena, Italy.

Patients treated with glucocorticoids (GCPs) present an increased risk of osteoporosis and fractures. However, in GCPs the reduction in BMD is smaller than that expected given their increased risk of fracture. A possible explanation is that DXA may fail to detect qualitative changes in bone. Quantitative ultrasonography (QUS) has been proposed as a technique that could theoretically provide information on bone structure. The aim of the present study was to investigate the usefulness of QUS, compared to DXA, for detecting bone status in GCPs.

We studied 179 GCPs (56.6±13.9 yrs, 127 women, 52 men) treated with oral prednisone or equivalent at a dose of ⩾7.5 mg/day for at least 1 year. In all we measured BMD at lumbar spine (BMD-LS) and at femoral subregions (femoral neck: BMD-FN, total hip: BMD-T, trochanter: BMD-TR, intertrochanter: BMD-ITR, Ward's triangle: BMD-W) by DXA (QDR 4500, Hologic), and ultrasound parameters at calcaneus: speed of sound (SOS), broadband ultrasound attenuation (BUA) and Stiffness (S), by Achilles plus (G.E.), and at phalanxes: amplitude dependent speed of sound (AD-SoS), and the parameters of the graphic trace by Bone Profiler (Igea). The data were analysed in all the population and after division in tertiles on the basis of cumulative dose (CD).

The T-scores were -2.2 for BMD-LS, -2.1 for BMD-FN, -1.2 for BMD-TR, -1.3 for BMD-ITR, -1.4 for BMD-T, -2.5 for BMD-W, -1.2 for SOS, -2.5 for BUA, -2.2 for S and -2.5 for AD-SoS. A significant correlation was found between CD and femoral subregions, the greatest value was for BMD-FN (r=-0.33, p<0.001). No significant correlations were found between CD and BMD-LS and QUS parameters. The analysis of variance ANOVA showed that femoral subregions and SOS and S were significantly different on the basis of CD. The discriminatory ability of different parameters to predict a BMD-FN T-score less than -1.5, assessed by ROC curves, resulted better for femoral subregions than for BMD-LS and QUS parameters; among QUS, S and AD-SoS showed the highest values. The analysis of the graphic trace showed a similar pattern with respect to postmenopausal osteoporosis.

We can conclude that: chronic treatment with corticosteroids is able to decrease in DXA and QUS parameters; trabecolar bone appears to be more affected with respect to cortical bone; BMD at proximal femur reflects better the skeletal damage produced by corticosteroids therapy. Among QUS parameters, S and AD-SoS show the greatest reduction in GCPs and the best ability to predict low BMD.

Disclosures: C. Cepollaro, None.


Effect of Soft Tissue on Ultrasonic Guided Wave Measurements in Bone.P. Moilanen*1, P. H. F. Nicholson*1, Q. Wang*1, J. Timonen*2, S. Cheng1. 1Department of Health Sciences, University of Jyvaskyla, Jyvaskyla, Finland, 2Department of Physics, University of Jyvaskyla, Jyvaskyla, Finland.

Current clinical techniques for ultrasonic assessment of long bones such as the tibia use an axial pulse transmission approach and measure the velocity of the first arriving signal. Such velocities are close to the bulk velocity for cortical bone (3600--4200 m/s). Elsewhere we have described a low frequency axial transmission method capable of measuring an additional “second” wave, consistent with a so-called Lamb A0 guided wave propagating in the cortex with a velocity ranging from 1300--2000 m/s. Guided waves are potentially sensitive to the material and geometric properties of the cortical layer, and may therefore offer an enhanced approach to long bone characterization. However, since the velocity of the second wave in bone overlaps that of soft tissue (1450--1600 m/s), it is possible that signals propagating through soft tissue may interfere with, or be mistaken for, a second wave in bone. In order to clarify this question, measurements were made on water or silicon rubber layers on top of solid substrates (metal, acrylic, animal bone), animal bone with and without skin, and the human tibia in vivo. Ultrasound velocity was measured separately in silicon rubber, and animal and human soft tissue. When the signal was mediated through water, no second wave could be observed. On the other hand, in all cases with soft tissue or tissue-mimics overlying the solid substrates, a second wave was observed. Comparing the second wave velocity to the tissue velocity confirmed, in most cases, that the second wave was consistent with a guided wave in the solid layer rather than a wave in the tissue. Tibial measurements were made in a group of 67 mature female subjects. Difficulties in obtaining a reliable determination of second wave velocity were encountered predominantly in overweight women (BMI>30). In normal women (BMI<30), satisfactory measurements were possible in 82% of subjects. In contrast, approximately 33% of the total group were overweight, and of these 41% could be reliably measured. In conclusion, whilst the in vitro results support the concept of exciting and detecting guided waves in bone through soft tissue, clinical measurements of the second wave using our current methodology are problematic in subjects with thick soft tissue. Options for modifying and improving the measurements with respect to clinical soft tissue effects are being investigated, but it should be noted that overweight women are not at high risk of osteoporosis. The idea of using guided waves for bone characterization remains an attractive potential adjunct to existing measurements.

Disclosures: P. Moilanen, None.


Performance Evaluation of the Achilles InSight: Precision, Accuracy, and Comparison to Central DXA.E. Hosszu*1, S. Meszaros*2, V. Ferencz*2, C. Horvath2. 1Department of Pediatrics, Semmelweis University, Budapest, Hungary, 2Department of Internal Medicine, Semmelweis University, Budapest, Hungary.

Heel ultrasound measurements have been shown to predict fractures in several prospective studies. However, not all heel ultrasonometers have equivalent clinical utility. Differences in technology, coupling, imaging capabilities, measurement time, precision, and relationship to central (spine and hip) measurements exist between systems. In this study, we evaluated a new imaging ultrasonometer, the Achilles InSight.

Fifty-two subjects referred for bone density evaluation were evaluated. Subject age ranged from 23 to 76 years, with an average of 55 ± 10 years. Each subject had spine and hip DXA measurements using the Prodigy (GE Medical Systems Lunar) as well as heel ultrasound measurements using Achilles InSight (GE Medical Systems Lunar). The InSight provides an ultrasound image of the heel to aid acquisition. In addition, the InSight is capable of using alcohol spray instead of ultrasonic gel for coupling, greatly decreasing measurement time. Precision (RMS standard deviation for the repeat measurements) was determined from a subset of 25 subjects measured twice each with both alcohol and gel. Sensitivity was assessed by comparing the InSight T-score results to the Prodigy values using a paired t -test. Using ISCD recommendations for peripheral screening, the 90% sensitivity cutpoint was determined for detecting osteoporosis (T-score ⩽ -2.5) at the spine or hip.

Average T-score for the InSight was -1.4 ± 1.1 using alcohol and -1.2 ± 1.3 using gel coupling. This difference, though of little clinical importance, was statistically significant (p < 0.01). There was no significant difference in precision error between the alcohol and gel (1.8% vs. 1.7%, p = 0.44). Fourteen of the 52 subjects were found to have osteoporosis at the spine or hip. From the ultrasound results, the 90% sensitivity cutpoint was found to be at a T-score of -0.6 for the alcohol and at -0.5 for the gel, with specificity of 32% and 35%, respectively.

We conclude that the Achilles InSight is an accurate and precise imaging ultrasonometer, with precision error less than 2% using either alcohol or gel. Using alcohol coupling, the InSight has significantly reduced preparation, measurement, and clean up time compared to gel-based ultrasonometers. The InSight can be used as an effective screening tool using a T-score cutpoint of -0.6, providing better than 90% sensitivity for detecting subjects with osteoporosis at the spine or hip. In situations where central DXA systems are not readily available, the InSight can be used to identify those who should be considered for spine and hip density measurements.

Disclosures: C. Horvath, None.


Performance of a Confocal Acoustic Mapping in Characterization of Trabecular Bone Quality in Human Calcaneus.Y. Xia*1, W. Lin*1, E. Mittra*1, B. Demes*1, B. Gruber2, C. Rubin1, Y. Qin1. 1Biomedical Engineering, SUNY Stony Brook, Stony Brook, NY, USA, 2Medicine, SUNY Stony Brook, Stony Brook, NY, USA.

Quantitative ultrasound (QUS) measurement of the calcaneus has been increasingly used to assess for osteoporosis. It provides both structural and strength information of bone in a manner which is non-invasive, non-destructive, repeatable, safe and relatively accurate. On this basis, a scanning confocal acoustic imaging system at the region of interest (ROI) has been developed. The objective of this work is to evaluate the performance of confocal ultrasound mapping in characterization of human calcaneus bone. 19 human calcaneus, harvested from cadavers with ages 66∼97 have been imaged. Broadband ultrasound attenuation (BUA) and ultrasound velocity (UV) determined from ROI have been performed. To evaluate the capability of high-resolution acoustic images in predicting bone's structural and strength properties, cylindrical samples (10 mm in diameter and 20 mm in medial-lateral length) at ROI are extracted from calcaneus and subjected to micro-CT (∼20 micron resolution) scanning and mechanical strength testing. Strong correlations were found between BUA and bone volume fraction (BV/TV) (R2 = 0.76), and between UV and bone's modulus (R2 = 0.53) (Table 1). The correlations are significantly improved (R2 > 0.64) using combined parameters of BUA and UV in linear regression analysis which ultrasound images determined parameters predict structural, e.g., structure morphological index (SMI) (R2 = 0.86), and strength modulus (R2 = 0.64) (Table 1).

These results suggest that high-resolution acoustic mapping is capable of predicting calcaneus bone quantity and quality non-invasively. Structural property parameters of trabeculi, e.g., BMD and BV/TV, is better represented by BUA, while ultrasonic wave velocity has a strong agreement with bone's strength property, e.g., modulus. This imply that overall bone quality is closely associated with both structure and stiffness of bone, in which combined BUA and UV has demonstrated significant performance in predicting mechanical characteristics of trabecular bone. Ultrasonic imaging has shown the great potential to be used as in vivo diagnostic modality for assessing skeletal disorder, i.e., osteoporosis. 

Table Table 1.. Correlation Coefficients (R⁁2) between US and Bone Quantity and Quality Parameters
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Disclosures: Y. Xia, None.


Comparison of Quantitative Heel Ultrasound with Osteodensitometry in Postmenopausal Women with Colles' Fracture and Evaluation of Secondary Osteoporosis.G. Tautermann*1, P. Langer*1, A. Gohm*2, R. Zinnecker*2, K. Benedetto*2, H. Drexel*1, G. Hoefle1. 1Endocrinology, Academic Teaching Hospital, Feldkirch, Austria, 2Traumatology, Academic Teaching Hospital, Feldkirch, Austria.

The aim of this study was to prospectively compare QUS of the calcaneus to BMD of the spine and femur using DXA in a cohort of postmenopausal women with Colles' fracture. The study population consisted of 64 women aged between 48 and 89 years who were referred to an osteoporosis screening programme after a wrist fracture.

Broadband ultrasound attenuation (BUA) and speed of sound (SOS) measurements as well as T score and stiffness evaluation were performed at the calcaneus using a Hologic Sahara ultrasound device.BMD of the lumbar spine, femoral neck and total hip was measured by DXA.Simultaneously bone turnover was assessed by measurement of serum ß- Cross-Laps. The incidence of osteoporosis using the WHO criteria was 27% at the calcaneus, 28 % at the spine and 7.3 % at the femoral neck. The incidence of osteopenia at the spine and femur as evaluated by DXA was 32 % and 40% respectively. Correlation coefficients for ultrasound and DXA T scores were in the range of 0.4.A similar result was obtained when SOS, BUA and stiffness were regressed against lumbar spine BMD and femoral neck BMD.Secondary osteoporosis accounted for 25 % of cases, the majority of which was attributed to alcoholism (7 of 16 individuals or 44%). An endocrinological cause due to primary/secondary hyperparathyroidism or hyperthyroidism was diagnosed in 25 % (4/16) of these patients.Glucocorticoid use was prevalent in 12.5% or 2/16 patients.Fifty nine percent of the study population had a pathologically elevated ß-CrossLap level. We conclude that QUS relates poorly to axial BMD measurements and that ultrasonographic diagnosis of osteoporosis was not reliable as a screening tool in our study population. The high incidence of secondary osteoporosis warrants full lab screening of all osteoporotic patients with Colles' fracture.

Disclosures: G. Tautermann, None.


Linkage Disequilibrium Analysis of the PTH/PTHrP Receptor Gene across different Human Populations.L. Fröhlich*, R. C. Gensure, H. Jüppner, M. Bastepe. Endocrine Unit, Massachusetts General Hospital/ Harvard Medical School, Boston, MA, USA.

The use of haplotype blocks in the human genome which contain polymorphisms that are in linkage disequilibrium (LD) could reduce the number of markers required for genome-wide scans of genetic linkage. This approach might provide an important tool in genetic studies to dissect complex multi-factorial diseases. To investigate LD within the human PTH/PTHrP receptor gene, we examined allelic association of four different single nucleotide polymorphisms (SNPs) at this locus. In addition to two previously described polymorphisms (Intron S/E1,BsmI (1): Exon M7, BsrDI(4)), two new SNPs were identified (Intron E1/E2: FokI (2), AvaI (3)) and genotyped for individuals of Caucasian, African-American and Asian origin. Expectation maximization algorithm (EH program) was employed to calculate chi-square values based on haplotype frequencies estimated by the allelic association hypothesis. 

Table  . LD analysis of the PTH/PTHrP receptor gene.
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Analysis of the four markers as a group in the overall population showed LD, which also appeared in the subpopulations. However, pairwise analysis of neighboring SNPs indicated no significant association between SNPs 1 and 2 in the overall population. In Caucasians, only SNPs 3 and 4 were in LD, and in African-Americans there was no LD. In Asians, on the other hand, the extent of LD was the same as that of the overall population. Because LD between SNPs 2 and 3 was evident only in the Asian population, we verified this association by direct sequence analysis of genomic DNA from a sample group of double heterozygous individuals. Our results thus demonstrate that LD in the region of the PTH/ PTHrP receptor gene varies between subpopulations, even for genetic markers located only 154 bp apart. Based on these results, it might be necessary to consider population-specific differences in the use of haplotype blocks for genome-wide linkage screens.

Disclosures: L. Fröhlich, None.


Allele Frequencies at 11 BMD-Affecting Loci Examined Are Different in Africans from Caucasians or Asians.G. Gong*, G. Haynatzki. Osteoporosis Research Center, Creighton University, Omaha, NE, USA.

To date, more than 30 candidate genes have been found to be associated with bone mineral density (BMD) in Asian and Caucasian populations. We have recently shown the importance of investigating African populations in the effort to identify susceptibility genes for common diseases such as osteoporosis. We have investigated the allele frequencies of 11 candidate loci in 86 recent immigrants from Africa (Sudanese) shown in the Table (n=86). 

Table  .  
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The numbers in the table are the frequencies of alleles with the restriction site. The allele frequencies of these candidate genes in the Sudanese immigrants are significantly different from those of either Asians or Caucasians or both except osteoprotegerin of which no published data are available for comparison. Notably, the allele frequency of COL1A1 is close to 0, similar to that seen in Asian populations. Also there is no polymorphism in TGFβ 1. Their potential association with BMD is under investigation.

Disclosures: G. Gong, None.


Association of Molecular Variants, Haplotypes, and Linkage Disequilibrium within the Tumor Necrosis Factor Receptor Associated Factor-Interacting Protein (I-TRAF) Gene with Adult Bone Mineral Density.R. Ishida*1, Y. Sudo*1, Y. Ezura1, H. Yoshida*2, T. Hosoi2, S. Inoue3, M. Shiraki4, H. Orimo2, H. Ito*5, M. Emi*1. 1Molecular Biology, Nippon Medical School, Institute of Gerontology, Kawasaki, Japan, 2Tokyo Metropolitan Institute of Gerontology and Geriatrics Hospital, Tokyo, Japan, 3Department of Geriatric Medicine, University of Tokyo, Faculty of Medicine, Tokyo, Japan, 4Research Institute and Practice for Involutional Diseases, Nagano, Japan, 5Orthopaedics, Nippon Medical School, Tokyo, Japan.

Osteoporosis is a multi-factorial common disease that results from interplay of multiple environmental and genetic factors. Signaling from tumor necrosis factor (TNF) receptor family is the most potent bone-resorbing system that regulates bone mineral status of individuals. Here in this study, we investigated TNF receptor associated factor-interacting protein (I-TRAF), an essential effecter of this system in genetic studies of 384 Japanese adult women. In the association study for age and body mass index adjusted radial bone mineral density (BMD), we found a significant correlation of the genotypes of a promoter variation -1542T/G with the adjusted BMD (r = 0.13, p = 0.012). Four promoter variations of I-TRAF gene including -525G/C were in strong linkage disequilibrium (LD) at the level of almost complete LD (D′ = 0.978, r2 = 0.917, Chi2 = 695.2, p = 3.4 × 10−153). When the adjusted BMD were compared among the three-haplotypic categories focusing on two exclusive haplotypes (-1542T/-525C, frequency 0.74 and -1542G/-525G, frequency 0.24), BMD was lowest among -1542G/-525C homozygotes (mean ± SD = 0.382 ± 0.060 g/cm2), highest among -1542T/-525G homozygotes (0.405 ± 0.051 g/cm2), and intermediate among heterozygotes (0.395 ± 0.056 g/cm2) (r = 0.11, p = 0.030). The observed trend supported co-dominant effect of the relevant haplotype of I-TRAF gene in determination of radial BMD. Functional promoter assay using luciferase reporter system, significant difference of two constructs harboring the corresponding nucleotides for the two haplotypes -1542G/-525C and -1542T/-525C was detected in osteoblastic MG63 cells. Sequential motif analysis for the transcription factor binding sites revealed that -1542T/G localized within the consensus GATA-2 binding site, which is abolished in the promoter carrying -1542G allele. These results suggested that variation of I-TRAF might be an important determinant for postmenopausal osteoporosis.

Disclosures: R. Ishida, None.


Polymorphisms in the RANK Gene Are Associated With Risk of Osteoporotic Fractures and Changes in Bone Mass.B. L. Langdahl1, L. B. Husted*1, L. J. Hocking2, L. Stenkjær*1, M. Carstens*1, S. H. Ralston2. 1Endocrinology & Metabolism, Aarhus University Hospital, Aarhus C, Denmark, 2Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.

RANK is the receptor for RANKL and therefore an important factor for osteoclast differentiation and activity. With this key role in the control of resorptive activity the RANK gene has been suggested as a candidate gene for genetic control of bone mass and fracture risk.

We examined the 10 exons with surrounding intron sequences of the RANK gene for polymorphisms in 50 osteoporotic patients. We found 13 polymorphisms, of which some have been reported earlier. Some of the polymorphisms are in complete or very strong linkage disequilibrium. We here report the analyses of the effects of one polymorphism in intron 5: TIVS5-39-A, four in intron 6: GIVS6+79-A and AIVS6+166- G, AIVS6-258-C and AIVS6-151-G and one polymorphism in exon 9: G932-A on prevalence of osteoporotic fractures, bone mass and bone turnover in 303 osteoporotic patients and 433 normal controls.

The GIVS6+79-A polymorphism was significantly associated with risk of osteoporotic fracture. The GG genotype was more common among fracture patients than in normal controls, 33% vs. 25%, p<0.05. Lumbar spine BMD (LS-BMD) was 0.883±0.169 g/cm2 in individuals with the GG genotype compared with 0.902±0.177 g/cm2 and 0.919±0.175 g/cm2 in individuals with the GA or AA genotypes, respectively, p<0.05. Regression analyses revealed that this polymorphism is significant associated with BMD at the lumbar spine, femoral neck and total hip. Biochemical markers of bone resorption were unaffected by this polymorphism, however, S-BAP was 69±26 U/l in individuals with the GG genotype compared with 60±28 U/l in individuals with the AA genotype, p<0.05. This polymorphism is in very strong LD (D′=1.0, p<10−6) with C575-T in exon 6, which causes a change from Alanine to Valine in position 192.

The A allele of the AIVS6--151-G polymorphism in intron 6, present in 40% of normal controls, is associated with increased BMD. LS-BMD was 0.920±0.180 g/cm2 in individuals carrying the A allele compared with 0.889±0.170 g/cm2 in individuals with the GG genotype, p<0.05. Although the A allele was less frequent in osteoporotic patients, the difference did not reach significance.

None of the other polymorphisms were significantly associated with fracture risk, BMD or biochemical markers of bone turnover.

In conclusion: We have identified 13 polymorphisms in the RANK gene and we have found that one of the polymorphism in intron 6 is associated with increased risk of osteoporotic fractures and changes in BMD and that BMD is influenced by polymorphisms in intron 6.

Disclosures: B.L. Langdahl, None.


VDR Polymorphisms and Musculo-Skeletal Parameters in Lebanese Adolescents.J. Maalouf*1, L. Zahed*2, R. Vieth3, M. Nabulsi*4, M. Choucair*5, G. El-Hajj Fuleihan1. 1Calcium Metabolism and Osteoporosis Program, American University of Beirut Medical Center, Beirut, Lebanon, 2Pathology and Laboratory Medicine, American University of Beirut Medical Center, Beirut, Lebanon, 3Mt. Sinai Hospital, University of Toronto, Toronto, ON, Canada, 4Department of Pediatrics, American University of Beirut Medical Center, Beirut, Lebanon, 5Department of Endocrinology, American University of Beirut Medical Center, Beirut, Lebanon.

Peak bone mass is a determinant of fracture risk at older ages. Environmental and genetic factors have been shown to influence it significantly. The list of candidate genes is long, however VDR polymorphisms are particularly relevant in our region in view of the high prevalence of vitamin D deficiency. It remains controversial whether VDR gene polymorphisms are associated with bone mineral density (BMD). Genetic associations may be stronger in younger age groups due to the lesser confounding effect of environmental factors. There are only a few studies investigating the association of VDR polymorphisms and BMD in children or adolescents.

We studied the association between VDR polymorphisms, bone mass and muscle strength using the baseline BMD data from a vitamin D-supplementation trial, enrolling 364 healthy adolescents, 184 boys and 180 girls, ages 10--17years, in a vitamin D supplementation trial. Muscle strength using a squeeze grip ball, and BMD at the lumbar spine, hip, forearm and total body by DEXA using a Hologic 4500A device were measured in all subjects. VDR polymorphism genotypes for two sites were determined by PCR and enzymatic digestion using two enzymes, TaqI and ApaI.

The distribution of VDR alleles for TaqI polymorphism were 51% Tt (+/−), 16% tt (+/+) and 33% TT (-/-) compared to 46% (+/−), 16% (+/+) and 38% (-/-) for ApaI. The Table below shows the results as mean (SD). There was a very small non-significant increase in BMD at all skeletal sites going from the TT, to the Tt to the tt genotype. The pattern was less consistent among the genotypes assessed by ApaI (data not shown). No relationship was found between grip (muscle) strength and VDR genotypes. Subgroup analyses by gender showed a similar pattern. 

Table  . VDR genotype (TaqI), BMD and Muscle Grip.
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There was no significant relationship between VDR genotypes and BMD or muscle strength in healthy adolescents. Whether VDR polymorphism affects BMD response to vitamin D supplementation is currently being evaluated.

Disclosures: G. El-Hajj Fuleihan, None.


Association of Estrogen and Vitamin D Receptor Gene in Elderly Osteoporotic Korean Women.D. J. Lee1, I. K. Han2. 1Rheumatology, Samsung Cheil Hospital, Seoul, Republic of Korea, 2Endocrinology, Samsung Cheil Hospital, Seoul, Republic of Korea.

One hundred women with normal spine BMD and one hundred women with T score below -2.5 were randomly enrolled to examined vitamin D receptor gene and estrogen receptor gene polymorphism.

One hundred and ninety five women, 93 normal BMD and 102 osteoporosis, completed study questionnaire and had drawn venous blood for their vitamin D(Bsm1) and estrogen receptor gene(PvuII) evaluation. Gene results were grouped according to the combinations of two genes.

The distribution of ER PvuII and VDR Bsm1 restriction fragment length polymorphisms was as follows: pp 23.7%,Pp 60.2%,PP 16.1% in normal BMD group, pp 45.1%,Pp 44.1%,PP 10.8%in osteoporosis group, bb 86.0%,Bb14.0%,BB 0% in normal BMD group, bb 87.3%,Bb 11.8%, BB 1.0% in osteoporosis group respectively. Using two gene polymorphisms participants were subgrouped as, pp-BB, pp-Bb, pp-bb, Pp-BB, Pp-Bb, Pp-bb, PP-BB, PP-Bb, PP-bb and the distribution was as follows: 0%, 2.1%, 32.8%, 0%, 7.2%, 44.6%, 0.5%, 3.6%, 9.2% respectively.

The prevalence of osteoporosis patients in the gene subgroups were 0%, 75%, 67%, 0%, 43%, 46%, 100%, 33%, 35% respectively. Gene polymorphism was independently associated with bone mineral density and thus the presence of any high risk gene polymorphism would result in higher prevalence of osteoporosis patients.

These results indicate that ER and VDR gene polymorphism influence development of osteoporosis in the elderly Korean women.

Disclosures: D.J. Lee, None.


Calcium-sensing Receptor Gene Polymorphism is not Associated with Bone Mineral Density in Italian Postmenopausal Women.F. Cetani1, E. Pardi*1, S. Borsari*1, E. Vignali1, G. Dipollina*1, A. Picone*1, V. Braga*2, S. Adami2, T. Giacomelli*1, A. Pinchera*1, C. Marcocci1. 1Endocrinology, University of Pisa, Pisa, Italy, 2Reumathology, University of Verona, Verona, Italy.

Calcium-sensing receptor (CaR) is a candidate gene for osteoporosis susceptibility. Several CaR polymorphisms have been identified and an association between the A986S genotype and serum calcium levels has been found in Canadian postmenopausal women. We investigated whether the presence of 986S allele was associated to BMD and osteoporotic fractures. The study group consisted of 164 Italian postmenopausal women without fragility fracture (Fx+) and 55 women with fracture (Fx). A fragment of exon 7 of CaR gene containing three polymorphisms (A986S, R990G and Q1011E) was amplified by PCR and sequenced. Antropometric characteristic and bone mineral density (BMD) were evaluated. The A986S polymorphism was the most commonly observed (27.9%), whereas the other two CaR polymorphisms, R990G and Q1011E occurred in a minority of cases (8.8% and 5.5%, respectively). There was no significant difference in the frequency distribution of any CaR allele between Fx and Fx+ patients. BMI was found to predict BMD at lumbar spine and femoral neck. The A986S polymorphism and YSM were not independent predictors of BMD at any sites. As far as fracture occurrence, there was no statistically significant difference in the prevalence of fractures between women carrying or not the 986S allele.

In conclusion, our data do not support a role of A986S CaR polymorphism on BMD and on the prevalence of fragility fractures in Italian postmenopausal women.

Disclosures: F. Cetani, None.


COL1A1 Sp1 Promoter Polymorphism Influences Serum N-terminal COL1A1 Propeptide (P1NP) Levels.L. J. Miles*1, J. Colley*2, A. Blumsohn3, R. Eastell3, E. L. Duncan4, M. Olavesen*2, J. A. H. Wass4, M. A. Brown*1. 1Spondyloarthritis and Bone Disease Research Group, Oxford University Institute of Musculoskeletal Sciences, Oxford, United Kingdom, 2Oxagen Ltd, Abingdon, United Kingdom, 3Bone Metabolism Group, University of Sheffield, Sheffield, United Kingdom, 4Department of Endocrinology and Metabolism, Nuffield Orthopaedic Centre, Oxford, United Kingdom.

Association between the Sp1 COL1A1 promoter polymorphism, vertebral bone mineral density (BMD) and hip fracture risk has been reported in many studies. The mechanism of this association is uncertain, but it is hypothesised that the polymorphism influences COL1A1 production. To test this hypothesis, we investigated association of the Sp1 polymorphism with a marker of COL1A1 production, serum P1NP.

Serum P1NP was measured by enzyme-linked immunoassay (Roche Elecsys) in 427 individuals belonging to 115 British Caucasian families with extreme low BMD (T-score <−2.5, Z-score <−2.0 at either the lumbar spine or femoral neck (FN)). Secondary causes of osteoporosis were excluded. BMD values were available from a further 168 individuals from these families. Genotyping of the COL1A1 Sp1 promoter polymorphism was performed using previously described methods1. Association was tested between the polymorphism and raw BMD and serum P1NP using the program QTDT2. Age, gender, height and weight were used as covariates.

Whilst no overall association was noted between COL1A1 and FN BMD and serum P1NP, association was seen when the parent-of-origin of the COL1A1 allele was taken into account. Considering P1NP levels, when parent-of-origin effects were considered, significant association was seen by total association (p=0.05) and by within-family methods (p=0.008). There was borderline within-family association of FN BMD with the COL1A1 Sp1 polymorphism when parent-of-origin effects were considered (p=0.06). A significant parent-of-origin effect was present (p=0.02), and significant association was seen in the maternally transmitted COL1A1 Sp1 alleles (p=0.01), but not with the paternal alleles. Significant association was noted between height and the COL1A1 Sp1 polymorphism (p=0.04), independent of age and gender.

These studies suggest that the COL1A1 Sp1 polymorphism influences BMD and fracture risk by effects on type 1 collagen production. A parent-of-origin effect was noted, consistent with the pattern of segregation of P1NP levels in these families, (Miles, ASBMR Conference Abstracts 2003). This suggestive finding may indicate the presence of imprinting at this locus.

1 Grant et al (1996) Nat Genet 14, 203--5; 2Abecasis et al (2000) Am J Hum Gen 66, 279--92.

Reagents for assay of PINP kindly supplied by Roche Diagnostics, Germany.

Disclosures: L.J. Miles, None.


Relation of Polymorphism in the CYP19 Gene to the Sex Hormone Level in Pre- and Early Pubertal Finnish Girls.A. Mahonen1, M. Suuriniemi2, A. Eriksson3, Q. Wang*2, A. Lyytikäinen2, V. Kovanen*2, M. Alen*4, C. Ohlsson3, H. Kröger1, S. Cheng2. 1University of Kuopio, Kuopio, Finland, 2University of Jyväskylä, Jyväskylä, Finland, 3Sahlgrenska University Hospital, Gothenborg, Sweden, 4Peurunka-Medical Rehabilitation Center, Jyväskylä, Finland.

Estrogens are the main sex steroids involved in skeletal maturation. The conversion of testosterone to estrogen by adipose and muscle tissue -catalyzed by aromatase (CYP19) is the major source of ovary-independent estrogen production. The purpose of this study was to evaluate the associations between a biallelic single nucleotide polymorphism in exon 3 of CYP19 gene and sex hormone levels and different bone properties in pre- and early pubertal Finnish girls. The subjects were healthy 10--12 year-old girls (n=214) with Tanner stage I-III, who enrolled in an intervention study (the CALEX-study). Genotyping of the CYP19 locus at the G/A polymorphic site in exon 3 was performed. Serum 17β-estradiol (E2), testosterone (T), and sex hormone binding globulin (SHBG) were determined using a time-resolved fluoroimmunoassay (Delfia, Wallac Oy, Turku). The SHBG was used to calculate the free E2 (fE) and T (fT). Bone properties were measured using different bone assessment modalities (DXA, Prodigy, Lunar; pQCT, XCT 2000, Stratec; QUS-2, Metra Biosystems; Omnisense, Sunlight). Our results showed that girls with the AA genotype had significantly higher T and fT level than girls with the GG genotype (p=0.017 and p=0.027, respectively). They also had higher T / E2 and fT / fE ratio than the GG girls (p=0.023). Girls with different genotype did not differ in E2 level, nor in Tanner stage, height, weight, or body mass index. The polymorphism was not significantly associated with bone mineral content, areal bone mineral density (BMD), volumetric BMD, cross-sectional area, or speed of sound at any measured site. However, girls with the AA genotype had significantly lower broadband ultrasound attenuation of the calcaneus than girls with the GG genotype (p=0.011). Our study suggests that the polymorphism in the CYP19 gene may affect the activity or the expression level of the aromatase enzyme.

Disclosures: A. Mahonen, None.


Lack of Association between Vitamin D Receptor or Estrogen Receptor Genotypes and Bone Mineral Content or Bone Mineral Density in Young Danish Females.S. Cusack*1, C. Mølgaard*2, K. F. Michaelsen*2, K. D. Cashman1. 1Food and Nutritional Sciences, University College, Cork, Ireland, 2Research Department of Human Nutrition, Centre for Advanced Food Studies, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark.

Peak bone mass (PBM) in early adulthood is considered an important determinant of osteoporosis risk later in life. Acquisition of bone mass during childhood depends on various factors, including genetic factors. Attempts to define genetic factors that influence bone mass have largely focused on adult populations. In contrast little is known about the genetic factors that influence bone density in children and adolescents. Therefore the aim of the present study was to investigate the relationship between polymorphisms in the vitamin D receptor (VDR) and estrogen receptor (ER) genes and bone mineral density (BMD) and bone mineral content (BMC) in a cohort of 222 randomly selected Danish girls, aged 11 - 13 years.

BMD, BMC, and total bone area of the total body and spine, fat content and lean mass were measured by dual energy x-ray absorptiometry (DEXA). The polymorphisms were examined by polymerase chain reaction-restriction fragment length polymorphism analysis (using Fok1 and Taq1, and PvuII and XbaI restriction enzymes) of genomic DNA isolated from peripheral blood leukocytes.

Differences in covariates between genotypes were tested by analysis of variance (ANOVA). Statistically significant different covariates were included in subsequent analyses, testing differences in BMD and BMC between genotypes. Size-adjusted and unadjusted BMD and BMC values were further analysed using 2-factor ANOVA to examine the effects of Fok1 and Taq1 genotype interactions.

The prevalence of FokI and Taq1 VDR gene and PvuII and XbaI ER gene polymorphisms in this cohort were 11.7% ff, 48.7% Ff, and 39.6% FF; 16.7% tt, 50.9% Tt, and 32.4% TT; 24.3% pp, 49.5% Pp, and 26.2% PP; and 29.8% xx, 48% Xx, and 22.2% XX. No significant association (P>0.05) was seen between BMD or BMC (adjusted and unadjusted) and any of the four polymorphic sites examined. Similarly, no significant interaction (P>0.05) between the combined VDR genotypes and BMC or BMD levels were observed. A previous report has indicated that differences of the order of 8.2% and 4.8% in total body BMD exist between FF and ff, and FF and Ff VDR genotype groups in children (Ames et al. 1999). The sample number in this study was sufficient to detect 6.0% and 3.3% differences in total body BMD between FF and ff, and FF and Ff VDR genotypes.

In conclusion, the findings of this study suggest a lack of relationship between polymorphisms of the VDR or ER genes and BMD or BMC levels in a healthy young, Danish girls.

Disclosures: S. Cusack, None.


VDR Gene Polymorphism and Bone Ultrasound Parameters in Newborns.D. Merlotti1, L. Gennari1, S. Gonnelli1, A. Montagnani*1, V. De Paola*1, A. Calabrò*1, S. Perrone*2, G. Martini1, G. Bonocore*2, R. Nuti1. 1Internal Medicine, Endocrine-Metabolic Sciences and Biochemistry, University of Siena, Siena, Italy, 2Pediatrics, Gynecology and Reproductive Medicine, University of Siena, Siena, Italy.

Bone metabolism is strongly influenced by heredity and environmental factors. Although some studies have reported a relationship between several candidate polymorphic genes and bone mineral density in adults little is known concerning the genetic factors influencing bone mass in children. Moreover the role of genetic polymorphism during intrauterine or early postnatal life is actually unknown. The aim of this study was to evaluate the role of the translation initiation site polymorphism in the vitamin D receptor gene (VDR) in newborns. The VDR genotypes, as detected by Fok I restriction endonuclease, were examined in 83 consecutive healthy full term newborns (43 males and 40 females; gestational age 39.5±1.5 weeks) and in their respective mothers (age range 24--38 years). Quantitative ultrasound (QUS) parameters at distal diaphysis of humerus were assessed within three days of birth by using a Bone Profiler (IGEA, Italy), after an appropriate modification of the calliper and software. Newborn's anthropometric data, such as weight, length, head circumference and APGAR score at 3rd and 5th minute after birth were also collected. Phalangeal QUS measurements were performed in all mothers. All the QUS parameters were slightly but not significantly higher in male than in female newborns. Moreover QUS parameters and anthropometric measurements were weakly correlated with gestational age and maternal calcium intake. A trend approaching statistical significance was observed between Fok I genotype in newborns and QUS parameters or anthropometric measurements at birth. By contrast, maternal Fok I genotype was not significantly correlated with maternal QUS at the phalanxes nor with newborns QUS parameters.

In conclusion, results from this preliminary study do not suggest a major contribution of Fok I polymorphism at the VDR gene and ultrasound bone parameters at birth.

Disclosures: D. Merlotti, None.


Lack of Association between the Leptin Polymorphism, Bone Mass or Body Composition in Young and Elderly Women.K. Akesson1, P. Gerdhem*1, M. Callreus*1, V. Lyssenko*2, O. Johnell1, K. Obrant1, L. Groop*2. 1Dept of Orthopedics, Malmö, Sweden, 2Dept of Endocrinology, Malmö, Sweden.

Background: Bone mineral density BMD is significantly related to total fat mass and body weight. Genes associated with obesity may therefore be explored for potential effects on bone mass. Recent evidence suggests high levels of gene expression of the leptin receptor (LEPR) in bone and that leptin directly stimulates osteoblasts. The LERP Gln223Arg polymorphism has been associated to BMD in men; in women, however, the association is unclear.

Aim: The aim of the present study was to investigate whether a LEPR 3′UTR insertion (I)/ deletion(D) polymorphism, which we have shown to be in linkage disequilibrium with Gln223Arg, was associated with bone mass in young women at peak bone mass and elderly women at high risk of fracture.

Material and methods: In this population-based study, 470 young women (age 25±0.1 yrs, BMI 23.0±3.7 kg/cm2) and 1044 elderly women, (age 75±0.1 yrs, BMI 26.2±4.2 kg/cm2) were recruited. The primary phenotype was BMD assessed by DXA. The 3′UTR polymorphism was genotyped by PCR and gel electrophoresis. In addition, body composition and calcaneus ultrasound were measured. In the elderly fracture data and bone markers (osteocalcin, CTX) were available.

Results: The LEPR genotype frequencies were I/I 3.0 and 2.6%, I/D 26.4 and 28%, D/D 69.4 and 70.6% in young and elderly, respectively. BMD measured at any site by DXA or calcaneus ultrasound, did not differ significantly between the different genotype carriers, neither between the young nor between the elderly. 

Table  . Bone Mineral Density according to LEPR Genotype in 25- and 75-year old Women
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The presence of the I-allele was not significantly associated with BMD (TB p=0.67 vs p=0.10, FN p=0.64 vs p=0.08, LS p=0.83 vs p=0.08, young vs elderly). We did not find any significant association between the LEPR polymorphism and variations in lean or fat mass, body weight or height, or in the elderly to bone turnover. In the elderly 24% of the I/ I, 34% I/D and 36% D/D genotype carriers had suffered from an osteoporotic fracture, most commonly of the wrist, between age 50 and 78(N.S).

Conclusions: Our results indicate that the LEPR 3′UTR insertion/deletion polymorphism is not significantly associated to variation in peak bone mass or bone mass and fracture at old age in women. However, there is a trend for an increasing effect of the I-allele with age.

Disclosures: K. Akesson, None.


Stimulation of Na-Dependent Phosphate Transport by Platelet-derived Growth Factor in Rat Aortic Smooth Muscle Cells.A. Kakita1, A. Suzuki2, Y. Ono*2, K. Nishiwaki*3, M. Kotake*2, Y. Miura*3, Y. Oiso*3, M. Itoh*2. 1Department of Internal Medicine, Hekinan Municipal Hospital, Hekinan, Aichi, Japan, 2Department of Internal Medicine, Fujita Health University, Toyoake, Aichi, Japan, 3Department of Metabolic Diseases, Nagoya University, Nagoya, Aichi, Japan.

We investigated the effect of platelet-derived growth factor B homodimer (PDGF-BB) on inorganic phosphate (Pi) transport activity, which has been reported to be involved in the mechanism of atherosclerosis, in A-10 rat aortic vascular smooth muscle cells (VSMCs). PDGF-BB time- and dose-dependently stimulated Pi transport in A-10 cells. Using Northern blotting analysis, the PDGF-BB-enhanced Pi transporter (PiT) in A-10 cells was identified as Pit-1 (Glvr-1), a member of the type III Na-dependent PiT. An inhibitor of PDGF receptor tyrosine kinase suppressed PDGF-BB-induced Pi transport. Both a PKC inhibitor calphostin C and PKC down regulation suppressed the stimulatory effect of PDGF-BB on Pi transport. On the other hand, inhibition of mitogen-activated protein (MAP) kinases by selective inhibitors did not affect Pi transport. Ly294002, a phosphati-dylinositol (PI) 3-kinase inhibitor, partially attenuated PDGF-BB-induced Pi transport. A selective inhibitor of S6 kinase, rapamycin, reduced this effect of PDGF-BB, while Akt kinase inhibitor did not. In summary, these results indicated that PDGF-BB is a potent and selective stimulator of Pi transport in VSMCs. The mechanism responsible for this effect is not mediated by MAP kinase, but involves activation of PKC, PI 3-kinase and S6 kinase.

Disclosures: A. Kakita, None.


Osteoformin Stimulates Osteoblast Differentiation.L. X. Bi, E. Mainous, H. M. Bahrani*, W. L. Buford*. Departments of Surgery and Orthopaedics, University of Texas Medical Branch, Galveston, TX, USA.

Our previous studies have shown that negatively charged resins increase bone formation and accelerate bone defect healing in vivo. In order to investigate potential effects of osteoformin, negatively charged peptide, on human preosteoblast, we examined expression of bone morphogenetic protein-7 [BMP-7] and type I collagen, alkaline phosphatase activity (ALP) and mineralization after treatment of cells with osteoformin. Human preosteo-blast cells were cultured in a-minimum essential medium [a-MEM] and 10% fetal bovine serum with or without osteoformin (5ug/ml) for 7, 10, 14 days, respectively. To determine mineralization, the cells were cultured in mineralizing-growth medium. The levels of ALP were assayed using a commercial kit (Sigma Chemical Co., St. Luis, MO). Expression of BMP-7 and type I collagen (polyclonal antibody, Santa Cruz Biotechnology, Inc. CA) was examined using immunoquantitative assay. The mineralization was assessed by Von Kossa technique. ALP activities were significantly elevated (69--109%, P<0.001) in osteoformin treated group, compared to control group. BMP-7 expression was increased (23% at day 10 and 69% at day 14, respectively, P<0.001) after osteofornin treatment compared to control. Expression of type I collagen was increased (45% at day 10 and 52% at day 14, respectively, P<0.001). There was significant increase in the levels of mineralization (2--3 fold, P<0.001) within 14 days of culture. We conclude that osteoformin significantly stimulates osteoblast differentiation in vitro. It might be an important regulator of bone formation by accelerating fracture and bone defect healing, and controlling bone diseases.

Disclosures: E. Mainous, None.


Induced Membranes Secrete Growth Factors Including Vascular and Osteoinductive Factors and Could Stimulate Bone Regeneration.P. Pelissier*1, R. Bareille*2, A. Masquelet*3, S. Mathoulin Pelissier*4, J. Amedee2. 1Service de Chirurgie Plastique, Hǒpital Pellegrin-Tondu, Bordeaux, France, 2INSERM U 577, Bordeaux, France, 3Service de Chirurgie Orthopédique, Hǒpital Avicenne, Bobigny 93009, France, 4Service de Biostatistiques Institut Bergonié, Bordeaux, France.

One of the used procedures for the treatment of extensive diaphyseal bone defects are the vascularized bone free transfer. A proposed procedure combining induced membranes and cancellous autografts have been developed. The first stage consists in the insertion into the defect of a cement spacer which is responsible for the formation of a pseudosynovial membrane. The second stage is the reconstruction of the defect, by an autologous cancellous bone graft. The aim of this study was to evaluate the histological characteristics of these membranes. This experiment was performed in New Zealand rabbit, four animals were sacrified after 2, 4, 6 and 8 weeks and the membrane surrounding the implants was removed. Four samples of subcutaneous tissue were taken as controls. The first part of the sample was fixed in formalin solution for histological study. The second part was frozen for protein extraction. Histological and immunochemistry studies were carried out after 2, 4, 6 and 8 weeks. Measurement of VEGF, TGFβ1 concentrations was performed using human VEGF EIA kit (Chemicon, USA), human TGFβ1 (Bender Medsystems, Austria), according to the manufacturer's protocol. The presence and the amount of BMP-2 into the induced membranes extracts were carried out with an ELISA technique using a monoclonal anti-human BMP-2 antibody (R&D Systems, USA). Rh- BMP-2 used for control (Genetics Institute Cambridge, USA). Finally, influence on human bone marrow stromal cells (HBMSC) differentiation (alkaline phosphatase measurement) and proliferation was studied. Results demonstrate that both VEGF and TGFβ1 identified by immunocytochemistry and quantified by EIA could be in part responsible of the HBMSC cell growth observed in presence of these membranes obtained after 2 or 4 weeks. More interestingly, the induced membrane obtained after 4 weeks exhibits osteoinductive properties due to the maximum secretion of BMP2. In conclusion, these membranes play a role of an in situ growth and osteoinductive factors-delivery system. Moreover, cancellous autograft usually included in this membrane could potentially be substituted by a biomaterial loaded with bone marrow stromal cells and then allowing both vascularization of the implant and activation of stromal cells in bone regeneration.

Disclosures: J. Amedee, None.


PKR is Regulated by Estrogen in Rat Bone.M. Zhang*, R. T. Turner, A. Maran. Orthopedic Research, Mayo Clinic, Rochester, MN, USA.

PKR is an interferon-inducible, double-stranded RNA-dependent protein kinase that mediates the anti-viral and anti-growth activities of interferon. In addition to its originally known function as a regulator of protein synthesis, PKR has recently been implicated in the regulation of apoptosis, differentiation, growth control, cell cycle progression, stress and transformation. Estrogen is essential for normal growth and remodeling of bone. Although the importance of estrogen in preventing bone loss in adults has been widely studied, the regulatory proteins associated with estrogen actions are incompletely known. The involvement of PKR in estrogen-mediated actions or in the skeletal system has not been previously investigated. In this report, we have investigated the effects of the gonadal hormone 17β-estradiol on PKR gene expression. Western blot analysis of the femoral metaphysis show that proximal tibiae metaphysis from three-month-old ovariectomized (OVX) rats have elevated levels (2.5-fold higher) of PKR protein compared to the intact animals. 17β-Estradiol decreased PKR protein expression in OVX rats at 8 hrs (1.5-fold), 16 hrs (1.2fold), 24 hrs (3-fold), and 32 hrs (3-fold). These studies demonstrate that PKR levels in bone are increased by OVX and are rapidly decreased following estrogen treatment. These findings suggest that PKR plays a role in mediating the actions of estrogen on bone metabolism.

Disclosures: M. Zhang, None.


Monocyte Chemoattractant Protein (MCP)1 Influences Osteoblastic Differentiation.E. A. Kaiser1, C. Gentzsch*1, U. Larsen*1, J. Plutat*2, R. Sellkau*2, J. Wodtke*2, G. Delling*1. 1Bone Pathology, Center of Biomechanics, Hamburg, Germany, 2Endoklinik, Hamburg, Germany.

Aseptic loosening is a major long term complication arising in 10% of all total hip replacements. The occurring periprosthetic osteolysis is the consequence of net bone loss, which result from an imbalance between bone resorption and new bone formation. Inflammatory mediators produced in the developing synovial membrane-like interfacial tissue (SMILT) play a key role in the osteolysis and implant loosening. The release of the CC chemokine monocyte chemoattractant protein (MCP1) from fibroblasts and osteoblast within the SMILT exposed to titanium and PMMA particles has been described. MCP1 has been demonstrated to be involved in monocytes/macrophage chemotaxis, but as its receptors CCR2 and CCR4 have also been observed on other cell types we were interested in MCP-1 effects on osteoblasts as they can limit osteolysis. Thus in the present study the cell line SAOS2 was differentiated in vitro with dexamethasone, b-glycerol phosphate and ascorbic acid while simultaneously treated with different concentrations of MCP-1 peptide (10, 50, 100 ng/ml). At 4, 7, 11, 14, 18, 21 days of culture proliferation and mineralization of the cell lines were assessed. In SAOS2 cells the differentiation-induced reduction of proliferation was more pronounced and could be perceived at an earlier time point (day 4) as compared to controls (day 7). Further, mineralization also initiated at an earlier time point, on day 14 as compared to day 18. The maximal MCP1 effect, inhibiting proliferation and promoting mineralization, could be observed using 10 ng/ml. Immuncytochemistry revealed a more pronounced CCR2 expression in dexamethasone stimulated cells. Immunohistochemical evaluation of SMILT sections exhibited MCP1 expression in areas where particles accumulated whereas CCR2 expression was distributed in cells surrounding these areas. These results suggest that in addition to the recruitment of monocytes/macrophages, MCP1 could force osteoblast into differentiation, thus potentially exhausting the osteoblast progenitor pool and thereby creating an imbalance between bone resorption and bone formation.

Disclosures: E.A. Kaiser, None.


Preptin, Another Peptide Product of the Pancreatic Beta Cell, is Osteogenic In Vitro and In Vivo.J. Cornish, K. E. Callon*, U. Bava*, M. Watson*, X. Xu*, J. Lin*, V. A. Chan*, A. B. Grey, G. J. S. Cooper*, I. R. Reid. Medicine, University of Auckland, Auckland, New Zealand.

Several hormones that regulate nutritional status also impact on bone metabolism. Prep-tin, a 34-amino acid peptide hormone that increases glucose-mediated insulin secretion, has been recently isolated from the same secretory vesicles that contain insulin and amylin from the pancreatic β-cells. Preptin corresponds to Asp69 - Leu102 of proIGF-II.

We have recently assessed preptin's activities on bone in a number of in vitro models. Prep-tin is anabolic to osteoblasts but, unlike amylin, does not regulate osteoclast activity. Prep-tin dose-dependently stimulated the proliferation of osteoblasts at periphysiological concentrations (>10−11M). In addition, thymidine incorporation was stimulated in murine neonatal calvarial organ culture, likely reflecting the proliferation of cells from the osteo-blast lineage. The mitogenic effect of preptin on osteoblasts depends upon signaling via p42/44 MAP kinases. Preptin also has anti-apoptotic effects in osteoblasts in vitro : at 10−8M there is a 22% reduction in the number of TUNEL-positive cells. Preptin not only stimulates osteoblast proliferation but also osteoblast differentiation at 10−8M, significantly increasing the number of mineralized bone nodules in long-term osteoblast cultures. These effects are also seen in vivo, when preptin is injected locally over the hemicalvariae of sexually mature male mice. After five daily subcutaneous injections of 16.5 micrograms of preptin, there was a significant increase in bone area, and mineralizing surface.

In conclusion, preptin, a peptide contained within proIGF-II, is anabolic to bone in in vitro and in vivo models. Since it is secreted from the pancreatic β-cell, it may act in concert with the other β-cell hormones, insulin and amylin, to stimulate bone formation in hyperin-sulinemic states, such as obesity. Preptin may also contribute to the osteosclerotic pheno-type observed in patients with chronic hepatitis C infection who have increased circulating levels of proIGF-II, which contains the preptin peptide. Thus, the anabolic effects seen in rodent models may be sufficient to influence bone density in humans in some situations.

Disclosures: J. Cornish, None.


VEGF Expression Is Altered in the Epiphyseal Cartilage Following Femoral Head Ischemic Injury.H. Bian*1, T. Randall*1, A. Garces*1, L. C. Gerstenfeld2, T. A. Einhorn2, H. K. W. Kim1. 1Shriners Hospital for Children, Tampa, FL, USA, 2School of Medicine, Boston University, Boston, MA, USA.

Ischemic necrosis of the femoral head is a serious condition that can arise following certain injuries or treatments of the pediatric and adult hip. Vascular Endothelial growth factor (VEGF) is an essential mediator of angiogenesis, however, its role in revascularization and repair of the infarcted femoral head has not been clearly defined. The purpose of this investigation was to determine whether a quantitative change in the expression level of VEGF occurs following ischemic necrosis and to correlate these changes with revascularization of the infarcted head. A piglet model of ischemic necrosis was used. Ischemic necrosis was induced surgically by placing a ligature tightly around the femoral neck in 30 piglets. At 2 days to 8 weeks following the induction of ischemia, epiphyseal cartilage was obtained from one half of the femoral head and total RNAs and proteins were isolated and used for western blot analysis and ribonuclease protection assay (RPA). The remaining half of the head was used for histology and immunohistochemistry. Western blot consistently showed increased VEGF expression on the infarcted side compared to the non-operated side at 4 and 8 weeks when revascularization and repair were observed. RPA demonstrated higher level of VEGF mRNA expression as early as 2 days following the induction of ischemia. Immunohistochemical assessment showed high VEGF immunoreactivity (IR) in the hypertrophic zone of the epiphyseal cartilage in the non-operated side. In the infarcted side, however, there was an absence of IR in the hypertrophic zone due to cartilage necrosis. Instead, an increased IR was seen in the proliferative zone of the cartilage. At 8 weeks, there was a persistence of increased IR in the proliferative zone with fibrovascular tissue invasion and resorption of the necrotic cartilage. In the areas where the necrotic cartilage was removed, restoration of endochondral ossification was observed. In conclusion, the level and pattern of VEGF expression was altered following ischemic necrosis. The findings suggest that increased VEGF expression in the proliferative zone induces fibrovascular tissue invasion of the necrotic cartilage that enables restoration of endochondral ossification. By understanding the key factors responsible for revascularization and repair following ischemic necrosis, new treatment strategies to stimulate the repair process can be developed.

Disclosures: H. Bian, None.


RANKL AutoVacTM Vaccine: A New Immunotherapeutic Approach for the Treatment of Bone Resorptive Disorders.A. C. Porchia*1, V. Nardi-Dei*1, M. Rask*1, T. Bratt*1, A. Neisig*2, J. Backlund*2, M. Ezban*1, M. Hertz2. 1Protein Chemistry, Pharmexa, Horsholm, Denmark, 2Immunology, Pharmexa, Horsholm, Denmark.

The TNF family member Receptor activator of NF-κB Ligand (RANKL) is a type II transmembrane protein found on osteoblasts which functions as a major determinant of osteoclast differentiation and activation. RANKL mediates bone homeostasis through binding to the cognate ligand on osteoclasts, RANK, and a soluble decoy receptor, osteoprotegerin (OPG). An imbalance of this cytokine system with an altered RANKL-to-OPG ratio has been implicated in the pathogenesis of animal models of rheumatoid arthritis, osteolytic tumor metastases, humoral hypercalcemia of malignancy, and various metabolic bone diseases. Furthermore, neutralizing RANKL is effective at reducing bone destruction in models of osteoporosis, RA, bone metastasis, periodontal disease and reducing markers of bone turnover in a phase I clinical trial of postmenopausal women. Since RANKL has emerged as an important therapeutic target, we are developing a RANKL vaccine based on the patented AutoVacTM technology to effectively neutralize RANKL by actively stimulating the endogenous immune system through vaccination. Thus, we have produced RANKL AutoVacTM proteins and verified their biological activity by immunizing rats and by testing the rat sera for their ability to neutralize native RANKL in a competition ELISA, in which sera competes for the binding of RANKL to the OPG receptor. RANKL AutoVacTM proteins were expressed in E.coli as insoluble (inclusion bodies) and soluble forms which were purified and characterized for biological evaluation. Our results demonstrate that sera from rats vaccinated with soluble RANKL AutoVacTM can inhibit the interaction between OPG and RANKL having a potential and positive clinical effect in bone loss diseases.

Disclosures: M. Hertz, Pharmexa A/S 1, 3.


OPG and TNF-Alpha Antibodies Prevent Inflammation Associated Bone Loss Through Distinct Mechanisms in a Collagen-Induced Arthritis Model.N. Saidenberg-Kermanac'h*1, A. Corrado*2, N. Bessis*1, M. C. de Vernejoul2, M. C. Boissier*1, M. E. Cohen-Solal2. 1UPRES EA-3408, Bobigny, France, 2INSERM U349, Paris cedex 10, France.

Rheumatoid arthritis (RA) is associated with focal and systemic bone loss involving several cytokines such as RANKL and TNF-alpha. Whereas osteoprotegerin (OPG) inhibits bone resorption and osteoporosis, TNF-alpha decreases articular inflammation and bone erosions, but its effect on bone loss remains to be elucidated. The aim of this study is was to evaluate the respective and combined effect of OPG and TNF-alpha on inflammation and on bone remodeling. We used a model of collagen-induced arthritis (CIA). DBA/1 mice immunized with bovine type II collagen. Mice were treated at the onset of arthritis with :1) OPG-Fc (10 mg/kg SC 3 times/week); 2) TNF-alpha antibodies (TNF-alpha Ab, 10 mg/kg IP 2 times/week); 3) both OPG-Fc + TNF-alpha Ab; 4) saline (placebo) and 5) one group of mice remained naive. Bone mineral density (BMD) was measured at the total body (Piximus Lunar) at baseline before immunization and at sacrifice allowing to measure bone gain in Bone Mineral Density (BMD). Deoxypyridinolin (D-Pyr) changes were measured in the urines. Histomorphometric parameters were measured at the femur metaphysis.

Clinical arthritic scores significantly improved in TNF-alpha Ab-treated group compared with placebo (p<0.02), whereas OPG had no significant effect on this score. OPG and TNF-alpha Ab increased BMD gain compared with placebo (39 ± 2% and 25 ±1% vs 15 ±3%, p<0.001 and 0.05 respectively), but OPG was more efficiency to increase BMD gain than TNF-alpha (p<0.003). There was no additive effect of both OPG and TNF-alpha on BMD gain (40 ± 3%). In addition, D-Pyr decreased by 65% with OPG compared to 7% with placebo (p<0.001) and13% when mice were treated with TNF-alpha Ab (p= NS). Compared with placebo, OPG induced increased Tb bone volume (8.4 ± 1.1 vs 13.4 ± 0.9, p<0.02) and decreased Tb spacing (345±91 vs190 ± 13 um, p<0.02) as well as decreased BFR (45 ± 8 um2/d vs 0, p<0.01). In contrast, TNF-alpha Ab-treated group showed no significant changes in bone volume (11.9 ± 1.5%) and Tb spacing (250 ± 30 um) compared with placebo. However, Tb thickness was higher in TNF-alpha Ab-treated mice than in placebo (30.4 ± 0.8 vs 23.9 ± 1.4um, p<0.02) and was close to those of naive mice (32.4 ± 2.3,p:NS), suggesting a protective effect on bone formation. There was no additive effect of OPG and TNF-alpha Ab in any parameters.

In conclusion, systemic administration of OPG and TNF-alpha Ab prevented bone loss in CIA-mice model through distinct mechanisms involving inhibition of bone resorption and formation. Combination of both treatment could be proposed for the prevention bone loss in inflammatory diseases.

Disclosures: M.E. Cohen-Solal, None.


Cortikal Osteoporosis and Reduced Bone Strength in the Interleukin-4 and Interleukin-13 Double-KO Mouse Phenotype.C. J. H. Silfversward*1, C. Ohlsson2, O. Nilsson*1, A. R. Frost1. 1Dept. of Orthopedic Surg., Surg sciences, Uppsala, Sweden, 2Dept. of Internal Medicine, Division of Endocrinology, Gothenburg, Sweden.

Bone loss (osteoporosis or osteolysis) can be caused by inflammation adjacent to bone through the actions of secreted inflammatory mediators. Previous in-vitro studies have revealed that the anti-inflammatory cytokines IL-4 and IL-13 inhibit proliferation of and stimulates IL-6 secretion by human osteoblasts.

In the present in-vivo study, using the IL-13 (-/-) knockout mouse and the IL-4/-13 (-/-) double knockout mouse, we have investigated the physiological role of these cytokines. The skeletal phenotypes of Balb/cJ knock-out mice were analyzed by DEXA, QCT and biomechanical testing and compared to WT-mice.

When sacrificed, at 20 weeks of age, the double-KO male mice presented with significantly thinner (DEXA and QCT) and weaker (three-point bending) cortical bone compared to the WT controls. A decrease in cortical BMC (tibia -8,2%; p<0,01 and femur -8,5%; p<0,01) together with cortical area (tibia -7,4%; p<0,01 and femur -7,9%; p<0,01) and thickness (tibia -3,7%; p<0,05 and femur -5,2%; p<0,01) was observed. At three-point bending there was a significant reduction of displacement (-11.4%; p<0,05), maximal load (-10.6%; p<0,01) and total energy (-29.4%; p<0,002) to failure.

As these effects were not seen in female IL-4/13 (-/-) mice we are now investigating the hypothetical protective role of estrogen.

In conclusion: IL-4/-13 double inactivated mice have a severe cortical bone loss, resulting in decreased mechanical strength. Thus, the TH2 derived cytokines IL-4/IL-13 are important regulators of cortical bone mass and may be potential targets for the development of new treatment strategies for osteoporosis.

Disclosures: C.J.H. Silfversward, None.


IL-12 and IL-18 are Both Required for Normal Bone Remodelling and Normal Trabecular Bone Mass.N. A. Sims1, D. Mirosavljevic*2, N. J. Horwood*2, M. J. Smyth*3, M. T. Gillespie2. 1Dept of Medicine at St. Vincent's Hospital, The University of Melbourne, Melbourne, Australia, 2St. Vincent's Institute of Medical Research, Melbourne, Australia, 3Peter MacCallum Cancer Research Institute, Melbourne, Australia.

Interleukin-18 (IL-18) is a pleiotropic factor that shares structural features with IL-1 and functional activities with IL-12. IL-18 is expressed by osteoblasts and has been reported to inhibit in vitro osteoclast formation by acting upon T cells to promote GM-CSF production. In addition to its ability to enhance GM-CSF production, it also increases IFN-γ, another osteoclast inhibitor. Contrasting with these osteoclast inhibitory actions, IL-18 has also been reported to enhance osteoblast proliferation, an action that appears to be independent of both GM-CSF and IFN-γ. Like IL-18, IL-12 also inhibits osteoclast formation in vitro as a result of its actions on T lymphocytes. When combined, IL-12 and IL-18 are powerful synergistic inhibitors of in vitro osteoclast formation. Since expression of both IL-12 and IL-18 are elevated in inflammatory responses, their postulated actions on osteoblast or osteoclast formation / activity have largely been considered only in the context of inflammatory conditions such as rheumatoid arthritis.

To determine whether IL-12 and IL-18 play are essential for normal bone development, growth and remodeling, we performed histomorphometric analyses of single IL-12−/−, IL-18−/− and double IL-12−/−IL-18−/− knockout mice. No gross skeletal alterations were evident in any of these mice. However, histomorphometry revealed a significant osteopenia in each of these knockout mice. Furthermore, mice deficient in both IL-12 and IL-18 demonstrated a similar phenotype to the single IL-12 or IL-18 null mice. At 10 week of age, both trabecular bone volume (BV/TV) and trabecular number (TbN) were lower than in wild type strain-matched controls (both were reduced to approximately 83%, 55% and 80% of wild type levels in IL-12−/−, IL-18−/− and IL-12−/−IL-18−/− mice, respectively). The osteopenia persisted, with the phenotype becoming more severe with age; at 6 months of age, both BV/ TV and TbN were less than half that of wild type controls in all three knockouts. This phe-notype appeared to result from an increase in osteoclast number (of approximately 46%, 40% and 17% over wild type levels in 10 week old IL-12−/−, IL-18−/− and IL-12−/−IL-18−/−mice, respectively), as predicted from in vitro experiments.

These results underscore a central role of T lymphocytes in normal skeletal development, in addition to their recognised role in inflammatory conditions such as rheumatoid arthritis and periodontal disease.

Disclosures: N.A. Sims, None.


Effects of Oncostatin M on Glucocorticoid Receptors and Cytokine Secretion in Human Osteoblasts.A. Dovio*1, M. Sartori*1, B. Ceoloni*1, L. Saba*1, S. Racca*1, A. Fazzari*2, A. Angeli1. 1Clinical and Biological Sciences, University of Turin, Orbassano, Italy, 2Clinical Pathophysiology, University of Turin, Turin, Italy.

gp130 family of cytokines includes interleukin (IL)-6, IL-11, leukemia inhibitory factor, oncostatin M (OSM), ciliary neurotrophic factor, cardiotrophin-1 e B-cell stimulating factor-3. Most gp130 cytokines and their receptors are expressed in the bone microenvironment, and are credited with osteoclastogenic activity. As far as osteoblasts are concerned, gp130 cytokines stimulate osteoblast differentiation and inhibit adipocyte differentiation of precursors. Moreover, we have previously demonstrated that IL-6 and IL-11 respectively up- and down-regulate glucocorticoid (GC) receptors (GR) in an autocrine-paracrine way in the human osteosarcoma cell lines Saos-2 and MG-63. GR is a key determinant of tissue sensitivity to GC. The aim of the present study was to extend our investigation to another osteotropic gp130 cytokine, OSM, in both Saos-2 and MG-63 cell lines and primary cultures of human osteoblasts. Cells were incubated with the cytokine (0.05--50 ng/ml) for 20 h; afterwards, supernatants were harvested, GR protein was assessed by whole cell radioligand binding assay followed by Scatchard analysis and western blot, and GR mRNA was measured by RT-PCR. OSM dose-dependently decreased GR number in MG-63 cells without affecting affinity; results were confirmed by western blot analysis. Decreased GR protein was parallelled by decreased GR mRNA. OSM did not exert any effect on GR number in Saos-2 cells, while it decreased GR mRNA in primary cultures of human osteoblasts. Since reciprocal regulation of gp130 cytokines has been previously reported, the effect of OSM on IL-6 and IL-11 release was investigated. OSM significantly increased IL-6 release in all models; on the contrary, IL-11 secretion was increased in Saos-2 cells and decreased in primary cultures, with unconsistent effect in MG-63 cells. No effect upon soluble IL-6 receptor was observed. Our data demonstrate a down-regulatory action of OSM on GR in human osteoblasts. Such action is likely not to be subserved by modulation of IL-6/IL-11 secretion. Lack of effect in Saos-2 cells is possibly due to low number of GR and different pattern of expression of OSM receptors. Further investigation is needed to elucidate the mechanisms of divergent effects on IL-11 release. Our results suggest that gp130 cytokines increase or decrease GC sensitivity of cells of the osteoblastic lineage by modulating GR. Such modulation could be of relevance in the pathogenesis of the biphasic bone loss observed in patients given GC therapy and having high concentrations of inflammatory cytokines in the bone microenvironment.

Disclosures: A. Dovio, None.


Bone Histomorphometry and IGF-1, IGFBP-3, and PTH in Young Women Osteoporosis.A. Z. Sawicki*1, A. Debinski*2, Z. Polowiec*3. 1Warsaw Osteoporosis Centre, Warsaw, Poland, 2Mineral Metabolism and Bone Disease, National Food and Nutrition Institute, Warsaw, Poland, 3Warsaw Osteoporosis Centre “Osteomed”, Warsaw, Poland.

The IGF-1 and its binding-protein 3 (IGFBP-3) as well as PTH plays very important role in regulation of bone formation and bone resorption.

The assessment of relationships between bone histomorphometry and IGF-1, IGFBP-3 and PTH in young women osteoporosis were the aim of the study.

Twenty five women aged between 20--39 (X±SD=32.1±6.5) years without known risk factors of osteoporosis underwent transiliac bone biopsy following tetracycline-labelling and bone markers studies. All of them had bone mineral density below -2.5 t-score, measured by DEXA method (Hologic QDR 4500A). None of them had hyperparathyroidism, renal disease, malabsorption and/or long-term corticosteroid treatment or other secondary osteoporosis risk factors. Histomorphometric assessment of their bone including trabecular volume, osteoid volume, osteoclast surface and osteoblast surface according to ASMBR were done. Normal values of static histomorphometric parameters were obtained from 13 women aged 20--39 years without bone disorders who died suddenly in traffic accidents. Serum IGF-1, IGFBP-3 and PTH levels were measured by RIA method. Normal values of IGF-1, IGFBP-3 and PTH levels were obtained from 16 health women aged 20--49 (X±SD=38±5.4) years.

In the study group mean bone volume was significantly decreased (15.80±3.83 vs 23.30±1.07; p<0.001), the mean osteoid volume without mineralization defect and mean osteoclast surface were significantly increased (3.96±2.72 vs. 1.98±0.45; p<0.01: 2.60±1.28 vs. 1.49±0.54; p<0.01 respectively). No significant difference in mean osteo-blast surface was found (2.84±2.22 vs 3.50±0.79; NS). In study group mean PTH level was significantly lower then in control group (22.89±9.55 pg/mL vs. 28.14±12.05 pg/mL; p<0.02). No significant differences, between study and control group, in IGF-1 and IGFBP-3 levels was found (307.5±97.8 ng/mL vs.263.4±66.3 ng/ml; NS, 3214.9±637.0 vs. 3481.1±989.2; NS respectively). Significant correlations between IGF-1 and osteoblast surface (R=0.613, p<0.01) as well as between PTH and osteoclast surface (R=0.498, p<0.05) were found. No significant correlations between IGFBP-3 and static histomorpho-metric parameters were found.

We conclude that high osteoclasts activity in young women osteoporosis is not related to hyperparathyroidism or osteomalacia.

Disclosures: A.Z. Sawicki, State Committe for Scientific Research 2.


CSF-1 Regulates Mast Cell Differentiation in Rat Tibia.G. L. Evans*1, P. R. Odgren2, C. A. MacKay*2, R. T. Turner1. 1Orthopedic Research, Mayo Clinic, Rochester, MN, USA, 2Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA.

In patients, osteoporosis can accompany Systemic Mastocytosis. In animals, the bone loss associated with ovariectomy is accompanied by an increase in mast cell number, and mutations that lead to mast cell deficiency often result in osteopetrosis. These observations suggest that mast cell and osteoclast differentiation are, in part, similarly regulated. Colony stimulating factor-1 (CSF-1) was shown to be essential for osteoclast differentiation and a CSF-1 inactivating mutation was recently identified in the toothless (tl) rat mutation. The purpose of the present study was to determine whether CSF-1 plays an equally important role in mast cell differentiation. Three groups of rats (3--6 w old) were studied; tl/tl (n=9--11), tl/tl treated for 1 w with (10--6 U/d) CSF-1 (n=3) and wild type (n=8--12). Measurements were performed in the proximal tibial metaphysis and osteoclasts were measured in tartrate resistant acid phosphatase stained sections and mast cells in Azure stained sections. As expected, the tl/tl rats had greatly reduced osteoclast number (1/mm2 tl/tl vs. 7/ mm2 wild type, p<.01) and CSF-1 was effective in reversing the defect (20/mm2 treated tl/tl, p<.001). Similarly, the tl/tl rats had greatly reduced mast cell number (1/mm2 tl/tl vs. 25/ mm2 wild type, p<.01). CSF-1 partially restored mast cell number (7/mm2, p<.001). These findings suggest that CSF-1 plays a role in mast cell differentiation. Mast cells produce many cytokines that influence bone resorption. Thus, parallel regulation of mast cell and osteoclast differentiation may be important to local regulation of bone resorption during normal growth, modeling and remodeling, as well as in disease.

Disclosures: G.L. Evans, None.


Chondrocytes Respond to the Low Intensity Pulsed Ultrasound by Calcium Influx Pathways.A. Miyauchi1, T. Sugimoto2, Y. Takagi1, K. Jinnai*1, Y. Yoshimoto1, K. Chihara*2, T. Fujita3, Y. Mikuni-Takagaki4. 1Medicine, National Hyogo-Chuo Hospital, Sanda, Japan, 2Division of Endocrinology/Metabolism, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan, 3Calcium Research Institute, Kishiwada, Japan, 4Oral Biochemistry, Kanagawa Dental College, Yokosuka, Japan.

Mechanical stimuli are essential in maintaining morphology and function of cartilaginous tissues. Low-intensity pulsed ultrasound (LIPUS), which accelerates fracture healing processes in the appendicular skeleton first by cartilaginous induction, provides non-invasive therapeutic treatment for fracture repair and distraction osteogenesis. While we have reported that osteoblasts but not osteocytes are the target cells of the anabolic LIPUS and that their response did not accompany calcium influx (J Bone Miner Res 18:360,2003). Parvizi et al. reported that rat chondrocytes responded to the LIPUS by increasing cytosolic Ca2+ ([Ca2+]i) and by proteoglycan synthesis. It suggests that the LIPUS sensing-mechanism of chondrocytes is distinct from that of osteoblastic cells. We, therefore, studied responses of chondrocytes to LIPUS as well as stretch loading. Human articular chondrocytes were preloaded with fura-2, and subcellular [Ca2+]i was monitored using a video image analyzer. Stretch loading by swelling the cells with hypoosmotic solution (182 vs. 363 mOSM) induced a rapid and progressive [Ca2+]i increase (65±6 nM above basal level, n=14). Since more than 90% of the hypotonically-induced [Ca2+]i increase was abolished either by 2×10−5M Gd3+, a selective inhibitor of stretch-activated cation channels (SA-Cat), or by Ca2+-free medium, the [Ca2+]i increase is likely caused by an influx of Ca2+ through SA-Cat. Meanwhile, LIPUS (90 mW/cm2) exposure induced a transient [Ca2+]i increase (63±5nM above basal level, n=10) followed by slow [Ca2+]i oscillation(1/150 c/s). A larger [Ca2+]i increase of approximately 90 nM was observed in the submembranous region than in the perinuclear cytoplasm (50 nM). The [Ca2+]i increase was abolished either by Gd3+ or Ca2+-free medium. Moreover, preincubation with thapsigargin was not effective and reduced the LIPUS-induced increase by only less than 20%. Taken together, our results indicate that the LIPUS-induced [Ca2+]i increase was primarily caused by an influx of Ca2+ through SA-Cat in human chondrocytes. This pathway may be an element in the signal transduction through which LIPUS stimulates chondrocyte maturation and fracture repair.

Disclosures: A. Miyauchi, None.


COX-2, but not COX-1, Is Involved in Mechanotransduction in Bone Cells.A. D. Bakker*, E. H. Burger*, C. M. Semeins*, J. Klein-Nulend. Oral Cell Biology, ACTA-VU, Amsterdam, Netherlands.

Cyclooxygenase (COX) is the key enzyme in the production of prostaglandins, which are essential for the anabolic response of bone to mechanical loading. There are two isoforms of COX, a constitutive isoform (COX-1) and an inducible form (COX-2). It is currently unknown which isoform regulates the prostaglandin response to mechanical loading. The aim of this study was therefore to test whether COX-1 or COX-2 determines pulsating fluid flow-induced prostaglandin production by primary bone cells in vitro.

Primary mouse and human bone cells were kept under static culture conditions, or were subjected to 1 h of pulsating fluid flow (PFF, 0.6±0.3 Pa at 5 Hz), in the presence or absence of either 10−7 M of the specific COX-1 inhibitor SC-560, or 10−5 M of the specific COX-2 inhibitor NS-398. After cessation of PFF, cells were post incubated under static conditions for an additional 24 h.

Both mouse and human bone cells reacted to PFF with an increased prostaglandin E2 production, which continued 24 h after cessation of PFF. NS-398 abolished the stimulating effect of PFF on PGE2 production both at 1 h and 24 h post-incubation, while SC-560 affected neither the early nor the late response to flow. PFF rapidly stimulated COX-2 mRNA expression at 1 h, but did not affect COX-1 mRNA expression. COX-2 mRNA expression was still significantly enhanced 24 h after cessation of PFF.

We conclude that mechanical loading-induced production of prostaglandins by bone cells is determined by COX-2, but not COX-1. These results explain the observation that COX-2 is essential for the anabolic response of bone to mechanical loading in vivo. 

Figure Figure:.

Effect of PFF, and NS-398 or SC-560, on PGE2 production by mouse bone cells, after 1 h of treatment (A), and during the 24 h post incubation period (B). Values are mean ± SEM. Stat, stationary culture; PFF, pulsating fluid flow. Stat and PFF, n=9; Stat + NS-398 and PFF + NS-398, n=5; Stat + SC-560 and PFF + SC-560, n=6. # Sign effect of PFF, * Sign effect of NS-398 or SC-560, p<0.05.

Disclosures: A.D. Bakker, None.


The Response of Bone Cells from Osteoporotic Donors to Mechanical Loading Is not Altered by Estrogen.A. D. Bakker*1, P. Lips2, R. Huiskes3, E. H. Burger*1, J. Klein-Nulend1. 1Oral Cell Biology, ACTA-VU, Amsterdam, Netherlands, 2Endocrinology, Vrije Universiteit Medical Center, Amsterdam, Netherlands, 3Biomed. eng., Technical University Eindhoven, Amsterdam, Netherlands.

Local bone mass and architecture are determined by mechanical loading, and the subsequent response of osteocytes to the loading-induced fluid flow through the lacuno-canalicular network. Estrogen also has a profound effect on bone mass. It has been suggested that the loss of estrogen during the menopause alters the response of bone cells to mechanical loading, thereby contributing to the rapid loss of bone. The present study aimed to determine whether estrogen modulated the mechanoresponse of bone cells from osteoporotic women in vitro.

Bone cell cultures from 11 osteoporotic women between 62 to 90 years of age were subjected to pulsating fluid flow (PFF, 0.6±0.3 Pa at 5 Hz) or static culture for 1 h, in the presence or absence of 10−11 M 17β-estradiol (E2).

PFF significantly stimulated PGE2 production after 5 and 60 min of application. Treatment with E2 enhanced basal PGE2 production by the bone cells, but did not affect the magnitude of the PGE2-response to PFF. E2-treatment stimulated endothelial nitric oxide syn-thase protein expression in the osteoporotic bone cells by 2.5-fold, but did not stimulate basal NO production. 1 h of PFF significantly stimulated NO production by approximately 5-fold. E2-treatment did not affect the magnitude of this NO-response to PFF.

These results suggest that E2 does not alter the response of bone cells from osteoporotic donors to mechanical loading. Thus, the rapid loss of bone during the menopause is unlikely to be caused by a change in mechanosensitivity of the bone cells. 

Figure Figure:.

Effect of E2 on the magnitude of the PGE2 and NO-response to PFF. Data are expressed as PFF-treated over static culture ratios. Bars are mean ± SEM. There was no difference in magnitude of the PGE2 or NO-response between E2 and vehicle-treated cells at any time point. * Signficant effect of PFF, p<0.05.

Disclosures: A.D. Bakker, None.


Modeled Microgravity Disrupts Integrin Expression and Function during Osteoblastogenesis of Human Mesenchymal Stem Cells.V. E. Meyers*1, M. Zayzafoon1, W. E. Gathings*2, J. M. McDonald1. 1Pathology, University of Alabama at Birmingham, Birmingham, AL, USA, 2Consortium for Materials Development in Space, University of Alabama in Huntsville, Huntsville, AL, USA.

Spaceflight leads to reduced bone mineral density in weight-bearing bones. This disruption of bone homeostasis is primarily attributed to decreased osteoblast function. Reduced differentiation of human mesenchymal stem cells (hMSC) into osteoblasts likely contributes to this phenomenon. Our lab has previously demonstrated reduced osteoblastic differentiation of hMSC following 7 days culture in modeled microgravity. One possible mechanism contributing to this decline in differentiation is reduced integrin-mediated signaling. When integrins bind extracellular matrix proteins, focal adhesion kinase (FAK) is activated and the transduced signals contribute to the survival and differentiation of hMSC. Here, we begin to characterize the effects of modeled microgravity on integrin expression and function during osteoblastogenesis of hMSC.

To model microgravity, we cultured hMSC for 7 days in a commercially available, NASA-approved rotary cell culture system (RCCS). Cells were seeded onto polystyrene microcarrier beads in either low adhesion 6-well plates, which serve as gravity controls, or 10 mL high aspect ratio vessels (HARVs) used by the RCCS to model microgravity. Cell/bead aggregates were allowed to form for approximately one week in DMEM containing 10% FBS. Osteogenic differentiation was then induced by the addition of β-glycerophosphate, ascorbic acid, and dexamethasone immediately prior to initiation of modeled microgravity. These conditions permit proper osteoblastic differentiation of control cells.

Seven days modeled microgravity was sufficient to cause a 4-fold increase in β1 integrin subunit protein expression. Increased expression of theα2 subunit, which dimerizes with β1 to form the primary functional receptor for type I collagen in osteoblasts, also occurs. However, despite increased integrin protein levels, autophosphorylation of FAK is reduced 4-fold following 7 days modeled microgravity. Protein expression of the α5 integrin sub-unit, which dimerizes with β1 to form a functional receptor for RGD-containing peptides, such as fibronectin, does not appear to be affected by modeled microgravity. In summary, integrin-mediated signaling is disrupted in modeled microgravity despite upregulated or unaltered integrin protein expression. Because integrin signaling is required for hMSC differentiation into osteoblasts, it is likely that this contributes to the reduction in functional osteoblasts and the resulting decline in bone mineral density experienced during spaceflight.

Disclosures: V.E. Meyers, None.


Hemichannels Formed by Connexin 43 (Cx43) are Essential for Prostaglandin E2 (PGE2) Production by Osteocytes in Response to Mechanical Strain.P. P. Cherian, X. Wang*, E. Sprague*, J. X. Jiang. Biochemistry, University of Texas Health Science Center, San Antonio, TX, USA.

Osteocytes embedded in the matrix of bone express large amounts of Cx43, the component of gap junctions, yet osteocytes are only in contact through the tips of their dendritic processes, an extremely small area compared to the total surface of the cell. These observations suggest that Cx43 may have other functions in addition to forming intercellular channels. As osteocytes are mechanosensory cells, it was hypothesized that Cx43 may mediate effects of mechanical strain on these cells. Similar to primary osteocytes, Cx43 is highly expressed in the osteocyte-like cell line MLO-Y4 compared to osteoblasts. Previous studies using MLO-Y4 cells have shown that shear stress induced by fluid flow increases functional intercellular coupling, in addition to increases in Cx43 protein expression and PGE2 production. PGE2 was demonstrated to be an essential mediator between mechanical strain and gap junction intercellular communication. However, it is not known if Cx43 is essential between mechanical strain, and the production and release of PGE2. In this study, MLO-Y4 cells were plated at various cell densities ranging from the lowest at which no cells were physically in contact with adjacent cells to the highest with considerable cell-to-cell contact. Plating at low density prevented the formation of intercellular gap junction channels. These cells were subjected to fluid flow treatment at 16 dynes/cm2 and conditioned media were assayed for PGE2. The MLO-Y4 cells with no or few intercellular channels produced significantly more PGE2 per cell than those at higher density. PGE2 production was exponentially increased at the lowest cell density of 1×103 cells/cm2, compared to the highest cell density of 1.1×105 cells/cm2. Both antisense Cx43 oligonucle-otides and β-glycyrrhetinic acid, a specific and reversible gap junction channel blocker that can also block hemichannels, significantly reduced PGE2 production induced by fluid flow at all cell densities tested, especially cells at the lowest density. Moreover, fluid flow shear stress rendered the Cx43 located at the cell surface to be resistant to Triton-X-100 extraction, suggesting the formation of insoluble protein plaques, similar to previously reported gap junctional plaques. Analysis of cell surface biotinylation revealed that fluid flow enhanced the phosphorylation of Cx43 on the cell surface making the molecule more susceptible to labeling by biotin. Our results suggest that hemichannels formed by Cx43, instead of intercellular channels, are likely to play a dominant role in regulating the responses of osteocytes to mechanical stimulation.

Disclosures: J.X. Jiang, None.


Zinc Transporter 5 (Znt5) Is an Essential Molecule for Mechanical Stress Signaling in Bone.K. Matsuda*1, K. Inoue*2, K. Yamakawa*1, H. Kawano1, T. Akune1, K. Hoshi1, Y. Nakamura*2, K. Nakamura1, H. Kawaguchi1. 1Orthopedic Surgery, Univ. of Tokyo, Tokyo, Japan, 2Institute of Medical Science, Univ. of Tokyo, Tokyo, Japan.

Mechanical stress is one of the most important factors for maintaining bone mass, although little is known about the molecular mechanism. We recently isolated by a differential display method a novel gene Znt5 whose expression increased in response to expansion load of rabbit aorta. Znt5 has 15 transmembrane domains with a cation efflux domain at its C-terminus, and functions as a zinc transporter. To learn the involvement of this molecule in the mechanical stress signaling in bone, we first examined the expression in bone responding to mechanical stress using several models. In the mouse tail suspension model, Znt5 mRNA level in tibiae and femora was decreased to about one third that of control after 3 weeks of unloading, and was restored to the normal level by reloading. In the culture of mouse newborn calvariae, Znt5 expression was induced by continuous tensile stress by an inserted spring for 9 h. When three kinds of osteoblastic cell lines, MC3T3-E1, MLO-Y4 and MLO-A5, were cultured with stretching stimulation by the Flexor Cell system, about 2-fold increase of Znt5 expression was seen only in MLO-Y4 cells that are known to be the most differentiated osteoblasts. To further investigate the physiological role of Znt5, we generated mice lacking the Znt5 gene (Znt5−/−). Znt5−/− mice exhibited slight growth retardation (-10% in length) as compared to wild-type (WT) littermates. Bone densitometry, histomorphometry and 3D-μCT analyses of the Znt5−/− bone showed a marked decrease in the volume of both trabecular and cortex bones (-30% in BMD, -60% in BV/TV) at 8 weeks of age. The bone loss seemed to be due to the impairment of bone formation (-60% in Ob.S/BS, -40% in MAR, -70% in BFR/BS) since bone resorption parameters were normal. Cultured primary osteoblasts derived from Znt5−/− calvariae showed markedly decreased differentiation and mineralization as determined by ALP and Alizarin red stainings, respectively; however, CFU-OB formation from bone marrow cells was normal, suggesting a dysfunction of osteoblasts but not of the precursor cells. When Znt5−/− mice were treated with tail suspension for 3 weeks, the decrease in bone density (-5%) was much less than that in WT (-15%). To explore the downstream signaling of Znt5, we compared the gene expression profile between Znt5−/− and WT, and found that several immediate early genes including Fos, Egr1 and heat shock proteins were decreased in Zn5−/−. We conclude that Znt5, whose expression is positively regulated by mechanical stress in mature osteoblasts or osteocytes, plays an essential role in the maintenance of bone volume by mechanical stress.

Disclosures: K. Matsuda, None.


Expression of P2Y Purinergic Receptors During Osteoblastic Differentiation.J. You, H. J. Donahue. Dept of Orthopaedics & Rehabilitation, Center for Biomedical Devises & Functional Tissue Engineering, The Pennsylvania State University College of Medicine, Hershey, PA, USA.

Accumulated evidence suggests that one of the ATP receptor subtypes, P2Y (G protein-coupled receptor), may play an important role in cell mechanosensing and differentiation. To date, four mouse P2Y purinergic receptors (P2Y1, P2Y2, P2Y4 and P2Y6) have been cloned. Recently we demonstrated that P2Y receptors (either P2Y2 or P2Y4) are responsible for oscillatory fluid flow-induced intracellular calcium (Ca2+i) mobilization in mouse MC3T3-E1 osteoblastic cells. The aim of this study was to identify the subtypes of P2Y receptors expressed in MC3T3-E1 cells that are responsible for flow-induced Ca2+i mobilization. Additionally, we examined whether the expression of P2Y receptors is changed during osteoblastic differentiation. MC3T3-E1 cells were cultured in normal media (MEMα+10%FBS) for 24 hours, then one half of the cultures were placed in differentiation (normal media + β-glycerophosphate + L-ascorbic acid phosphate) media for an additional 48 hours, while the other half remained in normal media. First, using RT-PCR, we examined the expression of all subtypes of P2Y receptors in cells cultured in normal media. Our results demonstrated that P2Y2 was the most abundant P2Y receptor subtype expressed in MC3T3-E1 cells cultured in normal media. We did not detect P2Y1, P2Y4, or P2Y6 receptors in MC3T3-E1 cells in normal media suggesting that these receptors may not be involved in flow-induced Ca2+i mobilization. In MC3T3-E1 cells cultured in differentiation media we were not only able to detect P2Y2 but also P2Y1, which was not detected in cells cultured in normal media. To examine the functional expression of receptors on MC3T3-E1 cells cultured in differentiation media, we exposed the cells to ADP (100μM), a P2Y1 agonist. ADP significantly induced Ca2+i mobilization in MC3T3-E1 cells cultured in differentiation media, but not MC3T3-E1 in normal media, suggesting the expression and function of P2Y1 receptors are dependent on cell differentiation. However, the Ca2+i responses of MC3T3-E1 cells to fluid flow were similar in cells cultured in normal and differentiation media suggesting that P2Y1 may not be involved flow induced Ca2+i mobilization. Our finding is the first evidence showing that cell differentiation may affect the expression of P2Y receptors in osteoblastic cells and also suggest that P2Y1 receptors are not involved in fluid flow-induced Ca2+i mobilization.

Disclosures: J. You, None.


Gene Expression Profile Comparisons between 2T3 Osteoblasts at Low Density and Confluent Stages with Osteocyte-like MLO-Y4 Cells at High and Low Densities.W. Yang*, M. A. Harris, D. Guo*, A. Krisnaswamy*, L. F. Bonewald, S. E. Harris. Dept. of Oral Biology, U. of Missouri at Kansas City, Kansas city, MO, USA.

The osteocyte is a terminally differentiated cell of osteoblast origin that only remains in physical contact with neighboring cells through the tips of dendritic processes. Markers for osteoblastic differentiation are well-known, but few are recognized for osteocytes. In this study, gene expression profiles for 2T3 cells at low density and confluent stages were compared to those for MLO-Y4 cells. It was postulated that MLO-Y4 cells at low density would have fewer functional gap junctions and therefore less cellular communication compared to cells cultured at higher density. The 2T3 osteoblast-like cells at low density expressed filopodia, reminiscent of early osteoblast precursors. Mouse 5k oligonucleotide microarrays were used to produce gene expression profiles. After triplicate sets of hybridization experiments for each condition, clusters of expression patterns that are characteristic of MLO-Y4 cells and 2T3 osteoblasts at different states were profiled. We then used global intensity and variance to normalize the original data, followed by statistical analysis to validate and exclude genes with high variance. Using percolation clustering and K-median clustering, we have and will continue to derive new biological hypotheses and insight from these data sets. We first selected a set that changed greater than 2-fold between cell lines or states. We then determined a set of genes in which the normalized SD between triplicates was less than 0.5SD. From about 700 genes, we then began our bioinformatics analysis. As might be expected, MLO-Y4 cells have much higher expression of genes that might be involved in the generation and maintenance of dendritic processes such as zyxin and CD44, which are known to interact with E11. MLO-Y4 cells also highly express chemotactic factors for osteoclasts such as Scya7 (MCP-3), VEGF, and TGFb1. Higher gene expression levels for Tob1, a suppressor of growth and BMP/TGFb signaling, Ncor2 (nuclear receptor co-repressor 2), Tgfbr3, and Hoxc10 was found in MLO-Y4 cells compared to 2T3 osteoblasts. We have over 300 genes that are at least 3 fold higher in MLO-Y4 cells than any of the two states of 2T3 cells. Experiments are underway comparing gene expression patterns in MLO-Y4 cells and 2T3 osteoblast that were subjected to 4 dynes/cm2 of fluid shear stress for 2 hrs followed by analysis at 2 and 24 hrs after stopping flow. Modeling of these gene expression patterns in the MLO-Y4 osteocyte model and 2T3 osteoblast model will be presented, as well as our preliminary studies on the response of these two cell types to mechanical loading.

Disclosures: W. Yang, None.


Ultrasound Mimics the Effect of Mechanical Loading in vivo on Rat Ulnae and Bone Marrow Cells.L. K. Parry*1, V. J. Burton*2, S. Gheduzzi*2, J. Beresford*3, M. J. Perry2, T. M. Skerry1, V. F. Humphrey*4. 1Veterinary Basic Sciences, Royal Veterinary College, London, United Kingdom, 2Orthopaedic Surgery, University of Bristol, Bristol, United Kingdom, 3University of Bath, Bath, United Kingdom, 4Physics, University of Bath, Bath, United Kingdom.

Mechanical loading influences bone strength, and it has been shown that high frequency low magnitude loads exert positive effects on bone. This experiment was designed to test the hypothesis that ultrasound stimulation exerts positive influences on bone.

Female Wistar rats were anaesthetized, the left ulna loaded cyclically 6 times on alternate days. 40 cycles of load were applied with peak strains of 3,000 microstrain at 0.12 per second, with a 10 second rest period between each cycle. In another group applied transcutaneous ultrasound stimulation consisted of a burst width of 200 microseconds containing 1MHz sine waves, with a repetition rate of 1 kHz and a spatial average-temporal average intensity of 150 mW/cm2 for the same duration as the loading. In a third group loading and ultrasound stimulation were applied concurrently. Fluorochrome labels were given at the start and 1day before the end of the experiment. The right leg served as a non-loaded control in each animal. At the end of the experiment, left and right ulnae were removed, and marrow cells removed by brief centrifugation before bones were processed for histomorphometry. Cells were cultured for 18 days then fixed and reacted to demonstrate expression of alkaline phosphatase positive colonies. Load induced bone formation on the medial periosteal surface was assessed by measuring interlabel distance.

Loading induced a significant periosteal response, increasing the interlabel distance by 300%, and reduced numbers of AP positive colonies significantly by 25%. The effect of ultrasound was to increase the periosteal bone formation by 50% and inhibit colony formation by the same amount as loading. The effects of loading plus ultrasound were indistinguishable from ultrasound alone.

These data suggest that ultrasound has the ability to induce changes in bone that appear to share common features with mechanical loading. The inhibition of colony formation by loading is part of the integrated structural response of the ulna to loading whereby medial periosteal osteogenesis is stimulated, but endosteal formation (which is part of the growth process in rats of this age) is inhibited to reduce the bone's curvature. The ability of concurrent ultrasound stimulation to suppress the effects of concurrent loading suggests that the interaction between the two stimuli is complex and not simply additive or synergistic. This non-invasive stimulation may induce bone formation safely in those with weak skeletons.

Disclosures: L.K. Parry, None.


A Novel Mechanical Stress-induced Signaling Pathway Leading to Smad1/ 5/8 Activation in Osteoblasts.S. Kido1, D. Inoue1, T. Imamura*2, K. Miyazono*2, T. Matsumoto1. 1Department of Medicine and Bioregulatory Sciences, University of Tokushima, Tokushima, Japan, 2Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo, Japan.

Mechanical stress to bone plays a critical role in maintaining bone mass and strength. We report here that mechanical loading to osteoblastic cells activates R-Smads 1/5/8, which act downstream of BMP and play an important role in bone formation. Fluid shear stress (FSS) induced endogenous Smad1 phosphorylation and Smad4 translocation to the nucleus within 30 min both in primary osteoblasts and MC3T3-E1 cells. Immunoprecipitation experiments with adenovirally introduced Flag-tagged Smads revealed that FSS also phosphorylated Smad5 and 8 to some extent, but not Smad2 or 3. We found that FSS induced expression of Smad7, an endogenous target of activated R-Smads, at both mRNA and protein levels. FSS also induced transcription from consensus Smad response elements. Moreover, when mice were mechanically loaded in rotating cages, Smad7 expression was induced in hindlimb bones. Therefore, mechanical loading to bone cells resulted in functional Smad activation both in vitro and in vivo. Interestingly, FSS-induced smad1 phosphorylation was inhibited by Smad6 or 7 over-expression, but not by dominant negative ALK3, suggesting that Smad activation by FSS was independent of BMP receptors. Furthermore, Smad activation was inhibited by EGTA or BAPTA, suggesting that it was dependent on extracellular calcium influx. Conversely, increasing extracellular Ca concentrations led to enhanced Smad phosphorylation by FSS. Smad activation by FSS was inhibited by PKC inhibitors or depletion by 24 hr treatment with 50 nM TPA, but not by inhibitors of ERK, p38MAPK or PKA, suggesting a role of PKC. In contrast, Smad activation by an authentic ligand, BMP-2, was not affected by PKC inhibitors. Moreover, we found that FSS activation of Smad was in part inhibited by calmodulin antagonists including W-12, W-13 and compound48/80, but not by a CAMKII inhibitor, KN-62. In vitro binding assay using calmodulin agarose beads revealed a Ca-dependent, direct physical interaction between calmodulin and Smad1/5/8. In summary, we have demonstrated for the first time that Smad1/5/8 lies downstream of mechanical stress-induced osteogenic signaling pathways. Further delineation of this novel signaling pathway may form a molecular basis to understand the mechanism of mechanical stress-induced bone formation and to identify a novel therapeutic target of immobilization osteoporosis.

Disclosures: S. Kido, None.


Stretch-induced Parathyroid Hormone-related Peptide (PTHrP) Gene Expression in Bone.X. Chen*1, C. M. Macica*1, G. Liang*1, B. E. Dreyer*1, K. W. Ng2, A. E. Broadus1. 1Internal Medicine, Yale University, New Haven, CT, USA, 2University of Melbourne, Melbourne, Australia.

Mechanical forces play a critical role in regulating skeletal mass and structure. PTHrP is expressed in bone cells under normal physiological conditions and is believed to function locally in an autocrine/paracrine fashion. PTHrP gene expression has been shown to be induced in response to mechanical stretch in several physiological systems. Although the role of PTHrP in bone is unknown, it has been well documented that PTH, which shares a common receptor with PTHrP, can have anabolic effect in bone. PTH has been reported to modulate stretch-activated cation channels (SA-cat) and L-type Ca2+ channels (VOC). However, PTH itself is unlikely to contribute to the local anabolic response induced by mechanical forces. We tested the possibility that PTHrP may act as a local mediator of mechanical stretch using UMR-201--10B osteoblast-like cells in response to variations in tonicity, as a surrogate for mechanical loading.

By RNase protection assay, a three-fold increase of PTHrP mRNA was observed within 30 minutes of a hypotonic challenge (240 mosm vs. 317 mosm isotonic control) in UMR-201--10B osteoblastic cells, and the peak level was reached by 1 hour of treatment. This effect was insensitive to both Gd3+ treatment and to the addition of the VOC inhibitor, nife-dipine, eliminating the possible role of both SA-cat and VOC channels in this effect. The increase in PTHrP message was also unaffected by omission of calcium in the incubation medium or depletion of intracellular calcium pool by thapsigargin, suggesting that neither extracellular nor intracellular calcium are involved.

Recent studies have shown that a class of tandem-pore potassium channels belonging to the TREK family are sensitive to stretch and intracellular acidosis but are insensitive to Gd3+. These channels have not been studied in bone cells. Using RT-PCR, we confirmed the presence of the TREK-2 isoform of stretch-activated K channels in UMR-201 cells. We also found PTHrP mRNA was increased in a dose-dependent manner upon intracellular acidification induced by sodium acetate in the medium, while intracellular alkalization by ammonium chloride had no effect.

We propose PTHrP as a candidate participant in the effects of mechanical loading on bone and that TREK-2 channels are involved in mediating this PTHrP response.

Disclosures: X. Chen, None.


Early Induction of Cyclooxygenase-2 in Osteoblasts by Fluid Flow Depends on the Protein Kinase C Signaling Pathway.S. Wadhwa, M. Mehrotra*, C. Alander, O. Voznesensky, C. C. Pilbeam. Medicine, University of Connecticut Heath Center, Farmington, CT, USA.

Mechanical loading of bone generates interstitial fluid flow within the mineralized bone matrix that exerts fluid shear stress (FSS) on cells, initiating signaling cascades leading to new gene transcription. One of the genes induced by FSS in osteoblasts is cyclooxygenase-2 (COX-2), which is essential for the anabolic effects of mechanical loading on bone. We previously characterized the maximal (late) COX-2 response to FSS in osteoblastic cells. This response occurred at 4--8 h and was dependent on the protein kinase A (PKA) but not the protein kinase C (PKC) signaling pathway. However, short exposures to FSS may be a better model of the effects of mechanical loading on bone in vivo than long exposures. Intuitively, flow duration in a single direction in vivo should be restricted by the geometry of the bone. Moreover, long durations of flow (greater than > 1 h) have been shown to cause cell damage. The purpose of this study was to characterize the early COX-2 response of osteoblasts to FSS. We used clonal MC3T3-E1 osteoblastic cells and primary osteo-blasts, sequentially digested from calvariae of CD-1 mice transgenic for 371 bp of the COX-2 promoter fused to a luciferase reporter (Pluc mice). Cells were plated on collagen-coated glass slides, grown to confluence, and subjected to 10 dynes/cm2 of steady laminar flow in a parallel plate flow chamber for 1 h. Controls were maintained in stationary culture. Unlike the late COX-2 mRNA response to FSS, Northern analysis showed that the early COX-2 mRNA response to FSS was independent of new protein synthesis. In both MC3T3-E1 and primary calvarial cells, the early COX-2 mRNA response to FSS was dependent on the PKC signaling pathway. GF109203X (1.25 μg/ml), an inhibitor of the PKC pathway, or 24 h of pretreatment with phorbol myristate acetate (PMA, 1 μM) to down regulate the PKC pathway decreased the FSS induction of COX-2 mRNA by 50--75%. In contrast, PKI, a specific inhibitor of the PKA pathway, had little effect on the early FSS induction of COX-2 mRNA. To see if the PKC-dependent effect was transcriptionally mediated, primary calvarial cells from Pluc mice were subjected to 1 h of FSS and then returned to stationary culture for 2 h before measurement of luciferase activity. FSS stimulated an increase in luciferase activity and this was blocked by GF109203X. Thus, we conclude that the early (primary gene response) and late (peak) induction of COX-2 mRNA expression by FSS occur by different signaling pathways. The activation of different signaling pathways may help to determine the optimal amount and duration of FSS needed to produce anabolic responses in bone.

Disclosures: S. Wadhwa, None.


Low Intensity, High Frequency Vibration May Increase Bone Formation in Aged Rats.H. Oxlund, B. S. Oxlund*, T. T. Andreassen. Dept of Connective Tissue Biology, University of Aarhus, Inst of Anatomy, Aarhus, Denmark.

The effect of low intensity, high frequency vibration on bone formation and bone strength was studied in an aged rat model. Twenty-three-month-old male rats were allocated randomly to the following groups: 1) start control, 2) mock vibrated control and 3) vibration at 45 Hz (3.0xg). Vibration was given 30 min/day for 90 days. During vibration the rats were placed in a box on top of the vibration motor. The amplitude of the vibration motor was 1.0 mm. The animals were labelled with calcein at day 79 and with tetracycline at day 86. The tibia mid-diaphysis was studied by mechanical testing and dynamic histo-morphometry. The vibration increased (2p=0.03) the periosteal bone formation rate (103x 1.66 +/− 0.24 cubic micrometer/day, mean +/− SEM) by 2 fold compared with the mock vibrated group (103x 0.82 +/− 0.27 cubic micrometer/day) at the tibia mid-diaphysis. Furthermore, the percentage labelled circumference of the vibrated group (35.5 +/− 4.3 %) was increased (2p<0.03) by 86 % compared with the mock vibrated group (19.1 +/− 5.4 %). These alterations did not result in significant increases in the breaking strength of the tibia diaphysis of these old rats. In conclusion, the results support the hypothesis of a possible beneficial effect of passive physical loading on the preservation of bone in aged animals.

Disclosures: H. Oxlund, None.


Physiological Levels of G-force Induces Cox-2 Gene Expression in MC-3T3-E1 Osteoblasts.M. Hughes-Fulford1, C. Li*2. 1Medicine, UCSF, Dept of Veterans Affairs and NCIRE, San Francisco, CA, USA, 2Lab of Cell Growth, NCIRE, San Francisco, CA, USA.

Physiological mechanical loading is required for maintenance of bone integrity and architecture. We have previously reported that as little as 15 minutes of physiological gravity force can induce MAPK and immediate early gene expression. Here we report that one hour after a 15 minute pulse of 120 μstrain of gravity force cox-2 was significantly induced over five fold. Levels of EP-1, 18s and cpla2 mRNA were not changed during this time-frame. The induction of cox-2 was inhibited 70% by MEK1/2 inhibitor U0126 (p<0.001) but was not effected by MEK1 or p38 MAPK specific inhibitors. Short-term physiological loading induced ERK 1/2 phosphorylation in a dose dependent manner with maximum phosphorylation saturating at mechanical loading levels of 12.g (p< 0.001) with no effect on total ERK. G-loading did not activate P38 MAPK or JNK. Additionally, a short duration gravity pulse resulted in the localization of phosphorylated ERK 1/2 to the nucleus while unloaded cells showed no concentration of pERK in the nucleus. The long-term consequence of a single 15 minute gravity pulse was a 64% increase in cell growth (p<0.001). U0126 significantly inhibited gravity induced growth by 50% (p<0.001). These studies suggest that physiological levels of compressive mechanical stress induce cox-2 message and protein synthesis as well as growth of MC3T3-E1 osteoblasts primarily through an ERK1/2 mediated pathway.

Disclosures: M. Hughes-Fulford, None.


T-Type Voltage Sensitive Calcium Channels Modulate the Mechanical Response of Osteoblasts to Fluid Shear.D. Liu1, E. C. Puente*2, J. J. Bergh2, Y. Shao*2, M. C. Farach-Carson2, R. L. Duncan1. 1Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN, USA, 2Biology, University of Delaware, Newark, DE, USA.

Osteoblasts express several different subtypes of voltage sensitive calcium channels (VSCC) that we postulate play significant roles in osteoblastic function. While we have previously shown that inhibition of L-type VSCC's significantly reduces bone formation in mice subjected to mechanical loading, this inhibition is incomplete. Patch clamp studies in MC3T3-E1 cells demonstrate the presence of a low voltage activated Ca2+ channels that exhibit kinetics consistent with those of the T-type VSCC. Immunocytochemistry and RT-PCR demonstrate the presence of both CaV3.1 (α1G) and CaV3.2 (α1H) subunits to the T-type VSCC in static MC3T3-E1 cells. To determine if activation of the T-type VSCC is important to the osteoblastic response to fluid shear, MC3T3-E1 cells were grown to confluency on collagen-coated (10 μg/cm2) glass slides, then subjected to 12 dynes/cm2 laminar shear using a parallel-plate flow chamber interfaced with a flow loop. One hour prior to shear application, cells were pretreated with either NiCl2 (5 mM) or mibefradil (1 μM) and these inhibitors were maintained in the medium for the duration of the experiment. Cells were exposed to 1 hour of fluid shear, followed by 3 hours post-incubation. After the first 30 minutes of post-incubation, medium samples were removed to assay for PGE2 release. After the 3 hour post-incubation, COX-2 expression and production were measured. Fluid shear produced a 5-fold increase in COX-2 production compared to static controls (p<0.001). NiCl2 significantly reduced this response, while mibefradil only moderately inhibited COX-2 production. Changes of mRNA expression of COX-2 with either NiCl2 or mibefradil inhibition reflected the protein results. PGE2 release in response to shear corresponded to the increase in COX-2 production compared to static controls (p<0.05), while both NiCl2 and mibefradil reduced this release (p<0.05). To examine the effect of shear on T-type VSCC expression, mRNA expression of α1H subunit was examined using RT-PCR. After 1 hour of post-incubation, α1H mRNA expression was enhanced 2.5-fold, with a further increase to 4.5-fold observed after 6 hours. By 24 hrs, the increase was 7.5-fold. These data suggest that fluid shear increases the expression and activity of T-type VSCCs (Cav3.1 and 3.2) and may contribute to the mechanical responses of osteoblasts.

Disclosures: D. Liu, None.


Comparison of Dentin Matrix Protein 1 (DMP1) Gene Expression with Experimental and Finite Element Strain Analysis in the Rat Ulnar Fatigue Model.J. Gluhak-Heinrich1, D. Pavlin1, S. P. Kotha*2, L. F. Bonewald3, M. B. Schaffler4, S. E. Harris5. 1Orthodontics, U. of Texas Health Science Center, San Antonio, TX, USA, 2Oral Biology, U. of Missouri at Kansas City, Kansas City, MO, USA, 3Oral Biology, U. of Missouri at Kansas City, Kansas City, MO, USA, 4Mount Sinai School of Medicine, New York, NY, USA, 5Oral Biology, U. of Missoui at Kansas City, Kansas City, MO, USA.

Osteocytes are thought to play critical roles in sensing and conveying messages to other osteocytes and to osteoblasts and osteoclasts. These candidate signals are thought to be required for bone homeostasis under various levels of mechanical and fatigue loading conditions. We have previously shown that the Dentin Matrix Protein I gene is highly and selectively expressed in alveolar osteocytes in vivo and is mechanically responsive in the tooth movement model (Gluhak-Heinrich et al, JBMR 2003). DMP1 function in osteocytes is most likely related to its role as a modulator and attenuator of mineralization. DMP1 therefore may play a role in modulating the local microenvironment within the osteocyte lacunae and canalicular walls. These modifications could then alter the dynamics of fluid flow through bone. In order to further explore the DMP1 gene as mechanically responsive in other bone loading systems, we investigated DMP1 expression in a rat ulnar fatigue model. Right ulnae were loaded at 4 Hz with a maximum force of 20N until there was a loss of 30% of the whole bone stiffness. These loading conditions cause considerable microdamage within the diaphyseal cortex. We then assayed for expression of DMP1 mRNA immediately after loading and 2 days later by quantitative in situ hybridization using P32 labeled RNA probes. In the ulnar midshaft, DMP1 expression was greatly increased on both the tension and compression sides of the diaphyseal cortex. However, a gradient of DMP1 expression in osteocytes near the periosteal surface as compared to osteocytes near the endosteal surface was observed upon quantitation of expression. This pattern of DMP1 expression reflects the known strain gradients in this bending model with higher strains expected near the surface and in the longitudinal axis near the midshaft region. We have now begun a detailed quantitative analysis of DMP1 expression in both the longitudinal axis at various distances from the mid-shaft and in the circumferential axis. By comparison of gene expression with experimental and finite element strain maps, we propose that levels of DMP1 gene expression may represent a sensitive readout of magnitudes of tensile and compressive strains, as well as torsional strains in bone.

Disclosures: J. Gluhak-Heinrich, None.


Tibial Cortical Strains Induced by Low-Level Vibrations Are Dependent on Genetic Make-up and Frequency of the Signal.S. Karnik*, E. Choi*, J. Jacobson*, B. Busa*, L. Donahue, C. Rubin, S. Judex. SUNY Stony Brook, Stony Brook, NY, USA.

Despite their low magnitude, vibrational mechanical stimuli induced at high frequencies (>20Hz) can be anabolic to trabecular bone. In genetically distinct strains of mice, we have previously demonstrated that the efficacy of these stimuli is dependent upon the genotype with trabecular bone of C3H/HeJ (C3H) mice being much less responsive than C57BL/6J (B6) mice. While these data may suggest that bone's mechano-sensitivity is regulated by the genome, the dramatic differences in bone mass and morphology between these two strains may alter the magnitude and pattern of the mechanical signals induced in the skeleton. Thus, it is possible that the differential tissue sensitivity is accounted for by interactions between genetic and mechanical factors. Here we collected preliminary data to investigate the different mechanical environments induced by vibrations in these two strains of mice. Single-element miniature strain gauges were implanted (cyanoacrylate) at the antero-medial surface of the proximal tibia of two B6 and two C3H mice. Strain data were recorded while the mice were subjected to a ground-based vertical oscillation at the frequencies of 22.5Hz, 45Hz and 90Hz, at accelerations of 0.2g. In low bone mineral density B6 mice, peak longitudinal normal strains generated in the tibial cortical shell decreased from 11.0 microstrain (μ) at 22.5Hz to 4.5μ at 45Hz to 1.1μ at 90Hz. Cortical normal strains induced in C3H mice at a frequency of 22.5Hz were much lower than in B6 (0.9μ) and were not affected by the frequency of the applied signal; normal strains at 45Hz and 90Hz amounted to 1.2μ. These data indicate a frequency dependency of vibrational mechanical deformations in B6 but not in C3H mice in which the generated strains were consistently low. While these data were collected from the cortical shell and we have only begun to extrapolate matrix strains and stresses to metaphyseal trabecular bone via finite element modeling, the differential mechanical environment induced by vibrations in these two mouse strains raises the possibility of both mechanical and genetic factors regulating trabecular bone adaptation.

Disclosures: S. Judex, None.


Cortical Bone Augmentation with Mechanical Loading Is Best at a Loading Frequency of 10 Hz.S. J. Warden*, C. H. Turner. Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.

Loading frequency is an important determinant of mechanically-induced adaptation in cortical bone. Researchers have shown an increase in bone formation with increasing loading frequency. This dose-response relationship is limited to loading frequencies below 10 Hz. Studies herein aimed to investigate cortical bone adaptation to loading frequencies of 1, 5, 10, 20 and 30 Hz. Two studies were completed in adult C57BL/6 mice using the ulna axial compression loading model. In the first study, the histomorphometric response of the ulna was studied when loaded for 120 cycles/day for 3 days at one of the five frequencies and one of three strain magnitudes (935, 1750 or 2566 microstrain). In the second study, the change in the ulna mechanical properties was studied following loading for 5 mins/day, 3 days/week for four weeks at one of the five frequencies and one of two strains magnitudes (935 or 1914 microstrain). Results of the first study revealed that loading at each frequency at 2566 microstrain increased all bone formation measurements. The increase in bone formation rate was greatest with loading at 10 Hz than at any other frequency. Results of the second study showed that loading at any frequency and 1914 microstrain increased the energy to failure by between 17.9% and 29.2%, and the ultimate force by between 5.8% and 17.7%. There was no effect of loading frequency on these changes. In both studies, loading at 935 microstrain had no effect irrespective of frequency. The combination of these results shows that cortical bone adaptation to increasing loading frequency does not show a simple dose-response relationship. The bone formation response appears to peak with a loading frequency of 10 Hz, and further increases in frequency do not result in subsequent increases in adaptation. Consequently, for optimal cortical bone building purposes loading should be performed at 10 Hz.

Disclosures: S.J. Warden, None.


Is Animal Age a Factor in the Response of Bone to Spaceflight?E. M. Morey-Holton1, L. P. Garetto*2, S. B. Doty*3, B. P. Halloran4, R. T. Turner5. 1NASA Ames Research Center, Moffett Field, CA, USA, 2Indiana University Schools of Dentistry and Medicine, Indianapolis, IN, USA, 3Hospital for Special Surgery, New York City, NY, USA, 4University of California and Veterans Affairs Medical Center, San Francisco, CA, USA, 5Mayo Foundation, Rochester, MN, USA.

The rodent bone response to spaceflight may be influenced by a multitude of factors including flight duration, strain (genetic background), and housing. Age may also play a role in the skeletal response. Weanling rats show fewer bone changes than older rats. To determine if the long bones of weanling rats are insensitive to weight-bearing, two spaceflight experiments and a hindlimb unloading experiment were conducted. The hindlimb unloading experiment with individually housed rats was conducted simultaneously with a 9d shuttle flight using 34d old group-housed male rats while 38d old male rats were individually housed during the 14d spaceflight. All animals were injected with fluorochromes before unloading and euthanized at various times (0--14d) following reloading. If no differences in body weight, bone length, or bone formation at the tibiofibular junction were noted at the different time points, data were combined for each group. No significant differences in body weight were found at any time period. The humerus, tibia, and femur elongated significantly during the flight period with no difference in lengths between groups. The group-housed flight rats showed no change in cortical bone formation rate compared to preflight values, flight controls, or vivarium controls. However, the hindlimb unloading group showed a significant 30% decrease in bone formation rate compared to all other groups. The individually-housed animals flown for 14d showed ∼10% suppression of cortical growth. Older singly-housed flight animals appear to show equal or greater bone changes compared to hindlimb unloaded rats. We speculate that the mechanical threshold required for cross-sectional bone growth is reached in group-house weanling rats during spaceflight, perhaps through physical interactions, and that weanling animals are sensitive to loading. However, the threshold is not fully reached in either singly-housed flight or hindlimb unloaded weanling rats. We conclude that age, housing, flight duration, and strain (genetic background) have important roles in rodent skeletal responses to spaceflight.

Disclosures: E.M. Morey-Holton, None.


Urinary 25-Hydroxyvitamin D Binding Activity Is Directly Associated with Urinary Sodium of African-American Men during Head-Down Tilt Bed Rest.M. Thierry-Palmer1, S. Cephas*1, H. R. Scott*1, P. Sayavongsa*1, M. Pasquali*2, E. Schwartz*2, R. Lapu-Bula*3, E. Ofili*3. 1Biochemistry, Morehouse School of Medicine, Atlanta, GA, USA, 2Pathology, University of Utah, Salt Lake City, UT, USA, 3Medicine, Morehouse School of Medicine, Atlanta, GA, USA.

The Dahl salt-sensitive rat, a model for salt-induced hypertension, excretes protein-bound 25- hydroxyvitamin D (25-OHD) into urine during low salt intake and excretion is markedly increased during high salt intake (J. Nutr. 133:187--190, 2003). Since the prevalence of salt sensitivity in the African-American population is greater than that in the Caucasian American population, we determined whether healthy normotensive African-American men (29 +/− 2 years old; 122+/− 3 mm Hg, n = 19) involved in a head-down tilt bed rest study excreted 25-OHD binding protein(s) into urine. The subjects were fed a low salt (50 mmol/day, 7 subjects) or high salt (200 mmol/day, 12 subjects) diet during seven days of head-down tilt bed rest. Plasma was collected at day 0 and day 7 and 24 h urine was collected at day 1 and day 7. Binding activity in the urine was determined by binding to radio-labeled 25-OHD3 (20,000 dpm) in the presence and absence of 200-fold un-labeled 25-OHD3. Urinary 25-OHD binding activity varied directly with urinary sodium (r = 0.70, P = 0.0002), as did urinary protein (r = 0.56, P = 0.0004). Baseline values for plasma 25-OHD (26 +/− 4 nmol/L), 24,25-dihydroxyvitamin D (2.4 +/− 0.4 nmol/L), and parathyroid hormone (30 +/− 3 pg/mL) were not significantly affected by seven days head-down tilt bed rest or by high salt intake. Plasma 1,25-dihydroxyvitamin D concentration of subjects on the low salt diet was significantly lower at day 7 than at baseline (55 +/− 17 vs. 120 +/− 10 pmol/L, P = 0.001). Urinary deoxypyridinoline, a marker of bone resorption, was significantly increased from day 1 to day 7 of head-down tilt bed rest (P = 0.01). In conclusion, increased pyridinium cross-links excretion was the major change related to bed rest. Low salt intake, in contrast to high salt intake, resulted in significantly decreased plasma 1,25-dihydroxyvitamin D concentration at the end of bed rest, compared with baseline. Further studies are needed to determine whether there is a contribution of bed rest, as well as salt, to the urinary excretion of 25-OHD binding protein(s).

Disclosures: M. Thierry-Palmer, None.


The Effectiveness of Osmotic Mini-Pumps to Deliver Fluorochrome Labels in Rats.L. L. Venton*1, G. Evans*2, E. Holton*3, E. Hill*4, R. Turner2, T. Wronski5, D. Bikle1, B. P. Halloran6. 1Division of Endocrinology, Veterans Affairs Medical Center, San Francisco, CA, USA, 2Mayo Foundation, Rochester, MN, USA, 3NASA Ames Research Center, Moffett Field, CA, USA, 4Lockheed Martin Space Operations, Moffett Field, CA, USA, 5Department of Physiological Sciences, University of Florida, Gainsville, FL, USA, 6Division of Endocrinology, Departments of Medicine and Physiology, Veterans Affairs Medical Center, University of California, San Francisco, CA, USA.

Automated delivery of bone fluorochrome labels could be invaluable in remote locations such as spaceflight. We developed an automated system using polyethylene tubing attached to Alzet osmotic minimpumps to deliver calcein subcutaneously on 5d5 of a 15d study (expt 1) or d12 of an 21d study (expt. 2). Polyethylene-60 tubing was cut to 6.55cm for d5 and 15.75cm for d12. Tubing was sterilized with ethylene oxide, rinsed with fetal bovine serum, and filled with sesame oil (24μl for d5 and 66μl for d12), a small bolus (6μl) of calcein (2.5mg/kg) and then a small bolus (6μl) of sesame oil. The calcein end of the tubing was attached to a saline filled osmotic pump. Male, Fischer 344 rats, 6 weeks or 6 months old, were either sham operated or implanted with minipumps. Three days after surgery, rats were divided into hindlimb unloaded or control groups. Demeclocycline was injected, s.c., on day 0, and calcein was delivered s.c. on day 5 (expt. 1) or 12 (expt. 2). Bone formation rate (BFR) was measured at the tibiofibular junction between the pump administered label (d5 or d12) and the periosteal surface (See Table). 

Table  . Periosteal Bone Formation Rate (um2/day ± SD)
  1. * ND - no detectable bone formation

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BFRs were not significantly different between the animals receiving calcein by conventional s.c. injection or by minipump. Young animals, as expected, showed less of a decrease in BFR in response to hindlimb unloading than did older animals. These data demonstrate that the minipumps can accurately deliver bone labels at the specified days and that the pump-tubing system could be utilized for many drugs/labels in various remote environments such as space.

Disclosures: B.P. Halloran, None.


A Dynamic Bone Architecture Produced by a Simple Reaction Diffusion Model.K. Tezuka1, Y. Wada*2, A. Takahashi*3, M. Kikuchi*3. 1Graduate School of Medicine, Gifu University, Gifu, Japan, 2Department of Mechanics and Systems Design, Tokyo University of Science, Suwa, Japan, 3Department of Science and Technology, Tokyo University of Science, Noda, Japan.

Bone is a complex system accompanied with adaptation and repair functions. To understand how bone cells can create a structure adapted to the mechanical environment, we propose a computer model for bone remodeling based on a reaction-diffusion system influenced by mechanical stress. In this model, a hypothetical stimulator of bone formation was produced in response to mechanical stress, and induced another hypothetical molecule that inhibited the activity of the stimulator. These two molecules interacted with each other, diffused independently, and formed a static distribution depending on the local stress value. Bone formation and resorption changed the shape of the model depending on the local concentrations of these two molecules. When an external mechanical stress was applied to a 2-dimensional model of human femoral neck, stimulated bone formation and subsequent activation of bone resorption produced an efficient adaptation of the internal shape of the model bone to a given stress, and demonstrated major structures of trabecular bone similar to those seen in real bone tissue (figure). Parameter analysis suggested that cell to cell communication via diffusion of a hypothetical stimulator of bone formation is important for the efficient adaptation of the entire system. Stepwise change of a parameter affecting the balance between formation and resorption demonstrated deformation of bone structure during osteoporosis. The loss of trabecular bone was much faster than that of cortical bone as observed in osteoporotic patients. Therefore, this simple model will give us an insight how bone cells create a sophisticated architecture adapted to mechanical environment, and serve as a useful tool to understand both physiological and pathological state of bone from structural information.

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Disclosures: K. Tezuka, None.


Interactive Effects of Parathyroid Hormone and Mechanical Stress on Nitric Oxide and Prostaglandin Production by Mouse Osteoblastic Cells.A. D. Bakker*1, E. H. Burger*1, M. Joldersma*1, C. Netelenbos2, J. Klein-Nulend1. 1Oral Cell Biology, ACTA-VU, Amsterdam, Netherlands, 2Endocrinology, Vrije Universiteit Medical Center, Amsterdam, Netherlands.

Local bone remodeling is regulated by mechanical usage, but the overall level of remodeling is determined by systemic hormones such as parathyroid hormone (PTH). Several animal studies suggest that there is an interaction of PTH and strain at the tissue level.

It is, however, still unclear how the effects of mechanical loading and PTH interact. To investigate whether PTH can modulate mechanotransduction by bone cells, we examined the effect of human PTH-(1--34) on fluid flow-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production by mouse osteoblastic cells in vitro.

Primary bone cell cultures were obtained as outgrowth from collagenase-stripped fragments of the long bones of adult mice. The bone cells were subjected to mechanical stress in the form of a pulsating fluid flow (PFF; 0.6 ± 0.3 Pa at 5 Hz), in the presence or absence of 10−9M human PTH (1--34). Medium samples were taken at 5 and 30 min after start of fluid flow, and nitric oxide (NO) and prostaglandin E2 (PGE2) production were determined as parameter of bone cell responsiveness. NOS activity was determined using a NOSdetect Assay Kit, in bone cell cultures that were subjected to 10−9 M PTH for 45 min.

PFF stimulated both NO and PGE2 production by 2-fold. In the absence of PFF, PTH also caused a 2-fold increase in PGE2 production, but NO release was not affected. Simultaneous application of PFF and PTH nullified the stimulating effect of PFF on NO production, while PGE2 production was only stimulated by two-fold. Treatment with PTH alone reduced NO synthase (NOS) enzyme activity to undetectable levels.

We speculate that PTH prevents stress-induced NO production via the inhibition of NOS, which will also inhibit the NO-mediated upregulation of PGE2 by stress, leaving only the NO-independent PGE2 upregulation by PTH. These results suggest that mechanical loading and PTH interact at the level of mechanotransduction. As both NO and PGE2 inhibit osteo-clast activity in short term experiments, we now propose that strain-derived NO and PGE2 serve to locally inhibit osteoclastic bone resorption. The opposite effects of stress and PTH on NO production as found in this in vitro study, may thus relate to their opposite effects on osteoclastic bone resorption in vivo.

We conclude that PTH and PFF interact in the production of PGE2 and NO, which may be part of the regulation mechanism of PTH and mechanical loading on bone turnover in vivo.

Disclosures: A.D. Bakker, None.


The Effect of Selective Agonist for Prostaglandin E Receptor Subtype EP4 (ONO-4819) on the Cortical Bone Response to Mechanical Loading.H. Hagino1, M. Kuraoka*2, Y. Kameyama*2, S. Fukata*2, T. Okano2, R. Teshima*2. 1Rehabilitation Division, Tottori University, Yonago, Japan, 2Department of Orthopedic Surgery, Tottori University, Yonago, Japan.

Purpose: The cortical bone response under selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) administration was evaluated using a 4-point bending device to clarify the relationship between the effect of ONO-4819 and mechanical loading.

Materials and Methods: Thirty-six 6-month-old female Wistar rats were used. Rats were randomized into 3 groups (N=12/group); EP4-low (6μg/kg BW/day), EP4-high (60 μg/kg BW/day), and EP4-v (vehicle). ONO-4819 was subcutaneously injected in the back twice a day for three weeks. Loads on the right tibia were applied in vivo by 4-point bending. The tibia was loaded at 35 N for 36 cycles at 2Hz three days a week for three weeks and then rats were sacrificed. Bone mineral content (BMC) of the whole body and bone mineral density (BMD) of the total and regional tibia (the region with maximal bending at the central diaphysis) were measured by dual energy X-ray absorptiometry. Histomorphometry was performed at the entire periosteal and endocortical surface of the tibiae.

Results: Whole body BMCs showed a significant difference between EP-v and EP4-high. Regional BMDs of the right tibia (loaded site) were higher in EP4 groups and loaded site, showing significant differences between loaded and non-loaded site and between EP-v and EP4-high. ONO-4819 showed an additive effect on loading at the regional BMD of the tibia. Histomorphometrically labeled surface was higher in EP4 groups than in the vehicle groups, and it was also higher at loaded site than non-loaded site. Bone formation rate was elevated by both EP4 administration and loading.

Conclusion: Selective agonist for PGE receptor EP4 (ONO-4819) has an additive effect on bone response to in vivo external loading by 4-point bending.

Disclosures: H. Hagino, None.


Side-to-side Difference in Bone Mineral Density of Tennis Players' Forearms: Determinant Parameters.G. Ducher*1, C. Jaffré*1, E. Lespessailles2, C. L. Benhamou2, D. Courteix1. 1Laboratoire de la Performance Motrice, UFR STAPS Orleans, Orleans, France, 2Ipros, CHR Orleans, Orleans, France.

A large side-to-side difference in bone mineral density (BMD) has been reported between both forearms in tennis players. It is assumed that the genetic, hormonal and nutritional influences are similar in the dominant and non-dominant forearms. Such difference is attributed to the mechanical loads encountered by the dominant arm during the tennis stroke. The aim of the study was to identify the training and muscular parameters explaining the greater BMD at this site.

Thirty male tennis players (age: 26.7 ± 6.9 years, total training time: 4337 ± 3801 hours) participated in this study. Grip strength (GS) was measured with a hand-held dynamometer. Lean tissue mass at the forearm and hand (LTM), bone mineral content (BMC), bone area and BMD were determined at the distal radius by DXA. The side-to-side differences were expressed as percentage of the non-dominant values (Δ).

BMD was higher at the dominant radius (p<0.0001), with the largest difference obtained at the ultra-distal region (+9.99 ± 5.81%). The BMC difference was greater (p<0.01), ranging from 15.14 ± 7.99% at the ultra-distal region to 17.30 ± 9.18% at the mid-distal region (p<0.0001). The side-to-side difference in bone area was larger (p<0.01) at the mid- and third-distal regions (+11.61 ± 6.65% and +9.67 ± 6.12%, respectively) than at the ultra-distal region (+4.72 ± 4.58%). BMC was still higher at the dominant side after adjusting the value for bone area, except at the mid-distal region. ΔLTM and ΔGS were 17.71 ± 10.33% and 14.34 ± 7.42% respectively (p<0.0001). ΔBMC and ΔBMD at the ultra-distal radius correlated with ΔLTM (r = 0.52, p<0.01 and r = 0.43, p<0.05, respectively). ΔBMC at the ultra-distal region correlated with ΔGS (r = 0.39, p<0.05). The starting age of playing was associated with ΔBMC at the ultra-distal radius (r = -0.42, p<0.05). The total training time correlated with ΔBMC at the third-distal region and ΔBMD at the mid-distal region (p<0.05).

The originality of these results was that a correlation between the side-to-side difference in grip strength and in BMC at the ultra-distal radius was observed in tennis players. Tennis playing seems to stimulate higher BMC at the dominant radius through muscular activity and mechanical vibrations. The higher BMC at the ultra-distal site was mainly related to an increase in BMD whereas the mid- and third-distal sites showed a preferential increase in bone area. The side-to-side difference in bone parameters appears to be greater in players who started tennis earlier and in those who have experienced a longer total training time.

Disclosures: C.L. Benhamou, None.


Greater Trabecular Bone Connectivity in the Knee of Female Collegiate Gymnasts.C. M. Modlesky*1, J. M. Slade*2, S. Majumdar3, A. Narasimhan*3, G. A. Dudley*2. 1Department of Health, Nutrition and Exercise Sciences, University of Delaware, Newark, DE, USA, 2Department of Exercise Science, The University of Georgia, Athens, GA, USA, 3Magnetic Resonance Science Center, University of California, San Francisco, CA, USA.

Frequent participation in high-load physical activity is associated with increased areal bone mineral density (aBMD); however, its connection to human trabecular bone microarchitecture, an important contributor to bone strength, has not been investigated. The purpose of this study was to determine if the apparent bone volume to total volume (appBV/ TV), trabecular number (appTb.N), and trabecular thickness (appTb.Th) are higher and trabecular separation (appTb.Sp) lower in the distal femur and proximal tibia of artistic gymnasts, a group that participates in a substantial amount of high-load physical activity, than controls. Seven female collegiate gymnasts (GYM) and 7 female controls (CON) not different in age, height and weight were tested. Axial images of the distal femur and proximal tibia were collected on a 1.5 T magnetic resonance imager (195×195×1000 microns) using a 3D fast gradient-echo sequence (TE 4.5 ms; TR = 30 ms; 40 degree flip angle) and measures of trabecular bone microarchitecture were assessed using previously described procedures. Results are listed in the table below. Higher appBV/TV (15.6 %, Cohen's d (d) = 1.15) and appTb.N (8 %, d = 1.53) and lower appTb.Sp (13.8 %, d = 1.36) were observed in the proximal tibia of GYM vs CON (P < 0.05). Although the differences were less pronounced and not statistically significant (P > 0.05), large effect sizes (d > 0.8) indicated a similar pattern in the distal femur, with higher appBV/TV (9.4 %, d = 0.87) and appTb.N (5.2 %, d = 0.91) and lower appTb.Sp (9.0%, d =0.93). The findings suggest that the high-load physical activity performed during gymnastics activity is associated with a greater connectivity of trabecular bone in the knee. Future studies assessing the effects of mechanical loading on trabecular bone structure in humans are warranted. 

Table  . Measures of Trabecular Bone Microarchitecture (*Significant group difference, P <0.05)
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Disclosures: C.M. Modlesky, None.


The Effects of Additional Weightbearing during Exercise and Estrogen on Bone Strength in Female Rats.A. M. Tromp*1, J. Skovhus Thomsen*2, L. Mosekilde*2, N. Bravenboer1, P. Lips1. 1Endocrinology, VU Medical Center, Amsterdam, Netherlands, 2Cell Biology, Institute of Anatomy, Aarhus, Denmark.

The aim was to investigate the effects of additional weightbearing during exercise and estrogen status on bone strength in female rats.

Sixty female Wistar rats were assigned to sedentary (SED), ovariectomized (SED+OVX), or ovariectomized with estrogen replacement (SED+OVX+E2) groups, or to the exercise groups EX, EX+OVX or EX+OVX+E2. Exercise consisted of running with a backpack (load 20% of bodyweight) for 19 weeks, 5 days/week, 15 min/day. Thereafter, rats were sacrificed and the femur and 4th lumbar vertebra (L4) were prepared for mechanical testing. Testing consisted of 3 point-bending of the femoral diaphysis, compression of the femoral neck and compression of L4. Load-deformation curves and data on maximum load (Fmax), maximum stress (σ max) and Young's modulus (E) were obtained. After testing, L4 specimens were ashed and apparent ash density (ρ) was determined.

In the 3 point-bending test of the femur, Fmax did not differ significantly between the groups (p>0.05). However, in the femoral neck test, Fmax was significantly lower in the EX+OVX and SED+OVX groups (p=0.025; fig. 1). For L4, Fmax (p<0.001) and σ max (p<0.001; fig.2) were significantly lower in the SED and SED+OVX groups but E was not (p>0.05). ρ was significantly lower in the EX+OVX and SED+OVX groups (p<0.001).

The strength of the femoral neck was decreased in case of estrogen deficiency, while the femoral diaphysis was not. It therefore seems, that the cortical bone of the femoral diaphysis is less sensitive to the effects of exercise and estrogen than the femoral neck which consists of both trabecular and cortical bone. In L4 however, bone strength was increased after exercise and in case of estrogen replacement in the sedentary state. Apparent ash density was decreased after ovariectomy. It therefore seems that, despite a lower ash density of the lumbar spine, exercise prevented loss of bone strength in the estrogen deficient state.

Figure Figure 1.


Figure Figure 2.


Disclosures: A.M. Tromp, None.


Body Weight Supported Treadmill Training in Individuals with Incomplete Spinal Cord Injury: Effects on Bone Mass and Body Composition.L. M. Giangregorio*1, C. E. Webber2, S. M. Phillips*1, A. L. Hicks*1, N. McCartney*1. 1Kinesiology, McMaster University, Hamilton, ON, Canada, 2Nuclear Medicine, Hamilton Health Sciences, Hamilton, ON, Canada.

The aim of the present study was to evaluate bone mass changes in men and women with an incomplete spinal cord injury (SCI) after 12 months of body weight supported treadmill training (BWSTT). Thirteen individuals (2 females, 11 males) who had sustained a traumatic, incomplete SCI participated in thrice-weekly BWSTT for a total of 144 sessions (∼12 months). The level of lesion ranged from C4 to T12, the average age of the participants was 29 years old, and average time post-injury was 7.70 years (range 1.17 to 24 years). Bone mineral densities of the proximal femur, spine, and whole body were measured using whole body dual-energy x-ray absorptiometry (DXA) scans at baseline and after completion of training. Significance was set at p < 0.05. The efficacy of BWSTT for improving walking ability in this group of participants has been published previously (Hicks, McCartney, et al. 2002). Repeated measures multivariate analysis indicated that significant changes occurred at the proximal femur and whole body after 12 months of BWSTT. Post hoc repeated measures analysis of variance revealed a significant decrease in total bone mineral content and area at both the right and left proximal femur, however bone mineral density at these sites was not significantly different after BWSTT. Whole body bone mineral density was significantly reduced after BWSTT, and whole body bone mineral content exhibited a trend towards a decrease (p<0.08). Participants did experience significant increases in lean mass, from 45.9kg±2.3 to 47.8kg±2.4 (mean ±SE) before and after training. On average, lean mass in males increased from 48.1kg±2.2 to 50.2kg±2.2, and in females from 33.9kg±3.0 to 34.7kg±1.9. Total body fat mass did not change significantly after BWSTT. Body weight supported treadmill training is an intervention that has been shown to improve walking ability in individuals with spinal cord injury, and may also be a promising intervention for increasing lean mass. However, our data suggests that it does not increase, or prevent the loss of bone in individuals with chronic incomplete spinal cord injury.

Disclosures: L.M. Giangregorio, None.


Effect of Impact Loading and Dietary Calcium on the Rat Ulna.J. M. Welch1, C. M. Weaver1, C. H. Turner2. 1Foods and Nutrition, Purdue University, West Lafayette, IN, USA, 2School of Medicine, Indiana University, Indianapolis, IN, USA.

The relationship between dietary calcium and impact exercise on bones is unclear. We investigated what effects impact loading and a diet marginally low in calcium (Ca) have on growing bones. Weanling F--344 female rats were randomized to either a low Ca (CaLow; 0.2%) or a control (CaCon; 0.5%) diet and were further divided into no impact (F0) or impact (F45) loading groups. Impact loaded rats were dropped from a height of 45 cm for a total of 400 impacts over 8 weeks. After completion of the trial, mechanical strength of each ulna was measured by axial compression test. Peripheral quantitative computed tomography (pQCT) was used to measure the cross-sectional area (CSA) and volumetric bone mineral density (vBMD) in whole (tot), cortical (cort), and trabecular (trab) bone at the olecranon process (87% distal) and shaft (45% distal). Mechanical tests indicated that dietary Ca did not significantly affect ulnar shaft strength in either F0 or F45 groups. However, impact loading improved ultimate force by 38.2% in the F45+CaLow group (p<0.01) and 32.5% in the F45+CaCon group (p<0.01) when compared to the F0+CaLow and F0+CaCon groups respectively. Analysis by pQCT also indicated that the shaft was affected by impact loading but not diet. At this site, vBMDcort was not affected, but CSA-cort of the F45+CaLow and F45+CaCon groups was 10.6% and 13.2% greater than that of the F0+CaCon rats (both p<0.001). In the olecranon, Ca intake affected only the F0 rats while impact loading influenced both the CaLow and CaCon groups. In the F0 rats, the CaLow diet produced a 5.1% lower vBMDtot (p<0.05) with a concomitant 2.4% decrease in vBMDcort (p<0.01). Impact loading increased vBMDtot in both dietary groups but the effect was greater in the CaLow (9.1%; p<0.001) than the CaCon rats (4.3%; p<0.05). This result was accompanied by a 2.6% increase (p<0.01) in vBMDcort of the CaLow group with impact loading. Although CSAtot did not differ between any treatments, impact loading increased the CSAcort of proximal ulna in both F0+CaLow (18.3%; p<0.001) and F0+CaCon rats (14.4%; p<0.01) while decreasing CSAtrab in the F0+CaLow (-45.3%; p<0.001) and F0+CaCon (-18.7%; n.s.) groups. Overall, impact loading increased the strength, CSAcort but not vBMDcort in the shaft, and the vBMDtot, vBMDcort, and CSA-cort in the proximal metaphysis of the growing ulna in both Ca sufficient and deficient rats. Bones of rats not subject to impact loading were adversely affected by a moderate calcium deficiency, but this effect on the skeleton was averted by impact loading. These results show that impact loading can provide considerable osteogenic stimulation to growing bones in the presence or absence of sufficient dietary calcium.

Disclosures: J.M. Welch, None.


The Aryl Hydrocarbon Receptor: What Is its Role in the Skeleton?J. E. Puzas1, E. Ryan*2, H. Drissi1, R. J. O'Keefe1, M. J. Zuscik1, R. N. Rosier1, E. M. Schwarz1, T. A. Gasiewicz*2. 1Department of Orthopaedics, University of Rochester School of Medicine, Rochester, NY, USA, 2Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY, USA.

The aryl hydrocarbon receptor (AHR) was discovered over two decade ago. Its role has been singularly characterized as a mediator of toxic molecules. Its ligands include agents such as dioxin, TCDD and other aromatic hydrocarbons. Moreover, it functions as a polymeric transcription factor for the activation of a number of genes such as the cytochrome p450 oxidases and cyclo-oxygenase 2. We have recently identified the presence of this receptor in developing skeletal elements and osteoblasts. However, its endogenous ligand and physiological role remain undiscovered.

We utilized cultures of osteoblasts derived from neo-natal rat calvaria as well as tissue specimens from both wild type and AHR -/- mice. AHR is expressed in primary cultures of osteoblasts that are induced to differentiate. Both the mRNA and protein levels of the receptor increase through day 15 at which point they begin to decline (Figure 1A). This pattern of expression parallels the induction of a number of early bone-specific genes involved in the maturation of osteoblasts. We also show by immunocytochemistry that the receptor is present in calvarial osteoblasts (Figure 1B). AHR is absent in AHR -/- mice. AHR levels are up-regulated by other toxic agents such as lead (Pb). Our data also demonstrate that the AHR functions as a transactivating receptor in osteo-blasts as evidenced by its ligand-dependent migration to the nucleus and its association with known DNA response elements. Dioxin exposure for 4 hours to osteoblasts in culture induces the expression of Cyp1A1 and Cox II protein levels.

Together these data imply that the AHR may not only mediate the effect of aromatic toxicants on bone cells but, with an as yet unidentified ligand, be involved in the differentiation pathways of the osteoblast.

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Disclosures: J.E. Puzas, Lilly Pharmaceuticals 8; Merck Pharmaceuticals 8; Procter & Gamble Pharmaceuticals 8.


Functional Elements for Transactivation by PTHrP Are Preserved in Both Human and Mouse RANKL Gene Promoters.R. Kitazawa, S. Kitazawa. Division of Molecular Pathology, Kobe University Graduate School of Medicine, Kobe, Japan.

The role of PTHrP in osteolytic bone metastasis has been linked to the promotion of osteoclastogenesis by the upregulation of RANKL expression on osteoblasts through type I PTH/PTHrP receptor. We have previously shown that PTHrP treatment of osteoblastic cell increases RANKL mRNA expression by Northern blotting as well as the transcription rate of the gene by nuclear run-on studies; forskolin (FK) shows the inductive effect, whereas H89 abolishes the effect of PTHrP. In this study, to elucidate the molecular mechanism transducing PTHrP signaling, the PKA pathway on human and mouse RANKL gene transcription was analyzed. In both human and mouse RANKL promoters, the basic structure composed of inverted TATA- and CAAT-boxes and VDRE was well preserved. Several CRE-like sequences, although not canonical, were detected in both promoters. By EMSA screening of CRE-like sequences, mouse -940/-933 (TGAGGTCA) and human -1559/-1552 (TAACGTCA) showed specific protein-DNA binding to the nuclear extracts from PTHrP-treated cells; the supershift with an anti-CREB1 antibody was demonstrated. In transfection studies using a series of deletion mutants of mouse (m) and human (h) promoters (0.5--2.3kb insert), PTHrP increased luciferase activity in m-1050-LUC (210%) and h-1974-LUC (180%), but not in m-723-LUC and h-1228-LUC, indicating that m-940/-933 and h-1559/-1552 could be functional CRE. Furthermore, FK enhanced promoter activity in m-1050-LUC and h-1974-LUC, and H89 abolished the inductive effects of PTHrP; inhibitors for p38MAPK (SB203580) and MEK (PD98059) did not suppress the effects of PTHrP. Moreover, reflecting the overlapping of CRE (-940/-933) by VDRE (-937/-922; AGGTCAGCCTGGTTCA) in the mouse RANKL promoter, co-treatment with PTHrP and 1alpha 25(OH)2Vitamin D3 did not exceed the maximum inductive effect of D3 alone. On the other hand, PTHrP and D3 additively increased promoter activity of the human RANKL gene where VDRE (-1584/-1570) and CRE (-1559/-1552) exist separately. These data suggest that 1) the PKA signaling pathway on RANKL gene transactivation is preserved in both mouse and human, 2) CRE and VDRE localize closely and comprise a crucial enhancer element in RANKL gene promoter.

Disclosures: R. Kitazawa, None.


Inhibition of Osteoblast Differentiation by TNF-α Is Associated with Suppression of Osterix Expression.L. C. Gilbert, X. He*, A. Kauppi*, J. Rubin, M. S. Nanes. Division of Endocrinology, Department of Medicine, Emory University, VA Medical Center, Decatur, GA, USA.

Tumor necrosis factor-alpha (TNF) plays an important role in the pathophysiology of osteoporosis and inflammatory arthritis by stimulating bone resorption and inhibiting new bone formation. We have previously shown that TNF is a potent inhibitor of osteoblast differentiation in experiments using fetal calvaria cells, marrow stromal cells, or clonal MC3T3E1 preosteoblasts. In addition, we have shown that TNF inhibits the expression of RUNX2 (Cbfa1/AML3/Pebp2alphaA), a critical transcription factor required for differentiation of osteoblasts from their pluripotent progenitors. Here, we test the hypothesis that osterix (Osx), another critical transcription factor acting downstream of RUNX2, is also regulated by TNF. Using the MC3T3E1 pre-osteoblast cell culture model, we used realtime PCR to measure the effect of TNF on steady state levels of Osx mRNA. MC3T3E1 cells were plated on day 0 in MEM + 10% FBS. L-ascorbic acid (50 μg/ml) was added to cells on day 1 and TNF (10 ng/ml) beginning on day 2. Replicate control and TNF-treated cultures were harvested for RNA at 4, 8, and 24 hours after addition of the TNF. TNF reduced the steady state level of Osx mRNA to 55% at 4 hrs, 23% at 8 hrs, and 5% of control cultures at 24 hrs. To determine if the effect of TNF was dose dependent, cells were treated on day 2 of culture with 0.1 to 10 ng/ml. The effect of TNF was dose dependent with an IC50 of 0.5--1 ng/ml, a value similar to that observed for inhibition of RUNX2 mRNA and for TNF inhibition of osteoblast differentiation. In order to determine if TNF reduced Osx mRNA by destabilization of the mRNA half-life, cells were treated with TNF and new RNA synthesis was inhibited by actinomycin D. RNA was isolated at time points between 2--14 hrs following addition of the actinomycin D and residual Osx mRNA was measured by real-time PCR. The half-life of Osx mRNA was short. TNF destabilized Osx mRNA, suggesting that this could contribute, in part, toward the reduction in Osx expression. We conclude that TNF inhibits the expression of Osx mRNA. TNF inhibition of transcription factors required for the differentiation of the osteoblast phenotype, including RUNX2 and Osx, may contribute to skeletal loss in the postmenopausal state and in rheumatoid arthritis.

Disclosures: L.C. Gilbert, None.


The Extracellular Matrix Protein hCYR61 (CCN1) Modulates Signal Transduction Pathways Associated with Proliferation and Cell Differentiation in Human Osteoblasts.N. Schuetze1, F. Graedler*2, M. Kunz*1, S. Balling*1, C. Hendrich*1, J. Eulert*1, J. Adamski*2, F. Jakob*1. 1Molecular Orthopaedics, Orthopaedic University Hospital, Wuerzburg, Germany, 2Institute of Experimental Genetics, GSF National Research Center for Environment and Health, Neuherberg, Germany.

The human cystein rich protein 61 (hCYR61) belongs to the CCN family of proteins. They act in cellular processes such as differentiation, proliferation, tissue regeneration and angiogenesis. Previously, we have shown that the hCYR61 protein expression in human bone correlates with conditions of remodeling and repair. Here we aimed to elucidate the hCYR61 function in human osteoblasts using cDNA microarray analysis. The protein was expressed in the baculovirus system as an IgGFc fusion protein and purified by affinity chromatography with protein G sepharose. Array analysis was performed to compare expression profiles of RNA from hFOB cells treated or not with rhCYR61 for 24 hours. The RNA samples were transcribed and indirectly labelled with the fluorescent dyes Cy3 and Cy5. “Colour flip” was done to aim for the identification of reproducibly regulated gene products only. Raw data were analyzed with the GenePix software and an in-house developed program for data normalization and extraction. RT-PCR was performed with the identical RNA and with RNA from additional time points of rhCYR61 treatment to validate array results and to find further targets. The rhCYR61 protein was detected as a single band in SDS electrophoresis followed by silver staining and western blotting. The chip contained 1920 PCR products spotted in duplicate for genes involved in bone cell development and function. Array analysis showed 28 reproducibly regulated gene products (= candidates; 13 up, 15 down, cut-off 2-fold regulation). Two thirds of the candidates proved to be regulated by rhCYR61 in RT-PCR analysis. Regulated gene products were membrane receptors (including Notch and Hedgehog signal pathway components) as well as intracellular signal molecules and transcription factors, mostly associated with cell proliferation and differentiation. Additional RT-PCR analysis identified downstream target genes of the Notch pathway to be regulated by rhCYR61. Results were coherent with the array analysis and indicate that downstream mediators (Hey and Hes genes) are regulated by rhCYR61. Interestingly, several regulated gene products belonged to the Hedgehog signal transduction pathway as membrane components or as downstream mediators, further strenghtening the validity of expression profiling with cDNA microarrays and RT-PCR. hCYR61 appears to be a control factor for proliferation and differentiation of human osteoblasts.

Disclosures: N. Schuetze, None.


Tob-Deficiency Preserves Bone Mass After Ovariectomy in Association with the Enhancement of Ovariectomy-Induced Osteogenic Gene Expression Levels in Bone Marrow.M. Usui1, Y. Yoshida*2, K. Tsuji1, I. Ishikawa*3, A. Nifuji1, T. Yamamoto*2, M. Noda1. 1Dept of Molecular Pharmacology, Tokyo Medical and Dental University, Tokyo, Japan, 2University of Tokyo, Tokyo, Japan, 3Periodontology, Tokyo Medical and Dental University, Tokyo, Japan.

Tob (transducer of ErbB2) belongs to a novel antiproliferative gene family, which include molecules containing a Tob-homology domain, such as Tob, Tob2, ANA/BTG3, BTG2/PC3/TIS21, PC3B and BTG1. Tob-deficiency (TD) increases bone formation without alteration in bone resorption to yield high levels of bone mass in adult. This enhancement in bone formation is based on the Tob action as an inhibitor against BMP (Yoshida Y. et al., Cell, 2000). We recently observed that TD preserved bone mass even after the bone loss due to ovariectomy (OVX). Bone histomorphometry revealed TD-enhancement in the levels of bone formation parameters but not bone resorption parameters after OVX. However, the alteration in gene expression underlying these TD-induced phenomena has not been known. The aim of this paper was to elucidate the molecular bases, which cause TD effects on OVX-induced bone loss. We carried out RT-PCR analyses on the transcripts of the genes expressed in the femoral bone marrow (BM) and calvaria of mice subjected to OVX. Two weeks after OVX, RNA was obtained from femoral BM and calvaria. In the BM obtained from wild-type (WT) mice, OVX up-regulated mRNA expression levels of bone formation (BF)-related osteogenic genes such as alkaline phosphotase, type I collagen, osteocalcin, Cbfa1 and osterix as well as those of bone resorption (BR)-related genes encoding RANK, RANKL and OPG as reported previously. Similar to BM in WT mice, expression of BR-related genes in the BM of Tob deficient (Tob KO) mice was up-regulated by OVX to the levels comparable to those in WT mice. In contrast, the levels of OVX-induced enhancement of BF-related gene expression in BM were significantly higher in Tob KO mice than those in WT mice. Furthermore, we also examined RNA prepared from calvariae, which mainly consist of cortical bone. Similar to BM, OVX increased mRNA expression of BF-related and BR-related genes in calvaria in WT mice. However, in contrast BM, the levels of OVX-induced enhancement of BF-related gene expression in WT mice and those in Tob KO mice were similar in calvaria. These data indicate that TD preserves bone mass after OVX through the enhancement of the expression of BF-related genes in bone marrow environment.

Disclosures: M. Usui, None.


Synergistic Effects of TGFβ and VEGF on Rat Bone Marrow Stromal Cells In Vitro.S. Kuroda, D. R. Sumner, A. S. Virdi. Department of Anatomy and Cell Biology, Rush University, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL, USA.

Bone regeneration is essential for healing bone defects. Growth factors have been increasingly investigated for their capacity to enhance the regeneration process. Recently, gene therapy has emerged as an effective approach to deliver therapeutic proteins in a more physiological and persistent manner. Transforming Growth Factor beta (TGFβ) has been implicated in osteoblast proliferation and differentiation. Vascular Endothelial Growth Factor (VEGF) plays an important role in angiogenesis and may be intimately related to bone development and healing as both intramembranous and endochondral ossification are associated with capillary development. In this study, we introduced transgenes for mouse TGFβ and/or mouse VEGF into rat marrow stromal cells (rMSC), and investigated their synergistic effects on proliferation, differentiation and osteogenic gene expression. rMSC were seeded at 1×105/well in 24-well plate and after 24 hours, transfer of 1 μg plasmid carrying mouse TGFβ and/or mouse VEGF full length cDNA was performed by LipofectAmine (Invitrogen) reagents. The cultures were assessed for alkaline phosphatase (ALP) staining and nodule formation, ALP activity and DNA content, and gene expression analysis by real-time PCR at d1, 2, 4, 7, 14 and 28. All cultures carrying transgenes showed less intensities for ALP staining at d7 and 14, which approached control (no transgene) levels by d28. At this time point more nodule formation appeared than in control. However, less mineralization was seen in each of the transgene carrying cultures when the medium contained dexamethasone (10−8) M. DNA assay showed higher proliferation in all cultures with transgenes by d28. While ALP levels also became higher, its levels per DNA were lower at the same time point due to the higher DNA levels. Most of 15 genes analyzed for expression, including: ALP, BMPs, Cbfa1, IGF1, RANKL and RANK, showed an increase due to each transgene at d2 or d4 with even greater effects in cultures transfected with both TGFβ and VEGF. These results demonstrated that transgenes for TGFβ andVEGF exhibited mitogenic effects and enhanced osteogenic differentiation of bone marrow stromal cells, and that TGFβ and VEGF act in a synergistic fashion.

Disclosures: S. Kuroda, None.


Analysis of Gene Expression During Osteogenic Differentiation of Human Periosteal Cells Using cDNA Array Technology.S. Honsawek*1, C. J. Osgood*2, L. Wolfinbarger3. 1Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand, 2Department of Biological Sciences, Old Dominion University, Norfolk, VA, USA, 3Research and Development, LifeNet, Virginia Beach, VA, USA.

The current study was undertaken to examine gene expression profile of osteogenic differentiation in human periosteal (HPO) cells using cDNA array analysis. HPO cell lines typically contain osteoprogenitor cells which can differentiate into osteoblasts. Numerous studies have demonstrated that demineralized bone matrix (DBM) comprises bone morphogenetic proteins (BMPs) that are essential regulators for inducing bone formation. We reported that DBM-conditioned media (DBM-CM) promoted osteoblast differentiation of periosteal cells in culture. In vitro alkaline phosphatase assay showed an increase in alkaline phosphatase activity in the DBM-CM treated cells. Additionally, histochemical detection of alkaline phosphatase revealed osteoblast-like cells in the HPO cells treated with DBM-CM. To attain a profile of gene expression during osteogenic differentiation, total RNA was harvested from HPO cells in the absence or presence of DBM-CM at day 7 and used as template for synthesis of biotin labeled cDNA probes. These cDNA probes were then hybridized to cDNA gene arrays. We demonstrated that several differentially expressed genes such as biglycan, TGF-β, and TGF-βR1 were up-regulated, whereas collagen14A1 was down-regulated. To verify the cDNA array results, RT-PCR analysis of parallel samples was performed on 2 selected genes, one was strongly up-regulated (bigly-can) and the other was highly down-regulated (collagen14A1). The results revealed identical expression pattern for selected genes as shown by the cDNA array analysis. To examine the temporal expression of biglycan and collagen14A1 in HPO cells untreated or treated with DBM-CM, poly (A)+ RNA was isolated for RT-PCR analysis at days 0, 4, 7, 10, and 14. The present study revealed that there was an increase in the level of biglycan expression and a decrease in the level of collagen14A1 expression following DBM-CM treatment. Our study demonstrated the potential of cDNA gene expression array technology to identify the gene expression involved in the cellular response to DBM-CM and to provide insight to the biological process of osteogenic differentiation.

Disclosures: S. Honsawek, None.


PTH Induces NGFI-B Nuclear Orphan Receptors in vivo.F. Q. Pirih*, O. Bezouglaia*, S. Tetradis. Dentistry, UCLA, Los Angeles, CA, USA.

Parathyroid hormone (PTH), a major regulator of osteoblastic function, induces transcription of several genes, including primary response genes (PRGs). Transcription factors are a subset of PRGs of particular importance since they propagate the activation signal downstream, by regulating secondary response gene expression. Among the transcription factors activated by PTH is the nerve growth factor inducible factor B (NGFI-B) family of orphan nuclear receptors, which is composed of three members: Nurr1, Nur77 and NOR-1. NGFI-B orphan receptors are constitutively expressed in the nervous system and can be induced in multiple tissues including PTH-treated osteoblasts. We have previously reported that PTH induces Nurr1, Nur77 and NOR-1 gene expression primarily through the cAMP protein kinase A pathway in vitro. We report here that PTH rapidly and transiently induced Nurr1, Nur77 and NOR-1 mRNA expression in bone and kidney in vivo. Unless otherwise specified all experiments were done using 1-month-old CD-1 male mice that received PTH (80μg/kg) i.p. injections. Nurr1, Nur77 and NOR-1 gene expression was assayed by semi-quantitative RT-PCR. PTH maximally induced Nurr1 and Nur77 from .5--1 h and returned to baseline levels at 2 h, while NOR-1 was maximally induced at 1--2 h and returned to baseline levels at 4 h in calvaria, long bone and kidney. Nur77 had the highest level of mRNA expression after PTH injection, followed by Nurr1 and lastly NOR-1. The magnitude and pattern of NGFI-B induction in vivo closely mimicked NGFI-B the induction in cultures of primary mouse osteoblast treated with 10nM PTH. To characterize NGFI-B expression at different stages of mouse development, 1-, 3- and 5-month old mice received PTH injections. There were no differences in NGFI-B mRNA expression among different stages of mouse development. To determine if Nurr1, Nur77 and NOR-1 gene expression is altered by intermittent administration of PTH, mice were injected with vehicle or PTH once a day for 1 wk. No difference was seen in the mRNA induction of NGFI-B members after freshly added PTH in calvaria and kidney. To determine which pathway mainly activates NGFI-B gene expression in vivo, mice were treated with 10--80 μg/kg of PTH (3--34), which does not activate PKA signaling but activates PKC and calcium pathways. This treatment did not induce NGFI-B mRNA levels, confirming our in vitro results with primary mouse osteoblasts. PTH-regulated induction of the NGFI-B family in vivo suggests that these genes may play an important role in the early response of osteoblasts to PTH.

Disclosures: F.Q. Pirih, None.


Mutations of Runx2 Binding Sites in the Rat Col1a1 Promoter Have Little Effect on Expression of Promoter-Reporter Constructs in Transgenic Mice.H. Chin*1, M. S. Kronenberg1, C. Wang*2, A. C. Lichtler1. 1Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA, 2Transgenic Core Facility, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Republic of China.

Runx2/Cbfa1 is a transcription factor that is necessary for formation of bone. It is expressed in osteoblasts, and has been shown to positively regulate expression of the osteocalcin promoter. Since induction of high levels of expression of type I collagen is necessary for synthesis of bone matrix, it would be expected that Runx2 should upregulate the Col1a1 promoter in osteoblastic cells. Despite this expectation, our studies, reported at previous ASBMR meetings, indicate that mutation of Runx2 binding sites in rat Col1a1 promoter-reporter constructs does not inhibit expression in transfected ROS 17/2.8 cells, and furthermore, overexpression of Runx2 inhibits expression of these constructs. To further investigate the role of the Col1a1 promoter's Runx2 binding sites in expression in osteo-blasts, we analyzed transgenic mice containing Col1a1 promoter-reporter constructs. The first series of experiments were designed to quantitatively compare expression of a wild type -1719 bp Col1a1 promoter fragment, fused to CAT, to the same promoter with one, three or five Runx2 binding sites mutated. In these experiments the constructs were injected into fertilized mouse eggs, transgenic animals were identified, and calvaria were analyzed for C AT activity. We analyzed at least three to six separate founder animals containing the wild type promoter or mutations in one, three or five of the Runx2 binding sites. Although there was significant variability between founder animals containing the same constructs, there was no apparent difference in expression between the wild type and the mutant constructs, supporting the observations that we have previously made using transfected ROS 17/2.8 cells. In a second series of experiments, the wild type and mutant constructs were linked to a GFP reporter gene to facilitate evaluation of the pattern of expression of the constructs in bone. Our evaluation of GFP expression in whole neonates indicates that there is no apparent difference in the pattern of expression of the wild type and mutant constructs. More detailed histological studies are underway. Thus our experiments in transgenic mice support our previous studies in transfected ROS 17/2.8 cells and fail to support the expectation that Runx2 is a direct inducer of the Col1a1 promoter during osteoblast differentiation.

Disclosures: A.C. Lichtler, None.


Interrelationship of Runx2 and BMP2 Signaling in Calvarial Bone and Suture Development.K. Choi1, S. Lee*1, M. Park*1, H. Shin2, S. Nam*3, Y. Kim*3, J. Wozney4, T. Komori5, H. Ryoo1, H. Kim*3. 1Department of Biochemistry, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea, 2Department of Oral Pathology, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea, 3Department of Pediatric Dentistry, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea, 4Genetics Institute, Inc., Cambridge, MA, USA, 5Japan Science and Technology Corporation, Suita, Osaka 565--0871, Japan.

Runx2, a key player of skeletogenesis, has been known as one of the downstream target genes of BMP signaling. However, little has been known their relationships in calvarial bone and suture development. In this study, we employed BMP2 bead and organ culture system using E15 mouse calvaria, and also used Runx2 knockout mice. The observation sites of the calvarial suture organ was osteogenic fronts, calvarial bones and sutural mesenchyme. In our previous study Dlx5, Msx2 and Runx2 have been known as downstream target genes of BMP2 in cell culture system. The BMP2 beads induced Dlx5 and Msx2 gene expression, however, it didn't induced Runx2 gene expression in the calvarial suture organ culture. To explore this discrepancy between ex vivo organ culture and in vitro cell culture system, we determined the expression of BMP2, Dlx5, Msx2 and Runx2 in both endogenous mouse calvaria itself by in situ hybridization. The expression of BMP2 and Dlx5 was observed at osteogenic fronts and calvarial bones and Msx2 was expressed at the sutural mesenchyme in wild-type and Runx2 heterozygous mice. However, neither BMP2 nor the Dlx5 and Msx2, downstream genes of BMP2, were expressed in Runx2 null mice. These results indicate that BMP2 is not an upstream signal of Runx2 gene at least in mouse calvarial bone and suture development. Conclusively, we suggest that Runx2 might be upstream gene of BMP2 signaling in mouse calvarial bone growth.

Disclosures: K. Choi, None.


SP1/SP3 and Myeloid Zinc Finger 1 (MZF 1)-like Factors Regulate Human N-cadherin Promoter Activity in Osteoblasts.S. Le Mée*, P. J. Marie. Hopital Lariboisiere, INSERM U349, Paris Cedex 10, France.

Recent studies indicate that N-cadherin plays an important role in cell-cell adhesion and osteoblast differentiation program. In this study, we have determined the molecular mechanisms of transcriptional regulation of N-cadherin expression in human osteoblasts. We isolated 3682 base pairs of 5′ flanking region of the previously characterized N-cadherin gene from a human BAC library. Nucleotide sequence analyss revealed 80% and 53% identity, respectively, between human/mouse and human/chicken. To characterize the DNA sequences involved in the activity of the basal N-cadherin promoter, a series of deletion mutants were made in pGL3 basic vector, and constructs were transiently transfected in Immortalized Human Neonatal Calvaria (IHNC) osteoblastic cells. The minimal sequence providing the highest luciferase activity covered 329 nucleotides upstream of the ATG. This region does not contain TATA-box nor CAAT-box but is characterized by a high content of GC and a GA-rich binding core that may potentially bind a transcription factor of the zinc finger family. Gel retardation analyses showed that two binding sites are involved in the regulation of the N-cadherin promoter. When nuclear extracts from IHNC cells were incubated with -305/-270 wild-type probe containing a putative SP1 binding site, two specific complexes, C1 and C2, were formed. These two complexes were not formed in the presence of a -305/-270 probe mutated in the SP1 binding site. Moreover, a consensus SP1 probe was as efficient as the -305/-270 wild-type probe in competition experiments whereas a -305/-270 mutated probe did not compete at all. Supershift EMSAs were conducted to identify the composition of the two complexes. The addition of anti-SP1 antibody reduced the formation of C1 whereas the addition of anti-SP3 antibody supershifted C2. When nuclear extracts from IHCN cells were incubated with -163/-131 wild-type probe containing the GA-rich binding core, one specific complex was formed. Moreover, the formation of this complex was abrogated in the presence of the -163/-131 probe mutated in the GA repeat. We used several consensus zinc finger probes to identify the nuclear factor involved in the formation of this complex. Only consensus oligonucleotide for Myeloid Zinc Finger 1 (MZF 1), a transcription factor expressed in hematopoietic progenitor cells, exhibited a clear competition for the complex. When SP and MZF 1 binding sites were mutated, the N-cadherin promoter activity decreased by 30% and 50%, respectively. These results identify for the first time critical regulatory regions for the human N-cadherin basal promoter activity in osteoblasts, and reveal a novel role for MZF 1 in the control of N-cadherin expression.

Disclosures: S. Le Mée, None.


Chromatin Remodeling of the Proximal Rat Osteocalcin Promoter is Dependent on Vitamin D Receptor Binding at the Distal Promoter Element.S. Gutierrez*1, A. Javed2, M. Montecino1, A. J. van Wijnen2, G. S. Stein2, J. B. Lian2, J. L. Stein*2. 1Departmento de Biologia Molecular, Universidad de Concepcion, Concepcion, Chile, 2Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, MA, USA.

Gene activation involves modifications in chromatin organization. Initiation of rat OC gene transcription is accompanied by changes in chromatin structure that are detected by nuclease accessibility. Two DNase I hypersensitive sites (DHS) are observed only in the actively transcribed OC promoter: DHS I (-600 to -400 bp) in the distal promoter encompasses the vitamin D response element (VDRE) and two flanking Runx sites; DHS II (-170 to -70 bp) in the proximal promoter spans Runx, C/EBP, OC-Box, and TATA elements. Vitamin D3 (VD) treatment results in transcriptional enhancement and intensification of the DHS. We addressed the role of the VDRE in maintaining chromatin architecture of the rat OC promoter, by introducing point mutations in the two steroid half elements of the VDRE (mVDRE) that do not affect binding of YY-1 or AP-1 to their sites which overlap the VDRE. This mVDRE OC promoter construct was stably integrated into ROS 17/2.8 osteoblastic cells and four monoclonal lines were studied. As expected, mutation of the VDRE results in a complete loss of VD responsiveness of OC activity. Our results show that in basal conditions the two DHS sites were not altered in the mVDRE promoter. However, in response to VD a decrease in DNase I accessibility of the mVDRE promoter was observed at the distal as well as the proximal DHS, reflecting a change in chromatin status. As another index of chromatin structure, we assayed restriction enzyme (RE) accessibility of the OC promoters. VD treatment increased RE accessibility in the distal endogenous (WT) promoter surrounding the VDRE but not in the distal mVDRE promoter. Surprisingly, the WT proximal promoter showed high RE accessibility, with a modest increase in response to vitamin D, and both of these effects were abrogated in the mVDRE promoter. These observations show for the first time that the VDRE influences chromatin structure at the proximal OC promoter. Increased histone H3 acetylation and association of RNA polymerase II were also observed with the mVDRE promoter compared to the WT gene, suggesting higher basal transcription in the absence of VD responsiveness. Thus binding of the VDR to the rat OC promoter appears to be required to complete the chromatin remodeling process across both the distal and proximal DHS domains associated with transcriptional activation of the OC gene.

Disclosures: S. Gutierrez, None.


Expression of LRP1 by Human Osteoblast-like Cells: A Mechanism for the Delivery of Dietary Lipids to Bone.A. C. Niemeier*1, J. Heeren*2, M. Kassem3, U. Beisiegel*2, W. Ruether*1. 1Orthopaedics, University Hospital Hamburg-Eppendorf, Hamburg, Germany, 2Molecular Cell Biology, University Hospital Hamburg-Eppendorf, Hamburg, Germany, 3Endocrinology, University Hospital Odense, Odense, Denmark.

Accumulating clinical and experimental data demonstrate the importance of dietary lip-ids and lipophilic substances, such as vitamin K for bone formation. However, little is known about the mechanisms underlying the delivery of lipids to bone. Thus, we investigated the expression and function of lipoprotein receptors in three different human osteo-blast-like cell lines: Osteosarcoma cell lines MG63, SaOS-2 and telomerase-immortalized human bone marrow stromal cells (hMSC-TERT) after 1,25(OH)vitaminD3 induction of osteoblastic differentiation, which was documented by increased osteocalcin expression (immunofluorescence and RT-PCR). In comparison to the human hepatoma cell line HuH7, human osteoblasts exhibited high levels of expression of known chylomicron remnant receptors : LRP1, VLDLR and LDLR proteins as shown by Western blot analysis and immunofluorescence. Uptake of Cy3-fluorescence-labeled chylomicron remnants by osteoblasts resembled the typical pattern of receptor-mediated endocytosis and was stimulated by the addition of exogenous apolipoprotein E and lipoprotein lipase. Quantitative radioactive uptake experiments with 125I-chylomicron remnants displayed that inhibitors of ligand binding to LRP1, such as RAP and lactoferrin, consistently reduced uptake by about 30%, suggesting that among the expressed receptors, LRP plays a predominant role. Immunohistochemistry of normal human bone sections showed strong LRP1 expression by osteoblasts and marrow stromal cells, but not by osteocytes. In conclusion, human osteo-blasts possess functional receptors of the LDLR family with a capacity for chylomicron remnant endocytosis, a novel mechanism for the delivery of dietary lipids and lipophilic vitamins to human bone.

Disclosures: A.C. Niemeier, None.


LIM Mineralization Protein-1 Stimulates Transcription of Procollagen Type I Alpha 2 Promoter in Human Osteoblasts by an AT-rich Cis-acting Element.T. A. Linkhart1, A. Delgado*2, C. Stivers*2, G. Gutierrez*2, D. D. Strong3. 1Pediatrics and Biochemistry, Loma Linda University and VAMC, Loma Linda, CA, USA, 2Loma Linda VAMC, Loma Linda, CA, USA, 3Medicine and Biochemistry, Loma Linda University and VAMC, Loma Linda, CA, USA.

LIM mineralization protein LMP-1 has been shown to induce osteoblast differentiation in vitro and increase bone formation in vivo. The mechanisms that underlie LMP-1 induction of bone formation, however are not clear. LMP-1 is a member of a diverse LIM domain protein family, and is identical to human Enigma. LMP-1 contains 3 LIM domains, named for homology to homeodomain proteins. We have identified human LMP-1 as a protein that interacted with IGF binding protein-6 in a yeast two-hybrid screen. To identify mechanisms of LMP-1 action on osteoblasts we investigated effects of LMP-1 expression on activity of the human Type 1 alpha 2 (hCol 1a2) and rat Type 1 alpha 1 (rCol 1a1) pro-collagen promoters. A hCol 1a2 promoter fragment (bp -267 to + 45) from genomic DNA was inserted into the pGL3Basic luciferase reporter gene vector. The rat Col 1a1 promoter was subcloned from ColCAT2.3 (D. Rowe U. Connecticut) into pGL3Basic. In transient transfection assays with TE85 human osteosarcoma cells, basal promoter activities were 40 fold greater than the pGL3 vector alone. To determine effects of LMP-1, TE85 cells were co-transfected with hCol 1a2 or rat 2.3 Col 1a1 promoter vectors and a human LMP-1 pcDNA3 expression vector (G. Gill, U.C. San Diego). LMP-1 expression resulted in reproducible 2 to 3 fold increases in activities of the collagen promoters but had no effect on the empty pGL3Basic vector. These results suggest that a function of LMP-1 in osteoblasts is to increase expression of Type I collagen. To identify promoter sequences involved in LMP-1 induction of the Col 1a2 promoter, we performed site directed mutagenesis of presumptive transcription factor binding sites in the promoter sequence and identified an AT rich site that may mediate transcriptional activation by LMP-1. Mutation of this site did not affect basal promoter activity but prevented activation by LMP-1. Overexpression of truncated LMP-1 (AA1--156) lacking the LIM domains failed to increase Col 1a2 promoter activity, suggesting an important role for the Lim domains. Summary: An AT rich sequence in the proximal region of the LMP-1 promoter sequence may be involved in mediating LMP-1 induction of Col 1a2 gene transcription. This transcriptional activation may require functions of the LIM domains.

Disclosures: T. A. Linkhart, None.


Inorganic Phosphate Regulation of a Discrete Set of Genes in MC3T3-E1 Osteoblasts Requires ERK1/2 and PKC.G. R. Beck. Center for Cancer Research, National Cancer Institute, Frederick, MD, USA.

The generation of inorganic phosphate by alkaline phosphatase during osteoblast differentiation represents a potentially important cellular signaling molecule although the consequences are relatively unknown. We have recently described a discrete set of genes both positively and negatively regulated by elevated phosphate in the murine pre-osteoblast cell line MC3T3-E1. Some of the increased genes include osteopontin, HMGA1, HMGA2, Fra-2 and Nrf2 a transcription factor involved in the regulation of many phase II detoxifying enzymes. The down regulated genes encode mostly for extracellular proteins including collagen type-1, periostin and thrombospondin among others. Because of the potential significance of phosphate as a signaling molecule in the process of osteoblast differentiation we were interested in defining possible mechanism for its ability to alter gene expression. Here we report that two families of signaling proteins, ERK1/2 and PKC are required for the regulation of genes responsive to inorganic phosphate. Analysis of the mitogen activated signaling kinases (MAPKs), ERK1/2, p38 and c-jun N-terminal kinase (JNK), reveal that specifically, ERK1/2 is required for the regulation of all phosphate responsive genes analyzed to date. Additionally we demonstrate that phosphate causes a biphasic phosphorylation of ERK1/2, with an initial activation at 15 to 45 minutes followed by a more gradual activation starting at 8 to 12 hours. Evaluation of other protein kinase signaling molecules reveals that PKC is also required in the phosphate-signaling pathway. Analysis of the timing of expression of phosphate responsive genes reveals that they can be separated into at least 2 groups, the first are those that respond early after phosphate treatment and correspond to the first ERK1/2 activation. This group consists mainly of transcription factors and includes Nrf2 and members of the AP-1 family of proteins. The regulation of the second group corresponds to the second ERK1/2 activation and includes osteopontin, HMGA1 and HMGA2 among others. The data presented here identifies ERK1/2 and PKC as important signal transduction molecules in the regulation of phosphate responsive genes in osteoblast cells.

Disclosures: G.R. Beck, None.


Pro-Osteogenic and Anti-Adipogenic Effects of Oxysterols in Marrow Stromal Cells are Mediated via COX/PLA2- and MAPK-Dependent Mechanisms.F. Parhami1, B. Basseri*1, H. T. Kha*1, D. Shouhed*1, J. A. Richardson*1, S. Tetradis2, T. J. Hahn3. 1Medicine, UCLA, Los Angeles, CA, USA, 2Dentistry, UCLA, Los Angeles, CA, USA, 3Medicine, West L.A. VA Medical Center, Los Angeles, CA, USA.

Identification of anabolic agents that induce osteogenic differentiation of osteoprogenitor cells, inhibit their adipogenic differentiation, and enhance bone formation is of great importance for improved prevention and management of osteoporosis. Oxysterols are naturally occurring products of cholesterol oxidation, have multiple biologic activities, and are made in part by cellular cytochrome P450 enzymes. We previously reported that specific combinations of oxysterols, namely 22(R)- or 22(S)-hydroxycholesterol with 20(S)-hydroxycholesterol have novel pro-osteogenic and anti-adipogenic effects when applied to pluripotent M2--10B4 (M2) mouse marrow stromal cells (MSC) in vitro. Osteogenic effects of these oxysterols were also induced in primary mouse MSC, C3H10T1/2 embryonic fibroblastic cells, MC3T3-E1 preosteoblastic cells, and SAOS-2 human osteosarcoma cells, as assessed by the induction of markers of osteogenic differentiation alkaline phosphatase (ALP) activity, osteocalcin (Osc) gene expression, and mineralization. Further studies demonstrated that oxysterol-induced ALP activity, Osc mRNA expression and mineralization in M2 cells were inhibited by cyclooxygenase 1 and 2 (COX-1 and COX-2) inhibitors, SC-560 (20 μM) and NS-398 (80 μM), respectively. Osteogenic responses of M2 cells to the oxysterols were also inhibited by phospholipase A2 (PLA2) inhibitors, ACA (25 μM) and AACOCF3 (20 μM). Prostaglandin E2 and arachidonic acid rescued the cells from the inhibitory effects of COX and PLA2 inhibitors, respectively. In addition, MAPK kinase (MEK) inhibitor, PD98059, inhibited oxysterol-induced mineralization, but not ALP activity or Osc expression in M2 cells. Ten minutes to 8 hours treatment with oxysterols induced a sustained phosphorylation and activation of Erk1/2. Moreover, the inhibitory effects of the osteogenic oxysterols on adipogenesis of M2 cells were completely inhibited by PD98059, but not by NS-398 or SC-560. These results suggest that the osteogenic and anti-adipogenic effects of oxysterols are mediated via COX/PLA2- and MAPK-dependent mechanisms. The osteogenic oxysterols also acted synergistically with BMP-2 and BMP-7 in inducing osteogenic differentiation of M2 cells. We suggest that oxysterols and their downstream targets may be important regulators of lineage-specific differentiation of MSC. The use of lipid-based rather than protein- or peptide-based molecules introduces a whole new strategy for creating therapeutics for osteoporosis intervention.

Disclosures: F. Parhami, None.


Near Infrared Light Irradiation Alters the Gene Expression of RANKL and OPG in Response to PTH.J. T. Ninomiya, S. K. Lee*, J. A. Struve. Department of Orthopaedic Surgery, Medical College of Wisconsin, Milwaukee, WI, USA.

Light irradiation in the near infrared region (NIR)has been shown to promote a variety of biologic effects, including cellular proliferation and wound healing. However, the effects of NIR light on bone are less well characterized. Therefore, we hypothesized that NIR light irradiation would enhance bone formation, and theorized that a potential mechanism of this action was through alteration of the gene expression of the proteins RANKL and OPG in response to stimulus with PTH.

Murine MC3T3-E1 cells were grown in tissue culture, and were irradiated with NIR light at 770, 830, and 880 nm at 4J. Controls consisted of non-irradiated cells. The cells received a daily dose of PTH (1--34), and following incubation the RNA was collected. Alterations in the gene expression of RANKL and OPG were determined by RT-PCR.

Parathyroid hormone increased RANKL gene expression in the osteoblasts. None of the three wavelengths altered RANKL or OPG gene expression in the absence of PTH. However, light of 830 nm produced a significant decrease in the RANKL/OPG ratio following the addition of PTH, and this was due to a decrease in RANKL gene expression.

We conclude that NIR light alters the gene expression of RANKL in osteoblasts that are stimulated with PTH. A decrease in the RANKL/OPG ratio would likely lead to less osteoclast maturation and possible less bone resorption. These findings have important implications for use in fracture healing and the prevention of bone loss during prolonged space flight.

Disclosures: J.T. Ninomiya, None.


Stimulation of in-vitro Osteoblastic Activity by Flavonoids.M. Gros-van Hest1, C. Beumer*1, Z. Dang2, K. C. T. Knipping*1, A. van Helvoort*1, I. Schoenmakers1. 1Biomedical Research, Numico Research, Wageningen, Netherlands, 2Endocrinology, Leiden University Medical Center, Leiden, Netherlands.

In the prevention of osteoporosis, components reducing bone loss and stimulating bone formation may be beneficial to maintain healthy bone mineral density. In the search of components stimulating bone formation, several flavonoids were tested on the mouse osteoprogenitor cell line KS483 and on the mouse pre-osteoblastic cell line MC3T3-E1.

KS483 cells were cultured in phenol red free alpha-MEM supplemented with 10% charcoal stripped FCS (M-FCSs) for 17 days. Three hours after seeding, half of the medium was replaced with medium containing 17ß-estradiol (10−7M), flavonoids (10−4 to 10−8M) and plant extracts (1--100μg/ml) containing these same flavonoids. Ascorbic acid (50μg/ml) and ß-glycerophosphate (10mM) were added to the culture medium from day 3 and 10, respectively.

MC3T3-E1 cells were pre-cultured in phenol red free alpha-MEM supplemented with 10% non-charcoal stripped FCS (M-FCSns), ascorbic acid and BMP-4 (10ng/ml) for 3 days. Thereafter, M-FCSns was replaced with M-FCSs containing ascorbic acid and ß-glycerophosphate and cells were exposed to 17ß-estradiol, flavonoids and plant extracts containing these same flavonoids for 7 days. All cells were refreshed twice a week.

Alkaline phosphatase activity, calcium deposition and DNA were determined and nodules were visualized with Alizarin staining.

At a concentration of 10−5M, one of the flavonoids increased the number and size of nodules formed by both KS483 and MC3T3-E1 cells. This was reflected in a significantly higher calcium deposition and alkaline phosphatase activity when compared to controls, ß-estradiol and the other flavonoids tested. Also the flavonoid containing plant extracts increased nodule formation, alkaline phosphatase activity and calcium deposition in MC3T3-E1 cells.

Toxicity of the flavonoids and flavonoid containing extracts was first observed at 10−4M and 100μg/ml respectively, as reflected by low DNA content and poor cell morphology. In vivo studies will have to prove the safety, bioavailability and efficacy of this component and/or its metabolites to protect against post-menopausal bone loss.

Disclosures: M. Gros-van Hest, None.


Traumatic Brain Injury Stimulates Systemic Bone Formation: Possible Role for Hypothalamic Leptin Signaling.Y. Tam*1, E. Regev*2, N. Casap*2, A. Shteyer*2, M. Attar-Namdar*1, A. Alexandrovich*3, O. Lahat*1, E. Shohami*3, I. A. Bab1. 1Bone Laboratory, Hebrew University of Jerusalem, Jerusalem, Israel, 2Oral and Maxillofacial Surgery, Hebrew University of Jerusalem, Jerusalem, Israel, 3Pharmacology, Hebrew University of Jerusalem, Jerusalem, Israel.

The common clinical finding of heterotopic ossification and enhanced fracture healing in traumatic brain injury (TBI) patients has attracted little attention and the underlying mechanisms are poorly understood. The consistency of the TBI-induced peripheral osteogenic effect lead us to initiate a study on its mechanisms in an established mouse model of closed head injury (CHI). Because leptin has been suggested as a master hormone, negatively regulating bone formation via a hypothalamic relay, we investigated the temporal relationship between peripheral osteoblastic activity and hypothalamic leptin signaling during a 16-day post-CHI period. CHI was induced in mice under ether anesthesia by a calibrated weight drop device resulting in a focal injury to the left hemisphere. The severity of injury and rate of recovery were assessed 1 hr -16 days post-CHI by a Neurological Severity Score, and the mice were sacrificed for in vivo assessment of bone formation and hypo-thalamic gene expression. Dynamic bone histomorphometric analysis showed stimulation of trabecular bone formation rate in the distal femoral metaphysis peaking on days 4 and 12 post-CHI, consequent to increases in both mineral appositional rate (MAR) and mineralizing perimeter (Min.Peri), suggesting a CHI-induced enhancement of both osteoblast activity and number. Real-time RT-PCR demonstrated impaired expression of ObRb, the signal-transducing form of the leptin receptor, 1.5 hrs post-CHI, followed by progressive recovery in spite of a sustained neurological damage. The ObRb expression exhibited a very high negative correlation (r= -0.9) with the MAR, but not not with the Min.Peri. The mRNA levels of other genes involved in leptin signaling was unaffected by CHI. Periosteal bone formation in the mid-femoral diaphysis was enhanced in almost all mice tested on day 4 after CHI. This was followed by a linear decrease in the number of such animals to control level by day 16 and was not related to ObRb expression. Endosteal bone formation was unaffected by CHI. Collectively, these data suggest an association between hypothalamic leptin signaling and TBI-induced enhancement in trabecular osteoblast activity. The involvement of other neuroendocrine systems, which regulate osteoblast number and periosteal bone formation, is also implied. The mouse model of CHI-induced increase in systemic bone formation constitutes a novel tool to study the central regulation of bone remodeling.

Disclosures: Y. Tam, None.


Osteoblasts Express Polycystin-1 and Polycystin-2.K. G. Oberman*, J. M. David*, D. C. Usher*, N. J. Karin. Biological Sciences, University of Delaware, Newark, DE, USA.

Autosomal dominant polycystic kidney disease (ADPKD) is a severe and common genetic disorder in humans caused by mutations in one of two genes, PKD1 or PKD2. ADPKD patients exhibit multiple fluid-filled cysts in their kidneys, frequently leading to end-stage renal failure. The physiological function of polycystin-1 (Pc-1) and polycystin-2 (Pc-2), the protein products of the PKD genes, is not clear in any tissue, however Pc-1 has similarities to cell adhesion molecules and Pc-2 is a cation channel with a high permeability to Ca2+. RT-PCR revealed that mRNA encoding polycystins is expressed in mouse MC3T3-E1 pre-osteoblastic cells and in primary rat calvarial osteoblasts. Quantitative RT-PCR measurements indicated elevated expression of both polycystins during in vitro differentiation of MC3T3-E1 cells. Pc-1 and Pc-2 transcripts were found in other mouse cells of mesenchymal origin, pre-adipocytes and C2C12 skeletal myoblasts, but developmentally-regulated expression was not detected. Sequence analysis revealed that MC3T3-E1 pre-osteoblasts also express a novel, alternatively spliced variant of the PKD2 gene product: a 108-base region of the Pc-2 mRNA encoding amino acids 117--152 is deleted in the pre-osteoblast transcript. The deleted region of the protein includes a consensus protein kinase C phosphorylation site. These data suggest a role for polycystins in osteoblast function and may be related to the skeletal deformities seen in PKD1-null mice [Boulter et al., PNAS 98:12174 (2001)].

Disclosures: N.J. Karin, None.


Molecular Nature of Functional Complexes of L-type Voltage-Sensitive Calcium Channels (VSCCs) in MC3T3-E1 Osteoblasts.Y. Shao*, J. J. Bergh*, K. Akanbi*, M. C. Farach-Carson. Biological sciences, University of Delaware, Newark, DE, USA.

Plasma membrane VSCCs serve as key regulators of Ca2+ permeability. In osteoblasts, the L-type VSCC consists of a pore forming α1 subunit, a disulfide-linked α2δ dimer, an intracellular β subunit, but no γ subunit (Bergh et al, in press). Subunit composition plays an important role in fine-tuning of VSCC expression and function. Ten α1, four β, and three α2δ subunits have been identified, giving 120 potential functional complexes even without a γ subunit. Previous studies in our laboratory have shown that the major functional α1 subunit present in proliferating osteoblasts is the α1c (Cav1.2). There are twelve functional complexes that could form with α1c subunit alone. Additionally, a novel 700KDa phosphoprotein AHNAK was recently identified that interacts with β2 subunits of Cav1.2 channels and links VSCCs to actin microfilaments via a region at the carboxyl-terminus of AHNAK. It is likely that AHNAK plays an important regulatory role linking VSCCs and the intracellular signaling pathways to a variety of protein kinases and actin-based cytoskeletal changes associated with Ca+2 signaling. We are using a combination of approaches to investigate exactly which auxiliary subunits form functional complexes with α1c in MC3T3-E1 cells and in bone sections. RT-PCR with subunit specific primers, multi-photon confocal co-immunolocalization studies using subunit-specific antibodies, and co-immunoprecipitation assays allow us to detect and identify the Cav1.2 channel complexes. Immunostaining and RT-PCR results reveal that MC3T3-E1 cells express β1, β2, β3, and α2δ1 subunits. β4 subunits are expressed at very low levels. RT-PCR also demonstrated significant amounts of AHNAK mRNA present in MC3T3-E1 cells. Immunostaining with antibodies against the repetitive sequence in the central conserved domain confirmed the subcellular localization of AHNAK mostly along the plasma membrane of MC3T3-E1 cells, consistent with its ability to interact with β2. Ongoing studies employ co-immunopre-cipitation and Western blotting to identify the direct interaction between the α1c subunit of L-type VSCCs and individual auxiliary subunits. Given the abundance and plasma membrane localization of the β2 subunit in mouse osteoblasts, we are investigating the potential functional interactions between osteoblastic α1c the β2 subunit and AHNAK. For the functional study of VSCCs, we have successfully designed the pU1RBα1c6 ribozyme plasmid that specifically knocks down α1c subunit expression and abolishes L-type depolarization. We will utilize the availability of these knockdown cells to determine if loss of α1c disassembles complexes containing β2, α2δ1 and AHNAK.

Disclosures: Y. Shao, None.


Uremic Serum Stimulates In Vitro Osteoblast Differentiation.A. Hufkens*, S. C. E. Verberckmoes*, M. E. De Broe, P. C. D'Haese*. Nephrology -Hypertension, University of Antwerp, Wilrijk, Belgium.

As a consequence of decreased phosphate excretion, disturbed parathyroid hormone (PTH) and vitamin D metabolism, end-stage renal failure patients may develop secondary hyperparathyroidism; a particular form of renal osteodystrophy, characterized by an increased bone turnover, resulting from an increased osteoblastic/osteoclastic activity. It is now well recognized that PTH plays a key role in this cellular response. However, to which extent other factors may trigger the increased bone cellular activity also, is not well known yet.

In the present study, an in vitro experiment, using primary osteoblasts, isolated from 20 day old fetal rat calvaria, was set up. At confluence, the cultures were divided into two groups that were further grown up with α-MEM medium supplemented with 50 μg/ml ascorbic acid, 2 mM β-glycerophosphate and either 10% heat inactivated human uremic serum (collected from patients prior to dialysis, N = 25) or 10% heat inactivated human normal serum (collected from age and gender-matched healthy volunteers, N = 9). The culture medium was changed every 2--3 days until 14 days post confluence. Alkaline phosphatase (ALP) activity, used as a marker of osteoblastic activity/differentiation was measured in the conditioned medium at the time points of medium refreshments. Fourteen days post confluence cells were von Kossa stained to identify the presence of calcified nodules. Number and size of nodules were counted by computer aided image analysis. Cellular samples were taken to determine total DNA-content. Data were then expressed per amount of DNA to correct for possible differences in cell number. Total nodular Ca-deposition was determined in a 0.6M HCl extract of the cultures. Uremic and normal sera were analysed for the most relevant biochemical parameters such as PTH, ALP-activity, urea and electrolytes. Area under the time curve (AUC) of ALP activity and total Ca-deposition were significantly higher in the uremic cell cultures vs. those grown in normal serum (AUC-ALP: 132.5 ± 22.2 vs. 61.3 ± 26.6 U/l; Ca-deposition: 139.3 ± 20.5 vs. 40.2 ± 27.8 μg Ca/well; p ⩽ 0.05). Quantification of the nodules revealed a significantly increased amount of nodules (number and size) in the uremic cultures vs. the normal serum group (number: 19.6 ± 2.3 vs. 12.8 ± 0.6 nodules/cm2; size: 209. 103 ± 19 103 vs. 130. 103 ± 24. 103; p ⩽ 0.05). These results allow us to suggest that an uremic environment stimulates the osteoblastic differentiation. Results obtained in the present pilot study are further investigated in detail using advanced techniques such as RT-PCR and proteomics.

Disclosures: S.C.E. Verberckmoes, None.


A Threshold Level of Hyaluronic Acid Is Critical for Osteoblast Differentiation: Insights into the Leukemia Inhibitory Factor-Mediated Arrest of Osteoprogenitor Cells Differentiation.D. Falconi, J. E. Aubin. Department of Medical Genetics and Microbiology, University of Toronto, Toronto, ON, Canada.

We have previously shown, using the rat calvaria (RC) cell culture system, that the gp130 family cytokine leukemia inhibitory factor (LIF) blocks osteoblast development in a differentiation stage-specific manner. This effect is also seen in vivo when neonatal rats receive sub-cutaneous injections of LIF above the calvaria, leading to dose-dependant widening of the sagittal suture. Using a differential display approach, we identified genes modified by LIF treatment of RC cells and found that hyaluronic acid synthase 2 (HAS2), an enzyme responsible for the synthesis and secretion of high molecular weight hyaluronic acid (HA) molecules, was up-regulated by LIF specifically during the osteoprogenitor inhibition-sensitive time window. Here we report the results of studies assessing the role of the HA/HAS2 system in osteoblast differentiation and the LIF-mediated arrest of osteoblast differentiation. Histochemical analyses with a biotinylated-HA binding protein (HABP) revealed that, in control-treated cells, high HA expression is restricted to the cells at the periphery of developing bone nodules, i.e. osteoblast precursor cells. However, in LIF-treated cultures, HA is expressed highly by all the cells within the few, immature bone nodules that formed, consistent with our previous finding that LIF blocks osteoblast differentiation at the late osteoprogenitor stage. Increasing the concentration of HA in the culture medium, either by adding exogenous HA or by overexpressing HAS2, lead to a dose-dependant decrease in bone nodule numbers and, importantly, the culture time for the HA-associated inhibition of bone nodule formation overlaps with the LIF-associated inhibition. Notably, however, decreasing the concentration of HA through the use of antisense oligo-nucleotides or hyaluronate lyase did not lead to an increase in bone nodule number or a reversal of the LIF effect. Instead, these treatments produced a decrease in bone nodule numbers in control and LIF-treated cells. Taken together, these results indicate that critical or threshold levels of HA are important for osteoblast differentiation and bone formation and that LIF abrogates the differentiation of osteoprogenitor cells by increasing the levels of HA through up-regulation of HAS2 expression.

Disclosures: D. Falconi, None.


Annexin 2 and Lipid Raft Involvement in Osteoblastic Mineralization.J. M. Gillette1, R. Globus2, S. M. Nielsen-Preiss3. 1Cellular and Developmental Biology, University of Colorado Health Sciences Center, Denver, CO, USA, 2Gravitational Research Branch, NASA Ames Research Center, Moffett Field, CA, USA, 3Orthopaedics, University of Colorado Health Sciences Center, Denver, CO, USA.

The experiments described herein were designed to elucidate the roles of lipid rafts and annexin 2 in the process of mineralization. Lipid rafts function to organize membranes into a series of discrete microdomains to facilitate protein interactions. Recently, our laboratory has established annexin 2, a cytoplasmic and membrane-associated calcium-binding protein, as a facilitator of osteoblastic mineralization, through a potentially novel mechanism. Annexin 2 overexpression in SaOSLM2 cells resulted in a two-fold increase in alkaline phosphatase activity and enhanced mineralization by SaOSLM2 cells. Futhermore, annexin 2 and alkaline phosphatase were localized to osteoblastic membrane microstructures (lipid rafts) which are cholesterol-mediated and triton X-100 insoluble. In separate experiments lipid rafts were isolated at days 3 and 12 during differention from primary fetal rat calvarial cells which were isolated by collagenase digestion. Lipid rafts were then assayed for annexin 2 and alkaline phosphatase activity. Prior to the onset of mineralization (day 3), annexin 2 was not detected in lipid rafts, although annexin 2 expression was present in a total cell lysate throughout the mineralization process. However, following the two weeks of differentiation, annexin 2 and alkaline phosphatase activity were co-localized to lipid rafts. Thus, the translocation of annexin 2 to lipid rafts in primary osteoblasts coincided with an increase in alkaline phosphatase activity during differentiation of primary osteoblasts. Together, our results support the hypothesis that lipid rafts provide a physical entity for the temporal and spatial regulation of proteins required to initiate mineralization.

Disclosures: J.M. Gillette, None.


Osteoactivin-Derived Peptides Induce Osteoblast Differentiation in MC3T3-E1 Cells.A. Selim*1, J. L. Castaneda*1, T. A. Owen2, S. N. Popoff1, F. F. Safadi1. 1Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA, USA, 2Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, CT, USA.

We previously identified novel gene called osteoactivin (OA) in bone. OA was identified by differential display using total RNA from wild type compared to osteopetrotic (op) long bone and calvaria. In this study, we examined the role of OA in osteoblast differentiation in vitro using two anti-OA antibodies: anti-OA antibody 27 (Ab-27) and anti-OA 551 (Ab-551). These antibodies were raised against different regions of the molecule; Ab-27 was raised against the N-terminus and Ab-551 was raised against the C-terminus, a sequence that contains an RGD motif. We found that only Ab-551 significantly decreased osteoblast differentiation including, alkaline phosphatase activity, nodule formation and matrix mineralization. In order to test the role of the RGD motif of OA protein in osteo-blast differentiation, we designed two peptides that mimic the sequence of the OA peptide used to generate Ab-551. The first peptide (OA-D) has the RGD domain and the second peptide (OA-E) has E (Glutamic acid) in the place of D (Aspartic acid). We examined the effect of these two peptides on osteoblast proliferation and differentiation in vitro. Although both peptides had no significant effect on osteoblast proliferation and/or viability, they significantly induced alkaline phosphatase activity, nodule formation and calcium deposition. Bioinformatic analysis of these peptides showed the presence of a serine residue that is potentially phosphorylated by casein kinase II (CK-II). Further analysis of other OA protein family members showed that there is conserved serine residue close to C-terminus, which matches the position of serine residue of the OA peptides. CK-II is known to phosphorylates many osteoblast-related proteins that regulate osteoblast development and differentiation such as osteopontin and vitronectin. Collectively, these data show that both OA-D and OA-E peptides significantly induced osteoblast differentiation in vitro and the effect of these peptides is RGD independent. Additional studies are warranted to determine if phosphorylation of the OA peptides by CK-II might be involved in their mechanism of action during osteoblast differentiation.

Disclosures: A. Selim, None.


Enhanced Bone Growth in Transgenic Mice Overexpressing Osteocrin, a Novel Secreted Bone Protein.P. Moffatt, F. Lafreniere*, M. Bessette*, K. Sellin*, E. Godin*, M. Gaumond*, C. Lanctot*, G. P. Thomas. Phenogene Therapeutiques, Montreal, PQ, Canada.

Previously we reported the identification of a novel bone protein, PGTI0306. Due to its highly bone specific expression pattern and putative pro-hormone like processing we have now termed the molecule “osteocrin”. Osteocrin is a 103aa-secreted protein with two conserved putative dibasic cleavage sites reminiscent of prohormones. Highly expressed in osteoblasts, osteocrin is down regulated in aged bone and in vitro is associated with matrix production. Treatment with 1,25(OH)2D3 down regulates osteocrin in a rapid dose-dependent manner further suggesting skeletal functionality.

To assess the role of osteocrin in bone we generated transgenic mice specifically expressing osteocrin in osteoblasts using 3.6Kb of the rat collagen type I promoter. Analysis by Northern blot demonstrated bone-specific transgene expression in three transgenic lines. In all transgenic lines, long bones were on average 7--10% longer than in wild-type littermates (P<0.001). The tails of the osteocrin transgenic mice were also significantly longer (10--13%, p<0.001) and the mice exhibited a marked kyphosis. DEXA analysis showed no differences in either BMD or BMC and there was no change in body weight. The increased bone length was apparent at 8 weeks of age and was maintained at 8 months of age suggesting a developmental or growth phenomenon.

Bone marrow stromal cultures from osteocrin transgenic mice expressed high levels of osteocrin protein from approximately day 8 in culture which increased when the cultures were differentiated with dexamethasone. Alkaline phosphatase and osteocalcin expression was significantly lower in osteocrin transgenic marrow cultures than in wildtype cultures. This correlates with our previous report of inhibition of alkaline phosphatase, osteocalcin and mineralisation in primary rat calvarial cultures chronically treated with osteocrin-conditioned media.

These data demonstrate osteocrin as an important anabolic factor in osteogenic regulation. Osteocrin may act directly on osteoblasts or indirectly via chondrocytes to stimulate bone growth. Continuing histological and histomorphometric analyses will further elucidate the mechanism underlying the elevated bone growth in these mice.

Disclosures: G.P. Thomas, None.


Direct Effect of an Angiogenesis Inhibitor on Osteoblast-like Cells and Bone Marrow Stromal Cells In Vitro.R. J. Majeska, M. B. Schaffler, M. R. Hausman*. Orthopaedics, Mount Sinai School of Medicine, New York, NY, USA.

The angiogenesis inhibitor TNP-470, which inhibits endothelial cell proliferation in vitro, impairs bone and cartilage formation in vivo. The purpose of this study was to test in vitro whether TNP-470 might also act directly on osteoblastic cells or their progenitors. Cultures of human endothelial cells (HUVEC), osteoblast-like cell lines (ROS 17/2.8, MC3T3) and mouse calvarial cells were plated at 0.5 - 1 ×104 cells/cm2, then treated with TNP-470 (25 pM - 0.25 mM). Cell growth and alkaline phosphatase (ALP) activity were measured at selected times between 4--14 days. Primary mouse bone marrow cell cultures were also tested for ALP+ colony formation in the presence of TNP-470. TNP-470 inhibited HUVEC growth (30 - 50%, p <0.05) at concentrations as low as 0.025 nM. TNP-470 also inhibited growth of ROS 17/2.8, MC3T3 and mouse calvaria cells to a similar extent, but at higher concentrations (0.25 - 2.5 nM). TNP-470 inhibition of osteoblastic cell growth was accompanied by inhibition of ALP activity. TNP-470 at 2.5 nM and 25 nM reduced the number of colonies formed by mouse marrow stromal cells by 50%, but had no effect on the % of ALP+ colonies. These findings support the concept that TNP-470 inhibition of cell proliferation is selective for vascular cells; however, proliferation of osteoblastic cells and their progenitors is also inhibited by TNP-470 albeit at higher concentrations. Whether the reduction in osteoblastic cell ALP activity is associated with lower cell growth is not clear; however, colony formation data indicate that TNP-470 does not appear to inhibit the growth of ALP+ and ALP- cells differentially. Thus TNP-470 can act directly to inhibit bone cell growth and function, but these effects are not likely to play a substantial role in the dramatic inhibition of skeletal development and repair produced by TNP-470 in vivo.

Disclosures: R.J. Majeska, None.


Connective Tissue Growth Factor (CTGF) Expression During Diabetic Wound Healing in a Tooth Extraction Model.R. A. Aswad*1, M. C. Rico*1, R. A. Kanaan*1, H. Devlin*2, F. F. Safadi1, S. N. Popoff1. 1Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA, USA, 2Restorative Dentistry, Temple University School of Dentistry, Philadelphia, PA, USA.

Delayed healing and infection often complicate healing of the tooth extraction socket in patients with diabetes mellitus. Streptozotocin, the diabetogenic drug of choice, produces in rats an elevated blood basal plasma glucose level and an impaired insulin response to glucose injections. The molar extraction socket of rats treated with this drug exhibits delayed healing with increased bone resorption. In calvarial defect, healing of streptozotocin-treated rats, exuberant formation of primitive bone was observed. It was concluded that uncontrolled diabetes exerts a direct influence on bone healing by inhibiting mineralization and remodeling. CTGF is a secreted, extracellular matrix-associated protein that regulates diverse cellular functions. CTGF mRNA and protein production has been demonstrated in multiple cell types including; fibroblasts, endothelial cells, osteoblasts and mesangial cells. CTGF has also been shown to be up-regulated in diabetic nephropathy. In this study, we examined CTGF expression in socket following tooth extraction in streptozotocin- treated and normal rats. Three and five days following extraction, CTGF was maximally expressed in fibroblasts, endothelial cells and osteoblast progenitors in sockets from diabetic rats when compared to normal. This expression level was decreased by 7 and 10 days in diabetic rats but not in the normal rats. These data suggest that CTGF plays a role in wound healing and is abnormally expressed in diabetic wounds. We next examined whether CTGF expression in osteoblasts is altered in response to glucose treatment. MC3T3-E1 cells were treated with different doses of glucose and examined for their proliferation and differentiation as well as CTGF expression. Glucose-treated cells showed an increase in cell proliferation, and decreased alkaline phosphatase expression and calcium deposition. CTGF expression determined by RT-PCR was increased in response to glucose treatment. These data suggest that glucose modulates osteoblast differentiation and this could be mediated through CTGF. Future studies are warranted to evaluate the mechanism by which glucose alters osteoblast differentiation by CTGF is yet to be determined.

Disclosures: R.A. Aswad, None.


Osmoadaptation Impairs Osteoblast Differentiation.L. R. McCabe, S. Botolin*. Physiology, Michigan State University, East Lansing, MI, USA.

Diabetes Mellitus type I is often accompanied with complications including bone loss and increased fracture rate. We hypothesize that osteoblast osmoadaptation to increased blood glucose and its associated increase in blood osmolarity results in impaired osteoblast function, ultimately contributing to the development of osteoporosis. To investigate this hypothesis we have taken in vitro and in vivo approaches. To examine in vitro effects, MC3T3-E1 osteoblasts were exposed to hyperosmotic media (320 mOsm) for short (1 hour) and long (1--7 days) periods of time. Exposure of osteoblasts to acute hyperosmotic stress was associated with significant changes in gene expression, cell signaling, cell shape and mechanisms necessary for osmoadaptation, such as connexin 43 and HSP27 expression. Here we show that during the process of long term osmoadaptation osteoblast expression of RUNX2 and osteocalcin, markers of osteoblast differentiation, are significantly suppressed. Expression of both markers decreased to 50--30% of control levels. Mannitol treatment, an osmotic control, caused the greatest suppression. To address in vivo effects of diabetes on osteoblast osmoadaptation and gene expression we developed a mouse model of long-term diabetes (4--6 weeks). We measured blood glucose and osmolarity and found an increase in both, by greater than 20 mM and 20 mOsm, respectively. Bone histomorphometry on these animals demonstrated significant changes. These in vivo and in vitro findings support our hypothesis that poorly controlled diabetes accompanied with increased blood osmolarity and glucose levels can contribute to the development of osteoporosis as a consequence of osteoblast dysfunction.

Disclosures: S. Botolin, None.


Osteoblast Lineage Differentiation Potential in the OIM Mice.I. Kalajzic*, X. Jiang*, K. Mack*, J. Delaney*, D. Rowe. Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA.

Osteogenesis imperfecta murine (OIM) is a mouse model that reassembles severe type III human OI. Recent studies suggested two pathophysiological mechanisms for the bone disease. The first is a state of high bone turnover secondary to the accumulation of defective bone matrix as a consequence of the underlying molecular mutation within the COL1A1 or Col1A2 genes. Second is an impairment of full osteoblastic differentiation in cells carrying an OI mutation. The combination of the two effects leads to diminished bone mass because bone formation cannot keep pace with bone resorption. We have previously developed mice transgenic for pOBCol3.6GFP and pOBCol2.3GFP and have shown that they can be used as visual markers of preosteoblast, early osteoblast and mature osteoblast differentiation. This study was designed to assess the validity of these two mechanisms using the visual transgenes that were breed into OIM mice. The analysis was performed on MSF cultures derived from the OIM/GFP mice using repetitive imaging for GFP expression of the same culture plate throughout the culture period from which the tempo and magnitude of lineage differentiation can be assessed. Cultures from the 2.3 oim/oim mice show a marked impairment in the number of cells with GFP expression relative to 2.3 +/+ litermates. The 3.6 oim/oim cultures showed a similar number of low intensity GFP+ cells (preosteoblasts) but an impaired number of high expressing GFP cells (early osteoblasts). Conventional markers of osteoblast differentiation confirmed the GFP data. Decreased AP histochemical activity and decreased expression of bone markers (Col1a1, BSP, OC) and mineralization became apparent in the oim/oim mice after 12 day of culture. Frozen decalcified sections of bone were prepared using the CryoJane tape system and the entire bone area was imaged and reconstructed as an intact bone section. The bone from the 3.6 oim/ oim mice showed a dramatic increase in the number and strength of GFP positive cells lining the endosteal, periosteal and trabecular surfaces compared to 3.6 +/+ mice. In contrast GFP expression was similar between 2.3 oim/oim and 2.3 +/+ mice. This data indicates that the lineage is under continuous stimulation that could result in premature senescence. Currently we are testing that possibility by evaluating the capacity of the osteoprogenitor lineage to generate mature osteoblasts with advancinmg age and by mechanical loading of aged GFP oim/+ mice.

Disclosures: I. Kalajzic, None.


Effects of Extracellular Matrix Proteins on Bone Differentiation around Titan Implants.C. S. Berntsen*, E. A. Riksen*, H. S. Berner*, J. E. Reseland*, I. Slaby*, D. Dutch*, J. E. E. Ellingsen*, S. P. Lyngstadaas*. Faculty of Dentistry, University of Oslo, Oslo, Norway.

In the enamel matrix amelogenin is the main component, comprising approximately 90% of all proteins secreted by ameloblasts. The others are amelin, enamelin, serum proteins, tuftelin and enzymes. Amelogenin in the form of Emdogain® (EMD) is a formulation of pig amelogenin proteins in polyglycol alginate. Amelogenin and amelin are involved in biomineralization of dental hard tissues, secreted by ameloblasts and odonto-blasts during tooth formation. The purposes of these studies were to investigate the effects of extracellular matrix proteins; EMD, rAmelogenin and rAmelin, on bone growth on titanium implants. Bone growth was studied in new zealand white adult female rabbits. Flat coin shaped implants are placed onto calibrated leveled defects made in the cortical bone of tibia. Proteins were applied on the implant site immediately before placement of implants. After eight weeks animals were sacrificed, implants exposed and tibia immediately placed in a pull-out test machine. During the pull out test implants were stressed with an increasing force perpendicular to the test surface, at constant speed, until detaching from the bone. A load versus time plot was recorded. After implant detachment, bone fluid from the implant site was collected for LDH activity analysis. Effect of matrix proteins on differentiation, proliferation and cell death were tested on osteoblasts (MC3T3) and osteosarcomas (SaOs), cultured on plastic, glass and titanium. The in vivo results demonstrated an positive effect of rAmelin, EMD, and to some extend rAmelogenin on bonding forces as compared to control. rAmelogenin had a 20% increased cytotoxicity, measured as LDH activity in bone fluid, whereas EMD and rAmelin were not different from control. We demonstrated that rAmelogenin and rAmelin were actively taken up by cultured osteo-blasts. No significant effect on proliferation of osteoblasts was observed by rAmelin and rAmelogenin. EMD, and to less extend rAmelogenin, enhanced differentiation as monitored by increased osteocalcin secretion to the media, whereas rAmelin had no significant effect. rAmelin significantly induced secretion of IL-6 to the media, whereas rAmelogenin had no significant effect. IL-6 may upregulate the activity of osteoclasts, and thus affect bone turnover. Pre-treatment of titanium implants with extracellular matrices proteins may be a strategy for promoting bone growth and inducing differentiation of osteoblasts.

Disclosures: C.S. Berntsen, None.


A Proton-Sensing G Protein-Coupled Receptor Expressed in Osteoblast-like Cells.M. Ludwig*, M. Vanek*, C. E. Jones*, U. Junker*, H. Hofstetter*, R. M. Wolf*, K. Seuwen*. Bone Metabolism, Novartis Institutes for Biomedical Research, Basel, Switzerland.

G protein-coupled receptors (GPCRs) represent the largest gene family in the human genome, with more than 800 members. These receptors respond to a variety of ligands including small molecule neurotransmitters, peptides, large proteins, lipids, but also calcium and photons. For many GPCRs ligands are still unknown. Working with recombinant cell systems expressing a specific orphan receptor (R412), we observed a strong apparent basal activity of the phosphoinositide signalling system which was not modulated by known GPCR ligands. Further experiments showed that the measured signal was strongly pH-dependent, close to zero at pH 7.8, maximal at pH 6.8, and independent of other assay buffer constituents. Activation at slightly acidic pH was strong, comparable to activation of other GPCRs by their cognate ligands. Inspecting the putative secondary structure of R412 we observed specific histidines at the extracellular surface of the receptor, that were most likely involved in pH sensing. Site-directed mutagenesis confirmed this hypothesis. The skeleton participates in pH homeostasis and osteoblasts were shown to respond to pH changes in the range of pH 6.8 -- 7.4. We detected expression of mRNA for R412 in MG63 osteosarcoma cells and in primary human osteoblast precursors isolated from trabecular bone. These cells showed strong pH-dependent inositol phosphate formation matching that observed in fibroblasts expressing ectopic R412. Our data suggest that R412 is a proton-sensing receptor involved in pH homeostasis and bone metabolism.

Disclosures: M. ludwig, None.


Role of Alternative Signaling Pathways in the Activation of a Cyclic AMP Response Element (CRE)-Luciferase Reporter System in an Osteoblast-like Cell Line.R. J. Murrills, B. M. Bhat, J. L. Andrews*, R. L. Rupert*, V. E. Coleburn*, F. J. Bex. Women's Health and Bone, Wyeth Research, Collegeville, PA, USA.

Parathyroid hormone (PTH) is known to activate both cAMP and calcium/PLC/PKC signaling pathways following binding to the PTH1 receptor in osteoblasts. In addition, the cAMP pathway can also activate the MAPK pathway. Previous work has shown that a discrepancy can exist between the potency of a truncated PTH peptide in stimulating cAMP and its activity on a CRE-Luciferase reporter, suggesting that other signaling pathways in addition to cAMP may be involved in the activation of the CRE. There is evidence, for example, from other systems that the PKC, calcium and MAPK pathways can each phosphorylate CREB and potentially activate CREs. We have constructed a CRE-Luciferase reporter containing multiple copies of the CRE and stably transfected it into the osteoblast cell line Saos-2. We then tested the ability of modulators of alternative pathways to either activate the CRE or block the PTH-induced activation of the CRE.

Forskolin, an adenylate cyclase activator, and the phosphodiesterase inhibitors IBMX and rolipram each activated the reporter, confirming the role of cAMP in the activation of the CRE. In addition, the protein kinase A (PKA) inhibitor H-89 blocked the effect of PTH in activating the CRE-reporter. Interestingly, a MAPK inhibitor PD-98059, which blocks a pathway outside of the main cAMP/PKA pathway, also partially inhibited the activity of PTH on the CRE reporter. Furthermore, phorbol myristate acetate, an activator of PKC, was capable of inducing a significant increase in CRE-Luciferase reporter activity, implicating the PKC pathway as an additional potential activator of the CRE. A23187, a calcium ionophore, had only a small effect on the reporter, which was not significant, while the calmodulin kinase II inhibitor KN-93 could not significantly block the effect of PTH.

We conclude that, in addition to the cAMP/PKA pathway, the PKC and MAPK pathways may also play a role in activating the CRE in this osteoblast-like system.

Disclosures: R.J. Murrills, None.


Stimulation of IL-6 Expression by TNFα in Osteoblast Does Not Depend on p38 Activation.J. C. Dai, X. Chen, E. M. Greenfield. Orthopaedics, Case Western Reserve University, Cleveland, OH, USA.

We have previously shown that TNFα stimulates biphasic expression of IL-6 mRNA and IL-6 protein in MC3T3-E1 osteoblastic cells (JBMR 17:S327, 2002). Thus, both IL-6 mRNA and protein are rapidly induced during the early phase of stimulation by TNFα (0--2 hours) followed by a period of declining IL-6 mRNA levels and little IL-6 protein secretion despite the continuous presence of TNFα. A late phase of increased IL-6 mRNA and protein expression occurs 12--24 hours after stimulation by TNFα, leading to a 5--10 fold increase in IL-6 protein secretion. Other investigators have shown that p38 MAP kinase is activated by TNFα in osteoblasts and that p38 inhibitors partially inhibit stimulation of IL-6 mRNA by TNFα. We therefore examined whether the early or late phases of stimulation by TNFα, or both, depend on activation of p38. Western blotting showed that p38 phosphorylation was rapidly stimulated by TNFα with maximal phosphorylation detected after 5 minutes of exposure. p38 phosphorylation slowly declined to a nadir at 8--12 hours and, then, increased at 20--24 hours. This biphasic pattern of p38 phosphorylation is similar to the biphasic expression of IL-6 induced by TNFα. Since the commonly used inhibitors of p38 have recently been shown to have potent non-specific effects, we used three analogues to determine whether p38 activation is responsible for stimulation of IL-6 expression by TNFα: SB202190, one of the commonly used inhibitors of p38; SB220025, a newly described p38 inhibitor that is more specific; and SB202474, an inactive analogue. As expected, both p38 inhibitors completely blocked the cellular phosphorylation of MAP-KAPK-2, a prominent p38 substrate, during both the early and late phases of stimulation by TNFα, while the inactive analogue had no detectable effect. Despite their similar effects on p38 activity, the two p38 inhibitors had divergent effects on TNFα-induced IL-6 expression. ELISA measurements showed that SB202190 strongly inhibited IL-6 secretion by 88% (p<0.0002) in the early phase and by 71% (p<0.001) in the late phase. In contrast, SB220025 and the inactive analogue had indistinguishable effects (p>0.5) in the early phase and SB220025 increased IL-6 secretion by 78% (p<0.001) during the late phase. Similar effects were also observed at the mRNA level. Since SB220025 inhibited TNFα-induced p38 activity without inhibiting expression of IL-6 mRNA or IL-6 protein, activation of p38 is not required for stimulation of IL-6 expression in either the early or late phases. Moreover, since SB220025 increased IL-6 expression during the late phase of stimulation by TNFα but not during the early phase, p38 activity may downregulate IL-6 expression specifically during the late phase.

Disclosures: J.C. Dai, None.


Parathyroid Hormone (PTH)-Induced Ca2+ Signaling in Osteoblasts is Modulated by the Level of PTH1R Expression.B. J. Votta, A. M. Rodriguez-Rojas*, S. M. Hwang*, D. J. Rickard, M. E. Nuttall, S. M. Blake, S. Kumar. MusculoSkeletal Diseases, GlaxoSmithKline, King of Prussia, PA, USA.

In osteoblasts PTH binds to the type 1 PTH receptor (PTH1R) and subsequently activates both the Gs/adenylate cyclase/cAMP/PKA and the Gq/Gi/PLC/IP3/Ca signaling pathways. The relative importance of these different signaling pathways in mediating the net physiological effects of PTH on bone remodeling remains unclear. In HEK293 cells stably expressing high levels of the human PTH1R, we have observed robust signaling via both the cAMP and [Ca 2+]i pathways in response to PTH[1--34]. In contrast, in SaOS-2, ROS 17/2.8, and primary rat osteoblasts expressing lower levels of the PTH1R, PTH[1--34] consistently elicited robust cAMP responses, but effects on [Ca 2+]i were barely detectable. In order to investigate the effect of the level of PTH1R expression on [Ca2+]i signaling SaOS-2 cells were transiently transfected with increasing amounts of a human PTH1R-enhanced green fluorescent protein (GFP) expression construct. The relative levels of PTH1R expression following transfection were quantitated using a 125I-PTH[1--34] radioligand binding assay. Gs-mediated signaling was monitored by assessing cAMP production (ELISA) in response to PTH[1--34] in the presence of IBMX. Gq/Gi-mediated signaling was monitored by assessing changes in [Ca 2+]i in Fura-2 loaded cells using a FlexStation. Cells transfected with the GFP-vector alone exhibited a transfection efficiency similar to those transfected with the PTH1R-GFP construct, but showed no increases in PTH receptor number, or PTH-stimulated cAMP or [Ca 2+]i compared to untransfected cells. Forty-eight hours following transfection with the PTH1R-GFP construct, SaOS-2 cells exhibited a 4-to 10-fold increase in receptor number. A similar increase in the maximal cAMP response to PTH[1--34], but not to Forskolin, was also observed. Interestingly, a corresponding increase in the magnitude of the [Ca 2+]i response was also observed indicating that efficient coupling of PTH1R to Gq/Gi, and hence PLC/[Ca2+]i, may require higher levels of receptor expression than does coupling to Gs. These data suggest that in addition to the mechanisms already described the level of PTH1R expression may qualitatively and quantitatively modulate PTH responsiveness in osteoblast-like cells.

Disclosures: B.J. Votta, None.


In Osteoblastic Cells, the JNK Pathway Mediates Cell Proliferation by Mitogens and Is a Negative Regulator of Early Differentiation.J. Caverzasio, J. Lemonnier*, C. Ghayor*. Dept of Geriatrics, Division of Bone Diseases, Geneva, Switzerland.

Mitogen-activated protein kinase (MAPK) cascades are central signalling transduction pathways induced by mitogens and stresses. At least four MAPK cascades exist, ERK, p38, JNK and ERK5. Recent studies indicate that various receptor systems can induce activation of the JNK (c-Jun N-terminal Kinase) cascade in mammalian cells including G protein-coupled receptors, BMP receptors as well as the Wnt/LRP receptors. In osteoblastic cells, recent studies suggest that the ERK pathway is implicated in mediating cell proliferation by mitogens whereas the p38 pathway is involved in regulating expression of alkaline phosphatase in response to various growth factors. The role of the JNK pathway in osteo-blastic cell regulation is not known and was investigated in the present study. We previously shown that JNK is activated by serum growth factors (SGFs) in MC3T3-E1 cells and recently observed a similar response in calvaria-derived osteoblastic cells. The role of JNK in mediating cell proliferation and differentiation by SGFs was studied in MC3T3-E1 cells using the selective SP600125 JNK inhibitor. We found that incubation of preconfluent cells with this inhibitor dose-dependently (10--25 microM) reduced DNA synthesis and cell number with a complete inhibitory effect at 25 microM. This dose of SP600125 had no effect on activation of ERK induced by SGFs. In early differentiating cells, treatment of cells with SP600125 surprisingly induced a time- and dose-related increase in alkaline phosphatase activity. With 20 microM SP600125, a significant effect was already observed after 48 h incubation with a pronounced and maximal effect after 5 days treatment (4 fold stimulation). This effect of SP600125 was associated with a significant increase in SGFs-induced activation of p38, a MAP kinase pathway that we previously found to be required for expression of ALP in MC3T3-E1 osteoblastic cells.

In conclusion, results presented in this study indicate that JNK plays an essential role in mediating cell proliferation by growth factors in osteoblastic cells. They also suggest that JNK is a negative regulator of alkaline phosphatase expression, an effect probably mediated by a yet unknown negative cross regulation of JNK on the p38 pathway. Taken together these data suggest that the JNK pathway is an essential signalling pathway for controlling the proliferation and early differentiation of osteoblastic cells.

Disclosures: J. Caverzasio, None.


Protein Kinase D Is an Essential Signalling Molecule for the Regulation of Osteoblastic Cell Growth and Differentiation.C. Ghayor*, J. Lemonnier*, J. Caverzasio. Dept of Geriatrics, Division of Bone Diseases, Geneva, Switzerland.

The development of osteoblasts is dependent of yet incompletely understood signalling cascades that support proliferation and differentiation. We recently described that BMP-2 induces activation of the MAP kinases p38 and JNK and that both pathways are implicated in BMP-2-induced osteoblastic cell differentiation. More recently, we provided compelling evidences that activation of p38 and JNK by BMP-2 involves protein kinase D (PKD). In the present study, we investigated the role of this new PKD/JNK-p38 pathway in mediating osteoblastic cell proliferation and differentiation by serum growth factors (SGFs). Exposure of unstimulated MC3T3-E1 cells to 10% FCS induced a rapid and transient activation of PKD with maximal activation/phosphorylation of PKD on Ser744/748 and Ser916 after 1 h incubation. Associated with this effect, there was a corresponding transient and maximal activation of JNK and p38. This latter effect was completely blocked by the selective PKD inhibitor G06976 (10 microM). We found that this new signalling pathway is also activated by SGFs in cultured mouse calvaria-derived osteoblastic cells with similar kinetic and potency of activation compared with that found in MC3T3-E1 cells. The role of PKD in mediating cell growth and differentiation was then analyzed in two clones of MC3T3-E1 cells stably expressing PKD antisense oligonucleotide (AS-PKD) and having a selective and markedly reduced expression of PKD compared with vector transfected cells (V-PKD). Results indicate that the size of AS-PKD growing cells appeared to be increased and cell number significantly reduced by 50 to 60 % compared with V-PKD cells (p<0.001). In confluent AS-PKD cells, expression of markers of osteo-blastic differentiation such as collagen I, alkaline phosphatase and osteocalcin were significantly (p<0.001) and respectively reduced by 40--50%, 60--70% and 70--80% compared with V-PKD cells. Associated with this effect, activation of JNK and p38 was nearly completely blunted in AS-PKD compared with V-PKD cells.

In conclusion, data presented in this study indicate that PKD is activated by serum growth factors in MC3T3-E1 and primary cultured calvaria-derived osteoblastic cells and that this signalling molecule is involved in regulating osteoblastic cell proliferation. They also suggest that PKD mediates activation of JNK and p38 and that this pathway is essential for the differentiation of osteoblastic cells. Together, these observations strongly suggest that PKD is an important signalling molecule for the regulation of the growth and differentiation of osteoblastic cells.

Disclosures: J. Caverzasio, None.


Bisphosphonates Affect Signaling Pathways Utilized by Teriparatide [rhPTH(1--34)].Q. Sun*1, R. W. Katz2, S. A. Morris3, J. P. Bilezikian1. 1Division of Endocrinology, College of Physicians and Surgeons, Columbia University, New York, NY, USA, 2School of Oral and Dental Surgery, Columbia University, New York, NY, USA, 3Aventis Pharmaceuticals, Bridgewater, NJ, USA.

There are questions about the antecedant use of bisphosphonates (BP) on the subsequent effects of teriparatide [PTH(1--34)] for the treatment of osteoporosis. This clinical question requires a more basic understanding of how various BP may influence PTH-dpendent signal transduction pathways, but also the reversibility of these effects. In this study, we compared the effect of Alendronate (ALE) or Risedronate (RIS) on the subsequent ability of PTH to stimulate adenylyl cyclase activity and inositol phosphate accumulation.

When a kidney cell line stably transfected with PTH type 1 receptor was exposed to either ALE or RIS (10 pM - 1 mM), maximal PTH dependent inositol phosphate formation was suppressed by ∼30--50% at a 0.1 mM concentration. Suppression was noted with 60 minutes of pretreatment; longer incubations did not increase or reduce suppression. Recovery from BP suppression was evaluated by removal of BP medium prior to PTH stimulation. After 24--48 hours of bisphosphonate exposure, the suppression of PTH inositol phosphate accumulation was completely reversible within two hours. Preliminary studies show that ALE caused more suppression than RIS at equivalent concentrations and that the recovery was faster with RIS than with ALE.

SAOS2 human osteosarcoma cells were exposed to graded concentrations of ALE or RIS (10 pM to 1 mM) for one hour and adenylyl cyclase activity (ACA) of the cell homogenate was determined +/− PTH. ALE did not reproducibly suppress PTH-ACA, but RIS at 0.1 micromolar suppressed PTH-ACA by ∼30 %. When cells were incubated for a longer period of time at a 1 micromolar concentration (24--48 hours), ALE and RIS appeared to weakly suppress PTH-ACA in an inconsistent fashion.

These findings confirm our hypothesis that bisphosphonates have measurable effects upon key signaling pathways stimulated by PTH, but these effects are subtle. The physiologic significance of the actual effects of these BP on PTH signaling are not known. However, the differences in reversibility of the PLC suppression by ALE and RIS may provide insight into the clinical impact of previous BP treatment followed by teriparatide therapy.

Disclosures: Q. Sun, Proctor and Gamble-Aventis Pharma R.


Role of Pten and Akt in the Regulation of Growth and Apoptosis in Human Osteoblastic Cells.S. M. Nielsen-Preiss1, S. R. Silva*1, J. M. Gillette2, S. Stroh*3. 1Orthopaedics, UCHSC, Denver, CO, USA, 2Cell and Developmental Biology, UCHSC, Denver, CO, USA, 3University of Colorado, Boulder, CO, USA.

Cancer cells are characterized by either an increased ability to proliferate or a diminished capacity to undergo programmed cell death. PTEN is instrumental in regulating the balance between cell growth and death in several cell types and has been described as a tumor suppressor. In a subset of human osteosarcomas the chromosomal arm on which PTEN is located has been deleted. Therefore, we predicted that the loss of PTEN expression was contributing to increased Akt activation and the subsequent growth and survival of osteosarcoma tumor cells. Analysis of several human osteosarcoma cell lines and normal osteoblasts revealed relatively abundant levels of PTEN expression. Furthermore, stimulation of cell growth or induction of apoptosis in osteosarcoma cells failed to affect PTEN expression or activity. Therefore, routine regulation of osteoblastic cell growth and survival appears to be independent of changes in PTEN expression. Subsequently, the activation of a downstream target of PTEN activity was analyzed.

Akt is a growth factor-responsive kinase that contributes to cellular growth and survival. We predicted that although we were unable to detect changes in PTEN levels, inappropriate activation of Akt could support uncontrolled cell growth and survival and thus still implicate this pathway in the osteosarcoma phenotype. Analysis of Akt expression in several osteosarcoma cell lines and normal osteoblasts also revealed uniformly low levels of activated (phosphorylated) Akt, even following growth stimuli. In addition, osteosarcoma cell growth was only minimally affected by high concentrations of inhibitors of phosphoinositol-3 kinase, an upstream activator of the Akt signaling pathway. These data further suggest that the Akt pathway in not the predominant signaling cascade required for osteoblastic growth.

Incidentally, inhibition of PTEN activity resulted in increased levels of Akt phosphorylation and enhanced cell proliferation, implying that the Akt signaling pathway is intact and functional, but normally suppressed. These data suggest that abundant levels of PTEN normally maintain Akt in an inactive form in osteoblastic cells. Suppression of PTEN, perhaps by chromosomal loss, can provide a mechanism for the activation of Akt and thus may contribute to osteosarcoma growth and survival.

Disclosures: S.M. Nielsen-Preiss, None.


Regulation of Phospholipase D in Osteoblastic Cells by Gα12 and Gα13, Mevastatin and Alendronate.A. T. Singh1, T. Voyno-Yasnetskaya*2, P. H. Stern1. 1Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA, 2Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA.

Phospholipase D (PLD) is an important activator of signaling in many cell types including osteoblasts, and leads to a wide range of biological responses. In UMR-106 osteoblastic cells, parathyroid hormone (PTH) stimulation of PLD activity leads to membrane translocation of PKCa and increased interleukin-6 expression and is dependent on the small GTP binding protein RhoA. Rho A can be activated through heterotrimeric G proteins of the Gα12/Gα13 class, and translocation of Rho A to the plasma membrane involves its modification by geranylgeranylation. The current studies were designed to determine the effects of Gα12 and Gα13 on PLD activity in UMR-106 osteoblastic cells, as well as to assess whether pharmacological agents that can decrease geranylgeranylation interfere with PTH stimulation of PLD activity. UMR-106 cells were cutured in DMEM/15% heat-inactivated horse serum/penicillin in 6-well plates. Phospholipase D activity was assessed by the transphosphatidylation of ethanol. Expression of constitutively active Gα12 and Gα13 in the UMR-106 cells markedly increased PLD activity. In contrast, activation of the PKA pathway by forskolin (0.04 mM, 3--30 min) did not affect PLD, and 60 min preincubation with the PKA antagonist PKI (0.01 mM) did not influence the PLD response elicited by 5 min treatment with 10 nM PTH. PLD activity was increased by geranylgeranyl pyrophosphate (0.01 mM, 60 min). The PTH-stimulated increase in PLD was inhibited by the HMGCoA reductase inhibitor mevastatin (0.2 - 5 μM) and the bisphosphonate alendronate (10μM), both of which can decrease the production of geranylgeranyl groups. Neither inhibitor affected basal PLD activity. The results indicate the involvement of heterotrimeric G proteins of the Gα12 and Gα13, but not the Gs class in the upstream activation of PLD in osteoblastic cells. Further, the findings identify PLD as a osteoblast target for statins and aminobisphosphonates, possibly through effects on Rho signaling.

Disclosures: A.T. Singh, None.


TNF-alpha Expression Is Transcriptionally Regulated by RANK Ligand.W. Zou*1, H. Drissi2, Z. Bar-Shavit1. 1Experimental Medicine and Cancer Research, Hebrew University, Jerusalem, Israel, 2Center for Musculoskeletal Research, University of Rochester, Rochester, NY, USA.

Tumor necrosis factor (TNF)-α is known for its osteoclastogenic and resorptive activities. Induction of osteoclastogenesis by receptor activator of NF-κB ligand (RANKL) is accompanied by increased TNF-α expression. In the present study we investigated the mechanisms by which RANKL induces expression of TNF-α in osteoclast precursors using murine bone marrow derived cells (BMMs) and the macrophage-like cell-line, RAW 264.7 (RAW). We found that RANKL is a potent osteoclastogenic stimulator in both models, whereas TNF-α exhibits very low osteoclastogenic activity. Anti-TNF-α antibody significantly inhibits RANKL-induced osteoclastogenesis while RANKL up-regulates TNF-α expression with similar kinetics in these two cell models. We first examined if RANKL-mediated increase in TNF-α expression, involves increased stability of its transcript. BMMs and RAWs were treated with or without RANKL for 20 minutes, and then a transcription inhibitor (DRB) was added. At different time points, TNF-α and L32 mRNA levels were examined. The rate of TNF-α mRNA degradation was not altered by RANKL indicating that this effect is not due to mRNA stability. We therefore measured the transcription rate of TNF-α by run-on assay in RAW cells after treatment with RANKL (50 ng/ ml, 25 minutes). RANKL increases TNF-α transcription rate by 2.5-fold in RAW cells. We further characterized this transcriptional induction of TNF-α by RANKL. Gel shift assays using nuclear extracts derived from RANKL-treated RAWs cells show increased specific NF-κB binding activity on the murine TNF-α promoter. We finally used 1260 bp of the murine TNF-α promoter fused to luciferase (1260TNF/Luc), as well as several 5′ deletion mutants of this promoter (containing 656, 529, 514 and 210 bp, 656TNF/Luc, 529TNF/ Luc, 514TNF/Luc and 210TNF/Luc, respectively) to stably transfect RAW cells. 1260TNF/Luc and 656TNF/Luc promoter activity was increased in response to RANKL, whereas treatment of 529TNF/Luc, 514TNF/Luc and 210TNF/Luc stable cell lines did not elicit significant reporter gene expression. In conclusion, RANKL induces TNF-α expression via a transcriptional mechanism, depending on some, but not all of the NF-κB sites in the TNF promoter.

Disclosures: W. Zou, None.


Tumor Necrosis Factor-alpha Inhibits the Formation of Osteoclasts in vitro through the p55 Receptor on Osteoblasts.R. Balga*1, C. Mueller*2, W. Hofstetter1. 1Department Clinical Research, University of Bern, Bern, Switzerland, 2Department for Pathology, University of Bern, Bern, Switzerland.

Tumor necrosis factor-alpha (TNFα) is a pleiotropic cytokine, acting in synergism with other cytokines such as IL-1 or receptor activator of NF-kappaB ligand (RANKL) during the recruitment of osteoclasts. It was suggested that TNFα plays a critical role in the induction of bone loss after estrogen depletion. We have found, however, that in mice deficient for TNFα or for the p55 TNFα receptor, ovariectomy induces a decrease in bone mass. To address this discrepancy, in the present study the effects of TNFα on the recruitment of osteoclasts in vitro were further investigated.

The role of TNFα in osteoclastogenesis was studied in two culture systems. Bone marrow cells (BMC) were cultured in the presence of colony stimulating factor-1 (CSF-1) and RANKL, or BMC were cultured with primary osteoblasts. In both culture systems the cells were grown in the presence or absence of TNFα. After 6 days, the cells were stained for tartrate resistant acid phosphatase (TRAP), and TRAP+ multinucleated osteoclasts (MNC) were counted. In cultures of BMC with CSF-1 and RANKL, the number of MNC was not affected by TNFα (mean ± SE: 106 ± 5 [-TNFα] vs. 100 ± 1 [+TNFα]). In co-cultures of BMC and wt osteoblasts, the formation of osteoclasts was found to be inhibited by TNFα (mean ± SE: 305 ± 28 vs. 1 ± 2). If wt BMC were cultured with osteoblasts lacking the p55 receptor, the inhibitory effect of the cytokine was blocked and the number of TRAP+ MNC formed was even increased (mean ± SE: 108 ± 8 vs. 188 ± 14).

To investigate, whether the effect of TNFα is mediated through the release of soluble factors, conditioned media (CM) from wt or p55-/- osteoblasts treated with the cytokine were added to cultures of wt BMC and osteoblasts from wt or p55-/- mice. CM from wt osteo-blasts treated with TNFα inhibited the formation of MNC (mean ± SE: 173 ± 6 vs. 26 ± 8), whereas CM from p55-/- osteoblasts treated with TNFα did not affect osteoclastogenesis (mean ± SE: 133 ± 1 vs. 133 ± 17). The effect of TNFα was dependent on 1,25(OH)2D3, since osteoblasts released the inhibitory activity on osteoclast formation only when cultured in the presence of 1,25(OH)2D3 and TNFα. To exclude toxic effects of the cytokine, the number of osteoblasts and the levels of transcripts encoding RANKL and osteoprotegerin (OPG) were determined. Both of these parameters were not affected by TNFα.

The results of this study show that the role of TNFα in bone is complex and not restricted to the stimulation of bone resorption. TNFα exerts an inhibitory effect on the formation of osteoclasts in vitro, this effect being dependent on the presence of functional p55 TNF receptors on the cells of the osteoblast lineage.

Disclosures: R. Balga, None.


Cytokine Imbalance in Celiac Disease and Parallel Direct Stimulation of Osteoclastogenesis and Osteoblast Activity in Vitro.A. Teti1, A. Taranta*1, D. Fortunati*1, M. Longo*1, N. Rucci1, S. Migliaccio1, M. T. Bardella*2, S. Saraifoger*2, A. Dubini*2, M. L. Bianchi2. 1Experimental Medicine, University of L'Aquila, L'Aquila, Italy, 2Bone Metabolic Unit, Istituto Auxologico Italiano, IRCCS, Milan, Italy.

Celiac disease is an auto-immune disorder characterized by atrophy of the intestine villi triggered by ingestion of gluten in genetically susceptible individuals. The association between celiac disease and low bone mineral density (BMD) has been recognized but the mechanisms of disturbance are poorly understood. We investigated 42 patients, 25 on gluten-free diet (GFD) (age 35.7+/−7.9, 19 females, 5 males), 17 not on GFD (age 41.3+/−10.8, 13 females, 4 males), and 21 normal controls (age 32.4+/−4.6, 17 females, 4 males). Patients presented a significant increase of the bone resorption marker N-terminal telopeptide of procollagen type I, but normal osteocalcin, calcium, PTH and 1,25(OH)2 D3 levels. IL-6, IL-1beta, TNFalpha and TNFbeta were similar to controls. The inhibitory cytokine IL-12 was reduced in all celiac patients, while IL-18 only in those on GFD. The RANKL/ OPG ratio was significantly higher (2.5-fold) in the celiac patients not on GFD, whereas it was not different from controls in the patients on GFD. Peripheral blood mononuclear cells from healthy donors cultured in media supplemented with 25 ng/ml M-CSF, 100 nM PTH, sub-optimal concentration of RANKL (0.5 ng/ml) and 5% sera of our patients not on GFD showed a dramatic increase of the number of TRAP-positive multinucleated cells relative to similar cultures supplemented with normal control sera. A lesser increase was instead observed with sera from celiac patients on GFD. The effect was clearly visible after one week of exposure and persisted throughout three weeks. Cultured human osteoblasts from healthy individuals were also subjected to incubation with 5% of our serum pools. No significant modulations of the stimulatory cytokines IL-6 and IL-1beta were observed, and TNFalpha was undetectable. The inhibitory cytokine IL-18, on the other hand, was found to be reduced by exposure to sera from all celiac patients, regardless of the diet regimen, whereas IL-12 was unremarkable. OPG expression was lower upon incubation with the sera from celiac patients not on GFD. RANKL and PTHrP could not be detected in any of our human osteoblast cultures. Proliferation, alkaline phosphatase and nodule mineralization were increased in osteoblast cultures containing sera from celiac patients, either on or not on GFD, but to a remarkably higher extent in the latter. We conclude that in celiac disease bone loss is likely to be due to an imbalance of factors affecting bone-turnover which may directly affect osteoclastogenesis and osteoblast activity.

Disclosures: A. Teti, None.


Role of Osteoclast Inhibitory Peptide-1(OIP-1/hSca) in Interleukin-12 Inhibition of Osteoclast Formation.N. Kawanabe1, M. Koide*1, G. D. Roodman2, S. V. Reddy1. 1Medicine-Hematology/Oncology, University of Pittsburgh, Pittsburgh, PA, USA, 2Medicine-Hematology/Oncology, University of Pittsburgh and Department of Veterans Affairs Medical Center, Pittsburgh, PA, USA.

Osteoclast formation and activity is regulated by local factors produced in the bone microenvironment. Immune cell products such as IFN-γ and IL-12 are potent inhibitors of osteoclast formation. Furthermore, IL-12 has been shown to stimulate IFN-γ production by T-cells. More recently, we found that IFN-γ stimulated osteoclast inhibitory peptide-1 (OIP-1/hSca) expression in osteoclast precursor cells, and that anti-OIP-1 c-peptide specific antibody significantly neutralized IFN-γs inhibition of osteoclast differentiation. However, it is unclear if OIP-1 represents a common mediator for IFN-γ and IL-12 inhibition of osteoclast formation. Therefore, we tested the capacity of OIP-1 c-peptide to inhibit osteoclast formation in IFN-γ receptor deficient mouse (IFN-γ R -/-) bone marrow cultures stimulated with RANKL and M-CSF. OIP-1 significantly inhibited osteoclast formation in IFN-γ R -/- mouse bone marrow cultures analogous to non-transgenic control mice. We further examined IL-12 regulation of OIP-1 expression using cycle-dependent RT-PCR analysis, which demonstrated that IL-12 treatment (24 hr) significantly enhanced OIP-1 mRNA expression in normal human bone marrow cells, but had no effect on highly purified osteoclast precursor cells. To determine the role of IFN-γ and OIP-1 in IL-12 inhibition of osteoclast formation, we tested the capacity of IL-12 to inhibit osteoclast formation in IFN-γ receptor deficient mice (IFN-γ R -/-) bone marrow cultures. Interestingly, in contrast to non-transgenic control mice, IL-12 (20 ng/ml) did not inhibit osteoclast formation in IFN-γ R -/- mice bone marrow cultures stimulated with RANKL and M-CSF. Furthermore, addition of a neutralizing antibody against IFN-γ to IL-12 treated control mice bone marrow cultures, completely abolished IL-12 inhibition of osteoclast formation. However, addition of OIP-1 c-peptide specific neutralizing antibody partially (50%) blocked IL-12's inhibition of osteoclast formation in normal mouse bone marrow cultures. In contrast, a non-specific IgG did not affect IL-12 inhibition of osteoclast formation in these cultures. Furthermore, IL-12 did not inhibit osteoclast formation in normal mouse bone marrow cultures that were depleted of T-cells using a Thy 1.2 antibody. These data suggest that IFN-γ and OIP-1 are responsible for the inhibitory effects of IL-12 on osteoclast formation.

Disclosures: S.V. Reddy, None.


gp130-Mediated Signals Play a Role in Osteoclast Differentiation and Activation.H. I. Shin1, E. K. Park2, S. Y. Kim2, T. Kobayashi3, P. Divieti3, F. R. Bringhurst3, H. M. Kronenberg3. 1Department of Oral Pathology, Kyunpook National University, Daegu, Republic of Korea, 2Skeletal Genome Research Center, Kyunpook National University Hospital, Daegu, Republic of Korea, 3Endocrine Unit, MGH, Harvard, Boston, MA, USA.

gp130, a common signal-transducing 130 Kd glycoprotein for gp130-associated cytokines such as IL-6, IL-11, LIF, OSM, CNTF, and CT-1, may play an important role in osteo-clastogenesis. However, the precise actions of gp130 that influence osteoclast differentiation and activation are not well understood. To address this issue, we analyzed the structural characteristics of osteoclasts in gp130-/- fetuses by staining for TRAP activity and by ultrastructural observation. We also studied in vitro osteoclastic induction by PTH(1--34), 1, 25 (0H)2 vitamin D3 and sRANKL using co-cultures, with analysis of enzymatic activity and resorptional activities for induced TRAP+ multinucleated cells. The gp130-/- TRAP+ osteoclasts in fetal tibiae were characteristically larger and more ovoid in shape with more nuclei/cell, than WT TRAP+ osteoclasts, which exhibited a small, flat triangular appearance. Ultrastructurally, the gp130-/- osteoclasts showed poor development of ruffled borders, suggesting dysfunctional bone resorbing activity. The in vitro induction of osteoclasts by co-culture of gp130-/- primary calvarial osteoblasts and wt adult bone marrow cells stimulated by either 10−7 M PTH(1--34) and 10−8 M 1, 25 (0H)2 vitamin D3 was significantly reduced when compared to cultures using WT calvarial osteoblasts, but the induction was closer to normal in response to exogenous sRANKL treatment. The TRAP+ multinucleated cells induced by gp130-/- primary calvarial osteoblasts in co-cultures showed disarrangement of actin filaments and weaker immunoreactivity using antiserum to TRAP and Cathepsin K. Furthermore, they did not form pits on dentin slices. These findings suggest that both PTH(1--34) and 1, 25 (0H)2 vitamin D3 require gp130-mediated signaling in osteoblast lineage cells to induce adequate RANKL for osteoclastogenesis and that gp130-mediated signaling is important for full osteoclastic activation through regulation of actin ring and ruffled border development, as well as TRAP and cathepsin K production.

Disclosures: H.I. Shin, None.


Gamma-Glutamyl Transpeptidase Protein Enhances RANK Ligand Expression and Osteoclastogenesis in Osteolysis.S. Niida1, T. Kondo*2, T. Hibi*1, K. Ikeda1. 1Geriatric Research, National Institute for Longevity Sciences, Obu, Japan, 2Department of Otolaryngology, Indiana University School of Medicine, Indianapolis, IN, USA.

γ-Glutamyl transpeptidase (GGT) is an ectoenzyme expressed in kidney, pancreas and liver, and used for a marker enzyme for several diseases. We identified GGT as a novel bone resorbing factor by using expression cloning system. Then, we examined the action of GGT in osteoclast formation. Addition of purified GGT (5--625ng/ml) to murine bone marrow cultures dose-dependently induced TRAP-positive cells, which expressed calcitonin receptor and pit forming activity, without 1,25(OH)2D3. Furthermore, we found that inactive form of GGT, whose enzymatic activity was blocked by chemical modification with acivicin, supported osteoclast formation. Taken together, it was demonstrated that GGT stimulated osteoclast formation independently of its enzymatic activity and may involved a putative receptor molecule. Native GGT and inactive GGT induced the expression of RANKL mRNA and protein in bone marow stromal cells, and OPG completely supressed GGT-induced osteoclast formation. In collagen-induced arthritis (CIA) mice and LPS-induced periodontal destruction, GGT expression was markedly increased in synovial tissue and tissue macrophages. Culture of isolated cells from arthritic paws in CIA mice spontaneously appeared the osteoclast-like cells, which was decreased by anti-GGT antibody. These phenomenon suggests that GGT is induced by inflammatory and stimulates osteoclastogenesis through the RANKL expression. Next, we examined effects of recombinat human GGT (rhGGT) on osteoclastogenesis in the co-culture system with bone marrow cells and ST2 cells. rhGGT also induced osteoclast formation and RANKL mRNA expression. Osteoclastogenetic activity of GGT is a novel function of this protein, and further investigation is required to identify the mechanism of induction of RANKL expression.

Disclosures: S. Niida, None.


Systemic TNFa Mediates an Increase in Peripheral CD11b-high Osteoclast Precursors by Inducing their Mobilization from the Bone Marrow.P. Li*1, E. M. Schwarz2, R. J. O'Keefe2, L. Ma*2, R. J. Looney*2, C. T. Ritchlin*2, B. F. Boyce2, L. Xing2. 1Dept. Microbiol. and Immunol., Univ. Rochester, Rochester, NY, USA, 2Center for Musculoskeletal Res., Univ. Rochester, Rochester, NY, USA.

Chronic exposure to TNFa enhances osteoclastogenesis by increasing the frequency of CD11bhi osteoclast precursors (OCPs) in the periphery. To elucidate the possible mechanism(s) involved (proliferation, survival, differentiation or redistribution from bone marrow), we used TNFa transgenic (TNF-Tg) mice and wild type (wt) mice injected with TNFa. TNF-Tg and wt mice were BrdU labeled for 24 hr and spleen cells were stained with antibodies to CD11b and BrdU. TNF-Tg mice had the expected increase in CD11bhi cells, but no increase in proliferation (% BrdU+ cells in the CD11bhi population was similar in TNF-Tg and wt mice:16.3% vs 18.3%). Next, TNF-Tg and wt splenocytes were analyzed for apoptosis by FACS using antibodies to CD11b, fluorescence-labeled annexin V and 7-AAD. In the CD11bhi population, the % of annexin V+/7-AAD- (apoptotic) cells was similar in TNF-Tg and wt mice (9.6% vs 9.5%). To determine if TNFa induces differentiation of CD11b-/lo cells to CD11bhi OCPs, wt splenocytes were cultured with 10 ng/ ml of TNFa for various times. No significant difference was detected in the % of CD11hi cells in control and TNF-treated cultures, determined by FACS after 24h, and no detectable induction of CD11b was found in CD11b mRNA expression by quantitative real-time PCR after 1, 4 and 24h TNF treatment. Similar results were obtained from CD11b- and CD11blo splenocytes sorted by FACS and treated with TNFa for 12h. To examine if TNFa affects the distribution of CD11bhi cells in vivo, wt mice were injected with BrdU for 3d to maximally label bone marrow CD11b+ cells (>96%) and given PBS or TNFa (1mg ip per injection.) either once and sacrificed 4h later or 4x/d for 3d. Bone marrow, spleen, and peripheral blood monocytes (PBMC) were harvested to determine the % of BrdU+/ CD11b+ cells by FACS. TNFa caused a rapid release of CD11b+ cells from bone marrow to the blood at 4h (% CD11b+/BrdU+ PBMC increased 4-fold), but significantly altered this population in the spleen only after treatment for 3d to a level similar to that observed in untreated TNF-Tg mice. Correspondingly, this treatment caused an increase in the osteo-clastogenic and CFU-M colony-forming potential of the splenocytes from these mice. Thus, we have identified a novel mechanism whereby TNFa causes a marked increase in circulating CD11bhi OCPs in the periphery to account for the increased osteoclastogenesis in patients with inflammatory arthritis: mobilization of OCPs from bone marrow without affecting their proliferation, survival, or differentiation.

Disclosures: L. Xing, None.


IL-3 Acts Directly on Osteoclast Precursors and Inhibits RANKL-Induced Osteoclast Differentiation.S. M. Khapli*, L. S. Mangashetti*, S. D. Yogesha*, M. R. Wani*. Laboratory-1, National Center for Cell Science, PUNE, India, India.

Osteoclasts, the multinucleated cells that resorb bone differentiate from hemopoietic precursors of monocyte/macrophage lineage. Interleukins produced by activated T cells, as well as by other cell types regulates osteoclastogenesis. However, it is not clear how osteo-clastogenesis is regulated by immune cell-derived cytokines. IL-3, a cytokine secreted by activated T lymphocytes stimulates the proliferation, differentiation and survival of pluripotent hemopoietic stem cells. Although osteoclast differentiate from hemopoietic stem cells the role of IL-3 in osteoclast differentiation is not clear. IL-3 has previously been shown to have both stimulatory and inhibitory action on osteoclast formation in complex culture systems.

In this study, we investigated the role of IL-3 in RANKL-induced osteoclast differentiation. We show here that IL-3 inhibits RANKL-induced osteoclast differentiation by a direct action on early osteoclast precursors. Anti-IL-3 Ab neutralized the inhibitory effect of IL-3 on osteoclast differentiation. In addition, IL-3 inhibits TNF-a-induced osteoclast differentiation in bone marrow-derived macrophages. However, IL-3 has no inhibitory effect on mature osteoclasts. In osteoclast precursors, IL-3 prevents RANKL-induced nuclear trans-location of NF-kB by inhibiting the phosphorylation and degradation of IkB. RT-PCR analysis revealed that IL-3 down-regulated c-Fos transcription. Interestingly, the osteoclast precursors in the presence of IL-3 showed strong expression of macrophage markers such as Mac-1, MOMA-2 and F4/80. Furthermore, the inhibitory effect of IL-3 on osteoclast differentiation was irreversible and the osteoclast precursors preincubated in IL-3 were resistant to RANKL action. Thus, our results first time reveal that IL-3 acts directly on early osteoclast precursors and irreversibly block RANKL-induced osteoclast differentiation by diverting the cells to macrophage lineage.

Disclosures: M.R. Wani, None.


Tyrosine-757 of gp130 Plays a Critical Role in Osteoclast Formation.J. M. W. Quinn1, A. Nakamura*1, B. Jenkins*2, N. A. Sims3, M. Ernst*2, T. J. Martin1, M. T. Gillespie1. 1Molecular Endocrinology, St. Vincent's Institute of Medical Research, Victoria, Australia, 2Ludwig Institute of Cancer Research, Victoria, Australia, 3Dept of Medicine, University of Melbourne, Victoria, Australia.

IL-6 and IL-11 play important roles in controling bone remodeling and both signal via the gp130 receptor subunit. Two major intracellular signaling pathways are activated by gp130: SHP2-mediated MAPK signaling cascade which emanates from the membrane proximal phospho-tyrosine residue 757, and the STAT-1/3 mediated pathways requiring membrane distal phospho-tyrosines in gp130. These pathways are under reciprocal negative feedback control. gp130deltaSTAT mice, which have a mutation that ablates STAT1/3 signaling, have normal bone mass while gp130Y757F knock-in mutant mice with a phenylalanine substitution of Y757F display osteopenia, with increased osteoclast and osteoblast numbers. This indicates an important role for gp130 tyrosine 757 in the control of bone remodeling.

In M-CSF-dependent colony formation assays and in cultures stimulated by M-CSF plus RANKL, gp130Y757F bone marrow resulted in greater numbers of colonies (180%) and osteoclasts (191%), respectively, compared to wild type bone marrow. Both IL-6 and IL-11 strongly inhibited colony and osteoclast formation from gp130Y757F bone marrow but had little effect on wild type bone marrow. In contrast, none of these effects were observed in gp130deltaSTAT bone marrow cells.

In co-cultures with osteoblasts, gp130Y757F bone marrow showed a strikingly different pattern of osteoclast formation to the M-CSF plus RANKL stimulated cultures. Wild type bone marrow cells formed 264±22, 406±63 and 309±70 osteoclasts/well in co-cultures stimulated by IL-11, PTH1--34 or 1,25 dihydroxyvitamin-D3 plus PGE2 (D3/PG) respectively. In contrast, gp130Y757F bone marrow cells formed fewer than 5 osteoclasts/well in IL-11 and D3/PG stimulated cultures and 69±3 osteoclasts/well with PTH treatment. This altered response of gp130Y757F bone marrow cells was also observed in co-cultures with gp130Y757F osteoblasts.

These results suggest that bone marrow cells from gp130Y757F mice contain more osteo-clast progenitors and thus form more osteoclasts (in response to RANKL) relative to bone marrow from wild type mice. However, the co-culture data suggests that osteoblasts may produce an activity that is inhibitory of osteoclastogenesis which is evident when signals emanating from gp130Y757 are ablated.

Disclosures: J.M.W. Quinn, None.


The Effect of Calcitonin-Gene-Related-Peptide (CGRP) on Osteoclast-like Cells (OCL) Differentiation and Function.A. C. Demulder1, E. D. Wittersheim*1, M. Guns*1, P. Fondu*1, P. Bergmann2. 1Laboratory of Hematology, Brugmann University Hospital, Brussels, Belgium, 2Laboratory of Experimental Medicine, Brugmann University Hospital, Brussels, Belgium.

We have shown previously that CGRP has in vitro a direct and dose dependent inhibitory effect on human OCL precursor that is at least in part mediated by cAMP. The direct action of CGRP on OCL precursors does not exclude an indirect action through the bone marrow microenvironment. The aim of the present study was to assess the effects of CGRP on bone resorption in human bone marrow cultures and to investigate the interaction of CGRP with the bone marrow microenvironment in this setting, particularly with the RANK-L/Osteoprotegerin (OPG) system. For this purpose, we used two different systems of human bone marrow cultures: the long term bone marrow culture (LTBMC) for OCL differentiation and the CFU-GM derived OCL cells cultivated on hydroxyapatite slices for bone resorption experiments. As previously shown, the formation of OCL in LTBMC was decreased when CGRP was added continuously during the first week of culture. This statistically significant inhibitory effect on proliferation was dose-dependent for 10−11 to 10−6 M concentration of CGRP. The conditioned media of these cultures were frozen for measurements of OPG and RANK-L by ELISA. Levels of OPG were low in control and CGRP treated cultures (100--250 pg/ml) and were not influenced by CGRP concentrations. RANK-L was undetectable in most cultures. Unexpectedly, despite the fact that OPG levels were low and did not differ between treated and controls, the addition of increasing concentrations of a neutralizing antibody directed against OPG (anti-OPG 1/200 to 1/50) restored the formation of OCL in CGRP treated LTBMC and increased OCL formation in control wells. When CFU-GM derived OCL were cultivated on hydroxyapatite slices, RANK-L stimulated bone resorption in a dose dependent manner. CGRP decreased consistently bone resorption by 30--50% when added together with RANK-L 20 ng/ml. In comparison, the addition of OPG at 50ng/ml totally abolished bone resorption. In conclusion, CGRP inhibits both differentiation and function of OCL. These effects do not seem to be mediated through the RANK-L/OPG system and result probably only of a direct action on OCL precursor. The very low or undetectable levels of RANK-L in LTBMC conditioned media could explain why these LTBMC derived OCL are unable to resorb bone.

Disclosures: A.C. Demulder, None.


The Relationship between Circulating Osteoprotegerin Levels and Bone Mineral Metabolism of Korean Women.K. Oh1, E. Oh*1, S. Moon*2, D. Lee*2, W. Lee*3, K. Baik*4, M. Kang4. 1Department of Internal Medicine, Miz Medi Hospital, Seoul, Republic of Korea, 2Department of Family Medicine, Miz Medi Hospital, Seoul, Republic of Korea, 3Department of Internal Medicine, Sungkyunkwan University School of Medicine, College of Medicine, Seoul, Republic of Korea, 4Department of Internal Medicine, Catholic University of Korea, College of Medicine, Seoul, Republic of Korea.

Osteoprotegerin (OPG) is a recently identified cytokine that acts as a decoy receptor for the RANK ligand. OPG has been shown to be an important inhibitor of osteoclastogenesis in animal models. The relationship between circulating OPG levels and female bone status in human populations is unclear. Thus, the aim of this study was to investigate the relationship between circulating OPG levels and bone mineral metabolism in Korean women. Subjects were 294 women aged 33--73 (mean age, 51.5 yr). Serum concentrations of OPG were determined by ELISA. Biochemical markers of bone turnover and follicular stimulating hormone (FSH) were measured by standard methods. Bone mineral density at femoral neck and lumbar spine was measured by dual energy x-ray absorptiometry. We observed a significant positive association between circulating OPG levels and urine deoxypyridinoline levels (r=0.125; p<0.05). And, there was a significant positive relationship between circulating OPG levels and urine calcium excretion (r=0.220; p<0.05). We found that mean OPG levels were about 10% greater in postmenopausal women (mean±SD, 1386.9±532.6 pg/mL) than in premenopausal women (1255.8±451.8 pg/mL; p>0.05). There was a significant positive relationship between circulating OPG levels and serum FSH levels (r=0.153; p<0.01). There was a no significant relationship between circulating OPG levels and bone mineral density at femoral neck and lumbar spine. In conclusion, our data shows that the circulating OPG levels are associated with the markers of bone resorption and serum FSH levels in Korean women. These data suggest that OPG may be an important paracrine mediator of female bone metabolism in human populations.

Disclosures: K. Oh, None.


TGFβ1 Upregulates CXCR4 Expression in Osteoclasts (OC): A Possible Mechanism to Enhance OC Survival and Bone Resorption.X. Yu, Y. Huang*, P. Collin-Osdoby, P. Osdoby. Department of Biology, Washington University, St. Louis, MO, USA.

Osteoclast (OC) bone resorption is essential for normal bone development and remodeling, but is excessive in skeletal pathologies such as inflammatory bone loss (rheumatoid arthritis, periodontal disease) or tumor osteolysis. OC precursors (pre-OCs) reside in the bone marrow and peripheral circulation, from which they migrate to bone sites for RANKL-mediated development into resorptive OCs. This process involves multiple factors including chemokines which affect cell migration, invasion, activation and survival. Previously, we showed that pre-OCs and OCs expressed the chemokine receptor CXCR4 and that SDF-1, the unique ligand of CXCR4: 1) increased pre-OC chemotaxis, 2) promoted pre-OC transcollagen migration via increasing MMP-9 activity, 3) stimulated the angiogenic-related recruitment of pre-OCs that developed into resorptive cells on bone in vivo, and 4) increased mature OC survival. TGFβ1 has also been found to increase OC survival. Because TGFβ1 induces CXCR4 expression in various cells, we investigated CXCR4 expression as a function of OC differentiation and TGFβ1 levels in two murine OC developmental models. Whereas CXCR4 expression declined during RANKL-induced OC formation from RAW 264.7 cells (RAW-OCs), it increased during OC formation from primary bone marrow cells (MA-OCs). This correlated with 2 to 3-fold higher basal TGFβ1 expression levels in primary MA-OCs (highly purified) compared to RAW-OCs. Addition of TGFβ1 restored CXCR4 levels in RAW-OCs and allowed high CXCR4 levels to persist during RAW-OC formation. In contrast, other factors known to increase CXCR4, such as VEGF, bFGF and IL-6, did not alter RAW-OC CXCR4 expression. Thus, higher TGFβ1 expression by primary MA-OCs may account for higher CXCR4 expression via an autocrine mechanism. Similar findings were observed during OC generation from human blood monocytes by M-CSF/RANKL: CXCR4 declined during human OC development in parallel with decreasing TGFβ1 expression, and the addition of TGFβ1 to differentiated human OCs upregulated their CXCR4 expression. TGFβ1 is both produced and activated by OCs, and is synthesized by many other cell types and released in abundant quantities from bone matrix during normal OC resorption. Furthermore, both TGFβ1 and SDF-1 are reportedly increased in rheumatoid arthritis, and a selective CXCR4 inhibitor efficiently suppresses bone loss in a murine model of this disease. Thus, in addition to promoting angiogenesis and OC recruitment, TGFβ1 may have an important role in regulating OC survival through elevating CXCR4 expression and thereby enhance OC bone resorption and remodeling in both physiological and pathological conditions.

Disclosures: X. Yu, None.


RANKL Regulates Fas Expression during Osteoclastogenesis.X. Wu1, G. Pan*1, M. A. McKenna1, J. M. McDonald2. 1Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA, 2Department of Pathology, University of Alabama at Birmingham; Veterans Administration Medical Center, Birmingham, AL, USA.

Osteoclast apoptosis is an influential determinant of osteoclast bone resorbing activity. Fas, the death receptor, is important in mediating apoptosis in osteoclasts, and Lpr and Gld mice with non-functional Fas and FasL, respectively, have decreased bone mineral density. Here, we report that RANKL regulation of Fas is biphasic, being increased during the early stage (1 day) of osteoclast differentiation and decreased during the late stage. To examine late stage of differentiation, monocyte-macrophage precursors isolated from C57BL/6 mouse bone marrow were differentiated with M-CSF or M-CSF plus RANKL for 6 days. Cells cultured with both RANKL and M-CSF had 42.7±10.5% (n=5) less Fas mRNA (semi-quantitative Real Time PCR) than cells differentiated in the presence of M-CSF only. Fas protein (Western blotting) was dose-dependently downregulated by RANKL with a maximal decrease of 44.9±17.1% (n=7). Flow cytometry confirmed decreased RANKL-induced surface Fas expression. Similar data were obtained with osteoclasts differentiated from RAW264.7. Both Fas expression and Fas-mediated apoptosis were downregulated by RANKL in a dose-dependent manner. Fas activating antibody induced 51% apoptosis, in the presence of 1.1nmol/L (50ng/ml) RANKL; in contrast, cells differentiated in 3.3nmol/ L RANKL were resistant to Fas antibody-induced apoptosis. To investigate the role of RANKL in the regulation of Fas expression during the early stage of differentiation, RAW264.7 cells were cultured with or without RANKL for 1 day. Fas mRNA (Real Time PCR) was increased by 30.4±9.9% (n=3) in cells cultured in the presence of RANKL. Surface expression of Fas was also increased by RANKL, as detected by flow cytometry. Using a dual luciferase report assay, Fas promoter activity was increased 2.4 ± 0.1 fold (n=9) in cells treated with 2.2nmol/L RANKL for 1 day, compared to untreated cells. Also, Fas promoter activity responded to RANKL treatment dose-dependently. Using the transcription inhibitor, actinomycin D, we demonstrated that Fas mRNA from RAW264.7 cells cultured with RANKL was more stable than Fas mRNA from control. These results demonstrate that RANKL upregulates Fas expression at an early stage, thus implicating RANKL in regulating osteoclast homeostasis by limiting the number of precursors entering the differentiation process. At the late stage of osteoclast differentiation, RANKL downregulates Fas expression and Fas-mediated apoptosis, which acts as a positive regulator for osteoclast function and activity.

Disclosures: X. Wu, None.


The Role of PTHrP in Regulation of OPG/RANKL in Cementoblasts.F. Boabaid*1, J. E. Berry*2, M. J. Somerman3, L. K. McCauley2. 1Federal Univ of Sao Paulo, Sao Paulo, Brazil, 2U of Michigan Dental School, Ann Arbor, MI, USA, 3U of Washington Dental School, Seattle, WA, USA.

Tooth eruption is a physiological event that involves epithelial-mesenchymal interactions, and PTHrP is well known to be involved in this process. In the tooth germ microenvironment PTHrP is produced by cells of the stellate reticulum (epithelial), and can bind to receptors present on follicle cells, cementoblasts and osteoblasts (mesenchymal). PTHrP has been linked with a variety of activities including recruitment of mononuclear cells, fusion of these cells into multinucleated osteoclasts, resorption of alveolar bone and creation of an eruption pathway. Studies in osteoblasts have shown that PTHrP promotes osteoclastogenesis by inhibiting expression of OPG, a decoy receptor for RANK, and by enhancing production of RANKL. However, little is known regarding the role of PTHrP in regulating cementoblast-mediated osteoclastogenesis or odontoclastogenesis. To analyze the effect of PTHrP on regulation of OPG/RANKL levels and, subsequently, promotion of osteoclastogenesis, three groups of cell cultures were analyzed; 1) OCCM-30 (immortalized murine cementoblasts) alone, 2) RAW 264.7 cells (murine myeloid cell line) alone, or 3) OCCM-30 plus RAW 264.7 cells. These groups were treated with vehicle alone, PTHrP (1--34) (10−7M) alone, RANKL (0.2ug/ml) alone or PTHrP and RANKL combined. Tartrate-resistant acid phosphatase (TRAP) staining for osteoclast detection and ELISA for OPG and RANKL (standardized to protein levels) were performed after 5d. The highest numbers of TRAP positive cells were found in the RAW cell only groups treated with RANKL, and PTHrP did not alter these numbers. In the combination OCCM & RAW cultures there were no TRAP+ cells in the vehicle group. PTHrP-treated cultures had few (6.3 ± 0.9), RANKL had a moderate number (17.0 ± 1.5) and the combination of PTHrP & RANKL had the highest (84.3 ± 8.6) TRAP+cells/well suggesting a synergistic response between PTHrP and RANKL. OPG levels in cell lysates and secreted in media were highest from OCCM cells and PTHrP significantly decreased these levels. In co-cultures, OPG levels were lower than OCCM alone and the PTHrP effect was diminished. In contrast, RANKL levels were low in OCCM cell lysates and PTHrP increased RANKL (9-fold) but in OCCM & RAW co-cultures, RANKL was high and PTHrP decreased levels (6-fold) in lysates but did not alter secreted RANKL levels. In conclusion, PTHrP can target cementoblasts, inhibit OPG, increase RANKL and consequently promote osteoclastogenesis, but this is dependent on the context of surrounding cells. Osteoclast-promoting actions of PTHrP may be important during the process of tooth eruption, and periodontal development and/or disease.

Disclosures: F. Boabaid, None.


MyD88 is Essentially Involved in Osteoclast Differentiation Induced by Lipopolysaccharide, Lipopeptide and IL-1.N. Sato*1, N. Takahashi2, K. Suda3, Y. Kobayashi2, K. Takeda*4, S. Akira*4, K. Shibata*5, T. Noguchi*1, N. Udagawa6. 1Periodontology, School of Dentistry, Aichi-Gakuin Univ., Nagoya, Japan, 2Institute for Oral Science, Matsumoto Dental Univ., Shiojiri, Japan, 3School of Dentistry, Showa Univ., Tokyo, Japan, 4Research Institute for Microbial Diseases, Osaka Univ., Suita, Japan, 5Oral Pathobiological Science, Hokkaido Univ. Graduate School of Dental Medicine, Sapporo, Japan, 6Biochemistry, Matsumoto Dental Univ., Shiojiri, Japan.

Lipopolysaccharide (LPS) is proposed to be a potent stimulator of bone resorption in inflammatory diseases caused by bacteria. Bacterial lipoprotein/lipopeptides are also pathogen-specific molecular patterns. Recently, toll-like receptor 4 (TLR4) was identified as the signaling receptor for LPS. In addition, TLR6 associates with TLR2, and the complex of TLR6 and TLR2 recognizes diacylated mycoplasmal lipopeptides. The signaling cascade of TLR is believed to be similar to that of IL-1 receptors (IL-1R), because both TLR and IL-1R use myeloid differentiation factor 88 (MyD88) as a common cytoplasmic signaling molecule. However, accumulating evidence also demonstrates the existence of MyD88-independent pathways, which may explain unique biological responses of individual TLR and IL-1R. Using MyD88-deficient (-/-) mice, we explored the involvement of MyD88-mediated signals in osteoclast formation. LPS, synthetic lipopeptide (FSL-1), IL-1α and 1,25(OH)2D3 all stimulated osteoclast formation in co-cultures of primary osteoblasts and bone marrow cells obtained from wild-type mice. Osteoprotegerin, a decoy receptor of RANKL, completely inhibited the osteoclast formation in the co-culture. In contrasts, LPS, lipopeptide and IL-1α failed to induce the osteoclast formation in the co-culture of MyD88 (-/-) mice-derived osteo-blasts and bone marrow cells, though 1,25(OH)2D3 stimulated osteoclast formation even in the MyD88 (-/-) co-culture. RT-PCR analysis showed that primary osteoblasts obtained from both wild type and MyD88 (-/-) mice similarly expressed TLR2, TLR4, TLR6 and IL-1R mRNAs. LPS, lipopeptide and IL-1α stimulated expression of RANKL mRNA within 24 hr in primary osteoblasts obtained from wild-type mice but not in those from MyD88 (-/-) mice. Similarly, LPS and IL-1α stimulated phosphorylation of ERK in wild-type osteoblasts but not MyD88 (-/-) osteoblasts. Hemopoietic cells obtained from MyD88 (-/-) mice and those from wild-type mice similarly differentiated into osteoclasts in response to RANKL and M-CSF. These results suggest that the MyD88-mediated signaling pathway is essentially involved in osteoclast formation induced by LPS, lipopeptide and IL-1α through the RANKL expression by osteoblasts.

Disclosures: N. Sato, None.


Transcription Factors Involved in Osteoclastogenesis.C. Day*1, M. Kim*1, G. Nicholson2, N. A. Morrison1. 1Health Science, Griffith University, Gold Coast, Australia, 2Department of Medicine, Geelong Hospital, Geelong, Australia.

Surprisingly, very few transcription factors have been implicated in osteoclast differentiation. Newly discovered upregulated transcription factors are obviously important in understanding osteoclast differentiation. However, we speculate that repression of transcription factor genes may also be necessary for osteoclast differentiation. In particular, we have looked for differential regulation of transcription factors between the two alternative states: macrophage and osteoclast. Two recent papers verified the role of NFATc1 in osteo-clastogenesis [1,2]; NFATc1 is required for the production of TRAP positive multinucleated osteoclast like cells (OCLs). We show here that NFATc1 and other transcription factors are strongly induced by RANKL in human osteoclast like cells. NFATc1 regulation steadily increases across time with a peak of 26 fold at three weeks post treatment with macrophage colony stimulating factor (M-CSF) and RANKL. Along with NFATc1 we have observed significant up-regulation of four other transcription factors: GA-binding protein a and b (GABP), early response growth factor 1 (EGR-1), and FUSE binding protein (FBP). The dynamics of regulation of ERG-1 and GABPa and b were identical to that of NFATc1. However FBP regulation peaks earlier and is of great magnitude. These data show that NFATc1 is not the only strongly regulated transcription factor in osteoclast differentiation and is later induced than FBP.

  • Ishida N, Hayashi K, Hoshijima M, Ogawa T, Koga S, Miyatake Y, Kumegawa M, Kimura T, Takeya T. 2002. J Biol Chem 277:41147

  • Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M, Yokochi T, Inoue J, Wagner EF, Mak TW, Kodama T, Taniguchi T. 2002. Dev Cell 3:889

Disclosures: C. Day, None.


RANKL Strongly Induces the GM-CSF Receptor during Osteoclast Differentiation but Continuous Exposure to GM-CSF Represses Osteoclast Formation.M. Kim*, C. Day*, N. A. Morrison. Health Science, Griffith University, Gold Coast, Australia.

The standard model of osteoclastogenesis uses M-CSF and RANKL to differentiate osteoclasts from peripheral blood mononuclear cells (PBMCs). Using real time PCR, we found that RANKL profoundly up-regulates the GM-CSF receptor in this model. This suggests that GM-CSF receptor might represent a target for regulation, whereby osteoclast precursors integrate RANKL and GM-CSF signals to either promote or inhibit differentiation. Exogenous GM-CSF was added to the in vitro osteoclastogenesis model to test these alternative hypotheses. Cells were examined through time, with TRAP staining, morphology and gene array analysis. Continuous exposure to GM-CSF totally represses osteoclast differentiation; and resulted in an alternative cell phenotype induced by GM-CSF in the presence of M-CSF and RANKL when compared to the alternative treatments.

A 19,000 gene microarray was used to compare GM-CSF+RANKL+M-CSF treated cells against osteoclasts. Microarray analysis showed GM-CSF mediated repression of osteo-clast differentiation was concurrent with suppression of osteoclast marker genes: cathepsin K; osteoclast specific H+ ATPase; surface marker, CD68; and transcription factors we have shown are up-regulated in osteoclasts, such as NFATc1. The inhibition by GM-CSF of known osteoclast markers indicates an alternative GM-CSF dependent differentiation pathway. Real-time PCR analysis of 7 regulated genes validated the array data (FBP, GABPa, GABPb, ILF3, Kox31, NFATc1 and SCYA2). These data support the hypothesis that GM-CSF receptor up-regulation by RANKL sensitises the cell for inhibition of differentiation. In the cytokine milieu of the bone marrow, the ratio of GM-CSF and RANKL may be an important determinant of osteoclast differentiation.

Disclosures: N.A. Morrison, None.


Over-Expression of TBP-2, a Protein Involved in Redox Regulation, Inhibits Osteoclastogenesis.C. J. Aitken*1, J. M. Hodge*1, T. Vaughan*2, D. E. Myers*1, N. A. Morrison2, G. C. Nicholson1. 1Clinical and Biomedical Sciences: Barwon Health, The University of Melbourne, Geelong, Australia, 2Genomics Research Centre, Griffith University, Gold Coast Campus, Australia.

Using gene array analysis, we found that thioredoxin (TRX) binding protein-2 (TBP-2) was down-regulated during osteoclast (OC) differentiation. TBP-2 is a negative regulator of TRX, a small protein with a redox-active dithiol active site. TRX enhances DNA binding of redox-sensitve transcription factors such as NFκB and AP-1.

OC were generated on dentine slices using human CFU-GM precursor cells treated with RANKL and M-CSF. At 4 days of culture, efficient (>80%) infection with adenovirus expressing β-galactosidase (AdLacZ) was achieved. Infection with adenovirus expressing TBP-2 (AdTBP-2) for 14 days resulted in 66% reduction in the total TRAP+ area and 50% reduction in OC numbers as compared to the AdLacZ control. The size of OC formed in the presence of AdTBP-2 was reduced by 66% and they contained fewer nuclei. Resorption of dentine was inhibited by 80%. In mature OC infected with AdTBP-2, RANKL-induced NFκB activation was reduced by 63% and Western analysis demonstrated markedly increased expression of TBP-2 protein. We have shown that the over-expression of TBP-2, a gene down-regulated during OC formation, inhibits OC differentiation and NFκB activation. These results are consistent with the known function of TBP-2 as a negative regulator of TRX and the importance of the redox-sensitive transcription factor NFκB in osteoclastogenesis.

Disclosures: G.C. Nicholson, None.


Expression of Regucalcin, a Novel Intracellular Signaling Regulatory Protein, in the Bone Marrow Cells of Normal and Transgenic Rats.N. Sawada*, M. Yamaguchi. Endocrinology and Molecular Metabolism, University of Shizuoka, Shizuoka, Japan.

Regucalcin, which was found by Yamaguchi et al., has been demonstrated to play a multifunctional role as regulatory protein in intracellular signaling process [Life Sci 66:1769--1780, 2000; BBRC 276:1--6, 2000]. The regucalcin gene is highly conserved in human and various vertebrate species [Int J Mol Med 6:191--196, 2000]. More recently, it has been shown that regucalcin is expressed in the femoral-diaphyseal and ---metaphyseal tissues of rats, and that bone loss is induced in regucalcin transgenic rats [Int J Mol Med 10:761--766, 2002]. Furthermore, the present study was undertaken to clarify whether regucalcin is expressed in bone marrow cells. We found that regucalcin mRNA is expressed in bone marrow cells of normal rats by RT-PCR and Western blot analyses. The expression of regucalcin in bone marrow cells was enhanced in the transgenic rats. The effect of regucalcin on osteoclastic formation is in progress.

Disclosures: N. Sawada, None.


Regulation of c-Fos Protein Expression in Osteoclast Lineage Cells: Involvement of Ubiquitin/Proteasome Pathway.Y. Ito*, D. Inoue, S. Kido, I. Endo*, T. Matsumoto. Department of Medicine and Bioregulatory Sciences, University of Tokushima, Tokushima, Japan.

Gene knockout studies have demonstrated that c-Fos, an AP-1 family transcription factor, is indispensable for osteoclast differentiation. So far, four members of fos family have been identified: fosB, c-fos, fra-1 and fra-2. Rescue experiments with c-fos -/- spleen cells and retroviral expression vectors of fos family members indicated that introduction of not only c-fos itself but also either one of the other three members was able to restore the ability of c-fos -/- spleen cells to differentiate into osteoclasts, suggesting a functional redundancy among the family members. On the other hand, in vivo gene deletion of other fos family members such as fosB and fra-1 did not result in osteopetrosis as observed in c-fos gene-deleted mice, indicating that only the c-fos gene has a unique function that cannot be compensated by the other family member genes. These enigmatic observations led us to hypothesize that specificity of c-fos resides not in its protein function but in its regulatory mechanisms of protein expression from the endogenous gene. In mouse osteoclast precursor cell lines, RAW264 and C7, serum and TPA induced expression of all the fos family members at both the mRNA and protein levels. Similar induction was observed in c-fos -/-primary spleen cells except c-fos, which is absent in these cells. We also confirmed that RANK expression was normal in c-fos -/- spleen cells. However, treatment with TPA in addition to RANKL and M-CSF did not rescue the defective osteoclast differentiation in c-fos -/- spleen cells. In RAW cells, TPA induction of c-Fos protein was well detectable at 3 hr, reached a peak within 8 hrs and was down-regulated by 24 hrs. In contrast, up-regulation of c-Fos protein expression by RANKL was much slower and more prolonged with a peak at 24 hrs or later. These results are compatible with an idea that stable c-Fos expression may be necessary for osteoclast differentiation. Therefore, we examined degradation pathways, especially focusing on the ubiquitin (Ub)/proteasome system. We found that a proteasome inhibitor MG132, but not a protease inhibitor E64, increased the level of c-Fos protein expression in RAW and C7 cells in a time- and dose-dependent manner. Immuno-precipitation with c-Fos antibody and subsequent blot with Ub antibody demonstrated that ubiquitinated c-Fos accumulated in the presence MG132. We observed similar effects on c-Jun, a known target of Ub. We conclude that c-Fos is a target of Ub/proteasome degradation pathway, which may play a role in unique regulatory mechanism of c-Fos expression in osteoclast precursors.

Disclosures: Y. Ito, None.


Prostaglandin E2 Stimulation of Osteoclast Formation in Hematopoietic Cell Cultures is Mediated by a Soluble T Cell Factor.H. Kaneko1, K. Ono2, S. K. Lee1, J. A. Lorenzo1, Y. Toyama*3, C. C. Pilbeam1, L. G. Raisz1. 1Medicine, University of Connecticut Health Center, Connecticut, CT, USA, 2General Medicine, National Defense Medical College, Tokorozawa, Japan, 3Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan.

Previously we found that PGE2 stimulated osteoclast-like cell (OCL) formation in spleen cell cultures treated with RANKL and M-CSF and that this stimulation was mediated by the EP2 receptor. Since T cells are possible regulators of bone resorption in inflammation, we examined their role in this response. Spleen cells from 6-week-old male C57BL/6 mice were plated at 2.5 × 105 cells/well (48 well plate) and cultured in αMEM containing 10% FCS with 10 ng/ml M-CSF and 30 ng/ml RANKL. At 5 to 9 d of culture adherent cells were stained for TRAP. TRAP(+) cells containing 3 or more nuclei were counted as OCL. Since T cells can produce RANKL, we first determined the dose response to RANKL. Spleen cells were cultured with 10 ng/ml M-CSF and 1--100 ng/ml RANKL. 10 to 100 ng/ml RANKL increased OCL number with the peak at 8d. There was no significant difference in OCL number between 30 to 100 ng/ml RANKL. In contrast, the mean effect of 10−6 M PGE2 added to 10 ng/ml M-CSF and 30 ng/ml RANKL at 8 d was a 48±9 % increase (p<0.01). T cell depletion using mouse pan T (Thy 1.2) immunomagnetic beads abrogated the stimulatory effect of PGE2. To determine whether a soluble product of T cells might mediate the effect of PGE2, we incubated T cells isolated from the spleen with or without 10−6 M PGE2 for 6 h, washed the cells, and then collected supernatants for the next 12 h of culture. This conditioned medium was then added to fresh spleen cultures. At 8 d, control cultures without supernatant produced 130±23 OCL, cultures with control T cell supernatant produced 252±20 OCL, and cultures with supernatant from T cells treated with PGE2 produced 353±17 OCL (p<0.01). These results suggest that T cells release stimulators of OCL formation and that PGE2 increases this activity. We speculate that the role of PGE2 in the bone loss associated with inflammation may be mediated by its effect on T cells.

Disclosures: H. Kaneko, None.


YY1 is Positively Involved in RANKL-Induced Transcription of Tartrate-Resistant Acid Phosphatase (TRAP) Gene.Z. Shi, Y. Liu*, A. Silveira*, P. Patel*, X. Feng. Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

Osteoclasts, the principal bone-resorbing cells, are derived from cells of monocyte/macrophage lineage. RANKL is an essential and potent activator of osteoclast differentiation. During RANKL-induced osteoclast differentiation, a variety of genes are activated, including the gene for Tartrate-Resistant Acid Phosphatase (TRAP), which is implicated in osteoclastic bone resorption. However, the molecular mechanism underlying the RANKL-induced TRAP expression has not been completely understood. Our previous characterization of the mouse TRAP promoter identified a 12-bp sequence AGCCACGTGGTG (-1220 to -1198, relative to the translation start site) that regulates RANKL-induced TRAP transcription by utilizing USF1 and USF2. Interestingly, during the course of the previous study, we also found that oligonucleotides (designated as Oligo IV) derived from a 50-bp TRAP promoter region (-1124 to -1074) bound RANKL-induced nuclear proteins from RAW264.7 cells. In the current study, we elucidated the identity of the nuclear protein binding to Oligo IV and further investigated the role of this nuclear protein in RANKL-induced transcription of TRAP gene. Given that RANKL activates NF-kB or AP1, we determined whether the nuclear protein binding to Oligo IV is NF-kB or AP1. Supershift assays with antibodies against p50, p65, c-fos and c-jun demonstrated that Oligo IV binds transcription factors other than NF-kB and AP1. To reveal the identity of the nuclear protein, we decided first to locate the specific sequence in Oligo IV that binds the protein. To this end, we performed EMSA/competition assays with shortened oligos derived from Oligo IV as competitors and these assays identified a 21-bp sequence TTATGATGGC-GAGGGGGAACT (-1097 to -1077) that specifically binds the nuclear protein. Computer analysis revealed that this sequence contains putative sites for both AP-2 and YY1. Subsequent competition assays with AP-2 and YY1 consensus sequences showed that only YY1 consensus sequence efficiently competed off the binding, suggesting that the nuclear protein binding to this sequence is YY1. Finally, supershift assays with antibody against YY1 confirmed that nuclear protein binding to the 21-bp sequence is indeed YY1. Importantly, mutation of the YY1-binding site resulted in a reduction in RANKL-induced TRAP transcription in RAW264.7 cells, indicating that YY1 is positively involved in RANKL-induced TRAP transcription. In summary, our data have not only established a functional role for YY1 in TRAP expression in osteoclasts but also defined a new transcriptional mechanism by which RANKL regulates gene transcription during osteoclast differentiation.

Disclosures: Z. Shi, None.


AZT-Associated Bone Loss in AIDS Therapy.G. Pan*1, X. Wu1, O. Mamaeva*1, M. A. McKenna1, J. M. McDonald2. 1Pathology, Uni. of Alabama at Birmingham, Birmingham, AL, USA, 2Pathology, Uni. of Alabama at Birmingham; Veterans Administration Medical Center, Birmingham, AL, USA.

Although highly active anti-retroviral therapy (HAART) has made a very significant advance in the treatment of AIDS, a variety of metabolic complications, including osteoporosis, osteopenia, and osteonecrosis, have been reported to be associated with this therapeutic regimen. To determine the effects of Zidovudine (AZT), a nucleoside reverse transcriptase inhibitor, on bone resorption and osteoclastogenesis, we cultured mouse osteoclast precursors, RAW264.7 cells, or mouse primary bone marrow macrophage-monocyte precursors with and without AZT in vitro. AZT significantly increases RANKL-stimulated differentiation of RAW264.7 cells, whereas AZT alone fails to increase osteo-clastogenesis. The maximum stimulatory effects of AZT, occurring between 10 and 40 μ M, increases TRAP-positive cells 10-fold over that obtained with RANKL alone (p< 0.05, t-test). Similarly, AZT increases relative TRAP activity in the primary bone marrow precursors by 2-fold compared to cells cultured without AZT. AZT is also capable of reducing the amount of RANKL required to induce maximal osteoclast differentiation from 50 ng/ ml to 12.5 ng/ml, indicating an increased sensitivity of osteoclast precursors to RANKL. To confirm the effects of AZT on TRAP promoter activation, an early key event in osteo-clast differentiation, a luciferase reporter assay containing the TRAP gene promoter was performed. AZT increases luciferase activity by up to 10-fold confirming the stimulatory effects of AZT on RANKL-induced osteoclastogenesis. More importantly, an animal model was generated to investigate the effects of AZT on bone in vivo. Mice treated with AZT (10mg/day) for 4 weeks show features of osteopenia as measured by DEXA, the bone mineral density (BMD) decreasing from 0.0462 ± 0.0015 g/cm2 in controls to 0.0416 ± 0.0001 g/cm2 in the AZT-treated animals (n=3, p< 0.05). The average mineralizing surface in the AZT-treated mice is increased from 1.75 to 4.28 mm, and the bone formation rate from 0.59 to 1.43 mm compared to the control (p< 0.05). The number of TRAP positive osteoclasts in tibial sections of AZT-treated mice is increased. In summary, AZT increases osteoclastogenesis and decreases bone mineral density by increasing RANK signaling in osteoclast precursors.

Disclosures: G. Pan, None.


Activation of Human Microvascular Endothelial Cells by IL-1 or TNF-α Enhances Transendothelial Migration and Chemokine (SDF-1) Recruitment of Precursors from Human Peripheral Blood that Develop into Osteoclasts.L. Blair*, L. Rothe, P. Osdoby, P. Collin-Osdoby. Department of Biology, Washington University, St. Louis, MO, USA.

Increased osteoclast (OC) bone resorption is prevalent in such pathological inflammatory diseases as rheumatoid arthritis and periodontal disease. OC precursors (pre-OC) are present in the bone marrow and peripheral circulation, from which they may be recruited to bone and develop into resorptive OCs. While much is known about osteoblast/stromal cell regulation of marrow OC development and function, little is understood about how circulating pre-OCs are recruited from peripheral blood into bone and to sites of resorption. We hypothesized that normal or pathological pre-OC recruitment may depend on bone microvascular interactions and particular combinations of chemoattractants, cell adhesion molecules, and matrix degradative enzymes. Therefore, we analyzed IL-1 and TNF-α effects on human microvascular endothelial cells (HMVEC) and their interactions with human peripheral blood monocytes (hPBMC) containing pre-OCs. Cell adhesion studies showed that HMVEC pretreatment (24 h) with IL-1 or TNF-α increased hPBMC adhesion to HMVEC (1 h). Consistent with this, microarray analysis of IL-1 treated (6 h) HMVEC revealed upregulated expression of cell adhesion molecules that could mediate such interactions, including ICAM-1, β1 integrin, and CD44. Previously, we showed that the hematopoietic cell chemoattractant and homing signal stromal-derived factor-1 (SDF-1): 1) increased the chemotaxis of hPBMC which subsequently developed into resorptive OCs following their culture with M-CSF/RANKL, 2) increased pre-OC transcollagen migration via increasing MMP-9 activity, and 3) stimulated the angiogenic-related recruitment of pre-OCs that developed into resorptive cells on bone in vivo. Here, we further showed that hPBMC transmigration through a confluent HMVEC monolayer, alone or in response to an SDF-1 gradient, was enhanced by IL-1 or TNF-α pre-activation (24 h) of HMVEC. Importantly, increases in transendothelial migration of hPBMC led to greater OC development following their culture with M-CSF/RANKL. Previously we found that IL-1 and TNF-α stimulate HMVEC release of multiple OC modulatory factors (IL-1, TNF-α, IL-6, M-CSF, IL-8, PDGF, bFGF) and raise RANKL production by HMVEC to promote human OC formation, resorption and survival. Therefore our findings suggest that inflammatory activation of the microvasculature may lead to increased local OC bone resorption through multiple mechanisms involving stimulation of pre-OC recruitment, adhesion to blood vessels, transendothelial migration, and development into bone-resorptive OCs.

Disclosures: L. Blair, None.


Osteoclasts Rely on Lipoproteins to Regulate Cellular Cholesterol Levels -- Relationship to Survival and Morphology.E. Luegmayr*, H. Glantschnig*, G. A. Rodan, A. A. Reszka. Bone Biology and Osteoporsis Research, Merck Research Laboratories, West Point, PA, USA.

Growing evidence suggests that abnormal lipid metabolism might be associated with the epidemiological correlation between osteoporosis and atherosclerosis. We demonstrate here that osteoclasts (OCL) depend on lipoproteins to maintain cellular cholesterol levels, which in turn control OCL morphology and survival.

Cholesterol was withdrawn from purified OCL maintained in media containing lipoprotein-deficient FBS (LPDS) by treatment with HDL or methyl-beta-cyclodextrin (MBCD). Rapid removal of cholesterol via MBCD induced OCL, but not osteoblast, apoptosis (>10-fold), with nuclear condensation, caspase activation and actin disruption. Meanwhile, cholesterol withdrawal induced HMG-CoA reductase to synthesize cholesterol in osteoblasts, but not in OCL.

Physiological, gradual removal of cholesterol from OCL with HDL was accompanied by gradual induction of apoptosis (up to five-fold, reaching 50% of OCL at 48 hr). In reciprocal experiments, LDL-mediated cholesterol delivery significantly suppressed spontaneous OCL apoptosis at 72 hr. Thus LDL and HDL have opposing effects on the levels of cellular cholesterol and on OCL survival.

We further examined apoptosis in OCL derived from LDL receptor knockout (-/-) mice relative to the (+/+) background strain (C57BL6). Purified -/- OCL were significantly more susceptible to spontaneous apoptosis, associated with an inability to gather LDL-cholesterol present in FBS.

Prior to purification -/- OCL also showed distinct morphological phenotypes associated with a significant 50% reduction in mean area. The number of large OCL decreased, while small multinucleate OCL increased. Interestingly, the morphology of -/- OCL was restored (2--4 fold) to the +/+ phenotype by daily cholesterol pulsing, using cholesterol-saturated MBCD. Reciprocally, +/+ OCL adopted the -/- morphological phenotype when differentiated in media lacking lipoproteins. This is consistent with the uptake of cholesterol as a primary driver for the phenotype.

In summary, these findings suggest an effect of extracellular cholesterol and lipoproteins on the regulation of OCL formation and function. This relationship might play a role in the epidemiological link between osteoporosis and atherosclerosis.

Disclosures: E. Luegmayr, Merck and Co., Inc. 3.


RANKL Induced iNOS/NO Acts as a Negative Feedback Inhibitor During Osteoclastogenesis Via an NF-kB and IFN-β-ERK-Stat1 Signaling Pathway.H. Zheng, X. Yu, P. Collin-Osdoby, P. Osdoby. Department of Biology, Washington University, St. Louis, MO, USA.

RANKL is essential for the formation of resorptive osteoclasts (OCs) via interaction with RANK on precursors to trigger multiple intracellular signal pathways (ERK, p38 MAPK, JNK, NF-kB). Recently, we showed that RANKL induces iNOS mRNA and NO production during OC differentiation and this NO functions as an autocrine negative feedback signal to regulate RANKL-mediated OC development from murine RAW 264.7 cells or primary bone marrow cells. Conversely, inhibitors of iNOS/NO enhanced RANKL-induced OC development and bone resorption. Similarly, RANKL-induced OC formation and resorption was greater in marrow cultures from iNOS -/- compared to iNOS +/+ mice. Here, we investigated signal mechanisms by which RANKL induces iNOS/NO to restrain OC development in RAW cells using inhibitors, western blots, and EMSAs. We found that the NF-kB inhibitor PDTC blocked both OC formation and iNOS/NO induction. In contrast, p38 MAPK (SB20358) inhibition blocked both OC formation and iNOS/NO induction, JNK (SP60025) inhibition blocked only OC formation, and ERK1/2 (PD98059) inhibition blocked only iNOS/NO induction which led to enhanced RANKL-mediated OC formation and resorption. Therefore, p38 MAPK, JNK, and NF-kB, but not ERK, are essential for OC formation. More importantly, ERK inhibition selectively abrogated the NO negative feedback signal pathway triggered by RANKL in OC formation, thereby mimicking the actions of iNOS inhibitors or iNOS -/- deficiency to enhance RANKL-induced OC formation. Because IFNβ was recently shown to be a RANKL-induced negative feedback signal during OC development, and IFNβ can induce iNOS/NO in other cells, we investigated if RANKL induction of IFNβ (and downstream phosphorylation of STAT-1) was involved in the RANKL stimulation of iNOS/NO to restrain OC formation. These studies showed that: 1) RANKL induced IFNβ (by 30′) and P-STAT-1 (by 4 h) in RAW cells, 2) RANKL stimulation of P-STAT-1 and iNOS/NO (but not IFNβ) required new protein synthesis (CHXs), was mimicked by exogenous IFNβ, and was inhibited by neutralizing Ab to IFNβ which enhanced OC formation, 3) NF-kB inhibition blocked RANKL induced IFNβ, and 4) ERK inhibition blocked IFNβ stimulated P-STAT-1 and iNOS/NO (but not NF-kB or IFNβ). These and other findings indicate that the novel auto-crine negative feedback pathway triggered by RANKL during OC development involves NF-kB activation, IFNβ induction, ERK activation, phosphorylation of STAT-1, and induction of iNOS and NO production. Specifically interfering with RANKL-mediated IFNβ or ERK activation prevents subsequent iNOS/NO induction and thereby enhances RANKL-induced OC formation and bone resorption.

Disclosures: H. Zheng, None.


Molecular, Biochemical and Functional Analysis of the Actin-Binding Site in the B Subunit of Vacuolar H+-ATPase.J. Zuo*1, S. Chen*1, M. R. Bubb*2, E. G. Yarmola*3, J. Jiang*4, I. R. Hurst*1, M. Lu*5, S. L. Gluck*3, L. S. Holliday1. 1Orthodontics, University of Florida College of Dentistry, Gainesville, FL, USA, 2Medicine/Rheumatology, University of Florida College of Medicine, Gainesville, FL, USA, 3Medicine, University of Florida College of Medicine, Gainesville, FL, USA, 4Endodontics, University of Florida College of Dentistry, Gainesville, FL, USA, 5Medicine/Nephrology, University of Florida College of Medicine, Gainesville, FL, USA.

Vacuolar H+-ATPase (V-ATPase) binds microfilaments in a regulated manner in osteoclasts suggesting that the interaction has a physiologic role. The B subunit binds actin and isolated V-ATPase can be competed from microfilaments by an excess of B subunit fusion proteins. The actin binding activity of B subunit maps to amino acids 23--67 of the B1 isoform and 29--73 of B2. High confidence models of the B subunit developed using 3D-PSSM (Imperial College, London) indicate that this region is divided into two surface exposed sections in the region of the B subunit furthest removed from the membrane, with a buried intervening fold. The exposed regions are AA 54--60 and 37--42 in B1. Each contains a number of exposed charged residues that are conserved in the B subunits of higher organisms. Replacement of 49--59 in B1, which has similarity to the actin binding site of profilin and which binds to actin as a 13-mer peptide, with sequence from an Archaebacteria, abolishes the actin binding activity of the resulting fusion proteins without disturbing the predicted structure. Modification of cysteine 40 with n-ethyl maleimide reduces the affinity for actin of a fusion protein containing B1 23--67. A polyclonal antibody against amino acids 1--20 of B1 does not interfere with the actin binding activity of the B1 1--106 fusion protein. Full length wild type B1 or B1 with the 49--59 substitution (B1(49--59)) were constructed and incorporated into an adeno-associated virus vector. By this means high level expression of B1 or the B1(49--59) has been achieved in OPGL-stimulated RAW 264.7 osteoclast-like cells and in mouse marrow osteoclasts. Preliminary data indicate that although B1 is not normally expressed in osteoclasts, it is incorporated into the holoenzyme and is transported to ruffled membranes. Efforts are now underway to characterize the phenotype of osteoclasts containing high levels of the actin binding deficient B1(49--59) construct and compare them with osteoclasts expressing similar levels of wild-type B1.

Disclosures: J. Zuo, None.


Role of Phosphatidylinositol 3-kinase (p110 alpha) in Osteoclasts.J. Jiang1, J. Zuo*2, Y. Gong*3, I. R. Hurst*2, L. S. Holliday2. 1Endodontics, University of Florida, Gainesville, FL, USA, 2Orthodontics, University of Florida, Gainesville, FL, USA, 3Pharmacy, University of Florida, Gainesville, FL, USA.

Phosphatidylinositol 3-Kinases (PI 3-Kinases) are enzymes that phosphorylate inositol lipids on the 3-position of inositol headgroup. Signal transduction by PI-3 kinases is known to be crucial for regulating osteoclastic bone resorption. We found that members of three distinct classes of PI 3-kinases were expressed in osteoclasts. The classes differ in structure, substrate specificity and targeting elements. To date, few data are available about the precise role of each type of PI 3-kinase in osteoclasts. In the present work, we investigated the role of a subclass of the class I PI 3-kinase (p110α) in regulation of osteoclasts. We produced a recombinant adeno-associated virus serotype 2 (AAV-2) containing CMV promoter, a 300-base pair reversed 5′-coding region of p110α (p110αAS), internal ribosomal entry site (IRES) and green fluorescent protein (GFP) gene (AAV2p110αAS-GFP). The efficiency of AAV2 gene transfer was examined with control virus (GFP AAV2 virus). Raw 264.7 cells stimulated with OPGL and primary murine osteoclasts were transduced with AAV2GFP 80%-90% of the total TRAP positive cells were GFP positive after three days of transduction. The transduced cells displayed normal actin ring structure when stained with TRITC conjugated phalloidin. Transduction with p110α antisense virus in OPGL-stimulated Raw 264.7 cells reduced the expression of endogenous p110α compared to cells transduced with GFP virus. P110α AS expression dramatically reduced osteoclast survival after 3 day of transduction. Osteoclasts transduced with p110α AS showed disrupted actin rings. These data suggest that p110α plays an important role in osteoclast survival and cytoskeletal organization.

Disclosures: J. Jiang, None.


Receptors for RANKL and M-CSF Are Induced during Monocytic Differentiation of Promonocytic U937 Cells via Protein Kinase Cδ- and p38 MAP Kinase-Dependent Pathway.E. Park1, H. Kang*1, K. Kim*1, J. Choi2, H. Shin3, S. Kim4. 1Skeletal Diseases Genome Research Center, Kyungpook National University Hospital, Daegu, Republic of Korea, 2Biochemistry, Kyungpook National University, Daegu, Republic of Korea, 3Oral Pathology, Kyungpook National University, Daegu, Republic of Korea, 4Department of Orthopedic Surgery, Kyungpook National University Hospital, Daegu, Republic of Korea.

Osteoclasts share common precursors with monocyte/macrophage lineage cells. However, molecular mechanisms underlying an osteoclastic commitment of precursor cells are not clear. In the present study, we have investigated signal transduction pathway leading to an osteoclastic commitment of promonocytes by analyzing an expression of RANK and c-fms, critical receptors for osteoclastic differentiation. RANK and c-fms are induced during monocyte/macrophage differentiation of U937 cells stimulated with PMA, alone or in combination with vitamin D3. Analysis of PKC isoforms reveals that most PKC isoforms are down-regulated whereas isoforms of PKCγ, δ and μ, are sustained throughout PMA stimulation. MAP kinase family members including ERK, p38 MAP kinase and JNK are also activated by PMA stimulation. These results suggest a possible involvement of some PKC isoforms and MAP kinase families in the expression of RANK and c-fms. Further analyses with pharmacological inhibitors revealed that Gö6983, a specific inhibitor for classical PKCs, PKCδ and γ, significantly reduced an expression of RANK and c-fms. Furthermore, Rottlerin, a PKCS-specific inhibitor, suppressed a PMA-induced expression of RANK and c-fms, but not that of CD11b, suggesting that PKCδ is involved in the PMA-induced expression of RANK and c-fms. While Gö6983 reduces the activities of both ERK and p38 MAP kinase, Rottlerin specifically suppresses the activity of p38 MAP kinase. Moreover, inhibition of p38 MAP kinase reduces an expression of RANK and c-fms, but not CD11b. Taken together, these results demonstrate that receptors for RANKL and M-CSF induced during monocytic differentiation of U937 cells are dependent on both PKCS and p38 MAP kinase.

Disclosures: E. Park, None.


Eosinophil Chemotactic Factor-L (ECF-L) Enhances RANKL Signaling.H. Y. Chung1, S. J. Choi1, G. D. Roodman2, 1Medicine-Hematology/Oncology, University of Pittsburgh, Pittsburgh, PA, USA, 2Hematology/Oncology, University of Pittsburgh and Department of Veterans Affairs Medical Center, Pittsburgh, PA, USA.

We recently identified ECF-L as a novel stimulator of osteoclast formation. ECF-L induces osteoclast formation independent of RANKL. Importantly, anti-ECF-L antibody blocks RANKL induced osteoclastogenesis, suggesting that ECF-L is a critical cofactor for RANKL induced osteoclast formation. ECF-L is a chemoattractant for osteoclast precursors, but its other effects on osteoclastogenesis are unknown. To further examine the role of ECF-L in osteoclastogenesis, the effects of ECF-L on intracellular signaling events after binding of RANKL to RANK, including the activation of the transcription factor NF-κB and the stimulation of the protein kinase JNK in osteoclast precursors were evaluated. RAW264.7 cells were treated with RANKL or conditioned media from 293 cells transfected with mouse ECF-L cDNA. ECF-L increased NF-κB binding activity by EMSA analysis after 30 minutes compared to basal levels. Furthermore, increased NF-κB binding activity in ECF-L treated RAW264.7 cells was further enhanced in the presence of low concentrations of RANKL (2.5 ng/ml). JNK activities were then measured using immunoprecipitation and Western blotting in human marrow cells. Human ECF-L increased JNK activity and acted synergistically with RANKL to increase JNK activity. In addition, the ECF-L induced DNA binding activity AP-1 was also increased and enhanced by JNK activity. Taken together, these data suggest that ECF-L supports osteoclast formation not only through chemoattraction for OCL precursors but also stimulation of cellular signaling responses necessary for osteoclast formation. In conclusion, these data demonstrate ECF-L is a previously unknown factor that is a potent mediator of OCL formation that enhances RANK signaling.

Disclosures: H.Y. Chung, None.


Lipid Rafts Are Important for the Akt Activation by RANK in Osteoclasts.Z. Lee1, H. Ha*1, H. Kim2. 1Microbiol. & Immunol., Chosun University College of Dentistry, Gwangju, Republic of Korea, 2Cell & Developmental Biology, Seoul National University College of Dentistry, Seoul, Republic of Korea.

TRAF6 and Src have been shown to play crucial roles in osteoclast function and RANK signaling. As Src family kinases preferentially segregate to raft microdomains, we sought to address the potential role of membrane rafts for signaling by RANK/TRAF6 in bone resorption function of osteoclasts. Our findings demonstrate that raft expression increases during the osteoclastogenesis and that TRAF6 is recruited to rafts by RANKL stimulation in osteoclasts. Further, we show that disruption of rafts interfere with a variety of parameters required for osteoclast function and differentiation. These include impeded RANK signaling to Akt, defective actin ring formation and resorption activity of osteoclasts, and the reduced survival of osteoclasts. Overall, our findings demonstrate for the first time, a crucial role for membrane lipid rafts in the function, and potentially differentiation, of osteoclasts.

Disclosures: H. Kim, None.


A Voltage-gated H+ Ghannel, a Powerful H+ Extruding Pathway, Operates in Murine Osteoclasts Stimulated by Phorbol Myristate Acetate in Association with Cell Acidosis.H. Mori*1, H. Sakai1, H. Morihata*1, J. Kawawaki*2, H. Amano3, T. Yamano*4, M. Kuno1. 1Physiology, Osaka City Univ. Grad. Sch. Med., Osaka, Japan, 2Central Laboratory, Osaka City Univ. Grad. Sch. Med., Osaka, Japan, 3Pharmacology, School of Dentistry, Showa University, Tokyo, Japan, 4Pediatrics, Osaka City Univ. Grad. Sch. Med., Osaka, Japan.

Diverse H+-translocating mechanisms are involved in regulation of osteoclast functions. Voltage-gated H+ channels are distinct in their surprisingly high H+ extrusion rate, ∼100 times as strong as the H+ pumps and transporters, and can compensate for pH imbalance rapidly. The H+ channel changes its activity by sensing the pH gradient across the plasma membrane (ΔpH), but its physiological relevance to osteoclast functions is unclear. Protein kinase C (PKC) is a strong activator for H+ channels and modifies various osteoclastic activities. To investigate how the channel operates in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA) on the H+ channel was examined in murine osteoclasts generated in the presence of M-CSF/CSF-1 and sRANKL. The H+ currents and intracellular pH (pHi) in clamped cells were recorded successfully in the permeabilized-patch. The reversal potential (Vrev) was a valuable tool for real-time monitoring ΔpH. PMA (10 nM - 1.6 μM) increased the current density and the activation rate, slowed decay of tail currents and shifted the threshold toward more negative voltages. Inward H+ currents were often recorded, suggesting that H+ could enter the cell. In addition, PMA caused a negative shift of Vrev, indicating intracellular acidification. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K+-rich alkaline solution probably via the activated H+ channel. Both cell acidosis and activation of the H+ channel by PMA were inhibited by staurosporine, a PKC inhibitor. In ∼ 80% of cells, the PMA-induced augmentation in the H+ current remained after compensating for the ΔpH changes, so that ΔpH-independent mechanisms also contributed to the channel activation. There was a correlation between Vrev and the membrane potential, indicating that the channel could control the membrane potential. PMA shifted the membrane potential shifted toward Vrev. These data showed that activation of PKC provided a functional state favorable for utilizing the H+ channel, by potentiating the channel activity and lowering the threshold, together with cell acidosis. Enhanced H+ efflux through the activated H+ channel may contribute to quick regulation of the pH environments and the membrane potential, to maintain osteoclast functions.

Disclosures: H. Mori, None.


Estrogen Modulates RANK-Mediated Signaling in the Rabbit Osteoclast.J. Shyu1, D. Sung*1, J. Wang*1, C. Lin*2, C. Shih1. 1Biology & Anatomy, National Defense Medical Center, Taipei, Taiwan Republic of China, 2Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan Republic of China.

The discovery of new members of the tumor necrosis factor (TNF) receptor ligand family has elucidated the precise mechanism by which osteoblasts/stromal cells regulate osteoclast differentiation and function. Osteoblasts/stromal cells express OPGL (ligand for OPG) as a membrane-associated factor. Osteoclast precursors, which possess RANK (receptor activator of NF-κB), recognize OPGL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts. Mature osteoclasts also express RANK, and their bone-resorbing activity is also induced by OPGL which osteoblasts/stromal cells possess. Thus OPGL, RANK, and OPG are three key molecules, which regulate osteoclast recruitment and function. However, the RANK-mediated signaling in osteoclasts is poorly understood. The major mechanism of the rapid phase of bone loss in post-menopausal osteoporotic women is estrogen deficiency. The exact mechanism of action of estrogen on bone is still unclear. We therefore examined the possibility that estrogen has its effect on osteoclasts by modulating the RANK-mediated signaling. Using rabbit osteoclast primary culture system, the molecules involved in the RANK-activated signaling was analyzed. Immunoprecipitation and Western blot analysis revealed that both RANKL and estrogen induced increased tyrosine phosphorylation of RANK. Estrogen induced transient increased associations between RANK and TNF-associated factor 6 (TRAF6). Estrogen also induced increased phosphorylation of P65 subunit of NF-κB. These effects of estrogen on RANK signaling appeared to have different time course. Both RANK-TRAF6 association and P65 phosphorylation were peaked at 5 min. The RANK-TRAF2 association was also increased after estrogen stimulation, but it appeared later and persisted for several hours. The increase of P65 phosphorylation also persisted for several hours. Both increases of RANK-TRAF6 association and P65 phosphorylation induced by estrogen were inhibited by PD98059 pretreatment. In conclusion, the current study provided evidences that estrogen modulated RANK-TRAF6- NF-κB signaling pathway, probably through Erk, in rabbit osteoclasts.

Disclosures: J. Shyu, None.


Osteoclast Apoptosis Is Driven by Mitochondrial Hydrogen Peroxide-Mediated Oxidative Damage and Degradation of PI3K/MEK/ERK Signaling Pathway Components.E. W. Bradley*1, S. L. Elfering*2, C. Giulivi*3, M. J. Oursler4. 1Biochemistry and Molecular Biology, University of Minnesota, Duluth, MN, USA, 2Chemistry, University of Minnesota, Duluth, MN, USA, 3Chemistry, Biochemistry and Molecular Biology, University of Minnesota, Duluth, MN, USA, 4Biology, Medical Microbiology and Immunology, Biochemistry and Molecular Biology, University of Minnesota, Duluth, MN, USA.

Bone resorbtion levels depend primarily on the number of osteoclasts present. Therefore, factors contributing to the survival of osteoclasts increase the amount of bone loss during both normal and pathological bone turnover. We have examined the mechanism of osteoclast survival using an in vitro generated mouse osteoclast model (mOCL). Survival of mOCLs is dependant on the activation of the PI3 kinase/MEK/ERK pathway. Inhibition of this pathway using kinase specific inhibitors as well as inhibition of protein synthesis leads to decreased mOCL survival. In this study, expression of downstream targets of the PI3 kinase/MEK/ERK pathway was examined for roles in osteoclast survival. Activation of Elk-1, a transcription factor for several Bcl-2 pro-survival genes, was detected five minutes after stromal cell removal. Using cDNA arrays, transcript levels for bcl-xL decreased 30 minutes after mOCL purification, whereas transcript levels for mcl-1 increased. Expression of Mcl-1protein increased 15 minutes after mOCL purification, followed by decreased expression within 45 minutes. Bcl-xL expression was detected at time zero and decreased until no expression was detected within 45 minutes after purification. Decreased levels of AKT, MEK, ERK and total cellular protein have also been observed 60 minutes after mOCL purification, suggesting general proteolysis. In addition, a decrease in the mitochondrial membrane potential and an increase in cytosolic cytochrome c were detected in cultured mOCLs. Evidence supports mitochondrial hydrogen peroxide production drives osteoclast apoptosis. These data include apoptosis repression when FCCP addition decreases mitochondrial hydrogen peroxide production and when supplementation with spin traps sequesters oxygen free radicals. We therefore conclude that the production of mitochondrial hydrogen peroxide is driving apoptosis via oxidative damage, leading to a decrease in downstream PI3 kinase/MEK/ERK targets and decreased osteoclast survival.

Disclosures: E.W. Bradley, None.


Exposure to Pasteurella Multocida Toxin Reveals an Inhibitory Role for the Small GTPase Rho in Osteoclast Differentiation.N. W. A. McGowan*1, D. Harmey2, G. Stenbeck*3, A. E. Grigoriadis1. 1Craniofacial Development, Kings College London, London, United Kingdom, 2The Burnham Institute, La Jolla, CA, USA, 3University College London, Bone and Mineral Centre, London, United Kingdom.

Rho GTPases belong to the ras superfamily of small GTP-binding proteins that are involved in multiple signal transduction pathways. Rho proteins are involved in the regulation of the actin cytoskeleton, and during the process of bone resorption osteoclasts are critically dependent on rapid cytoskeletal re-arrangements, for the maintenance of the F-actin podosomal ring and subsequent ruffled border formation. However, the role of Rho in osteoclast differentiation is unknown.

In this study osteoclast differentiation was examined in the presence of Pasteurella Multocida Toxin (PMT), a bacterially-produced toxin that causes the porcine bone resorbing disease, atrophic rhinitis. In addition to inducing actin rearrangements through Rho, PMT is a potent mitogen and stimulates phospholipase C, leading to activation of protein kinase C, and increases in inositol phosphates and intracellular calcium. PMT also signals indirectly through the Ras/MAP kinase pathway. Previous work by our laboratory has established that PMT inhibits osteoblast differentiation, in part via activation of Rho and Rho kinase (ROK).

In RANKL- and MCSF-based cultures of murine bone marrow cells or human peripheral blood monocytes, PMT dose-dependently inhibited osteoclast formation and resorption, and pulse studies demonstrated that this effect was manifested predominantly in the early stages of culture. To investigate the signalling pathways responsible for the inhibitory effects of PMT, we used inhibitors. Treatment of murine bone marrow cultures with the ROK inhibitor Y-27632 partially rescued the inhibition in the number of TRAP-positive osteoclasts, whereas Y-27632 alone had little or no effect. These data suggest an important inhibitory role for Rho and its downstream effector, ROK, in osteoclast differentiation. Interestingly, the inhibition of differentiation was in contrast to the effect of PMT on differentiated osteoclasts where treatment of human osteoclast cultures after day 10 failed to inhibit resorption. Rather, preliminary experiments suggest that addition of PMT to mature osteoclasts increased the proportion of F-actin ring-containing vitronectin receptor-positive osteoclasts, with a concomitant increase in resorption. Thus, although Rho activation is apparently required for osteoclast activity, this pathway negatively regulates osteoclast precursor differentiation. Current studies using PMT will dissect the signalling pathways used by osteoclasts during differentiation and bone resorption.

Disclosures: N.W.A. McGowan, None.


Characterization of an Anti-rat αvβ3 mAb which Blocks Differentiation of Osteoclasts.L. An*1, S. Langermann*1, J. Suzich*2, S. Mao2. 1Cell Biology, MedImmune, Inc., Gaithersburg, MD, USA, 2Molecular Biology/Biochemistry, MedImmune, Inc., Gaithersburg, MD, USA.

αvβ3 integrin is highly expressed on osteoclasts and is crucial for mediating their attachment to bone matrix to allow active bone resorption. We have characterized a rat αvβ3-specific mAb, VNR149, and tested its effects on rat tumor cell attachment and osteoclast differentiation in vitro. VNR149 binds to the αvβ3 integrin of rat and mouse, but not human. Replacement of either rat αv or β3 subunit by its human homologue prevents binding of the antibody. This property was used for selection so that VNR149 does not cross react with other rat integrins containing either αv or β3 subunit. Binding studies showed that the antibody has a high affinity (KD = 3 nM) for rat αvβ3, and binds mouse αvβ3 with an affinity 3-fold lower than that for rat analog. In this study, rat osteoclasts were differentiated in vitro from bone marrow cells. The precursor cells were cultured with MCSF and RANKL for 7 days in the presence or absence of VNR149. The osteoclasts were identified as multinucleated, TRAP-positive cells. VNR149 inhibited osteoclast differentiation in a dose-dependent manner, with complete inhibition observed at doses greater than 7 nM. In separate experiments, VNR149 was shown to block the attachment of αvβ3-positive rat glioma cells to vitronectin. Approximately 35% less cells became attached after 1 hour in the presence of 0.7 nM of the mAb. Taken together, our results indicate that VNR149 is effective at inhibiting osteoclast differentiation and tumor cell adhesion mediated by αvβ3 integrin; thus, it is a useful reagent for studying the role of αvβ3 integrin in various rodent models of human diseases.

Disclosures: S. Mao, None.


P2Y6 Nucleotide Receptors Signal through Nuclear Factor κB in Rabbit Osteoclasts.J. Korcok*, L. N. Raimundo*, X. Du*, S. M. Sims*, S. J. Dixon. CIHR Group in Skeletal Development and Remodeling, The University of Western Ontario, London, ON, Canada.

ATP and UTP are released from cells at sites of inflammation or in response to mechanical stimuli. Extracellular nucleotides can then act through P2 cell-surface receptors, which are subdivided into P2X (ligand-gated cation channels) and P2Y (G protein-coupled receptors) families. Previous studies by others have identified expression of P2Y1 and P2Y2 receptors in osteoclasts. Moreover, ADP acting through P2Y1 receptors stimulates osteoclast formation and resorptive activity (FASEB J 15: 1139, 2001). RANK ligand is a potent activator of osteoclast formation and bone resorption - effects that are mediated at least in part through activation of nuclear factor KB (NF-κB). The aim of this study was to determine whether P2Y receptors also signal through NF-κB in osteoclasts. Osteoclasts were isolated from long bones of neonatal rabbits. In osteoclasts further purified by micromanipulation, transcripts encoding P2Y1, P2Y2 and P2Y6 receptors were identified by RT-PCR. Immunofluorescence was used to detect the p65 subunit of NF-κB, which upon activation translocates from the cytosol to the nucleus. In control samples, 7 ± 2% of osteoclasts demonstrated nuclear localization of NF-κB. Exposure to the P2Y6-selective agonist UDP (10 μM) for 3 h resulted in nuclear translocation of NF-κB in 34 ± 5% of osteoclasts. In contrast, there was no significant response to the highly selective P2Y1 agonist, 2MeSADP (10 μM) or to the P2Y2 agonists ATP or UTP (10 μM). Thus, NF-κB appears to be activated selectively by P2Y6 receptors. To monitor the concentration of cytosolic free calcium ([Ca2+]i), osteoclasts were loaded with the Ca2+-sensitive dye fura-2. All nucleotides tested (10 μM) induced a transient rise of [Ca2+]i, consistent with the presence of functional P2Y1, P2Y2 and P2Y6 receptors. Moreover, the coupling of multiple P2Y receptors to Ca2+ signaling indicates that elevation of [Ca2+]i alone is not sufficient to activate NF-κB. Pretreatment with osteoprotegerin (OPG, a decoy receptor for RANK ligand) inhibited activation of NF-κB by RANK ligand, but not by UDP, establishing that UDP-induced translocation of NF-κB does not involve RANK ligand. These findings demonstrate that osteoclasts express functional P2Y1, P2Y2 and P2Y6 nucleotide receptors and that, of these receptors, NF-κB is activated selectively by P2Y6. Upon release, ATP and UTP can activate P2Y2 receptors and subsequently breakdown to form ADP and UDP, which can then activate P2Y1 and P2Y6 receptors. NF-κB signaling through P2Y6 receptors may contribute to the regulation of osteoclast formation and activity.

Disclosures: J. Korcok, None.


Regulation of Bone Resorption by c-Cbl: Increased Bone Resorption in Osteoclasts Expressing v-Cbl, an Oncogenic Form of c-Cbl, and Inhibition by C-Terminal Mutants.C. Itzstein, T. Miyazaki, W. C. Horne, A. Sanjay, R. Baron. Yale University - School of Medicine, New Haven, CT, USA.

Several studies have indicated that c-Cbl, an adaptor protein with no kinase activity, can be both a positive and a negative regulator of bone resorption. c-Cbl−/− osteoclasts exhibit decreased migration and lack of c-Cbl phosphorylation in src−/− leads to a decrease in bone resorption. Thus, as a positive modulator, c-Cbl forms a tri-molecular complex with Pyk2 and Src in osteoclasts (OCs) promoting OC motility and bone resorption. However, and in contrast, c-Cbl also mediates the down-regulation of several tyrosine kinase receptors, including M-CSF, and non-receptor tyrosine kinases such as Src itself, by promoting their ubiquitination. The structure-function relationship that allows c-Cbl to act both as a positive and a negative regulator of signaling events is however still unclear. Structurally, the N-terminal half of c-Cbl includes a tyrosine kinase binding (TKB) domain and a RING finger, responsible for the ubiquitin-ligase activity of c-Cbl, while the C-terminal half contains a proline-rich region and several regulatory tyrosines, responsible for SH2 and SH3 mediated protein interactions. To further investigate the functional roles of various protein binding domains of c-Cbl, we have expressed myc-tagged wild-type (WT) c-Cbl, the C-terminal half of c-Cbl (Cbl-CT) or c-Cbl containing a mutated Src SH3 binding site (Cbl6PA) or a mutated PI3-kinase binding site (CblY731F) in murine OCLs using the adenovirus system, and evaluated their effects on OC activity in a bone resorption assay. Neither WT c-Cbl nor Cbl-CT affected pit formation. However, over-expression of Cbl6PA or CblY731F inhibited bone resorption by 25% (P<0.05) and 70% (P<0.001) respectively, demonstrating that interaction between Cbl and Src or Cbl and PI3-kinase play positive roles in OC activity. In contrast, v-Cbl (containing only the TKB domain) increased pit formation by 30% (p<0.05), an effect that was even more pronounced in c-Cbl−/− OCLs, increasing pit formation by three-fold. These results suggest that v-Cbl acts in a dominant negative fashion by displacing full-length Cbl proteins from phosphotyrosine binding sites on other key signaling protein. This demonstrates that the N-terminal region of Cbl is involved in the down-regulation of bone resorption. Thus, c-Cbl participates both in stimulatory and in inhibitory regulatory events in bone resorption. Binding of Cbl proteins to specific phosphotyrosines in signaling molecules is required for the down-regulation of resorption-induced mechanisms in OCs, whereas SH2 and SH3-mediated interactions with its C-terminal domain are required for their activation.

Disclosures: C. Itzstein, None.


Comparison between 60 Matched Pairs of Postmenopausal African American and Caucasian Women: Analysis of Risk Factors Related to Bone Loss.J. E. Ballard1, L. S. Wallace2, D. Holiday*3, K. Wells*1. 1Health and Kinesiology, University of Texas at Tyler, Tyler, TX, USA, 2Family Medicine, University of Tennessee Graduate School of Medicine, Knoxville, TN, USA, 3Biostatistics, University of Texas Health Center at Tyler, Tyler, TX, USA.

The purpose of this study was to compare known or suspected risk factors (gynecological, nutritional, and/or medical variables) related to bone loss between 60 African American (AA) and 60 Caucasian (C) postmenopausal women. The two racial groups had been matched one for one on selective anthropometric variables [age (yrs), standing height (cm), and body weight (kg)] in order to equate age and body size between groups. Information on risk factors was obtained from an orally administered questionnaire and body composition variables (in addition to those used for matching) were assessed by anthropometry and total body DEXA. Four skinfold sites (chest, triceps, mid-axillary, and abdomen) were measured with Harpendon calipers and four body circumferences (chest, forearm contracted, waist, and gluteal) were assessed with a Gulick tape. DEXA whole body measurements were obtained on a Hologic QDR-2000 with software version 7.20. The same technician administered the oral questionnaire to all subjects. Results of the questionnaire revealed that the C women reported significantly higher proportions of alcohol use, family history of broken bones, and a greater utilization of hormones, calcium and vitamins than did the AA women. On the other hand, the AA sample reported a higher proportion who had undergone extensive dental work and a greater numbers who had other diseases (i.e., overactive thyroid, diabetes, rheumatoid arthritis, or kidney stones). On the basis of these data, it was concluded that part of the difference often observed in bone density between AA and C postmenopausal women may be due to lifestyle factors.

Disclosures: J.E. Ballard, None.


Knowledge about Osteoporosis in the German Population - Perception and Reality.F. Raue1, J. Scheld*2, S. Schmitt2, B. Tischer*3. 1Practice for Endocrinology & Genetics, Heidelberg, Germany, 2Procter & Gamble Pharmaceuticals, Weiterstadt, Germany, 3TNS EMNID, Pullach, Germany.

Awareness for and knowledge about specific diseases, their risk factors, outcome and treatment possibilities are strong factors for the patients support for diagnostic interventions, treatment adherence and long term compliance. However, there is little data on the impact of global or local initiatives on awareness of osteoporosis.

Objective of this research executed by an independent market research institute, TNS EMNID, was to assess awareness, knowledge, attitudes and medication habits regarding osteoporosis in the German population and compare it with the awareness of other common diseases.

A telephone survey was performed in an adult German population between 20 and 80 years in January 2003. 22 mainly closed questions were presented to a representative sample of 2309 Germans in an omnibus study. 53% of the participants were female, 33% aged 50+, 6% had been previously diagnosed with osteoporosis.

Unaided awareness of osteoporosis as being a chronic disease was only 3%, significantly lower than awareness for asthma (27%), diabetes (20%) and similar to the awareness of urinary tract infection (3%). Knowledge about osteoporosis was also rather low, even in the population at highest risk: 55% of the men aged 50+ and 63% of the women aged 50+ perceived the progression of osteoporosis after diagnosis as slow. Only 17% of the men aged 50+ and 12% of women aged 50+ associated an increased risk to die with osteoporosis. Information about illness, progression and mortality risk worsens with increasing age. Only 12% of the population aged 50+ feared to suffer osteoporosis in the future, other diseases like cancer (38%) or cardiac infarction (28%) were perceived to be of greater threat. Consistent with this, 71% of the total population assessed themselves not being at risk for osteoporosis. 37% of the total population and 54% of people aged 50+ suffered from any chronic disease, accompanied by regular intake of medication. Regarding the most convenient dosing frequency, 55% of the total population preferred daily intake vs. 35% weekly. However, for a medication with intake instructions like bisphosphonates, 60% preferred weekly medication vs. 32% for daily intake.

We conclude that ignorance and misinformation about osteoporosis is still common both amongst the total population and the higher risk group of elderly aged 50+. An effective communication framework between physicians, patients and their organisations is required to change this situation. Preference for intake of medications depends on the mode of administration and the habits of the respective patients, with preferences for either daily or weekly intake.

Disclosures: F. Raue, None.


Osteoporosis Health Belief Scale: Psychometric Properties by Telephone Administration.S. M. Cadarette, D. E. Beaton*, G. A. Hawker. Health Policy, Management & Evaluation, University of Toronto, Toronto, ON, Canada.

The Osteoporosis Health Belief Scale (OHBS) is a 42-item self-administered questionnaire designed to measure 7 constructs: susceptibility, seriousness, calcium benefits, calcium barriers, exercise benefits, exercise barriers and health motivation. Susceptibility measures the perceived risk of developing osteoporosis; seriousness measures the perceived threat from having osteoporosis; benefits focus on the belief in the effectiveness of specific behaviors to prevent the occurrence of osteoporosis; barriers measure beliefs about the negative components of the behaviors required to prevent osteoporosis; and health motivation measures the tendency to engage in healthy behaviors. Understanding current levels of osteoporosis health beliefs in a population of older women who are entering the highest risk for osteoporosis-related fracture may facilitate the development of interventions to improve osteoporosis prevention. Therefore, we conducted this study to examine the psychometric properties of the OHBS by telephone administration in a cohort of older women. Ethical approval for this study was provided by the Health Sciences II Review Committee of the University of Toronto Office of Research Services (protocol reference # 8376). A convenience sample of 425 women aged 61--93 years (mean=73.6) participating in a longitudinal arthritis study was recruited by telephone. Item clarity was evaluated and 22 additional items were considered to supplement or replace existing scale items. Multi-trait scaling techniques and exploratory factor analysis were used to test scale structure. A few modifications to the OHBS scale were suggested based on these data, reducing the scale by 5 items, rewording 1 item and moving 1 item to a different subscale. The modified 37-item OHBS had a 7-factor uncorrelated solution explaining 48% of the model variance with internal consistency ranging from 0.73 to 0.88. In summary, this study found that minor changes to the OHBS results in reduced redundancy and improved internal structure of the scale for telephone administration among women over 60 years of age. However, given that a convenience sample of women participating in a longitudinal study of arthritis was used to test the psychometric properties of the scale, the study should be repeated in other cohorts to confirm these findings. Thus, we recommend that the reworded item: “taking in enough calcium cuts down the chances of getting osteoporosis” be added to the end of the current 42-item OHBS in future telephone administration to permit full confirmatory analysis of both the original and the modified scale.

Disclosures: S.M. Cadarette, None.


Low Calcium Intake Magnifies the Bone Loss Seen with Low Dietary Protein Intake in Elderly Men and Women.M. T. Hannan1, K. L. Tucker2, B. Dawson-Hughes2, I. Chazaro*3, D. Kiel1. 1Harvard Med Sch, Hebrew Rehab Ctr for Aged, Boston, MA, USA, 2USDA HNRC, Tufts Univ, Boston, MA, USA, 3BUSPH, Boston, MA, USA.

Our prior work showed that low protein intake resulted in greater bone loss in elderly men and women in the Framingham Study, suggesting that protein intake is important in minimizing bone loss. It is unclear if this effect in elders depends on calcium (CA) intake. Thus, we examined the relation between baseline dietary protein and subsequent 4-year change in femoral bone mineral density (BMD) according to CA levels for 392 women and 224 men from the population-based Framingham Osteoporosis Study. BMD was assessed in 1988--89 and in 1992--93 at the femoral neck (FN), trochanter (TR), and Ward's area, using Lunar DPA & DXA densitometers adjusting for shift in technology using published corrections. Dietary protein was determined using the Willett food frequency questionnaire and expressed as percent of energy from protein (%Protein). We also evaluated the ratio of animal to vegetable protein (A:V Ratio). BMD loss over 4-y was regressed on protein adjusting for baseline age, sex, weight, height, smoking, alcohol use, physical activity, total CA intake, total energy intake and for women, estrogen use. Protein was evaluated as a continuous variable and as quartiles for the whole sample, as well as by total CA intake group (<800 mg/d=low CA, 800+ mg/d=high CA) and the group not using supplemental CA (n=490).

Mean age (SD, range) at baseline was 74y (4.4, 68--90), 18% used supplemental CA, mean protein intake was 68 g/d (23.5, 16--152) and %Protein was 16% (3.3, 8--27). Lower %Protein was significantly related to bone loss (all sites p<0.05) and this association was stronger when limited to subjects with low CA (FN, p=0.03; TR, p=0.02). %Protein quartiles for the whole sample and low CA subset showed again subjects with low CA and low protein intakes had the greatest bone loss (see table, model FN p=0.02, TR p=0.003),. Similar results were seen for those not using CA supplements and at other BMD sites. A:V Ratio showed no relation to bone loss for the whole sample (all p>0.24) or subsets of subjects. Thus, in elderly men and women, low CA, in addition to low protein intake, leads to even greater bone loss than low dietary protein alone. Our results suggest that protein intake may be even more important for elderly persons with low CA intakes than those with high CA intakes to minimize bone loss. 

Table  . Adjusted Mean FN-BMD (SD, p-value for quartile comparison) by Quartile of Protein Intake
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Disclosures: M.T. Hannan, None.


Prevalence and Seasonal Variation of Hypovitaminosis D and its Relationship to Bone Metabolism in Healthy Postmenopausal Hungarian Women.H. P. Bhattoa, P. Bettembuk, A. Balogh. Regional Osteoporosis Center, Department of Obstetrics and Gynecology, University of Debrecen, Debrecen, Hungary.

Hypovitaminosis D can result in low bone mass. The prevalence of hypovitaminosis D has public health implications, especially where data are lacking. Since diet and sunlight are the two sources of vitamin D, the results obtained in one geographical region may not be universally applicable.

The aim of this study is to characterize the prevalence and seasonal variation of hypovitaminosis D and its relationship to parathyroid hormone (PTH), dietary calcium intake, biochemical markers of bone turnover and BMD at the L2--L4 lumbar spine (LS) and the femur neck (FN) in healthy community dwelling postmenopausal women in Hungary. We determined serum levels of 25 hydroxyvitamin D (25-OH-D), PTH, osteocalcin (OC) degradation products of C-terminal telopeptides of type-I collagen (CTx), dietary calcium intake and BMD at LS and FN in 319 ambulatory postmenopausal women. The prevalence of hypovitaminosis D (serum 25-OH-D ⩽ 50 nmol/l) was 56.7%. On comparing the patients with normal and low 25-OH-D, significant difference was found in age (67.3 ± 9.9 years vs. 61.6.3 ± 8.5 years; p < 0.001), FN BMD (0.744 ± 0.125 gm/cm2 vs. 0.802 ± 0.123 gm/cm2; p < 0.001) and dietary calcium intake (607.9 ± 233 gm/day vs. 714.4 ± 199.4 gm/day; p < 0.001). Osteoporotic patients had a significantly lower 25-OH-D (37.6 ± 19.8 nmol/l vs. 56.4 ± 24 nmol/l; p < 0.001) and dietary calcium intake (781.2 ± 164.3 mg/day vs. 519.2 ± 244.5 mg/day; p < 0.001). After controlling for all other variables 25-OH-D was found to be significantly associated with age, the average hours of sunshine in the three months prior to 25-OH-D level determination and dietary calcium intake (R2 = 0.190; p < 0.001). For FN BMD significant independent predictors were age, body mass index, 25-OH-D and dietary calcium intake (R2 = 0.435; p < 0.001). The prevalence of hypovitaminosis D during spring, summer, autumn and winter was 71%, 46.3%, 49.4% and 56.7%, respectively. There was significant seasonal variation in 25-OH-D, PTH, OC, calcium intake and FN BMD.

There is a high prevalence of hypovitaminosis D in the healthy postmenopausal Hungarian women, and FN BMD is associated with serum 25-OH-D and dietary calcium intake. We believe that universal vitamin D and calcium supplementation, especially in winter, can be beneficial and cost effective at a population level.

Disclosures: H.P. Bhattoa, None.


Green Tea Drinking Is Associated with Increased Bone Mineral Density.S. Muraki*1, S. Yamamoto2, H. Ishibashi*2, T. Horiuchi*2, T. Hosoi*2, T. Suzuki*3, H. Orimo2, K. Nakamura1. 1Orthopaedics, Tokyo University School of Medicine, Tokyo, Japan, 2Tokyo Metropolitan Geriatric Medical Center, Tokyo, Japan, 3Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.

Tea drinking is associated with increased bone mineral density (BMD), as demonstrated by previous studies, suggesting that flavonoids, contained in tea, may partly account for it. However, to the best of our knowledge, this study serves as the first to investigate the relationship between consumption of green tea, which also contains flavonoids, and BMD. The aim of this study is to determine whether green tea drinking is associated with increased BMD. This study included six hundred and fifty-five women aged 60 years and above (mean age 71.6 psul/minus 7.6 years) visiting the Osteoporosis Outpatient Clinic in Tokyo Metropolitan Geriatric Medical Center. At entry to this study, body height and weight were measured, and body mass index (BMI) was calculated. Subjects were interviewed their lifestyles by means of a questionnaire which includes consumption of 10 dietary items such as green tea, milk, cheese, yogurt, fish, vegetable, tofu, natto, meat and coffee as well as their history of smoking, alcohol consumption, their level of physical activity, and the use of osteoporosis medicaton. For each dietary item, subjects were divided into two groups: consuming five or more days per week, and less than five days per week. BMD of lumbar spine, serum calcium, serum phosphorus, serum parathyroid hormone, serum alkaline phosphatase, serum osteocalcin, serum 1,25-dihydroxyvitamin D and urine deoxypyridino-line were measured. Statistical analysis was performed using a non-paired t test and multiple regression analysis. Among six hundred and fifty-five subjects, six hundred (91.6%) consume green tea five days or more per week and their mean BMD was 0.808±0.199 g/ cm2, and fifty-five (8.4%) consume green tea less than five days per week and their mean BMD was 0.738±0.174 g/cm2. The BMD of the former subjects was significantly higher than the latter. The correlation between green tea drinking and BMD was independent of age, BMI, other dietary items, their history of smoking and alcohol consumption, their level of physical activity and the use of osteoporosis medication. No significant correlation was observed between green tea drinking and any serum or urinary levels. Estrogenic effect induced by flavonoids or apoptosis of osteoclasts induced by (-)-epigallocatechin-3-gallate, one of flavonoids, may account for the significant increase of the BMD. We conclude that green tea drinking is associated with increased BMD among elderly women.

Disclosures: S. Muraki, None.


Carbonated Beverage Consumption and Bone Mineral Density.K. L. Tucker1, L. Troy*1, K. Morita*1, L. A. Cupples*2, M. T. Hannan*3, D. P. Kiel*3. 1USDA HNRCA Tufts University, Boston, MA, USA, 2Boston University, Boston, MA, USA, 3Hebrew Rehab Center for Aged, Boston, MA, USA.

Soft drink consumption has been thought to have negative effects on BMD, but studies have shown mixed results. Carbonated soft drinks often displace milk in the diet and introduce phosphoric acid (H3PO4) without calcium. Since the phosphorus content of regular cola is 44--62 mg, and of diet cola 27--39 mg, per 12 oz serving, while most other carbonated beverages contain no phosphorus, we hypothesize that consumption of these specific soft drinks would be associated with lower BMD in adult participants in the Framingham Offspring Study. Valid dietary data and BMD measurements were collected for 1672 women and 1148 men from 1996 to 2001. BMD was measured at the spine and 3 hip sites using a Lunar® DPX-L. Dietary intake was assessed with a semi-quantitative food frequency questionnaire that specifically queried respondents on the number of servings of cola and other carbonated beverages consumed, and differentiated between regular, caffeine-free and diet beverages. We regressed each BMD measure onto various measures of soft drink consumption, adjusting for BMI, height, age, energy intake, physical activity score, smoking, alcohol use, use of osteoporosis medication, use of calcium or vitamin D supplements, intake of calcium and vitamin D from diet and, for women, menopause status and estrogen use. There were no significant relationships between total carbonated beverage consumption or non-cola carbonated beverage consumption and BMD at any site. Women, but not men, consuming more than three servings of cola (all types)/d had significantly lower BMD at each of the three hip sites, relative to those consuming less than one serving/d: 2.3% lower at the trochanter (p=0.05), 3.3% lower at the femoral neck (P<0.001), and 5.1% lower at Ward's area (P<0.0005). For the spine those with the highest cola intake had BMD 1.2% lower than those consuming less than one serving/d, but this was not significant. This same pattern of results was generally seen for non-caffeinated and diet cola. Adjusting for caffeine did not change results and there was no significant interaction with calcium intake. These results suggest that cola, but not other carbonated soft drink consumption, contributes to lower BMD in adult women, after adjusting for calcium and caffeine intake. Because similar results were seen with diet and non-caffeinated cola, these associations may be due to the phosphoric acid content of cola.

Disclosures: K.L. Tucker, None.


Cortical Bone Response to Long-term Smoke and Ethanol Exposure.D. M. Cullen1, J. Carter*1, S. Morgan*1, M. Gentry-Nielsen*2, M. P. Akhter1. 1Osteoporosis Research Center, Creighton University, Omaha, NE, USA, 2Medical Microbiology, Creighton University, Omaha, NE, USA.

Smoking and drinking behavior often coincide and have been associated with poor bone mass. In human studies, the effects of these activities can not be separated. In a previous study we showed negative effects of 36% EtOH on cortical bone formation. In this study we examined the dose response of EtOH combined with smoking. Young male Sprague-Dawley rats (120g, n=96 or 12/group) were either Nonexposed or Smoke Exposed to mainstream and sidestream smoke for an hour (30 cigarettes) twice/day weekdays and once/day weekends for 12 weeks. For the last 5 weeks, the Nonexposed and Exposed groups were randomized to liquid diets with 0, 16, 26, or 36% of the calories from EtOH. Bone formation was labeled with calcein (6mg/kg). Tibiae were dehydrated, embedded in methyl-methacrylate, and sectioned at 70mcm. Tibial periosteal and endocortical surfaces were measured by histomorphometry for mineralizing surface (MS), mineral apposition rate (MAR), bone formation rate (BFR), resorption surface and intracortical bone formation. Difference due to Smoke and EtOH were tested by two-factor ANOVA and reported below as mean (SD).

Smoke effects were seen on the periosteal surface where MS and BFR were elevated in smokers. A similar pattern was seen for intracortical MS in smokers. There were no smoke effects seen on the endocortical surface. Although no differences in periosteal resorption surface were detected, the increase in intracortical MS suggests that elevated formation may be secondary to resorption. Smoking had no effect on periosteal MAR. Alcohol effects were seen only on the endocortical surface. In both smokers and nonsmokers, 36% EtOH suppressed MS, MAR, and BFR. There were no effects of either 16% or 26% EtOH on bone formation parameters. There was no interaction between smoke and alcohol. In conclusion, in young growing male rats smoking stimulated periosteal bone formation, while high alcohol consumption suppressed endocortical formation. 

Table  .  
  1. adif Nonexposed vs Exposed P=0.02; bdif Chow vs EtOH P<0.001

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Disclosures: D.M. Cullen, None.


Bone Density Measurement Positively Influences Postmenopausal Women to Improve Osteoporosis Prevention Habits.C. S. Cowgill, D. Krueger, S. Zeldin*, N. Vallarta-Ast, N. Kaech*, K. Hansen, M. Drezner, N. Binkley. Osteoporosis Clinical Research Center and Research Program, University of Wisconsin, Madison, WI, USA.

Results of the Women's Health Initiative prompted many postmenopausal women to discontinue estrogen, thereby ending effective bone preserving therapy. However, controversy exists regarding the need to measure bone mineral density (BMD) after discontinuing estrogen. We hypothesized that BMD measurement will encourage postmenopausal women to increase bone prevention activities or seek pharmaceutical intervention when appropriate.

Community-dwelling postmenopausal women were invited through newspaper advertising to have a free BMD measurement. Proximal femur and lumbar spine BMD was measured by dual x-ray absorptiometry in 215 women utilizing GE Lunar DPXIQ or Prodigy densitometers. All participants discussed their results with a physician or nurse practitioner immediately after the scan; osteoporosis prevention or intervention was suggested as appropriate. Subsequently, 75 days after their BMD test, a 25-question survey was mailed to the women who agreed to be contacted. Information on what changes, if any, these women implemented is reported from 50 completed surveys received from the 74 women contacted to this time. The survey consisted of questions about osteoporosis related lifestyle and overall health habits before and after their “DXA Day” scan. Of 50 responders, 17 had normal BMD, 26 were osteopenic and 8 osteoporotic. 90% of these women correctly remembered their BMD classification.

When asked why they elected to have a BMD measurement, 73% listed concern for personal health and 48% because they recently stopped estrogen. Lifestyle changes were made by 90%; specific interventions included increasing exercise (48%) increasing calcium or vitamin D (42%/36%) reducing caffeine intake (28%) seeking medical treatment (20%) and discontinuing smoking (4%). Of the 45 women reporting lifestyle changes, 47% made unsolicited comments indicating that the interaction with a clinician was important.

In conclusion, BMD measurement immediately followed by osteoporosis education is effective in motivating 90% of women to favorably alter lifestyle factors which reduce osteoporotic fracture risk. Perhaps osteoporosis education should become part of routine bone mass measurement.

Disclosures: C.S. Cowgill, None.


Prospective, 2-Year Study in Postmenopausal Women Reveals Negative Association of Vitamin A and Bone Mineral Density of Various Skeletal Sites.J. Z. Ilich1, R. A. Brownbill1, H. C. Furr*2. 1School of Allied Health, University of Connecticut, Storrs, CT, USA, 2Craft Technologies, Inc., Wilson, NC, USA.

Recent epidemiological studies suggest that higher vitamin A intake is associated with lower BMD and higher risk of hip fractures. It is not clear what level of vitamin A intake becomes detrimental for bone health. The purpose of this study was to evaluate relationship between dietary vitamin A and BMD of various skeletal sites in over 100 postmenopausal women followed for 2 y. Subjects were generally healthy, free of chronic diseases or medications known to affect bone (including HRT) and participants of a larger clinical trial examining the effect of sodium (Na) on bone. BMD at various skeletal sites was measured by LUNAR DPX-MD and dietary intake was assessed by 3-day dietary record, both at baseline and 6,12,18 and 24 months into the study. The diets were analyzed using Food Processor® and mean daily intake, including total energy and all other nutrients was calculated. The intake of supplements was recorded as well, and included in the nutrient analysis. Vitamin A was evaluated both as continuous and categorical variable, the latter one by stratifying subjects below and above the median of intake. Repeated measures ANCOVA (adjusted for age, BMI, energy, and Ca, Na intake) were used to compare groups over time. Multiple regression models were utilized at each time point to assess relationships between vitamin A metabolites and BMD or BMC.

The average intake of vitamin A (diet and supplements) in our population at each visit was above the tolerable upper limits (10000 IU/day), with over 50% of subjects taking vitamin A supplements (from 1000 to 20000 IU/day) throughout the study period. The average cumulative intake at 24 months was 10034±4352 IU/day. The results reveal that higher intakes of vitamin A were significantly associated with decreased BMD of the femoral shaft and total femur, as well as total body and forearm. This trend was noticeable at each time point and as a cumulative effect throughout 24 months of follow-up.

Based on our data, intakes of vitamin A above 9000--10000 IU/day, may be detrimental for bone health and elderly women should use caution when selecting/consuming vitamin A supplements. Several foods, such as milk, cereals, and some juices are currently fortified with vitamin A and their re-evaluation may be warranted.

Disclosures: J.Z. Ilich, None.


Relationship of Life Style, Dietary Factors, Serum PTH and 25-Vitamin D3 Level with Lumbar Spine BMD in Premenopausal and Postmenopausal Women in Korea.S. K. Lee1, I. S. Yeo*2, K. S. Jeon*2, S. W. Kim*3, H. W. Baik*1, C. Kim*1, J. K. Oh*1, K. S. Park*1. 1Eulji University School of Medicine, Daejeon, Republic of Korea, 2Eulji University Hospital, Daejeon, Republic of Korea, 3Food and Nutrition, Joongbu University, Daejeon, Republic of Korea.

Bone mineral density (BMD) is determined by the combined effects of genetic factors and environmental factors such as life style and dietary facotrs. The present study was to evaluate the relationships of life style, dietary factors, serum intact PTH (iPTH) and 25-vitamin D3 (25-Vit D3) level to lumbar BMD in Korean women. We investgated 95 premenopausal (PRE) and postmenopausl (POST) women who had no confounding diseases except hypertension, visiting Eulji University Hospital, South Korea in Winter. We used Questionnaire about physical activity, quality of life (QOL), food habit. We measured various blood parameters, serum iPTH & 25-Vit D3 level and spine L1-L4 BMD using DXA. Mean (±SD) of each variable in PRE normal BMD group and PRE osteopenia & osteoporosis group was Age (42±7 vs 43±7 yrs), Height (158±6 vs 153±6 cm), Weight (58±6 vs 54±6 kg), serum iPTH (28.8±9.3 vs 40.8 ±22.7 pg/mL) and 25-Vit D3 (59.5±18.0 vs 53.5±22.0 nmol/L). There was a significant difference only in height, but no difference in physical activity score, QOL score, food habit score, smoking, alcohol intake, dietary calcium intake, Ca/P ratio in diet between PRE normal BMD and PRE osteopenia & osteoporosis group. Mean (±SD) of each variable in POST normal BMD, POST osteopenia and POST osteoporosis group was Age (56±5 vs 58±6 vs 62±7 yrs), Height (157±4 vs 154±4 vs 151±4 cm), Weight (68±10 vs 62±10 vs 57±9 kg), Fat mass (24±7 vs 21±6 vs 19±6 kg), Menopause Duration (5±5 vs 10±7 vs 13±8 yrs), serum iPTH (35.0±3.1 vs 31.7±11.3 vs 35.9±17.7 pg/mL), serum 25-Vit D3 (94.0±32.3 vs 78.8±21.8 vs 62.3±20.8 nmol/L). There were significant differences in age, height, weight, fat mass, menopause duration, serum 25-Vit D3 between POST normal BMD and POST osteoporosis group. However, there were no significant differences in life style & dietary factors including dietary calcium intake, Ca/P ratio in diet among POST three groups. Correlations of BMD with age, height, weight, fat mass, menopause duration, serum 25-Vit D3 were significant in POST three groups. In conclusion, life style & dietary factors, serum iPTH level had no relationships to lumbar BMD in this limited Korean women. Age, body composition, menopause duration and serum 25-Vit D3 level had relationships to lumbar BMD only in postmenopausal women. Subjects in this study had relatively low calcium intake, low Ca/P ratio in diet, irrespective of lumbar BMD. Population-based study and genetic study is needed.

Disclosures: S.K. Lee, None.


High Vitamin A Intake Is Not Associated with Low Bone Mineral Density in Older Men.K. L. Stone1, T. Blackwell*1, E. S. Orwoll2, J. A. Cauley3, E. Barrett-Connor4, D. Sellmeyer1, S. R. Cummings5. 1University of California, San Francisco, San Francisco, CA, USA, 2Oregon Health Sciences University, Portland, OR, USA, 3University of Pittsburgh, Pittsburgh, PA, USA, 4University of California, San Diego, San Diego, CA, USA, 5San Francisco Coordinating Center, San Francisco, CA, USA.

Vitamin A in high doses stimulates bone resorption and inhibits bone formation. However, epidemiologic studies of the relationship between Vitamin A intake and bone mineral density (BMD) or fracture risk have been conflicting. Similar studies based on serum retinol concentrations are also conflicting.

We tested the relationship between intakes of Vitamin A, retinol and beta-carotene and BMD among older men participating in the multi-center Osteoporotic Fractures in Men (MrOS) Study. During the baseline examination, a 70-item food frequency questionnaire (Block Dietary Data Systems) was administered in 5995 men aged 65 and older. BMD of the whole body, total hip and total spine were assessed by DXA (Hologic QDR 4500W). Food questionnaires were analyzed to obtain estimated daily intake of nutrients from both diet and supplements. Intakes were categorized as low (lowest quintile), medium (2nd through 4th quintile) and high (upper quintile) for the analyses. All results were adjusted for age, body weight, physical activity, and total energy intake. Further adjustment for race, smoking, alcohol, caffeine, health status, calcium and vitamin D did not change the results. As shown in the table below, there is a trend towards increasing total hip BMD across levels of intake of dietary vitamin A and beta-carotene. This trend becomes statistically significant for total intake of Vitamin A (diet + supplement): men with the highest intakes have the highest BMD (p=.003). On the other hand retinol intake shows no relationship with BMD. Results are similar for other BMD sites, or when analyzed as dietary intake excluding vitamin A supplement users. 

Table  . Adjusted mean total hip BMD (g/cm2) by levels of dietary intake.
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We found no evidence of lower BMD among men consuming the highest amounts of Vitamin A. Although vitamin A consumption may be lower in our study population than in others, our highest category of intake is similar to others in which lower BMD has been reported. It remains to be seen whether higher vitamin A intakes will be associated with subsequent rates of bone loss, or fracture risk.

Disclosures: K.L. Stone, None.


Type 2 Diabetes and Non-Traumatic Fractures in Older White and Black Men and Women.E. Strotmeyer1, J. Cauley1, A. Schwartz2, H. E. Resnick*3, D. C. Bauer2, F. Tylavsky4, N. De Rekeneire*5, T. Harris*5, A. Newman*1. 1University of Pittsburgh, Pittsburgh, PA, USA, 2University of California, San Francisco, CA, USA, 3MedStar Research Institute, Hyattsville, MD, USA, 4University of Tennessee Health Science Center, Memphis, TN, USA, 5National Institute on Aging, Bethesda, MD, USA.

Older white women with type 2 diabetes may have increased fracture rates despite higher bone mineral density (BMD) but little information exists for men and older black adults. The Health, Aging, and Body Composition Study included white and black, physically able, men and women age 70--79 years, followed for health events including incident non-traumatic fractures verified by radiology reports. Falls in year prior to baseline were assessed by self-report. BMD, total fat and total lean mass were measured with DXA (QDR 4500A, Hologic Inc, Bedford, MA) and visceral fat area by CT scans. Diabetes was defined as reporting diabetes, diabetes medication use, or fasting glucose of ⩾126 mg/dl. Exclusion criteria were oral steroid use (n=69), diabetes diagnosis at age <20 years (n=5), or missing diabetes status (n=22). Of the remaining 2979 participants (mean age 74±3 years; 49% men; 42% black), 566 (19%) had diabetes at baseline. Participants with diabetes were more likely men (57% vs. 47%; p<0.001) and less likely white (43% vs. 62%; p<0.001) than those without diabetes. Diabetic participants had higher total hip BMD than non-diabetic participants (0.96±0.17 vs. 0.87±0.17 g/cm2, p<0.001). A total 164 non-traumatic fractures were observed in 4.5±1.1 years. Non-traumatic fracture incidence was 11.7 per 1000 person-years for those with type 2 diabetes and 12.3 for those without diabetes (p=0.80). Race and gender specific rates are shown below. 

Table  . Non-traumatic fracture incidence/1000 person-years
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Diabetic black women had almost a two-fold higher incidence of fractures than non-diabetic black women (p=0.07). In cox regression models controlled for gender, race, site, age, hip BMD, falls, weight change history, HbA1C, diabetes and bone-active medications, visceral fat, total fat and lean mass, baseline diabetes was significantly related to non-traumatic fractures (RR=1.6; 95%CI: 1.1--2.5). Type 2 diabetes was independently associated with a 60% increased risk of fracture in this cohort of white and black men and women despite higher BMD in diabetic participants at baseline.

Disclosures: E. Strotmeyer, None.


Assessment of Women after Colles Fracture for Osteoporosis and Hip Fracture Risk using the ‘Black Fracture Score’.L. Dolan*1, S. Califf*2. 1Rheumatology, Queen Elizabeth Hospital, Woolwich, London SE10, United Kingdom, 2Rheumatology, Queen Elizabeth Hospital, Woolwich, London, United Kingdom.

Colles Fractures are early low impact fractures and a ‘red flag’ for identifying osteoporosis. Fracture clinics have been poor at this. We use Fracture Intervention nurses to identify and arrange DEXA on these patients.

This study assesses whether Colles patients are at imminant risk of more severe fractures. The ‘Black Fracture Score’1 is a well validated tool to assess future hip fracture risk, based on age, previous fracture, maternal hip fracture, smoking and inability to rise from a chair.

Method. Sequential women attending the fracture clinic at Queen Elizabeth Hospital were identified by a fracture nurse and referred for DEXA. They completed a risk factor questionnaire. The ‘Black fracture score’ was calculated.

Results; 118 women with Colles fracture were identified over 6 months. Median age 67 yrs, range 35--83yrs. 97.5% Caucasian. 36.4% had spine BMD T score <−2.5. 37.2% had spine BMD T score <−2.5 or z <−1. 17.7% had hip BMD T score <−2.5. 18.6% had hip BMD T score <−2.5 or z <−1. 40% satisfied hip or spine BMD criteria for treatment. Risk factors were early menopause (33%), low BMI (8.4%), previous falls (8.4%), use of arms to stand from chair (36.4%), smoking (18.6%). Few had previous major fractures (4 hip, 2 pelvic, 4 vertebral) but 26% had other peripheral fractures. Previous use of osteoporosis treatment was low. Black fracture score showed 33 had a raw score of ⩾ 5 (28%)(ie 5yr hip# risk of >8.3%); 22 had fracture score including BMD of ⩾ 8 (18.6%) (ie 5yr hip # risk of >8.7%). 

Table  .  
  1. P = comparison of >75 yr with rest, using Chi sq test

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Comment: Bone densitometry in Colles patients identified a significant number satisfying BMD criteria for treatment (40%) and supports a strategy of case finding by a fracture intervention nurse. Few had hip or vertebral fracture, confirming Colles fracture as an early low-trauma fracture. More women with colles # have reduced spine BMD than hip. The discrepency between the raw score and that with may reflect a tendency to spine osteoporosis, so fewer are at risk of hip fracture. Imminent fracture risk increases with age, whilst low spine BMD incidence is stable.

Reference; 1 D Black, Osteoporosis Int. 2001 12 519--28

Disclosures: L. Dolan, None.


Impact of Height Loss Due to Vertebral Fractures on Body Mass Index.K. S. Davison*1, K. Siminoski2, C. Chik*3, H. Jen*4, R. Warshawski*4, K. Lee*4. 1Medicine, McMaster/Laval Universities, Hamilton/Quebec City, ON, Canada, 2Radiology and Medicine, University of Alberta, Edmonton, AB, Canada, 3Medicine, University of Alberta, Edmonton, AB, Canada, 4Radiology, University of Alberta, Edmonton, AB, Canada.

Body mass index (BMI; weight in kg divided by the square of height in m) is frequently used to define clinical obesity. Vertebral fracture, most often due to osteoporosis, can result in height loss and a falsely elevated BMI. The objective of this investigation was to determine the effect of vertebral fractures on BMI (1) theoretically, by modeling, and (2) empirically, by assessing height loss attributable to vertebral fracture to determine the actual effect on BMI. Empirical data was derived from the study of 458 women (mean age 56 years) referred for osteoporosis assessment. Height loss was calculated as the difference between recalled tallest height and current height (by stadiometer). Lateral radiographs of the thoracic and lumbar spine were assessed by quantitative morphometry (T4--L4) with fractures defined as reduction in vertebral height ratio of >20%. The amount of height loss attributable to vertebral fractures was determine by linear regression modelling.

The theoretical relationship between percent change in height and percent change in BMI can be described by: ΔBMI% = {(1/[1-(0.01 x Δheight%)]squared)-1} x 100. While the relationship is curved, height loss up to 5% leads to a percentage increase in BMI that is about 2.1 times the percentage loss of height.

Among the study population, 34.3% had one or more vertebral fractures. After correction for height loss due to aging, each fracture was associated with a height loss of 1.0 cm (r = 0.56, p<0.001). Thoracic vertebral fractures led to height loss of 0.76 cm (r = 0.33, p<0.001; corrected for age and presence of lumbar fracture), while each lumbar vertebral fracture produced height loss of 1.5 cm (r = 0.36, p<0.001; corrected for age and presence of thoracic fracture). When the calculated percent height loss per fracture was applied to the average height of the non-fractured patients (164.3 cm), the theoretical percent change in BMI ranged from 1.2% for one fracture to 12.1% for nine fractures. The increase in BMI for a patient with two vertebral fractures would be 2.5% on average, 1.9% if both fractures were thoracic, or 3.7% if both fractures were in the lumbar spine.

We conclude that (1) height loss due to fractures can significantly alter apparent BMI, (2) that the average height loss per fracture is 1.0 cm; per thoracic fracture is 0.76 cm; and per lumbar fracture is 1.5 cm, and (3) patients with known vertebral fractures should have their heights corrected for loss due to fractures, and corrected height should be used to calculate a corrected BMI.

Disclosures: K.S. Davison, None.


Evaluation of Mechanical Competence of Radius in Women with Colles Fracture.P. Pludowski*1, R. Bienkowska*2, R. S. Lorenc1. 1Dept. of Biochemistry and Exp. Medicine, Children's Memorial Health Institute, Warsaw, Poland, 2Szpital Kolejowy, Warsaw, Poland.

Distal radius was evaluated with use of pQCT concerning bone mineral content, density, geometry and bone strength aimed to discriminate between fractured and non-fractured individuals. The study population comprised 76 wrist fractured female aged 46--70 years and 144 healthy female the same ages. Using logistic regressions, we tested how well the data approached the probability of a given individual to belong to fractured or non-fractured group. Odds ratios (OR) were derived from the data corresponding to the inflexion points of logistic curves. ROC was performed to calculate AUC and “optimal cut off” values, leading to evaluate the most efficient discriminator of fractured subjects. The results of logistic regressions (Chi-square, p values, OR) and ROC (AUC, cut off values) are presented in decreasing order of discriminatory power in Table 1 and as follows: (pQCT measured; Chi-square; p; OR; AUC%; cut off;) (TotalBMD; 9.8; <0.01; 1.72; 63.2; 347.3; CorticalBMD; 4.5; <0.05; 1.87; 55.2; 571.4; Trabecular area; 1.8; ns; -; 52.9; 129.7; Periosteal circumference; 0.3; ns; -; 46.5; 62.3; Total area; 0.3; ns; -; 46.4; 296.0). As shown, indicators of mineral content ranked higher than respective volumetric bone densities. Within each type of bone (total, trab, cort) the assessments of bone mass provided higher chi-squares indicating better-adjusted logistic curves, higher OR and larger AUC than volumetric density. The geometric indicators, except Cortical area, did not discriminate fractured subjects. The SSI-X, SSI-P ranked higher than mass and density parameters itself (except Trabecular), indicating that compilation of structural distribution (CSMI) and vBMD into single number, as SSI, provided valuable estimation of bone strength. The results of logistic regressions and ROC pointed out, that mass and density indicators of Trabecular compartment were the most efficient discriminators between fractured and non-fractured women. It seems to be surprising, because long bone fractures are caused by stresses on the periosteal surface. However, the large proportion of trabecular bone in distal radius probably influences the mechanical competence of bone by supporting the cortical shell. It can be concluded that pQCT offer significant informations of mechanical status of human radius. 

Table  . 1
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Disclosures: P. Pludowski, None.


Nationwide Hip Fracture Survey in Japan.H. Hagino1, T. Nakamura2, K. Sakamoto*3. 1Rehabilitation Division, Tottori University, Yonago, Japan, 2Department of Orthopedic Surgery, University of Occupational and Environmental Health, Kitakyushu, Japan, 3Department of Orthopedic Surgery, Showa University, Tokyo, Japan.

Purpose: To elucidate the current status of hip fracture incidence and treatment in Japan, Japanese Orthopedic Association (JOA) conducted a tally of hip fractures in JOA related hospitals in Japan.

Patients and Methods: A tally of all hip fractures in patients from 1998 to 2001 was conducted in JOA-authorized hospitals and in Japanese Clinical Orthopedic Association (JCOA) hospitals. There were 2270, 2264, 2312, and 2291 JOA-authorized hospitals and 1529, 1430, 1512, and 1493 JCOA hospitals in 1998, 1999, 2000, and 2001, respectively. Registration forms were sent to these hospitals by mail and registration was performed by doctors at each hospital according to their hospital records.

Results: Response rates were 48.4%, 55.1%, 47.0%, and 53.0% in 1998, 1999, 2000, and 2001, respectively. The survey found a total of 155,216 new hip fractures aged 35 year-old and over during the survey years. The number of women patients was 3.7 times that of men. Dividing by fracture types, 86,558 were trochanteric fractures, 66,880 neck fractures, and 1,778 were unclassified fractures during the three-year survey period. Age- and gender- specific number of patients increased with age and peaked at the age 80--84, then leveled off after 85 years of age. The number of patients with femoral neck fractures exceeded that with trochanteric fractures before 75 years of age and these figures became inverted thereafter. The number of patients per month was highest in January (15,027), and lowest in June (11,004) during the four observation years, showing a significant monthly variation. More left hips were fractured than right in all survey years; however, the difference was not statistically significant. The most common cause of hip fractures was a simple fall, and 73% sustained fractures in-doors. In patients aged 90 years old and over, simple fall was the cause in more than 80%. Ninety-three percent in patients with femoral neck fractures and 94% in patients with trochanteric fractures were treated surgically. About 3/4 were treated with hemi-arthroplasty among patients with femoral neck fractures. The mean hospitalization period was 55.7 days during the observation period.

Conclusion: The data from a survey covering more than 40% of all hip fractures occurring during 1998 to 2001 in Japan showed patient distribution by age and fracture type, cause of fracture, selection of the treatment, and duration of hospitalization.

Disclosures: H. Hagino, None.


Prevalence of Vertebral Fractures in Mexico: A Population-Based Study.P. Clark1, M. Deleze2, F. Cons-Molina*3, J. Salmeron*4, L. Palermo*5, S. R. Cummings5. A. The LAVOS Study Group*6, 1Clinical Epidemiology Unit, Centro Medico Nacional, IMSS-Faculty of Medicine UNAM, Mexico City, Mexico, 2Clinica de Osteoporosis, Puebla, Mexico, Mexico, 3Unidad de Diagnostico de Osteoporosis, Mexicali, Mexico, 4Epidemiology & Health Systems Unit, IMSS, Morelos, Mexico, 5SF Coordinating Center, San Francisco, CA, USA, 6Clinical Epidemiology Unit, Centro Medico Nacional, IMSS, Mexico City, Mexico.

The rate of vertebral fractures in Latin America has never been studied in population-based samples. We designed the Latin American Vertebral Osteoporosis Study (LAVOS) to determine the prevalence of vertebral fractures in women over 50 years in several countries. We report here preliminarily results from the Mexico survey. An age-stratified sample of 400 randomly selected women from Puebla Mexico was surveyed in a face-to-face interview. A questionnaire to get information on demographics, OP conventional risk factors, and some life styles were applied. BMD in two regions and lateral dorsal/lumbar X rays were obtain in all cases accordingly with international protocols to be able to have cross-national comparisons. Digital Morphometry was used to determine vertebral deformities by Eastell criterion. The overall prevalence of vertebral fractures was 17.5% and increased exponentially with age. Comparing to studies that used very similar methods and criteria, the rate in Mexican women is very similar to that in Caucasian women and higher then the prevalence found in African American and Chinese women. This first population-based study of radiographically confirmed vertebral fractures in a Latin American country that shows indicates that Mexican women have a risk of vertebral fracture that is similar to white US women and greater than the risk of Chinese and African-American women. Treatments to prevent vertebral fracture as important for Mexican as for US women. 

Table  .  
  1. *> 65 years

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Disclosures: P. Clark, None.


Irregularity of the Thoracolumbar Curvature Is a Sensitive and Specific Indicator of Structural Failure of the Spine.R. M. D. Zebaze*1, G. Maalouf*2, E. Seeman1. 1Endocrinology, Austin and Repatriation Medical Centre, University of Melbourne, Australia, 2Saint George University Hospital, Beyrouth, Lebanon.

The credibility of comparisons of vertebral fracture (fx) rates between sexes, races, locations, times and clinical trials depends on there being accurate, consistent and reproducible methods of quantifying spinal structural failure. Quantitative vertebral morphometry (QVM) is not an optimal option. It focuses on vertebral body (VB) shape but this varies unpredictably across individuals, sexes, races, and time. Moreover, ‘fracture’ defined by QVM as loss of VB height is difficult to distinguish from the necessity for there to be differences in anterior and posterior VB heights to form a thoracolumbar curvature (TLC): a biomechanical necessity, critical in providing ‘shock absorber’ function to maintain cephalic stability during bipedal gait. Resemblance and consistency of an individual's adjacent VB dimensions produces the regularity in the TLC. Rather than considering structure failure of vertebral elements separately as done in QVM, we quantified this regularity as a single continuous mathematical function expressing how well adjacent vertebrae fit a sector of a circle, a function sensitive to structural failure because of the resemblance of adjacent vertebrae, but insensitive to anatomical variation (often called a ‘fracture’ using QVM). We expressed the deviation from regularity of the TLC as a Spinal Curvature Irregularity index (SCII = 100 - x%). Spine BMD and VB heights were measured using DXA in 697 Lebanese women (20--87 yrs). Deformities (fx by QVM) were assessed by QVM. In pre-menopausal women, the SCII was independent of age, height and weight but correlated with BMD (r= - 0.22) and had a mean of 10% (range 4--15%). In postmenopausal women, the mean SCII was unchanged but the scatter increased (4--30%). The SCII correlated with height (r = - 0.23), BMD (r = - 0.18), and the number of fx by QVM (0.31--0.60) (all p lower than 0.001). SCII was better correlated with indicators of spinal fragility (age, height loss, BMD) than number of deformities. Only 0.8% of women had an SCII of > 17% (the value defining fx by SCII) before menopause, but this proportion increased exponentially to 15% after age 80 (r = 0.97). They had lower BMD (- 1.01 SD) and height (- 0.49 SD) and 3 to 9 times more fx by QVM than those with no fx by SCII (< 17%). Women with fx by SCII but no fx by QVM had lower BMD and height, whereas women with no fx by SCII but fx by QVM had normal BMD and height. The SCII (i) can be used as a continuous variable to monitor structural failure (ii) is sensitive and specific, and more so than QVM (iii) is easy to compute in an individual (iii) does not require a reference range and (iv) can be a reliable alternative to QVM.

Disclosures: R.M.D. Zebaze, None.


What Is a Vertebral Fracture?R. M. D. Zebaze*1, G. Maalouf*2, J. Wehbe*2, A. Nehme*3, N. Maalouf*4, E. Seeman1. 1Endocrinology, Austin and Repatriation Medical Centre, Melbourne, Australia, 2Saint George University Hospital, Lebanon, Lebanon, 3Centre Hospitalier Rangueil, Toulouse, France, 4University of Texas Southwestern Medical Center, Texas, TX, USA.

Credible inferences regarding the burden of vertebral fractures (VF) within and between sexes, races, placebo arms of clinical trials, countries and decades cannot be made until there is a globally accepted method of defining ‘fracture’. Differences in vertebral heights are essential for there to be a thoracolumbar curvature. These differences do not necessarily reflect structural failure but are used to define ‘fracture’ based on reductions in intra- or inter- vertebral body (VB) height ratios, by 15 or 20%, or 3--4 SD below the mean. As there is no gold standard to distinguish anatomical variation from fractures, group differences in the VF prevalence may reflect differences in methodology, not differences in fragility.

A quantitative definition of VF was developed based on (i) a new method of defining threshold values and (ii) the requirement for two abnormal height ratios which reduces the chance of false positives ten fold. BMD and vertebral heights were measured using dual X-ray absorptiometry (Lunar Expert-XL) in 697 Lebanese women (age 20--89 years). VF prevalence ranged from 7 to 70 percent using published cut-offs defining ‘fracture’. At 3 SD, the prevalence of deformities was 70--80% in older women (aged 50 and over) and women with lower BMD, but ∼65% in younger women (aged 20--49) and women with normal BMD; 4 SD or 15% cut offs produced similar high sensitivity and poor specificity. The 20% cut off produced a 40--50% prevalence in older women and women with low BMD, a 25--35% prevalence in young women and women with normal BMD. The 30% cut off produced a low prevalence which decreased with age. The new classification produced prevalence figures of 3.3% in younger and 14% in older women, 20, 10, and 7% in the lower, middle and upper BMD tertials respectively. ‘Deformities’ detected in younger women were not associated with height loss or low BMD. Subtracting the prevalence of deformities in young from older women resulted in a decrease the disparity from 10 to 2 fold between methods. Current morphometric criteria for VF produce prevalence figures which are unrelated to age or low BMD and are likely to capture anatomical variation rather than structural failure. A standardized definition of deformities is needed before credible comparison across epidemiological studies can be made.

Disclosures: R.M.D. Zebaze, None.


Diabetes Mellitus and the Risk of Non-Vertebral Fractures.L. A. Ahmed*1, R. M. Joakimsen2, G. K. Berntsen1, V. Fønnebø1. 1Institute of Community Medicine, University of Tromsø, Tromsø, Norway, 2Department of Internal Medicine, University Hospital of Tromsø, Tromsø, Norway.

The aim of this study was to assess the relation between diabetes mellitus and non-vertebral fractures. Methods: this is a population based study where we followed up 12 270 persons, who attended both the second (1979/80) and third (1986/87) surveys in the Tromsø Study, from 1988 to 1995 with respect to non-vertebral fractures. At baseline, 1988, the age range for women was between 29 to 58 years, and for men was between 29 to 63 years. Diabetes mellitus cases were defined by self-report, which could be validated by information in medical records. All non-vertebral fractures, which occurred in the follow up period, were registered by computerized search in radiographic archives in the sole provider of radiographic service in the area. This method was shown to identify 97% of all forearm fractures in the original cohort. Adjustment for possible confounding by age, body mass index (BMI) from the third survey, weight loss (between the two surveys), physical activity, smoking (previous and current), milk consumption and self-reported health status was performed. Results: we identified 72 cases of diabetes mellitus, and 935 non-vertebral fracture cases (491 women, 444 men). During the follow up period, 6 out of 22 diabetic women and 3 out of 50 diabetic men had fractures. The crude relative risk (RR) of fracture was 4.7 (95% confidence interval (CI) 2.1--10.4) and 0.9 (95% CI 0.3--2.9) for women and men respectively. Among women, age-adjusted relative risk of fracture was 5.7 (95% CI 1.8--17.8) in type I diabetics, and 1.2 (95% CI 0.2--8.7) in type II diabetics compared to non-diabetics. There was a significant interaction between diabetes and Body mass index (BMI) on fracture risk. Women with BMI less than 25 had a relative risk of 14.3 (95% CI 5.9--34.5). The relative risk was not significant among women with BMI of 25 or more, (RR 1.0 (95% CI 0.1--7.3)) or among men. Adjustment for Age, BMI from the third survey and weight loss gave a relative risk of 10.6 (95% CI 3.9--28.7) and 1.1 (95% CI 0.2--7.7) for diabetic women with BMI less than 25 and more than 25 respectively. For men, the adjusted relative risk was 1.3 (95% CI 0.3--5.1) for those with BMI less than 25, and 0.6 (95% CI 0.1--4.5) for those with BMI of 25 or more. Conclusion: women with type I diabetes and women with diabetes and a BMI less than 25 had increased the risk of non-vertebral fractures. Male diabetics and female diabetics with BMI of 25 or more had no such increase in fracture risk.

Disclosures: L.A. Ahmed, None.


Prevalence of Vertebral Fractures in Postmenopausal Glucocorticoid-treated Women: a National Study.A. Angeli1, G. Capelli*2, S. Giannini3, G. Guglielmi4, M. Bevilacqua*5, L. Moro6, L. Sinigaglia*7, D. de Feo*8, A. Giustina*9. 1Internal Medicine, University of Torino, Orbassano, Italy, 2Public Health, University of Cassino, Frosinone, Italy, 3Internal Medicine, University of Padova, Padova, Italy, 4Radiology, Scientific Institute Hospital “CSS”, Foggia, Italy, 5Endocrinology, Sacco Hospital, Milano, Italy, 6Bbcm, University of Trieste, Trieste, Italy, 7Rheumatology, Gaetano Pini, Milano, Italy, 8Procter & Gamble, Rome, Italy, 9Internal Medicine, Brescia University, Brescia, Italy.

Glucocorticoids (GCs) administration in post-menopausal women accelerates bone loss and increases fracture risk. GIOVE (Glucocorticoid-Induced Osteoporosis Vertebral Evaluation) is a national study aimed at measuring the prevalence of vertebral fractures (VFs) in a sample of postmenopausal women treated with GCs. Forty-three University and Hospital centers in Italy participated in the study. A total of 979 ambulatory female subjects (aged 45+ yrs, 1+ yrs in menopause, treated with systemic GCs for at least 6 months, cumulative dosage of GCs > 1.350 mg of prednisone or equivalent) affected by Reumathoid Arthritis (RA), Lupus (SLE), Vasculitis/Connectivitis, Polymyalgia rheumatica (PMR), Asthma/COPD were enrolled. All subjects had undergone an X-ray evaluation of thoracic and lumbar spine. Films were centrally digitized: T4--L4 vertebral heights ratio were measured using a morphometry software (Spine-X Analyzer; CAM Diagnostics, Milan, Italy). The VFs were defined as: mild, moderate or severe, based on a height ratio decrease of 20%-25%, 25%-35% and more than 35% respectively. Currently data on 485 of the women are under revision mainly due to low-quality radiographs. The table below shows the age-adjusted prevalence VFs rates (%) calculated in the women grouped by each major disease. 

Table  .  
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About 38% of patients had one VF and about 14% had more than one VF. PMR, Vasculitis/ Connectivitis and Asthma/COPD affected patients showed the highest rates of VFs prevalence. Even if PMR patients were generally older and SLE patients were younger than others, age adjustment did not significantly change the pattern. This study confirms that the use of GCs is associated with a higher risk of developing single or multiple VFs.

Disclosures: S. Giannini, None.


The Influence of Socioeconomic Deprivation on the Incidence of Fractures & on the Uptake of Post-fracture Osteoporosis Assessment by a Fracture Liaison Service.A. R. McLellan1, M. Fraser*1, S. J. Gallacher2, I. Baxter*3, C. Morrison*3. 1Medicine & Therapeutics, Western Infirmary, Glasgow, United Kingdom, 2Medicine, Southern General Hospital, Glasgow, United Kingdom, 3Greater Glasgow NHS Board, Glasgow, United Kingdom.

Socioeconomic deprivation (SED)has implications for disease and health-care access; whether this is true for fractures (fx) is unclear. SED adversely influences access to osteoporosis assessment when fx case-finding involves Primary Care (Gallacher JBMR 2002;17supp1, absF269). A prospective audit of incident fx was undertaken to assess whether SED influences fx incidence and access to assessment by the North Glasgow (NG) Fracture Liaison Service (FLS) that covers a population of 500K. The FLS offers osteoporosis assessment and treatment for the 2y prevention of fx to all >50yr with any new fx (non-RTA); Case-finding of inpatients & outpatients with fx, does not require participation of Primary Care, and is achieved by dedicated nurse practitioners. To investigate the influences of SED, all 3190 incident Fx cases presenting to NGFLS in 2002 were reviewed; deprivation category (depcat)(Carstairs 1991) was allocated to each patient based on their postal code of residence. Depcat is a 7 point score; depcat 1 being most affluent & depcat 7 least affluent. Fx incidence at all sites expressed as age /sex adjusted incidence rate (ASAIR) per 10K by depcat from 1 to 7 was 176.2, 134.8, 93.8, 146.9, 209.6, 170.7 & 190.5 resp. Data for the 4 commonest fx sites are shown in table 1; those for hand/foot(n=364) and all other fx(n=395)are not shown, but trends are similar to ankle and wrist fx resp. Depcats 2 or 3 have the lowest ASAIRs for fx at any site. Depcat 7 had the highest ASAIR for fx of hip and humerus, while depcat 1 had the highest incidence of fx of ankle and hand/foot. 

Table  . ASAIR for Fx per 10K by Depcat
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The FLS offers assessment to all fx cases >50yr with fx irrespective of depcat; however, uptake is inversely related to depcat; for depcat 1 to 7, 18%,19%,28%,20%,22%,21% and 31% resp. either decline assessment or do not attend. SED contributes to fx risk at hip and humerus. The FLS model achieves its aim of offering post-fx assessment to all fx, regardless of depcat, but uptake of that offer is lower with SED. For optimal 2y prevention of fx, a FLS must address the disadvantages of SED that contribute to fx risk and that are also barriers to accepting opportunities to reduce future fx risk.

Disclosures: A.R. McLellan, None.


Risk Factors that Predict New Non-Vertebral Fracture in Postmenopausal Women: The Canadian Multicentre Osteoporosis Study (CaMos).A. Papaioannou1, G. Ioannidis1, J. P. Brown2, C. Berger*3, D. A. Hanley4, J. C. Prior5, L. Joseph*3, W. P. Olszynski6, T. M. Murray7, A. Tenenhouse3, T. Anastassiades*8, S. Kirkland*9, C. Joyce*10, S. Poliquin*3, N. Kreiger*7, K. Siminoski11, J. D. Adachi1. 1McMaster University, Hamilton, ON, Canada, 2Laval University, Ste-Foy, PQ, Canada, 3McGill University, Montreal, PQ, Canada, 4University of Calgary, Calgary, AB, Canada, 5University of British Columbia, Vancouver, BC, Canada, 6University of Saskatchewan, Saskatoon, SK, Canada, 7University of Toronto, Toronto, ON, Canada, 8Queen's University, Kingston, ON, Canada, 9Dalhousie University, Halifax, NS, Canada, 10Memorial University, St. John's, NF, Canada, 11University of Alberta, Edmonton, AB, Canada.

The purpose of this study was to determine the association among various potential risk factors and new non-vertebral fractures in a three-year prospective cohort study of menopausal women who were enrolled in CaMos. CaMos is a nation-wide, randomly selected sample of the Canadian population with extensive osteoporosis related data. Participants were classified into three groups according to their new non-vertebral fracture status: those without a new fracture (No-Fx, n=4829), those with a new non-vertebral fracture at the wrist, hip, humerus, pelvis or ribs (Main-Fx, n=163) and those with a new non-vertebral fracture at any skeletal location (All-Fx, n=280) during the study period. Follow-up was calculated as time from baseline examination to the date of the new fracture or three years. We preformed Cox multivariate survival analysis on several potentially important risk factors. Final model selection was conducted using the Bayesian Information Criterion technique. Relative risk and 95% confidence intervals (CI) were calculated. Results indicated that higher quality of life (measured by the SF-36 physical component summary scale) and lumbar spine and femoral neck BMD was associated with a decrease risk of sustaining a new Main-Fx 0.969 (CI: 0.952, 0.986), 0.162 (CI: 0.034, 0.766) and 0.065 (CI: 0.007, 0.605); and a new All-Fx 0.967 (CI: 0.954, 0.980), 0.161 (CI: 0.046, 0.566) and 0.089 (CI: 0.016, 0.504). In addition, a previous minimal trauma forearm fracture after the age of 50 years (2.078; CI: 1.202, 3.593), and weight loss (1.020; CI: 1.002, 1.037) were associated with an increase risk of sustaining a new Main-Fx. Inflammatory bowel disease (1.683; CI: 1.084, 2.613), kidney disease (3.084; CI: 1.560, 6.099), height (1.039; CI: 1.015, 1.064) and diuretic use (0.550; CI: 0.304, 0.994) was associated with All-Fx. In conclusion, several important historical and medical factors are related with non-vertebral fractures. These should be assessed in osteoporosis management.

Disclosures: A. Papaioannou, None.


Participant Characteristics that Predict New Clinically Recognized Vertebral Fracture in Postmenopausal Women: The Canadian Multicentre Osteoporosis Study (CaMos).W. P. Olszynski1, G. Ioannidis2, C. Berger*3, A. Papaioannou2, J. C. Prior4, L. Joseph*3, D. A. Hanley5, T. M. Murray6, J. P. Brown7, A. Tenenhouse3, T. Anastassiades*8, S. Kirkland*9, C. Joyce*10, S. Poliquin*3, N. Kreiger*6, K. Siminoski11, J. D. Adachi2. 1University of Saskatchewan, Saskatoon, SK, Canada, 2McMaster University, Hamilton, ON, Canada, 3McGill University, Montreal, PQ, Canada, 4University of British Columbia, Vancouver, BC, Canada, 5University of Calgary, Calgary, AB, Canada, 6University of Toronto, Toronto, ON, Canada, 7Laval University, Ste-Foy, PQ, Canada, 8Queen's University, Kingston, ON, Canada, 9Dalhousie University, Halifax, NS, Canada, 10Memorial University, St. John's, NF, Canada, 11University of Alberta, Edmonton, AB, Canada.

Utilizing participants from CaMos, a nation-wide, random sample of the population, we performed a three-year prospective cohort study in community dwelling menopausal women to examine the relationship among various participant characteristics and clinically recognized new vertebral fractures as reported on the yearly follow-up questionnaire. Participants were classified into two groups according to their new fracture status: those without new fractures during the study period (n=4829), and those with a new minimal trauma vertebral fracture (n=34). Follow-up was calculated as the time from baseline examination to the date of the new fracture or three years. Characteristics examined in the Cox multi-variate survival analysis included age, prevalent vertebral fracture status, change in height, current height, change in weight, current weight, body mass index, SF-36 physical component summary score, and baseline lumbar spine and femoral neck bone mineral density (BMD). Final model selection was conducted using the Bayesian Information Criterion technique. Relative risks and 95% confidence intervals (CI) were calculated. The mean (standard deviation) age of the participants was 66 (10) years, and 74 (10) years for those without new fractures and for those with a new vertebral fracture. The relative risk of sustaining a new vertebral fracture was negatively associated with the SF-36 physical component summary score (0.962; CI: 0.930, 0.996) and femoral neck BMD (0.0002; CI: 0.000, 0.010). A prevalent vertebral deformity (2.357; CI: 0.920, 6.039) and a loss of height (1.089; CI: 0.993, 1.194) tended to be positively associated with new fracture status but further evidence will need to be collected to verify these findings. Women with lower physical quality of life measures and femoral neck BMD were at higher risk for sustaining a new vertebral fracture. The identification of postmenopausal women at risk is important given that proven therapies are available.

Disclosures: W.P. Olszynski, None.


Most Fractures are Incidental with No Lasting Medical or Social Consequences.A. Hoiseth. Sentrum Roentgeninstitutt, 0155 Oslo, Norway.

To assess the seriousness of fractures, historical fracture data was obtained from 3997 women, consecutively having routine BMD-measurements. Lasting consequences of the fractures, namely pain, social and/or medical consequences were recorded. Mean age of the subjects was 63 years (sd 12). 1780 had had a fracture; 789 at least one fore-arm fracture, 271 at least one vertebral fracture, 179 at least one femoral fracture and 888 at least one “other” type of fracture. 944 reported having had one fracture, 445 two fractures, 188 three fractures and 203 at least four fractures. Only 331 (19%) reported lasting consequences of the fractures; 13% of those with one fracture, 21% with two fractures, 23% with three fractures and 39% with at least four fractures. Further, only 7% with one fore-arm fracture only had som lasting sequel, this increased to respectively 19%, 9%& and 42% in those with one, two and three additional fractures. For those with one vertebral fracture and additional fractures the figures were respectively 17%, 38%, 40% and 43%. But, for those with femoral fractures the values were respectively 43%, 41%, 33%, 53% and for “other” types of fractures, 8%, 21%, 20% and 40%. The majority of fractures seemed to have no lasting or permanent consequences, even in those with just one vertebral fracture the number of patients reporting lasting consequences was small. However, having had at least a femoral fracture or multiple fractures left nearly half of the women with some lasting sequel. This corresponded to the group of women with low BMD in the femur and spine. A rational approach to osteoporosis intervention seems to be BMD assessments to find those at risk of having at least one femoral, more than one vertebral or multiple other fractures. Single, or event two non-vertebral or non-femoral fractures do not seem to be indicate a serious condition and should probably not be used as an indication for medical intervention without a BMD based assessment of the risk of additinal fractures.

Disclosures: A. Hoiseth, None.


Prospective Study of Health-Related Quality of Life in Women with Hip Fractures.A. Cranney, D. Coyle*, W. Hopman*, V. Hum*, P. Tugwell*. Medicine, Queen's University, Kingston, ON, Canada.

To prospectively assess Health-related quality of life (HRQoL) in women with recent hip fractures. Forty women over age 50 were identified within one month of their hip fracture. Study interviews were conducted at baseline, 3 and 9 months. Utility values were elicited using direct methods; with the Feeling thermometer (FT), (Current health and 5 osteoporotic health states) and the Standard gamble (SG). Indirect elicitation methods included use of the HUI II. Health status was measured using the SF-36. Baseline measurements were taken in a group of 40 controls without a history of hip fracture. 35 women with hip fractures completed 3 visits; 3 women died, 1 woman completed the initial visit and 1 dropped out after second visit. The mean age of controls and hip fracture patients was 72 and 80 years respectively. Twenty-five percent of hip fracture patients were on osteoporosis medications. The SF-36 scores were lower for the hip fracture patients as compared to controls and age and sex-matched normative Canadian data. Utility values were significantly lower in hip fracture patients when elicited using the HUI II and FT, but not with the SG. There were progressive improvements in the Physical Component Scale, Role Physical, Body Pain and Physical Functioning domains of the SF-36 over the 9-month period. There were similar improvements in the HUI II, and FT, but the SG values did not change over the 9 month period. Health states that included side effects were rated lower than a similar health state without side effects. There was a significant correlation between the FT and HUI results. There was also a significant correlation between the SF-36 scores and the HUI. The standard gamble may not be sensitive to changes in HRQoL in women with hip fractures. Women with hip fractures have lower HRQoL in comparison to non-fracture controls, but show good improvement over time. 

Table  .  
  1. ** significant difference from baseline value P<0.001, * P=0.01

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Disclosures: A. Cranney, None.


Health Related Quality of Life After Osteoporotic Fractures.I. Hallberg*1, A. Rosenqvist*2, L. Kartous*2, O. Löfman*3, O. Wahlström*4, G. Toss1. 1Endocrinology, Medicine and Care, Health Faculty, Linköping, Sweden, 2Geriatrics, Jönköping, Sweden, 3Centre for Health Sciences, Public Health, Linköping, Sweden, 4Orthopedics, Linköping, Sweden.

In order to compare the impact on Health Related Quality of Life (HRQOL) of different types of fractures we examined consecutive women 55--75 years old with a new low-energy fracture of forearm (n=171), proximal humerus (n=37), vertebra (n=55) or hip (n=40). Anti-osteoporotic treatment was given according to local guide-lines. Calcium 500 mg, vitamin D 10 mikrogram and cyclic etidronate were the most common treatment in all fracture groups. HRQOL was evaluated by the SF-36 questionnaire and compared with a large age matched local populations sample. A baseline examination was performed 82 days (59 --103, IQR) after fracture, and a second examination two years later.

At baseline women with forearm fracture had reduced scores for role function (-physical causes), bodily pain, general health and role function (-emotional cause), but not for physical function, vitality or mental health. Two years after forearm fractures all the eight scores were normal. Women with fracture of the proximal humerus showed a similar pattern as the forearm fracture group.

At baseline after hip fracture scores for physical function, role function (-physical cause), bodily pain, vitality, social function and role function (-emotional cause) were lower than after forearm or humerus fractures. After vertebral fracture at baseline scores for physical function and role function (-physical cause) were slightly less reduced than after hip fracture while bodily pain, general health and vitality were more severely reduced. Two years after vertebral fracture improvements were seen for some scores, but all scores were still significantly below normal and lower than after hip fracture. Patients with one or more fracture before the index fracture had lower HRQOL than those with no previous fracture. Patients with osteoporosis had lower scores than those with normal BMD.

In conclusion complete normalization of HRQOL as measured by SF-36 was seen two years after forearm and humerus fractures, while vertebral fractures had a pronounced impact on HRQOL even two years after the fracture, even more than hip fractures. HRQOL should be taken into account when evaluating new treatment methods for fractures and osteoporosis.

Disclosures: G. Toss, None.


Relation Between Previous Fracture and Loss of Teeth Independent of Bone Mineral Density: A Population Study of 566 - 70 Year Old Women, The Nordos Study.T. Österberg*1, U. H. Lerner2, O. Johnell3, D. Mellström1. 1University of Gothenburg, Department of Geriatrics, Gothenburg, Sweden, 2University of Umeå, Department of Oral Cell Biology, Umeå, Sweden, 3University of Lund-Malmö, Department of Orthopedic Surgery, Malmö, Sweden.

Previous studies have shown a correlation between low bone mineral density (BMD) and loss of teeth. Other studies have indicated a relation between previous fracture and prospective fracture, independent of BMD. The aim of this study was to examine the relation between previous fracture and loss of teeth. The question is if loss of teeth can be an indicator of osteoporosis and fractures?

A population based sample of 566 - 70 year old women were examined with a health interview, including questions about dental status. Bone densitometry was performed using a Hologic 4500A.

Result: In the sample, 12% had no own teeth and 25% reported previous fracture(s). Tooth-lessness was related to increased risk for previous fracture; odds ratio 1.75 (1.10--2.78). Loss of teeth was not related to BMD in spine or hip, but to BMD in whole body (p < 0.01) and manifest osteoporosis; odds ratio 2.07 (1.13--3.78). Women with a normal BMD according to WHO had a lower risk for loss of teeth compared to women with osteoporosis or osteopenia. Loss of teeth was more prevalent in shorter and smoking women. A logistic regression model showed that loss of teeth was an independent predictor for previous fracture; odds ratio 1.25 (1.04--1.50). Other significant predictors for previous fractures in the model were BMD in hip, body height and BMI.

Conclusion: there is a relation between loss of teeth and previous fracture independent of bone mineral density.

Disclosures: U.H. Lerner, None.


Risk Factors Related with Quality of Life (QOL) in Postmenopausal Women With Prevalent Vertebral Fractures: The Canadian Database of Osteoporosis and Osteopenia (CANDOO).D. A. Hanley1, G. Ioannidis2, R. J. Josse3, T. M. Murray3, J. P. Brown4, R. J. Sebaldt*2, C. H. Goldsmith*2, A. Papaioannou2, W. P. Olszynski5, A. Petrie*2, K. S. Davison*2, J. D. Adachi2. 1University of Calgary, Calgary, AB, Canada, 2McMaster University, Hamilton, ON, Canada, 3University of Toronto, Toronto, ON, Canada, 4Laval University, Ste-Foy, PQ, Canada, 5University of Saskatchewan, Saskatoon, SK, Canada.

Several risk factors may be related to QOL in patients who have sustained vertebral fractures. Thus, the purpose of this cross-sectional study was to examine a broad variety of general health risk factors associated with QOL in postmenopausal women with vertebral fractures who were registered in CANDOO. CANDOO is a prospective, observational database designed to longitudinally capture a standardized and comprehensive set of clinical information that includes data regarding demographics, medications, illnesses, fracture history, medical and family history, dietary and lifestyle factors, bone mineral density results and laboratory investigations. The mini-Osteoporosis Quality of Life Questionnaire was used to measure QOL. The instrument is self-administered and consists of 10 items (total score) subdivided into five domains (symptoms, physical functioning, emotional functioning, activities of daily living, and leisure). Higher scores indicate improving QOL. A total of 1129 women with a mean (standard deviation) age of 67.2 (11.9) years, height 155.4 (12.5) cm, and weight 64.7 (17.6) kg were evaluated. These women had 2.2 (1.6) vertebral fractures (confirmed by x-ray or medical report). Multivariable linear regression analysis was conducted to examine the relationship between potential risk factors and QOL (total score). Variable selection for the final model was determined using Mallows C(P) statistic. Regression coefficient estimates and 95% confidence intervals (CI) were calculated. Adjusted results indicated that previous surgeries of the hip or spine (-0.94; CI: -1.47, -0.41) and prior cardiovascular diseases (-0.74; CI: -1.15, -0.33) were associated with decreased QOL. The number of hours spent exercising per week (0.08; 0.03, 0.13), those working full time (0.41; CI: 0.04, 0.77), and those with a longer duration between their last non-vertebral fracture and baseline assessment (0.01; CI: 0.00, 0.02) were associated with increased QOL. In conclusion, improving QOL is an outcome of primary importance. In osteoporotic women with vertebral fractures, a comprehensive patient evaluation is necessary to accurately assess QOL.

Disclosures: D.A. Hanley, None.


The Human Cost of Fractures in Community Dwelling Older Adults.A. Papaioannou, M. Bedard*, A. Uppaluri*, G. Ioannidis, J. D. Adachi. Medicine, McMaster University, Hamilton, ON, Canada.

The purpose of our study was to examine the potential impact of fractures on pain, functional status and quality of life in the elderly from the epidemiological population based Canadian Study on Health and Aging (CSHA-2).

Data were obtained from the second wave of the CSHA study on dementia. The population included 5393 survivors from the first wave between 1996--97. Cognitive status was determined using the expanded version of Mini-Mental State Examination (3MS). Participants were interviewed regarding their living arrangements, need for supports around the house and transportation, and number of fractures in the year prior to the interview. Fractured cases (n=262) were compared to controls matched on age, gender, cognition, vision problems, living arrangements, use of supports, help with transportation and help around the house. Cases and controls were compared based on activities of daily living (ADL) and pain interference. The McNemar test for dichotomous data was used and continuous data were examined using paired t-tests or analysis of variance.

Mean age (SD) for cases and controls was 81 years (6.0), and 76% of the cohort was female. In the matched sample of 262 cases, a total of 294 fractures were reported for the year prior to interview. Predominant fracture types included: hip (17.3%), wrist (14.6%), rib (9.9%) and ankle (8.5%). Unmatched cases in the sample had 52 fractures and more likely to be older, more cognitively impaired with vision problems. 3MS scores were 86.74 (10.8) for cases and 87.01 (9.98) for controls. Dementia was determined if 3MS scores < 78. Higher dependence was reported for instrumental (IADL) such as shopping (p=0.001) and housework (p=0.007) among the cases as compared to the controls. Individuals with fractures also demonstrated higher dependence in basic activities of daily living (ADL) such as bathing (p=0.001), dressing/undressing (p=0.007) and grooming (p=0.008). The presence of pain was noted in 47% cases and 41% of controls. A higher proportion of cases (31%) reported moderate to severe pain as compared to controls (21%) (p=0.032). Self-reported pain interference measures showed a severe effect on mood in cases as compared to controls (p=0.031). Individuals with fractures have increased pain that interferes with their mood. In addition, they have difficulties with ADL and IADL that persist a year post fracture.

Disclosures: A. Papaioannou, None.


Calcium, Multivitamin and Osteoporosis Medication use in Women and Men with Recent Fractures.A. Pro-Risquez*1, S. S. Harris2, E. Ross*3, S. Rudicel*3, B. Barnewolt*3, B. Dawson-Hughes4. 1Brookline Associates of Internal Medicine, Brighton, MA, USA, 2New England Research Institutes, Watertown, MA, USA, 3Tufts-New England Medical Center, Boston, MA, USA, 4Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA.

Fractures are associated with a substantial morbidity and mortality and they predict future fractures. For this reason, evaluation and treatment with calcium (Ca), vitamin D (vit D) and prescription medications is important in fracture patients. This study was conducted to assess and compare Ca and vit D intake and bone medication use at the time of an acute fracture and 6 and 12 mo later. We studied 106 patients, 69 female and 37 male, mean age 66.7±10.3 yrs. Medical history and diet questionnaires were administered at enrollment (in an urban hospital) and again 6 and 12 mo later (by telephone). Of 86 patients who could be contacted 6 mo after their fracture, 36.2% of the women and 7.4% of the men had recently discussed osteoporosis with their primary care doctor. At 6 months, 24.2% of the women and 3.6% of the men were taking bone medications (compared with 27.8% and 3.6% before the fracture, ns). At 6 mo, 52.6% of the women and 10.7% of the men indicated that their doctor had recently recommended Ca or vit D. Overall, Ca supplement use rose from 53.4% at baseline to 74.1% at 6 mo (P= 0.004) in the women and from 14.8% to 17.9% in the men (ns). Among the women who had recently been advised by their primary care doctor to use Ca or vit D, supplement use increased from 63.3% to 90.0% (P= 0.021) and dairy food intake increased from 1.5 +/− 1.1 to 2.4 +/− 1.9 servings/d (P= 0.016). Only 3 men received this advice and 2 of them heeded it. Among women and men not receiving this advice, there was no significant increase in calcium supplement use or dairy food intake. There was no increase in multivitamin use after the fracture in the women or the men. Of the 76 patients who could be contacted 12 mo after the fracture, prescription medication and supplement use was not different from use at 6 mo. Of note, 7.9% had sustained another fracture during that year. In conclusion, the occurrence of a fracture did not increase likelihood of pharmacologic treatment for osteoporosis and osteoporosis was rarely discussed with men with fractures. After their fractures, women did increase their intake of calcium supplements and dairy foods particularly when it was recommended by their doctor. This suggests that the primary care physician is well positioned to bring about much needed change in the quality of care of fracture patients.

Disclosures: A. Pro-Risquez, None.


Prevalence and Identification of Vertebral Fractures: Comparison of Visual Inspection, Digital Computerized Morphometry and Visual Semiquantitative Assessment.G. Di Fede1, N. Napoli1, G. Guglielmi2, S. Bucchieri*1, C. Sferrazza*1, R. Giorgino3, E. Carmina*1, G. Rini1. 1University of Palermo, Palermo, Italy, 2Scientific Insitute Hospital “CSS”, San Giovanni Rotondo, Italy, 3Novartis Pharma, Origgio, Italy.

The aim of this study is to determine how many vertebral fractures elude X-ray reports and to estimate the accuracy of a digital morphometry system used for X-ray analysis of vertebral body heights. We studied 233 postmenopausal women (aged 63.9± 0.5) who underwent clinical observation in a Bone Metabolic Diseases workut setting. A lateral and posterior-anterior spine radiograph of both thoracic and lumbar vertebrae was performed in each subject. Reports from inspection of radiographs showed the presence of at least 1 vertebral fracture in 12% subjects. We analyzed each T4--L4 film by a digital computerized morphometry [DCM] (Spine-X Analyzer, CAM Diagnostics, Milan, Italy), which calculates vertebral height ratios based on a standard 6-point identification method of the 3 vertebral heights. A 20% reduction of any vertebral height ratio was chosen as threshold for vertebral deformity definition. A 46.25% prevalence of fractures was observed. In addition, X-ray films were blindly analyzed by an expert radiologist using the Genant's semi-quantitative grading scheme for the assessment of vertebral fractures [VQA]. A 49.75% prevalence of fractures was observed in this phase. Both methods showed that most fractures occurred in the T6 and T9 interval. End-plate fractures were the most common (68.9% by DCM, 58.4%, by VQA). Good agreement (0.839) between VQA and DCM was found using κ score. Sensitivity (0.655) and specificity (0.957) of vertebral fractures identified by VQA, were calculated considering DCM method the gold standard.

The present study shows that in clinical practice vertebral fractures are often undiagnosed and not adequately reported by inspection of radiographs and that both DCM and VQA methods demonstrate the high prevalence of vertebral fractures and represent a valid tools in clinical research.

Disclosures: G. Di Fede, None.


Racial and Ethnic Disparities in Length of Stay and Discharge Disposition of Patients with Hip Fracture.S. L. Silverman1, D. Zingmond*2. 1Cedars-Sinai/UCLA, Beverly hills, CA, USA, 2UCLA, Los Angeles, CA, USA.

There is a need for understanding the costs of hip fracture in racial and ethnically diverse populations in the United States. The state of California has an ethnically diverse population and has maintained detailed data documenting hip fractures over the past two decades. The greatest cost of hip fracture is the direct cost of hip fracture hospitalization. Methods: We studied length of stay (LOS) and discharge disposition (DD), two surrogate measures of cost and utilization, associated with hospitalization following hip fracture requiring surgery in California in 2000 as an index year among four different racial/ethnic groups: Nonhispanic White (NHW); African-American (AfrA); Hispanic American (HispA) and Asian American (AsA). We used the California Office of Statewide Health Planning and Development annual hospital Patient Discharge Database (PDD), to identify all hospitalizations for acute hip fracture among individuals at non-Federal California general acute care hospitals from 1983 to 2000. Individual patient race/ethnicity, gender, and age are reported for each hospital discharge. We report length of stay (LOS) since charges are not available for those in Kaiser hospitals, representing care for one quarter of insured Californians. LOS is highly correlated with charges reported at non-Kaiser hospitals.

Results: Mean length of stay in 2000 was 10.2 days for NHW, 10.4 days for HispA, 12.4 days for AsA and 12.9 days for AfrA. In 2000 only 13.2% of NHW were reported to have routine discharge to home, while 18.6% of AsA, 22.7% of AfrA, and 24.4% of HispA were discharged to home. The numbers discharged to home with arranged home health care visits were similar among the four groups: 12.6% of NHW, 13.2% of AfrA, 14% of HispA, and 13.2% of HispA. 60% of NHW were discharged to skilled nursing facilities while 51.4% of AsA, 45% of HispA and 46% of AfrA. Less than 10% of all ethnic groups were discharged to inpatient rehab settings. There were no significant differences in incidence of death during hospitalization among the four groups (overall < 3%). Female patients predominated (72% NHW, 61% AfrA, 65% HispA, and 71% AsA). The median age range of NHW and AsA was somewhat greater (80 to 84 years old) than for AfrA and HispA (75 to 79 years old).

Conclusions: Racial and ethnic disparities do exist in California during hospitalization for hip fracture. Further studies are needed to determine which factors result in the variation in LOS and DD in California ethnic groups. Potential factors include age, prior residential status, comorbidities and socioeconomic status.

Disclosures: S.L. Silverman, Procter and Gamble 2.


Hip Fractures in Lebanese Patients: Determinants and Prognosis.H. Hreybe*1, M. Salamoun*1, M. Badra*2, N. Affeich*2, O. Baddoura*2, S. Boulos*2, R. Haidar*2, S. Lakkis*2, R. Musharrafieh*2, A. Nsouli*2, A. Taha*2, A. Tayim*2, G. El-Hajj Fuleihan1. 1Calcium Metabolism and Osteoporosis Program, American University of Beirut, Beirut, Lebanon, 2Orthopedics Department, American University of Beirut, Beirut, Lebanon.

Due to demographic explosion worldwide and especially in the Middle East, the human and economic toll of osteoporosis will increase substantially. Hip fractures are among the most costly of all osteoporotic fractures, but very little is known about the determinants of this fracture in our region.

We evaluated all hip fracture (hip fx) patients above 50 years of age admitted to our institution from 1992--2002. Patients with osteoarthritis admitted during the same period and with the same age for total hip replacement were used as controls (C). Information on gender, age, type of fracture, co-morbid conditions and medications use was obtained. Patients or their families were called on the average 5±2.5 years post fracture to assess mortality. Numbers are expressed as mean (± SD).

There were a total of 274 hip fx patients and 112 C. The mean age for hip fx patients was 71(4) years and 72(9) for C, 62% of hip fx patients were women compared with 67% of C. Fractures were 59% intertrochanteric, 34% femoral neck and 7% sub-trochanteric, with no gender differences. 13% of hip fx subjects suffered from neurological disorders compared with 4% of C (p=0.01), 6% had renal disorders compared to 1% of C (P=0.04) and 11% had a history of prior fracture compared with 2% of C, p<0.0001. The higher prevalence of neurological and renal disorders among hip fx patients was mostly due to the high prevalence of these disorders in male subjects with hip fx. Less than 10% of hip fx patients received any calcium, vitamin D or osteoporotic therapy, either on admission or upon discharge post- hip fx. Follow up information was available on 1/3 of all subjects. There was no significant difference in the clinical characteristics, including age, gender, co-morbid conditions (except for neurological diseases) between patients with follow-up and those lost to follow up. The mortality rate among hip fx patients was 47% compared to 18% among C, p<0.001. 70% of hip fx subjects who died did so within the first year post fracture, compared with 66% of C, p=0.02. Gender, but not fracture type, was a predictor of mortality post hip fracture. The mortality in male subjects was 73% compared with 28% in female subjects, p=0.0004.

Risk factors for hip fx in Lebanese subjects include gender, history of prior fracture, renal and neurological disorders. Few hip fx subjects received therapy for osteoporosis. Compared with western counterparts, Lebanese patients with hip fx are significantly younger, and experience a higher mortality rate.

Disclosures: G. El-Hajj Fuleihan, Merck Sharp and Dohme 2.


Predicting Previous Fracture With The Health Utilities Index (HUI) Mark II and III Systems in Both Women and Men: The Canadian Multicentre Osteoporosis Study (CaMos).G. Ioannidis1, J. P. Brown2, C. Berger*3, D. A. Hanley4, J. C. Prior5, L. Joseph*3, W. P. Olszynski6, L. Pickard*1, T. M. Murray7, A. Tenenhouse3, T. Anastassiades*8, W. Hopman*8, S. Kirkland*9, C. Joyce*10, A. Papaioannou1, A. Cranney8, O. Johnell11, E. A. Papadimitropoulos*12, J. D. Adachi1. 1McMaster University, Hamilton, ON, Canada, 2Laval University, Ste-Foy, PQ, Canada, 3McGill University, Montreal, PQ, Canada, 4University of Calgary, Calgary, AB, Canada, 5University of British Columbia, Vancouver, BC, Canada, 6University of Saskatchewan, Saskatoon, SK, Canada, 7University of Toronto, Toronto, ON, Canada, 8Queen's University, Kingston, ON, Canada, 9Dalhousie University, Halifax, NS, Canada, 10Memorial University, St. John's, NF, Canada, 11Malmo University Hospital, Malmo, Sweden, 12Eli Lilly and Company, Toronto, ON, Canada.

The purpose of the study was to examine the relationship between a prior minimal trauma fracture and health related quality of life (HRQL), as measured by the HUI Mark II & III. Participants were included from CaMos, a nation-wide, randomly selected sample of the Canadian population, and were classified into three groups according to their previous fracture history: those with any prior fracture (ANY), those with a prior main fracture at the wrist, hip, clinical spine, pelvis or ribs (MAIN), and those with only a prior wrist/forearm fracture (WRIST). A total of 1598, 760 and 558 women and 535, 210 and 134 men 50 years of age and older had a minimal trauma ANY, MAIN, and WRIST. HRQL was measured using the HUI Mark II & Mark III. The Mark II and III multi-attribute utility scales (global health) are both classified such that the score for 0=dead and the score for 1=perfect health. Multivariable logistic regression analyses were performed to determine the association between prior fracture types and HRQL. All analyses were conducted separately for women and men. Final model selection was conducted using the Bayesian Information Criterion technique. From these analyses, we calculated odds ratios (OD) and 95% confidence intervals (CI) for all parameters. In women, adjusted OD indicated that higher Mark II HRQL was associated with a lower likelihood of having a prior ANY (0.45; CI: 0.27, 0.74), and MAIN (0.45; CI: 0.25, 0.84). Higher Mark III HRQL was associated with a lower likelihood of having a prior ANY (0.49; CI: 0.35, 0.69), and MAIN (0.53; CI: 0.35, 0.81). Higher Mark II (0.51; CI: 0.26, 1.01) and Mark III HRQL (0.66; CI: 0.41, 1.06) tended to lower the likelihood of having a prior WRIST but further evidence will need to be collected to verify these findings. In men, our results had wide CI and are difficult to interpret. In conclusion, HRQL as measured by the HUI was negatively associated with previous fractures.

Disclosures: G. Ioannidis, None.


Patterns in Loss to Follow-up Can Influence Estimates of Risk Reduction of Fracture.D. M. Black1, D. E. Thompson2, C. Teutsch2, A. E. de Papp2. 1University of California San Francisco, San Francisco, CA, USA, 2Merck & Co., Inc., West Point, PA, USA.

When exact times or intervals containing exact time of fracture are known, survival analysis methodology is appropriate to describe and infer about time-to-event data. In clinical trials when two groups are compared, there are three components to the analysis: estimating cumulative incidence within each group, making inference about differences between two cumulative incidence curves, and quantifying difference in terms of relative risk. Each component uses different methodology: Kaplan Meier (KM) estimation of cumulative incidence, log rank test, and Cox's proportional hazard model, respectively. The focus of this paper is to comment on issues that can influence the first component of the analysis, specifically, can patterns in loss to follow-up influence estimates of cumulative incidence derived from KM methodology? KM methodology attempts to estimate proportion of patients with a specific event during the study. It takes into account that not all patients will complete the study. Patients who are lost to follow up (e.g., died) are considered “censored” and are included in the denominator at each event only prior to being censored. When dropouts are low and occur at a uniform rate, difference between KM estimate at study end and observed proportion at study end is small. When dropouts occur late in the study they have little effect on estimates of true cumulative incidence. However, when dropouts are large and occur very early in the study, the KM estimated cumulative incidence in early periods have a much greater carryover effect on cumulative incidence at the end of the study. Specifically, early effects are given more weight than late effects. We examine loss to follow-up in two (FIT I and VERT MN) recent osteoporosis clinical trials and possible effect this loss could have on the calculated fracture risk reduction. In both studies an intention-to-treat analysis was employed. In VERT MN the loss to follow-up was large (approximately 40%) and appeared to be early. KM estimates of the cumulative incidence at study end were 29 and 18% in the placebo and treated groups, respectively. Observed simple %'s which assumes no dropout were 25.7% and 15.4%, respectively. Corresponding relative risk reductions (RRR) were 49% and 39%. In FIT I, loss to follow-up was less than 3% and corresponding RRR were 51 and 52%. If we simulate a 50% loss to follow-up, RRR would be 63%. Loss to follow-up can bias the true relative risk reduction. If there is significant loss to follow-up, particularly early in the trial, the statements that investigators conducted an “intention-to-treat analysis” generally provide little assurance about the accuracy of the risk reduction.

Disclosures: D.E. Thompson, Merck & Co., Inc. 3.


Hyperkyphosis Predicts Mortality Independent of Vertebral Osteoporosis in Older Women.D. M. Kado1, G. A. Greendale1, L. Lui2, K. E. Ensrud3, H. A. Fink3, T. Hillier4, S. R. Cummings2. 1Medicine, University of California, Los Angeles, Los Angeles, CA, USA, 2San Francisco Coordinating Center, San Francisco, CA, USA, 3University of Minnesota, Minneapolis, MN, USA, 4Kaiser Center for Health Research, Portland, OR, USA.

Only about half of hyperkyphosis, or excessive curvature of the thorax, is due to vertebral fractures. Vertebral fractures are associated with increased overall mortality. Whether hyperkyphosis, also predicts increased mortality, independently of vertebral fractures, is unclear. To answer this question, we studied a consecutive sample of 610 women, aged 67--93 years, who had kyphosis measured using a flexicurve (Milne and Lauder, Ann Hum Biol, 1974). Prevalent vertebral fractures at baseline were defined by morphometry, and mortality was assessed during an average follow-up of 11 years. In age-adjusted models, each SD increase in kyphosis carried a 1.19-fold increased risk of mortality (95% C.I.: 1.04--1.36, p = .01). Those in the top quartile of kyphosis had an age-adjusted risk of 1.38 (95% C.I.: 1.02--1.87, p = .04) compared to the lower three quartiles. Adjusting for age, self-reported health, physical activity, weight change, lumbar bone mineral density, and number of prevalent vertebral fractures did not change the relationship between poorer kyphosis and increased mortality (RH per SD increase in kyphosis: 1.19; 95% C.I.: 1.01--1.39, p = .03). We conclude that older women with hyperkyphosis are at increased risk of mortality that is not due to underlying spinal osteoporosis.

Disclosures: D.M. Kado, None.


Evaluation of Decision Rules for Referring African-American Women for Bone Densitometry by Dual-Energy X-Ray Absorptiometry.L. Wallace1, J. Ballard2, D. Holiday*3, P. Cussen*4, L. Turner*5. 1Family Medicine, University of Tennessee Graduate School of Medicine, Knoxville, TN, USA, 2Health & Kinesiology, University of Texas at Tyler, Tyler, TX, USA, 3Biostatistics, University of Texas Health Center at Tyler, Tyler, TX, USA, 4Radiology, University of Texas Health Center at Tyler, Tyler, TX, USA, 5Health Science, University of Arkansas, Fayetteville, AR, USA.

Osteoporosis is an increasingly extensive, chronic, metabolic bone disease characterized by decreased bone mass and increased susceptibility to fractures. It is widely accepted that bone mineral density (BMD) measurements form the basis for the diagnosis of osteoporosis. The purpose of this study was to assess criterion validity of 6 decision rules--Age, Body Size, No Estrogen (ABONE), National Osteoporosis Foundation (NOF) practice guidelines, Osteoporosis Risk Assessment Instrument (ORAI), Osteoporosis Self-Assessment Tool (OST), Simple Calculated Osteoporosis Risk Estimation (SCORE), and body weight less than 70 kg (Weight Criterion)--for selecting postmenopausal African-American women for dual-energy x-ray absorptiometry (DEXA). Chart abstractions from 174 African-American women (mean age=59.4±12.5 years) with BMD testing results by DEXA were completed. Sensitivity, specificity, 95% confidence intervals (CIs), and area under the receiver operating characteristic (AUROC) were used to measure the overall ability of each decision rule to discriminate between women with varying degrees of low BMD. The percent of women with a BMD T-score less than -1, less than -2, and no more than -2.5 were, 25.3%, 14.9%, and 14.9% respectively. Sensitivity for identifying women with a BMD T-Score of less than -2.0 ranged from 31.2--62.5%, while specificity ranged from 62.9--93.7%. Sensitivity for identifying women with a BMD T-Score of no more than -2.5 ranged from 32.1--61.3%, while specificity ranged from 84.2--96.2% (Table). The AUROC curves were greatest using SCORE, OST, and Weight Criterion for BMD T-Scores of no more than -2.5. The OST and Weight Criterion are easiest to calculate therefore may be most useful in clinical practice. 

Table  . Sensitivity (SEN) and Specificity (SPC) of Decision Rules by Femoral Neck BMD T-Scores
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Disclosures: L. Wallace, None.


Prevalence of Osteoporosis in Patients Undergoing Evaluation for Lung, Liver, and Heart Transplantation.P. M. Camacho1, B. Pisani*2, S. Bhorade*3, S. Creech*4, F. Nabhan*1, P. Sapountzi*1, S. Hou*5, G. Sizemore1, D. Van Thiel*6. 1Endocrinology and Metabolism, Loyola University of Chicago, Maywood, IL, USA, 2Cardiology, Loyola University of Chicago, Maywood, IL, USA, 3Pulmonary and Critical Care Medicine, Loyola University of Chicago, Maywood, IL, USA, 4Biostatistics, Loyola University of Chicago, Maywood, IL, USA, 5Nephrology, Loyola University of Chicago, Maywood, IL, USA, 6Gastroenterology and Hepatology, Loyola University of Chicago, Maywood, IL, USA.

This study aimed to determine the prevalence of osteoporosis and osteopenia among patients undergoing evaluation for liver, lung and heart transplantation at Loyola University Medical Center between 1998--2003. 187 patients with pretransplant DEXA scans out of 314 candidates were identified. The group was comprised of 71 liver, 61 lung, and 55 heart transplant candidates. The World Health Organization criteria was used to define osteopenia (<−1 to -2.4) and osteoporosis (<=-2.5). The mean age in years of the liver group was 51.5 +-11.4, lung was 48.7 +-11.6, and heart was 53.3 +-11.2. Females comprised 32% of the liver, 63% of the lung and 20% of the heart transplant group.

Overall 66 % of patients had osteoporosis or osteopenia before transplantation. Prevalence of osteoporosis was highest in the lung transplant group, followed by the liver and heart groups (24%, 16%, and 11% respectively). The rates of osteopenia in the heart, lung and liver groups were 45%, 44%, and 31% respectively.

Mean lumbar spine bone mineral density (LS BMD) and lumbar spine T scores (LS T) were similar between the groups. Mean femoral neck bone mineral density (FN BMD) (0.866 vs. 0.977 vs. 0.982 gm/cm2, p=<.001) and T scores (FN T) (-1.1 vs. -.49 vs.-.554, p=.015) were significantly lower in the lung than liver and heart transplant groups.

In the liver transplant group, patients with primary biliary cirrhosis had significantly lower LS BMD than those with alcoholic cirrhosis and chronic viral hepatitis (0.965 vs. 1.135 vs. 1.20 gm/cm2, p= .030), as well as lower LS T (-1.93 vs. -.786 vs. -.19, p= .042), FN BMD (0.790 vs. 0.984 vs. 0.977 gm/cm2, p= .003) and FN T (-1.5 vs. -.46 vs. -.58, p= .013). In the lung transplant group, cystic fibrosis patients had significantly lower LS BMD (0.984 vs.1.109 vs.1.201 gm/cm2, p=.005) than those with emphysema and idiopathic pulmonary fibrosis. LS T, FN BMD and FN T scores were similar in these groups. In the heart transplant group, no significant differences in bone density and T scores were seen between patients with dilated cardiomyopathy and ischemic cardiomyopathy.

Patients undergoing heart, liver and lung transplantation are at increased risk of bone loss. Proper pre-transplant screening is advised in this high risk population.

Disclosures: P.M. Camacho, None.


Hip Structural Geometry Changes During and After Lactation.M. A. Laskey*1, B. C. C. Khoo*2, R. I. Price2, T. J. Beck3, A. Prentice1. 1MRC Human Nutrition Research, Cambridge, United Kingdom, 2Sir Charles Gairdner Hospital, Perth, Australia, 3Johns Hopkins University, Baltimore, MD, USA.

Lactation is associated with temporary decreases in hip BMC. This study investigates whether these changes are accompanied by alterations in hip structural geometry that may affect bone strength. DXA measurements were made longitudinally during 65 lactations in 47 breast-feeding women (BF). Nonpregnant nonlactating women (NPNL, n=24) were studied as controls. Women had hip DXA scans (Hologic QDR-1000W) at baseline (2 wk post-partum (pp)), peak-lactation (3mo pp if BF for 6mo), post-lactation (1yr pp or 3mo post-lactation if BF >9mo) and follow-up (at least 2 year pp). Hip scans were analysed using the hip structural analysis (HSA) program which measures BMD and structural geometry at the narrow neck (NN), intertrochanter (IT) and proximal shaft (S).

No significant differences from baseline were observed in HSA measurements at any time-point or hip site in NPNL women. In contrast for BF women, at peak lactation, BMD, cross-sectional area (CSA) and average cortical thickness (AvCT) had decreased from baseline values at NN (-2.8%, P<0.001; -3.0%, P<0.001, -2.9%, P<0.001), IT (-2.7%, P<0.001; -2.0%, P<0.001; -2.7%, P<0.001) and shaft (-1.3%, P=0.01; -0.9%, P=0.09; -1.78%, P<0.01). Bone width was unchanged at all sites but endosteal diameter (ED) increased at S (1.6%, P=0.03) and IT (1.1%, P<0.001). These changes resulted in an increase in buckling ratio (BR, index of cortical stability) at S (2.4%, P<0.01) and IT (3.1%. P<0.001) and a trend to a decrease in section modulus (Z, indicator of bending strength) at NN (-2.4%, P=0.08). By post-lactation most HSA measurements were not significantly differently from baseline. Small nonsignificant increases in HSA measurements were observed at NN and S at follow-up. In contrast, at IT, there were significant increases above baseline in BMD (2.5%, P<0.001), CSA (3.2%, P<0.001), Z (5.0%, P<0.001) and AvCT (2.2%, P=0.01) and a significant decrease in BR (-2.2%, P<0.01).

These results suggest that during lactation decreases in HSA measurements are significant but temporary. The alterations occurred mainly at internal surfaces. As these are close to the neutral axis, the effect on fracture risk is minimal. Changes in cortical stability (BR) may be minimal in young women because BR describes a threshold instability that is unlikely to be exceeded in young women. At follow-up, the positive changes in IT hip geometry, above baseline may reflect the return to the prepregnant state and/or indicate a long-term benefit of childbearing on hip structure.

Disclosures: M.A. Laskey, None.


Parkinson's Disease Is Associated with Low Bone Mineral Density in Older Men.H. A. Fink1, M. A. Kuskowski*1, K. E. Ensrud2, E. S. Orwoll3, J. A. Cauley4, S. R. Cummings5. 1For the MrOS Research Group, GRECC, VA Medical Center, Minneapolis, MN, USA, 2University of Minnesota, Minneapolis, MN, USA, 3Oregon Health Sciences University, Portland, OR, USA, 4University of Pittsburgh, Pittsburgh, PA, USA, 5UCSF Coordinating Center, San Francisco, CA, USA.

The high risk for fractures associated with Parkinson's disease (PD) is usually attributed to an increased risk for falls. Limited case-control data suggest a possible association between PD and bone mineral density (BMD).

Utilizing data from MrOS, a multi-center, prospective cohort study of osteoporosis in 5995 men aged ⩾65 yrs, we examined the association between PD and BMD. At MrOS baseline, self-reported data collected included demographics, lifestyle factors, medications, and medical history, including report of physician diagnosed PD (n=52). Clinic measurements included anthropometry and physical performance. BMD was measured with DXA at the total hip (TH), femoral neck (FN) and lumbar spine (LS). For each skeletal site, ANOVA and ANCOVA were used to assess the unadjusted, age-adjusted and multivariate-adjusted association between PD and BMD. The multivariate models for all sites included age, height, height loss since age 25, usual walking speed and maximum grip strength. In addition, the LS model included SF-12 physical component summary score, and both hip models included PASE physical activity score.

Results are presented as the mean percentage difference in BMD in men with PD vs. men without PD. Negative values indicate lower BMD in the PD group. 

Table  . Mean Difference in BMD between Men with PD vs. without PD
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Older men with PD have substantially decreased BMD at the hip and lumbar spine. The association of PD with low BMD was only modestly altered by adjustment for age and multiple covariates. Our results suggest that older men with PD should be targeted for osteoporosis screening and possibly considered for pharmacologic intervention to reduce their elevated risk for fractures.

Disclosures: H.A. Fink, None.


Depression Contributes Substantially to Osteoporosis Among Elderly Men. Results from Mr. Os Hong Kong, the First Prospective Cohort Study of Osteoporosis in Asian Men.S. Y. S. Wong1, E. M. C. Lau1, J. Woo*2, K. Ng*1, H. Lynn*1, K. L. Stone*3, S. R. Cummings*3, E. Orwoll*4. 1Jockey Club Centre for Osteoporosis Care and Control, School of Public Health, Chinese University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China, 2Department of Community and Family Medicine, Chinese University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China, 3San Francisco Coordinating Center, California, CA, USA, 4Oregon Health and Science University, Portland, OR, USA.

Depression causes biological changes, such as decreased testosterone and increased cortisol levels, that may influence bone mass and fracture risk. To test the hypothesis that men with depression have lower bone mass, we used data from the baseline examination of Mr. Os Hong Kong, a prospective study of 2000 Hong Kong men age 65 to 92 recruited from community-based sources. Depression was diagnosed by face-to-face interview, using a validated Chinese version of the Geriatric Depression Scale (GDS), with depression being defined as a cut-off of 8 or more. Bone mineral density (BMD) of the total hip was measured by dual X-ray densitometry (DEXA) using the Hologic QDR-4500W. In the study sample, 8.5% of men were found to be depressed; and the BMD at the hip in these subjects were 2.1% lower than non-depressed subjects (95% CI -4.1 to -0.13); after adjusting for body weight, medical history, calcium intake and physical activity. Depression was associated with a 1.4-fold (95% CI=1.00 to 2.08) relative risk (RR) of osteopenia or osteoporosis. Study subjects from Mr. Os were healthy volunteers, and population-based surveys have found that the prevalence of depression in elderly men in Hong Kong was 29.2%. Based on this figure, we estimated that 9.8% of ‘osteopenia and osteoporosis’ among elderly Hong Kong men could be attributed to depression (Population Attributable Risk, PAR). We conclude that depression is associated with lower BMD and accounts for a substantial percentage of osteoporosis and osteopenia among elderly Chinese men.

Disclosures: S.Y.S. Wong, None.


Risk Factors for Low Bone Mass in Elderly Men -- Mr. Os (Hong Kong, the Largest Cohort Study in Asian Men).E. M. C. Lau1, S. Y. S. Wong1, H. Lynn*1, J. Leung*1, P. C. Leung*2, K. L. Stone*3, S. R. Cummings*3, E. Orwoll*4. 1Jockey Club Centre for Osteoporosis Care and Control, School of Public Health, Chinese University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China, 2Department of Orthopaedics and Traumatology, Chinese University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China, 3San Francisco Coordinating Center, California, CA, USA, 4Oregon Health and Science University, Portland, OR, USA.

There has been no comprehensive study of risk factors for low bone mass in Asian men. Mr. Os (Hong Kong) is the largest and only cohort study to address this issue. Two thousand community dwelling and ambulatory Hong Kong Chinese men aged 65 to 92 years were recruited from the community and risk factors for osteoporosis were assessed by face-to-face interviews using a standardized, structured questionnaire. Bone mineral density (BMD) was measured by DEXA (Hologic, Inc). Factors found to be significant in regression was put into a final multiple regression model, which is described below. 

Table  . Percentage difference in BMD (95% confidence interval) for significant risk factors for hip BMD
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Model adjusted for dietary intake of protein and calcium, hypertension and thiazide diuretic intake.

These results suggest that ideal body weight, avoiding smoking and maintaining muscle strength are important in preventing osteoporosis in men. However, lifestyle and anthropometric risk factors could only account for 30% of the individual variability in BMD, and cannot replace BMD measurements.

Disclosures: E.M.C. Lau, None.


Risk Factors for Low Bone Mass in Elderly Asian Women -- Ms. Os (Hong Kong, a Large Cohort Study in Asian Women).D. T. K. Choy*1, E. M. C. Lau1, J. Leung*1, P. C. Leung*2, J. Woo*3, A. Hong*1. 1Jockey Club Centre for Osteoporosis Care and Control, School of Public Health, Chinese University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China, 2Department of Orthopaedics and Traumatology, Chinese University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China, 3Department of Community and Family Medicine, Chinese University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China.

Little is known about risk factors for low bone mass in elderly Asian women. Ms. Os (Hong Kong) is a large cohort study to address this issue. Eight hundred community dwelling and ambulatory Hong Kong Chinese women aged 65 to 94 years were recruited and followed up. Risk factors for osteoporosis were assessed by face-to-face interviews using a standardized, structured questionnaire. Bone mineral density (BMD) was measured by DEXA (Hologic, Inc). Factors found to be significant in regression was put into a final multiple regression model, which is described below. 

Table  . Percentage difference in BMD (95% confidence interval) for significant risk factors for hip BMD
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Model adjusted for hypertension, GI surgery, Diabetes mellitus, grip strength and walking. The results suggest that maintaining body weight, avoidance of cigarette smoking and maintaining calcium intake will be important in preventing osteoporosis in elderly Chinese women. However, lifestyle factors only account for 35% of the variability in BMD, and could not replace BMD measurement.

Disclosures: D.T.K. Choy, None.


Hyperkyphosis Increases Body Sway, Gait Unsteadiness and Risk of Falls in Osteoporosis.M. Sinaki1, R. H. Brey*2, C. A. Hughes*3, K. R. Kaufman*3. 1Physical Medicne and Rehabilitation, Mayo Clinic, Rochester, MN, USA, 2Vestibular and Balance Laboratory, Mayo Clinic, Rochester, MN, USA, 3Motion Analysis Laboratory, Mayo Clinic, Rochester, MN, USA.

This controlled trial was designed to investigate the influence of osteoporosis-related thoracic hyperkyphosis (OP-THK) on gait unsteadiness, body sway and falls. Thirteen OP-THK women (Cobb angle 50 -- 65° measured from spine radiographs) and 13 healthy women serving as controls (C) were enrolled. Mean age of the OP-THK group was 76 (±5), height 160 cm (±4), and weight 60 kg (±8) and C group means were age 71 (±5), height 160 (±4), and weight 68 (±13). Isometric strength data was collected using a Quantitative Muscle Assessment (QMA) System. Gait was monitored during unobstructed level walking and while stepping over an obstacle of four different heights randomly assigned (2.5%, 5%, 10%, and 15% of the subject's height). A ten-camera Real Time system was used to collect 3-D marker trajectory at 60 Hz during gait from 28 reflective markers. A repeated measures ANOVA was used for the statistical analysis with the significance level set at p < 0.05. Balance was objectively assessed using Computerized Dynamic Posturography (CDP) consisting of two components, the Sensory Organization Test (SOT), and the Motor Control Test (MCT). For this study, a composite score calculated from 14 scores of SOT was used. Statistical analysis was done using a student's paired t-Test with significance level set at p < 0.05.

The OP-THK subjects were significantly weaker than C subjects in all lower extremity (LE) muscle groups (p < 0.05) except the right ankle plantar flexors. There was a significant difference in the anteroposterior (A/P) and mediolateral (M/L) displacements and velocities. The OP-THK subjects had less A/P displacement, greater M/L displacement, reduced A/P velocity, and increased M/L velocity when compared to the C subjects. This was true for all conditions of unobstructed and obstructed level walking. There was a significant effect of obstacle height on all center of mass (COM) parameters. Even though COM displacement differences were detected, there was no significant difference in the right or left single support time nor the step width, which are usual indicators of balance problems. The OP-THK subjects had statistically significantly greater balance abnormalities on CDP compared to the C group (p=0.0006).

Our data show that thoracic hyperkyphosis plays a significant role in increasing body sway, gait unsteadiness and risk of falls in osteoporosis.

Disclosures: M. Sinaki, None.


Relationships Between the 4 Major Risk Factors for Hip Fracture and QUS Parameters from 5 Devices: The OPUS Study.A. Stewart*1, F. Thomasius2, D. Felsenberg2, D. M. Reid1, R. Eastell3, C. Roux4, C. C. Glüer5. 1Osteoporosis Research Unit, University of Aberdeen, Aberdeen, United Kingdom, 2Free University Berlin, Berlin, Germany, 3University of Sheffield, Sheffield, United Kingdom, 4René Descartes University, Paris, France, 5University Hospital Schleswig-Holstein, Kiel, Germany.

Previous studies have identified 4 major risk factors for hip fractures. We examined these risk factors in the Osteoporosis and Ultrasound Study (OPUS) which is a large European population of 2374 postmenopausal women aged 55 to 79 years who were chosen at random. Each answered a risk factor questionnaire and had QUS scans of the heel (Lunar Achilles + (Ach.), UBIS 5000 (UBIS), OSI/Osteometer DTU-One (DTU), Quidel/Metra QUS-2 (QUS-2)) and of the fingers (IGEA DBM Bone Profiler (IGEA)) measuring broadband ultrasound attenuation (BUA), speed of sound (SOS), amplitude-dependent speed of sound (AdSOS), ultrasound bone profile index (UBPI) and bone transmission time (BTT). Comparing the number of risk factors with the QUS results we found significant relationships for all measurements (all p<0.01), with those with no risk factors having higher QUS results compared to those with 4 risk factors. Table 1 shows the results taking each of the risk factors individually. Any previous fracture; yes n = 1016, no n = 1323: all QUS parameters p < 0.001. Weight; lowest quartile n = 530, upper three quartiles n = 1844: all QUS measurements (p<0.01) except for IGEA BTT and IGEA UBPI. Smoking; ever smoked n=1016, never smoked n=1336: significantly different QUS for smokers for DTU SOS, UBIS SOS, IGEA Ad-SOS and IGEA UBPI. Maternal hip fracture; yes n =242, no n = 2095: none of the QUS parameters were significantly different. In conclusion there does appear to be similar relationships between the 4 major risk factors and QUS parameters in this European population. However the different QUS scanners do not all give the same results. 

Table  .  
  1. * p < 0.05

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Disclosures: A. Stewart, None.


Two Types of Osteoporosis and Fracture: “A” -- BMD-dependent, and “B” -- BMD-independent. A New Insight Into Old Classification.J. E. Badurski. Polish Foundation of Osteoporosis & Center of Osteoporosis and Osteoarticular Diseases, Bialystok, Poland.

Mean age of population of adult polish women in two large epidemiological studies BOS (1) and NEPOS (2) are 59 and 56 respectively. Prevalence of osteoporosis (Hip T-score -2.5) in BOS is 15% and 18% in NEPOS (Forearm T-score -2.5). The number of fractures in BOS is twice as much as number of women with Hip T-score -2.5. Mean Hip T-score of women with previous fractures is -1.5. It contrasts with epidemiological studies conducted in older segment of female population (3), but is in accordance with that performed in all-adult ones (4). Comparative analysis of BOS, NEPOS and others (5) with known distinguishing factors shows a possibility of existence of two forms of osteoporosis and fractures, which is a proposal for clinical practice: 

Table  .  
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Overlap of both types increases risk fracture. Mostly epidemiological and observational data of BMD-dependent and BMD non-dependent fractures allows to see Melton's and Riggs's classification (6) in light of difficulties in evaluation of fracture risk and treatment of women with T-score between -1.0 and -2.0, in which antiresorptive treatment is questionable.

References: 1. Nowak NA, Badurski JE, et co: Post Osteoartrol 2003,14:1--2. 2. Dobrenko A, Badurski JE, et co.: Post Osteoartrol 2003, 14:1--2 (in press). 3. Newitt MC, Johnell O, et co.: Osteoporos Int 1994,4:325. 4. Burger H, de Laet CE, et co.: Am J Epidemiol 1998,147: 871. 5. Cummings SR, Melton III LJ: The Lancet 2002,359:1761. 6. Melton III LJ, Riggs BL: in Avioli LV Edr. The osteoporotic syndrome. Grune-Stratton Inc. Orlando 1987,1

Disclosures: J.E. Badurski, None.


Prevalence of Glucocorticoid Treatment in Kiel, Germany: Results From the Population-based PSIO-D Study.C. C. Glüer1, C. Nöldeke*1, R. Barkmann1, M. G. Glüer1, W. Timm*1, D. Felsenberg2, P. Martus*3, M. Heller*1. 1Diagnostic Radiology, University Hospital Schleswig-Holstein, Kiel, Germany, 2Diagnostic Radiology, University Hospital Benjamin Franklin, FU, Berlin, Germany, 3Medizinische Informatik, University Hospital Benjamin Franklin, FU, Berlin, Germany.

Glucocorticoid (GC)-induced osteoporosis is a widespread skeletal disease with substantial impact on health and quality of life. In Germany as well as in other countries few population-based data are available. The “Prevalence of steroid induced osteoporosis in Germany” (PSIO-D) study was designed to survey use of steroids in a population-based setting (survey phase) and subsequently study skeletal and health status in a subset of subjects undergoing extensive evaluations by state-of-the-art diagnostic tests (clinic phase). Here we report data for the survey phase from one center in Kiel, Germany.

30,876 subjects age 55 to 80, representing about 54% of the population of Kiel in this age segment were contacted by mail. 22,944 (74.3%) responded by returning the survey questionnaire. The response rate was similar for men and women and across age groups.

4,458 subjects, i.e. 19,4% (16% for men, 22% for women) reported current or previous GC treatment excluding topical applications. The rate slightly decreased with age ranging around 20% at age 55 down to 16% at age 80. In a random subset of 2,091 respondents that had reported use of GC, details of the kind of GC use was assessed. 49% of these subjects reported a history of oral GC treatment, 25% with a duration of at least 3 months. For current oral treatment the percentages decreased to 11% and 10% (of which 75.3% reported a daily dose of at least 2.5mg prednisolone equivalent), respectively. Based on these data, 4.7% of the general population reported a history of oral GC use exceeding 3 months, with a point prevalence of 1.9% for current use of GC exceeding 3 months, and of 1.4% for current medication of at least 2.5mg/day for at least the previous 3 months. Of the 2,091 individuals, 18% stated that they had been diagnosed with asthma (20% for men, 17% for women) and 19% with rheumatic disorders (14% for men, 22% for women). A diagnosis of osteoporosis was reported by 8.3% of those that stated that they never had taken GC treatment, by18% (6% for men, 24% for women) of subjects with a history of GC use, and 20% if GC use exceeded 3 months.

In conclusion, if our data from Kiel are representative we estimate a point prevalence of 1.4% in the general German population age 55--80 for continuous oral use of GC with a duration of at least 3 months and a daily dose of at least 2.5 mg prednisolone equivalent.

Consequently, approximately 300,000 subjects of age 55--80 in Germany may be at increased risk for osteoporotic fracture.

Disclosures: C.C. Glüer, Procter&Gamble Pharmaceuticals 2.


Prevalence and Predictors of Osteoporosis and Fractures in Type 1 and Type 2 Diabetes Mellitus.S. Finger*1, T. Bruckner*2, J. Schneider*1, C. Scheidt-Nave*2, C. Kasperk1, R. Ziegler1, G. Leidig-Bruckner1. 1Internal Medicine, University Heidelberg, Heidelberg, Germany, 2Clinical Social Medicine, University Heidelberg, Heidelberg, Germany.

The study aimed to investigate the prevalence and possible predictors of osteoporosis and related fractures in type 1 and 2 diabetes.

We investigated 398 consecutive patients from the outpatient clinic (diabetes type 1: 79 men (42 +/− 12 years), 76 women (46 +/− 13 years); diabetes type 2: 115 men (63 +/− 9 years), 128 women (63 +/− 9 years)). All patients received a standardized questionnaire on diabetes history, risk factors for osteoporosis and insufficieny fractures. Diabetes related vascular complications were documented from the patients records and actual HbA1c was determined. Bone mineral density (BMD) was measured using DXA (Hologic 4500) at the lumbar spine (LS) and femoral neck (FN). Relationship between BMD/osteoporosis and possible risk factors (age, duration of diabetes, body mass index (BMI), HbA1c and vascular complications) was assessed by correlation analyses and logistic regression models.

Osteoporosis (t-score <−2.5 SD) at the LS (FN) was found in 5 (11) % of men and 5 (9) % of women with type 1 and in 6 (13) % of men and 9 (22) % of women with type 2 diabetes. Osteopenia (t-score between -1 and -2.5 SD) at the LS (FN) was found in 32 (44) % of men and 30 (41) % of women with type 1 and in 30 (36) % of men and 27 (31) % of women with type 2 diabetes mellitus. 12% of all patients suffered from 1 or more insufficiency fractures (13% in type 1 and 11% in type 2). FN-BMD was negatively correlated to duration of diabetes in type 1 (men: R=-0.25, women: R=-0.3) but not in type 2 diabetes. FN-BMD was positively correlated to BMI in type 1 (men: R=0.28, women: R=0.23) and in type 2 diabetes (men: R=0.37, women: R=0.44), but no relationship was found between BMD and HbA1c. The presence of vascular complications from diabetes was not significantly related to BMD or risk of osteoporosis. Multivariate analysis revealed only BMI to be predicitve in respect to occurrence of osteoporosis in type 1 and type 2 diabetes.

The prevalence of osteoporosis and fractures was similar in type 1 and type 2 diabetes without sex difference, although type 1 patients were about 20 years younger. Our data indicate that osteoporosis is a clinical significant and commonly underestimated problem especially in patients with type 1.

Disclosures: S. Finger, None.


Body Weight per se can Be used in Case-Finding for Bone Density Measurement in Asian Men -- Findings from Mr. Os (Hong Kong).H. Lynn*1, E. M. C. Lau1, S. Y. S. Wong1, P. C. Leung*2, P. Mannen*3, S. R. Cummings*3, E. Orwoll*4. 1Jockey Club Centre for Osteoporosis Care and Control, School of Public Health, Chinese University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China, 2Department of Orthopaedics and Traumatology, Chinese University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China, 3San Francisco Coordinating Center, California, CA, USA, 4Oregon Health and Science University, Portland, OR, USA.

The National Osteoporosis Foundation had clear guidelines for bone mineral density in older women. However, this does not apply to Asian men. The objectives of the current study were to evaluate if body weight could be used to identify older men for BMD measurement, and ascertain the value of adding other risk factors for case-finding. Mr. Os (Hong Kong) is the largest cohort study on osteoporosis in older Chinese men. Two thousand community-dwelling and ambulatory men aged 65 to 92 were recruited. Life style factors, anthropometry and BMD (DEXA, Hologic, Inc) were obtained by standard procedures.

Risk factors were identified by logistic regression, and ROC analysis was performed on a training sample of 1300 and a validation sample of 700. Using body weight less than 62 kg as a case-finding criteria for a BMD T-score of ⩽ -2.5 at the hip or spine, a sensitivity of 82%, specificity of 57% and an area of 0.77 under the ROC curve was obtained in the validation sample. When cigarette smoking (for 40 years or more) was added, the sensitivity was 87%, specificity was 51%, and the area under the curve was 0.79. In comparison, when history of wrist fracture was added the sensitivity was 83%, specificity was 57%, and the area under the curve was 0.78.

We conclude that body weight provides a simple and satisfactory case-finding criteria for bone density measurement in Asian men.

Disclosures: H. Lynn, None.


Risk Factors for Osteoporosis in Postmenopausal African American Women.G. C. Woodson. Atlanta Research Center, LLC, Decatur, GA, USA.

Although postmenopausal (PM) African American (AA) women are at lower risk for all categories of osteoporosis-related fragility fractures compared with Caucasian (CA) women, osteoporotic fractures in AA women are associated with significantly higher morbidity and mortality. Therefore the early diagnosis and treatment of osteoporosis (OP) in this population is at least as important as for other ethnic groups and is worthy of the attention of their primary care physician, non-profit voluntary health organizations, and governmental agencies responsible for healthcare policy.

This retrospective case control study investigates risk factors (RF) for OP in population of PM AA women having bone density testing at an outpatient community-based OP specialty center between 1992 and 2002. The subjects were either self-referred or referred by their physician for testing. Spine and hip DXA (Hologic, Bedford, MA) bone density testing was performed on each subject. Patient medical history, family history, past and present pharmaceutical use, and dietary and exercise habits were collected using a patient self-administered osteoporosis RF questionnaire. The DXA manufacture's AA normative database was used for calculation of the patient's t-score and the diagnostic class was determined using the WHO's t-score based OP classification system as applied to either the L1--L4 PA lumbar spine or the total hip sites.

Results showed that 56 patients found to be osteoporotic, 99 were osteopenic, and 46 were diagnosed as having normal BMD. RFs more commonly found in the osteoporotic group compared to the normal group are displayed in table 1. Factors or conditions that were more common in the normal group versus the osteoporotic group are found in table 2. 

Table  . Results Table 1
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Table  . Results Table 2
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In summary, while the incidence of osteoporotic fractures is 60% lower for PM AA women compared with their CA peers, the health consequences of these fractures is significantly greater. In this study, RFs for OP in PM AA women using a race specific normative database were found to be similar to those reported for PM CA women. These common RF can be used to help select AA women appropriate for bone density testing using central DXA. Treatment of PM OP diagnosed by DXA in AA women using US FDA registered therapies is indicated to prevent the consequences of this disease.

Disclosures: G.C. Woodson, None.


Relationship between Bone Mass and Cognitive Function in Postmenopausal Women.R. A. Brownbill1, J. Z. Ilich2. 1School of Allied Health, University of Connecticut, Storrs, CT, USA, 2School of Allied Health, University of Connecticut, Storrs, CT, USA.

It has been suggested that bone loss and cognitive decline are co-occurring disease states, likely due to their relationship with estrogen. Few studies have examined the relationship between cognitive function and bone mineral density (BMD), a possible marker of cumulative estrogen exposure. We therefore were interested in determining if BMD and bone mineral content (BMC) measured from both cortical and trabecular sites, are positively related to cognitive function in 97 healthy postmenopausal women (ages 59.4--85.0 years). BMD and BMC from the whole body, lumbar spine, femur and forearm were measured with a Lunar DPX-MD instrument. Cognition was assessed using the mini mental state examination (MMSE). The MMSE is a widely used screening tool to assess cognitive impairment. It consists of various graded questions and tasks totaling to a maximum of 30 possible points, with 27 as a cut-off for normal, and less than 27 indicating impaired cognitive abilities. Subjects MMSE scores ranged from 22--30, indicating all subjects had either mild cognitive impairment or no cognitive impairment. Subjects were also divided into two groups, above and below the mean MMSE score (27.9). MANCOVA (adjusted for age, BMI, physical activity and lifetime exposure to smoking) reveled a significant moderate effect of MMSE on BMD, p<0.05, indicating 22% of generalized variability in BMD from the whole body, spine, hip and forearm was accounted for by differences in MMSE scores. ANCOVA follow-up tests did not reveal significant group differences for individual bone sites, though Group 2 (>27.9 score) had higher adjusted means for all sites except for the femoral neck, Ward's and trochanter. We hypothesize statistical significance was not reached because the average score on the MMSE of 27.9 was considered normal cognitive function based on MMSE criteria, indicating on average our subject population was not cognitively impaired. ANCOVA follow-up tests to MANCOVA did become significant when BMI was removed as a covariate. Groups were found to be significantly different for both total body BMC (p=0.018) and right shaft BMC (p=0.01). In conclusion, it appears mild degrees of cognitive impairment may be associated with lower levels of cortical bone specifically in the hip and whole body regions. Individuals with cognitive impairment should be screened for potential bone loss.

Disclosures: R.A. Brownbill, None.


Womens' Knowledge about Osteoporosis Does Not Influence Health-related Behavior.B. J. Edwards1, S. Desai*2, J. Feinglass*1. 1Medicine, Northwestern University, Chicago, IL, USA, 2Medicine, University of Illinois, Chicago, IL, USA.

Methods: 102 women seen at the McGaw Medical Center admitted with minimal trauma (MTF) hip fracture (n=44, 43%), or outpatients at the Geriatrics (n=19, 18%), or Osteoporosis Clinics (n=39, 38.2%) were interviewed. 58 (57%) had a history of prior fracture, 81 (79%) were on calcium and vitamin D, and 69 (69%) had a prior bone density test (DEXA). Subjects received education about osteoporosis risk factors, and osteoporosis diagnosis and treatment. Subsequently they underwent a survey exploring prior knowledge about osteoporosis and attitudes towards benefit of osteoporosis treatment.

Analysis: descriptive statistics, and correlations were performed.


Table  . Measures of Osteoporosis Knowledge and Attitudes Toward Benefit of Treatment
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Overall Knowledge is assessed on a scale (1--6) based on respondents knowledge and understanding of medications.

Motivation to change scale is calculated by a number of lifestyle changes respondents are willing to make (Scale 0--4)

Importance of Osteoporosis Scale is calculated by reversing the rankings given to osteoporosis by respondents (scale 1--5)

Overall attitude/behavior is assessed by combining the scales of benefits gained from the information and importance of osteoporosis for the respondents (Scale 2--10) 

Table  . Predictors of Knowledge and Attitude Levels
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High knowledge is defined as respondents indicating yes to all six knowledge items. Subjects attending the Ostoporosis Clinic exhibited positive attitude level as compared to other patient groups. Subjects with hip fractures exhibited high knowledge but low positive attitudes/behavior with regard to osteoporosis. The lowest knowledge and attitude/behavior toward osteoporosis was seen in Geriatric patients.

Conclusions: Our results indicated that attitudes toward osteoporosis are not greatly influenced by knowledge about this disease. In addition patients at high-risk for fracture (Geriatrics & MTF hip fractures) exhibit low health-related behavior about osteoporosis. Greater patient awareness is necessary to address the growing problem of osteoporotic fractures.

Disclosures: B.J. Edwards, Merck 2, 5; Procter & Gamble 2, 5; Eli Lilly 8; Wyeth 8.


Thyroid Functions, Bone Mass and Body Composition in a Population Study of 482 70-Year Old Women. The Nordos Study.M. Stenstrom*1, E. Nystrom*2, S. Jansson*3, D. Mellstrom1. 1Geriatric Medicine, Goteborg university, Goteborg, Sweden, 2Dep of Endocrinology, Goteborg university, Goteborg, Sweden, 3Dep of Endocrine Surgery, Goteborg university, Goteborg, Sweden.

Excess thyroid function increase bone turnover and the risk for osteoporosis. The question is if thyroid function within the normal reference range are related to bonemass and boby composition. An other question is if thyroxin treatment is related to low bone density in a representative population?

A random sample of 482 70 years old women participated in the study. Bone mass was measured with Hologic 4500A in hip, spine, forearm and whole body. Blood samples were taken in the morning under fasting conditions. Free thyroxineand TSH.

S FT 4 was not related to BMI r 0.05 but was related to fat mass r 0.125 p < 0.01 and indirectly to lean mass r -0.100 p< 0.02 and indirectly to whole body BMD r -0.095 p < 0.02. FT4 wasrelated to s-calcium, r 0.18 and s-alcaline phosfatase activety r 015 p < 0.01. After exclusion of women treated with thyroxin and controling for BMI these correlations were strengthen further and s FT4 correlated inversily to hip BMD r -0.083 p < 0.05. Women who were treated with thyroxin, 13.1 per cent, had a non significant trend to higher BMD in spine and hip compared to non-treated in spite of a significant higher s FT4 and a significant lower TSH. There was not any difference in prevalence of previous fracture between thyroxin treated and controls. In conclusion we found a direct correlation between sFT4 and fat mass and a indirect correlation to lean mass and whole body BMD. Thyroxin treated women did not had lower bone mass in spite of significant higher s FT4 and lower TSH.

Disclosures: M. Stenstrom, None.


Awareness, Knowledge, Risk Factors and Current Treatment of Osteoporosis in a Brazilian Cohort of Elderly Subjects.R. Restituti*, C. H. M. Castro, M. M. Pinheiro*, V. L. Szejnfeld. Rheumatology, Universidade Federal de Sao Paulo, Sao Paulo, Brazil.

We evaluated awareness, knowledge, risk factors and current treatment of osteoporosis in 54 consecutive seniors attending an educational/social program for elderly people and 32 elderly subjects living in a nursing home, in Sao Paulo, Brazil. Our objective was to determine differences between the two cohorts and detect differences between men and women, as well. Subjects included were 70 women and 16 men, with an average age of 76 years. All participants answered an interviewer-administered questionaire regarding osteoporosis awareness, risk factors and treatment. Most of the subjects from the nursing home (44%) had finished high school, while the majority of seniors attending the educational program (55.5%) had not completed elementary school. Ninety six percent of all subjects were aware of osteoporosis and 49% gave the correct definition. In spite of the different education backgrounds, awareness of osteoporosis and its correct definition were not different between the two groups, neither between men and women. Television, friends and physicians were identified as the main source of information, especially among women. Most of the subjects (78% and 79%, respectively) were aware that osteoporosis could affect men and that diet was important. In all, 80% knew that osteoporosis could be prevented, and this was less in the nursing home group (p<0.001). Women believed that they could get osteoporosis more than men (p<0.001) and, in fact, a previous diagnosis of osteoporosis was more prevalent among women(40%) compared to men(6%; p<0.05). Besides old age and estrogen deficiency associated with menopause, the most prevalent risk factors included familial history of fractures-FHF (19%), past smoking (30%) and previous fractures (13%). FHF and previous fractures were both more common among seniors attending educational program than between subjects from a nursing home (p<0.05) and among women compared to men (p<0.05). Only 25% percent of the subjects were in use of specific treatment for osteoporosis and were taking calcium supplements. Calcium supplements use was more prevalent among women (30%) than men (6%; p<0.01). Our results demonstrate a high level of awareness and accurate definition of osteoporosis in a Brazilian cohort of elderly people. The study also shows that educational programs can provide specific information for elderly people that might influence the management of osteoporosis. Specific therapy and prevention measures for osteoporosis were inappropriately low for this group of subjects at high risk of osteoporosis.

Disclosures: C.H.M. Castro, None.


Response of Cancellous Bone to Estrogen Depletion in Three Strains of Mice.U. T. Iwaniec, D. Yuan*, R. A. Power, T. J. Wronski. Physiological Sciences, University of Florida, Gainesville, FL, USA.

The purpose of this study was to characterize the skeletal response to estrogen depletion (ovariectomy) in three strains of mice (129P3, C57BL/6, and B6129PF2) commonly used in transgenic and/or knockout studies. The mice were ovariectomized (ovx) or sham-operated at 4 months of age. At this time, femora were collected from baseline control groups to determine strain-specific bone mass and turnover prior to estrogen depletion. The remaining mice were sacrificed at 1 or 3 months postOVX. Distal femora were processed undecalcified for collection of histomorphometric data, including cancellous bone volume, and osteoclast, osteoblast, and osteoid surfaces. The data are expressed as mean ± SD.

The 3 strains of mice differed in cancellous bone mass at baseline. Cancellous bone volume (BV/TV) was significantly greater in 4 month old 129P3 (12.9±4.0%) mice than in 4 month old C57BL/6 (5.2±1.3%) and B6129PF2 (5.5±1.4%) mice. Differences in osteoclast surface (Oc.S) were not detected among the 3 murine strains at baseline. However, osteo-blast surface (Ob.S) and osteoid surface (OS) tended to be greater (P<0.1) in C57BL/6 and B6129PF2 mice than in 129P3 mice, suggesting increased bone turnover in the former 2 strains. Ovariectomy decreased bone volume and there was also a significant strain by ovx interaction. At 1 month postOVX, BV/TV was approximately 40% (P<0.05), 50% (P0.1) lower in ovx 129P3, C57BL/6, and B6129PF2 mice compared to age-matched sham mice, respectively. At 3 months postOVX, BV/TV was approximately 60% (P<0.05), 40% (P>0.1), and 60% (P>0.1) lower in ovx 129P3, C57BL/6, and B6129PF2 mice compared to age-matched sham mice, respectively. Although Oc.S, an index of bone resorption, increased with ovx and there was a significant strain by ovx interaction, Oc.S was significantly higher in ovx 129P3 mice than in sham 129P3 mice at 1 month postOVX only and significant differences in Oc.S were not detected between ovx and sham C57BL/6 or B6129PF2 mice. Ob.S and OS also tended to increase with ovx by approximately 20%. However, there was no significant strain by ovx interaction and significant differences in Ob.S and OS following ovx were not detected in any of the 3 strains. In conclusion, 129P3 and C57BL/6 mice lose bone following ovx, while only a trend for postOVX bone loss occurs in the B6129PF2 strain. In comparison to mice, rats ovx at 3 months of age lose approximately 80% of cancellous bone in the proximal tibia in the first 3 months postOVX and exhibit a 7-fold increase in Ob.S at 5 weeks postOVX (Wronski et al., CTI 43:179, 1988). Therefore, estrogen depletion appears to induce a greater increase in cancellous bone turnover in the rat skeleton than in the mouse skeleton.

Disclosures: U.T. Iwaniec, None.


Structure Analysis of the Distal Radius Using the Scaling Index Algorithm in the Prediction of Osteoporotic Spine Fractures.D. Mueller*1, T. M. Link1, R. Monetti*2, J. Bauer*1, E. J. Rummeny*1, C. W. Raeth*2. 1Department of Radiology, Technische Universitaet Muenchen, Muenchen, Germany, 2Institut fuer extraterrestrische Physik, Max-Planck-Society, Garching, Germany.

Purpose: In this study we are using a newly developed 3D-based scaling index method in comparison with standard 2D-techniques to analyze High resolution magnetic resonance (HR-MR) images of the distal radius to investigate the trabecular structure in patients with and without osteoporotic spine fractures.

Methods and materials: Axial HR-MR images of the distal radius were obtained at 1.5 T in 46 women (22 postmenopausal women with osteoporotic spine fractures and 24 post-menopausal controls). A three-dimensional gradient-echo sequence was used with a slice thickness of 500 μm and in plane spatial resolution of 195×195 μm. Structure analysis was performed using algorithms based on a local 3D scaling index method as well as morphological 2D parameters. In addition BMD measurements of the spine using quantitative CT (QCT) and dual energy X-ray absorptiometry (DXA) were obtained in all patients.

Results: Significant differences between both patient groups were obtained using structure analysis and spine BMD (p<0.05). Receiver operating characteristics (ROC) analyses were used to determine the diagnostic performance in differentiating both groups. In comparison with BMD of the spine (Area under curve (AUC) =0.72) and the 2D based structure parameters (AUC up to 0.70) the best results were found for the local 3D scaling index method (AUC=0.85, Fig. 1).

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Conclusion: The results of our study show that trabecular structure measures derived from HR-MRI of the radius using a newly developed algorithm based on a local 3D scaling index method can improve the diagnostic performance in differentiating postmenopausal women with and without osteoporotic spine fractures.

Disclosures: D. Mueller, None.


Bone Material Properties and Hip Fracture: Regional Decreases in Hardness and the Association with Bone Remodelling.E. Follon*, N. Loveridge, J. Power*, B. Bonfield*. University of Cambridge, Cambridge, United Kingdom.

Although differences in bone mass and structure between cases of intracapsular hip fracture and controls have been documented, there is little data on the material properties of the femoral neck bone or its relationship to either habitual load or the degree of bone remodelling. A micro-indentation technique was used to determine the regional hardness of the femoral neck cortex in cases of hip fracture (3F 1 M; ages 60--95y) in comparison with age/gender matched controls (4F, 1M; 52--91y).

After embedding, biopsies were ground and polished then indented using a Mitutoyo MVK-H2 microhardness tester with a Vickers tip. A 50g load was applied for a period of 10s. Each section was indented 200 times, with 50 indents in each of the four regions (anterior (A), inferior (I), posterior (P), and superior (S)); 30 in osteonal bone and 20 in interstitial bone. Data were converted to GPa and analysed using regression modelling in which region, bone type (interstitial/osteonal) and case/control status were considered as independent variables. Cortical remodelling was assessed on histological sections as the proportion of canals with osteoid (forming) or crenellated (resorbing) surfaces.

Interstitial bone was harder than osteonal bone (+10%; p<0.0001) and hardness was related to the proportion of canals with new bone formation (Osteonal: GPa=0.63--0.025*ln%osteoid; Adj.r2=0.20, p=0.005; Interstitial: GPa=0.7--0.026*ln%osteoid; Adj.r2=0.22, p=0.003). There were marked regional differences in the hardness of both osteonal and interstitial bone (Osteonal: P: 0.61±0.011 and I: 0.60±0.010 > A: 0.56±0.008 and S: 0.55±0.006. Interstitial: P: 0.68±0.011 and I: 0.66±0.009 > A: 0.062±0.007 and S: 0.62±0.004). Fracture cases had significantly lower hardness values in the anterior (-3.9%, p=0.005) and inferior (-2.5% p=0.049) regions for osteonal bone and in the anterior region (-5%, p=0.0005) for interstitial bone. After adjustment for the indices of remodelling the case-control differences were non-significant but the regional differences remained.

In conclusion, there are marked variations in the hardness of bone in the femoral neck, both between osteonal and interstitial bone and between cortical regions subjected to varying habitual strain modes. Bone habitually loaded in compression (I) was harder than that loaded in tension (S), and interstitial bone was harder than osteonal bone. Finally, hardness was lower in the anterior regions of the cases of hip fracture for both bone types, and lower in the inferior for osteonal bone, associated with increased remodelling.

Disclosures: E. Follon, None.


Periosteal Bone Turnover at the Femoral Neck in Non-Human Primates.M. Bliziotes1, J. Sibonga2, R. Turner2, E. Orwoll1. 1Oregon Health and Science University, Portland, OR, USA, 2Mayo Clinic, Rochester, MN, USA.

Bone size is an important determinant of bone strength, and modeling at the periosteal surface alters bone dimensions in adults as well as during growth. Nevertheless, periosteal biology remains poorly understood.

Therefore, the proximal femurs of 16 adult non-human primates (Rhesus (Macaca mulatta, n=9) and Japanese Macaque (Macaca fuscata, n=7)), were analyzed after double-labeling with two discrete intervals of tetracycline (250 mg bid for 3 days separated by a 14 or 15 day interlabeling period). Necropsy was performed 2--7 days following the last administration of tetracycline. Histomorphometric measurements of fluorochrome labels and of stained sections complied with standardized procedures, formulae and nomenclature. Statistical comparisons between multiple groups were analyzed by 1-way ANOVA followed by a Fisher PLSD post-hoc test after determination of significance.

Both bone resorption and formation were present on the femoral neck periosteal surface. Multinucleated, acid phosphatase positive osteoclasts were present in typical Howship's lacunae. This osteoclastic activity was not the result of the emergence of intracortical tunneling at the bone surface. The periosteal eroded surface of the femoral neck was significantly greater than in the marrow compartment (p<0.0001) or on the femoral shaft periosteum (p<0.0001), although osteoclast number was similar at all three surfaces. Mineral apposition rate on the periosteal surface of the femoral neck was greater than on the shaft but less than on cancellous surfaces. Femoral neck periosteal bone formation rate was 2.5% of tissue volume per year, approximately 10-fold more than observed in the femoral shaft.

Thus, not only have we documented intramembranous periosteal bone formation in the femoral neck in a series of non-human primates, we have also found that the tissue level bone formation rate was sufficient to add substantively to femoral neck size over time. We also documented active periosteal bone resorption at the femoral neck. The turnover rate in the femoral neck periosteum was intermediate between femoral shaft periosteum and femoral neck cancellous bone. In conclusion, bone formation and resorption are active in the femoral neck periosteum and have the potential to exert meaningful effects on femoral neck size. The modeling rate at the femoral neck is different from femoral shaft periosteal and cancellous bone sites, suggesting distinct regulatory influences.

Disclosures: M. Bliziotes, None.


Regional Trabecular Anisotropies Suggest a ‘Two-Domain’ Loading Regime in the Proximal Femur.J. G. Skedros. Ortho, U of Utah, Salt Lake City, UT, USA.

Conventional wisdom teaches that the human proximal femur is adapted for transmission of tension/compression stresses associated with habitual bending. This appears to be embodied as arched trabecular patterns. However, recent 3-D finite-element analyses (FEAs) of physiologic loading suggest an alternative interpretation for stress transfer across this region [Lotz et al., 1995, Osteopor. Int.]. These analyses suggest that the ‘expanded’ trochanteric region allows for the appropriate mechanical advantage and attachment site for muscles rather than for structural integrity of the bone itself. Studies of patterns of predominant collagen fiber orientation (CFO) in cortical bone suggest that prevalent bending occurs in the subtrochanteric region, and prevalent torsion/compression at mid-neck. These data suggest that the trochanteric region may separate two loading ‘domains’ (subtrochanteric and femoral neck (FN)), each with important differences in stress distribution and transfer. This issue is relevant in understanding normal loading conditions across the hip and bone mass/quality changes that occur with age and implantation of intramedullary prostheses, and may have implications for age-related changes in the prevalence of FN vs. intertrochanteric fractures. Rigorously examining this ‘two-domain’ hypothesis is difficult because it requires the application of in vivo strain gauges. In this study we examined this question using patterns of trabecular architecture. 5mm thick sections of 15 Caucasian human femora (age range 20--70) were obtained in the plane of ante-version. Using radiographs of each specimen, obvious arched trabecular tracts were traced in the FN and lesser trochanteric (LT) regions. Cartesian data of each tract (superior & inferior in FN; lateral & medial in LT) were fit to linear or non-linear equations (r⁁2 > 0.95). Intersection angles at ‘arch’ apices were measured. Results (Table) demonstrate that the LT trabecular tracts are symmetric, and FN tracts are non-symmetric. The LT region exhibits intersections that are nearly orthogonal, in contrast to the non-orthogonal intersections in the FN (p<0.05). These data are consistent with the ‘two-domain’ hypothesis, as suggested previously by CFO data and FEAs showing that non-orthogonal, non-symmetric trabecular tracts are optimal for habitual torsion [Pidaparti and Turner, 1997; J Biomech]. 

Table  .  
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Disclosures: J.G. Skedros, None.


Structural and Biomechanical Basis for Differences in Vertebral Fragility in Chinese and Caucasians.Y. Duan1, X. Wang*1, C. H. Turner2, C. Fong*1, E. Seeman1. 1Endocrinology, Austin & Repatriation Medical Centre, The University of Melbourne, Melbourne, Australia, 2The Biomechanics and Biomaterials Research Centre, Indiana University, School of Medicine, IN, USA.

We hypothesized that the structural abnormalities predisposed to vertebral fracture are similar in Chinese and Caucasians, accounting for the similar vertebral fracture rates between races. We studied 687 healthy Chinese (449 females) and 1088 healthy Caucasians (738 females) aged 18 to 92 yrs. Vertebral body (VB) cross-sectional area (CSA) and volumetric BMD (vBMD, excluded posterior elements) were measured using dual x-ray absorptiometry by postero-anterior and lateral scanning. We calculated VB stress (load/ CSA) and FRI (load/strength) during bending forward. In young adulthood, VB stress did not differ by race in either sex because the lower load (10--14%) in Chinese was distributed on a proportionately lower CSA (13--14%) than in Caucasians. However, vBMD was 9--13% higher in Chinese than Caucasians, conferring 12--19% lower FRI in Chinese men and women. Ageing was associated with increased CSA in both Chinese and Caucasian men and women. However, racial differences in periosteal expansion were minimal, increasing by 8.7% and 11.8% in elderly Chinese and Caucasian men, and increasing by 8.6% and 5.7% in elderly Chinese and Caucasian women (both no significant different to each other). VB stress decreased similarly in Chinese and Caucasian men (13.3% vs 13.7%) but decreased more in Chinese than Caucasian women (10.0% vs 5.5%, p < 0.01). Net decline in vBMD was greater in elderly Chinese than Caucasian women (33% vs 27%, p < 0.01) but similar in Chinese and Caucasian men (11% vs 12%). These structural changes were captured by FRI; a similar proportion of elderly Chinese and Caucasians men (5% vs 6%) and women (25% vs 29%) had the FRI >= 1. The results are consistent with the notion that vertebral fractures occur more commonly in women than in men but similar proportions of Chinese and Caucasians (of either sex) sustain fractures.

Disclosures: Y. Duan, None.


Structural Basis for Differences in Femoral Neck Fragility in Chinese and Caucasians.X. Wang*1, Y. Duan1, T. J. Beck2, E. Seeman1. 1Endocrinology, Austin & Repatriation Medical Centre, The University of Melbourne, Melbourne, Australia, 2Radiology, The Johns Hopkins University, School of Medicine, Baltimore, MD, USA.

We hypothesized that structural characteristics may be better maintained in Chinese than Caucasians in old age, accounting for the lower hip fracture rates reported in epidemiological studies. A faster rate of periosteal apposition maintains bending strength, while a slower rate of periosteal expansion with a slower rate of endocortical resorption should reduce the increased risk of buckling with age. We measured femoral neck (FN) dimensions and bone mass using DXA, estimated endocortical diameter, cortical thickness, section modulus (a measure of bending strength), and buckling ratio (subperiosteal radius/ cortical thickness) in 738 Chinese (490 females) and 1181 Caucasians (788 females) aged 18 to 93 years. In young adult women, after adjusting for racial differences in height and weight, FN axis length and diameter remained 4--8% lower in Chinese, while cortical thickness and vBMD were no different by race. Thus, growth produced racial differences in FN geometry; the same cortical thickness was distributed further from the FN neutral axis conferring 22.3% greater bending strength in Caucasians than Chinese. However, buckling ratio was 5.2% lower in Chinese than Caucasian women. In young adult men, bending strength was 12.5% lower while buckling ratio was no different in Chinese compared to Caucasians. From young (∼30yrs) to old age (∼70yrs), FN periosteal diameter (height and weight adjusted) increased less in Chinese than Caucasian men (1.0% vs. 9.1%), but increased similarly in Chinese and Caucasian women (4.6% vs. 3.3%). Endocortical diameter also increased less in Chinese than Caucasian men (2.6% vs. 12.5%), but similarly in Chinese and Caucasian women (8.5% vs. 6.5%). Consequently, bending strength decreased by 6.9% in Chinese men but maintained in Caucasian men, while bending strength decreased similarly in Chinese and Caucasian women (4.0% vs 6.9%). Buckling ratio increased less in Chinese than Caucasian men (14.5% vs 28.4%) but increased similarly among Chinese and Caucasian women (28.8% vs 31.2%). These changes produced 17.4--25.0% lower bending strength and 6.9--8.7% lower buckling ratio in elderly Chinese than Caucasians in both sexes. We concluded that despite the smaller FN diameter and lower bending strength, the relatively thicker cortex and narrower diameters in elderly Chinese suggest a lower risk of structural failure by local buckling than Caucasians. These structural differences in Chinese and Caucasians are likely to be established during both growth and aging.

Disclosures: Y. Duan, None.


Longitudinal Changes in 3D Microarchitecture of Human Iliac Crest Bone Biopsies: Transmenopausal versus Postmenopausal.J. J. Zhao1, Y. Jiang1, R. R. Recker2, M. W. Draper3, E. F. Eriksen3, H. K. Genant1. 1Osteoporosis and Arthritis Research Group, University of California, San Francisco, CA, USA, 2Osteoporosis Research Center, Creighton University, Omaha, NE, USA, 3Lilly Research Laboratories, Indianapolis, IN, USA.

This study compares true longitudinal transmenopausal (TransM) changes with longitudinal postmenopausal osteoporotic (PostM) changes in 3D trabecular (Tb) structure. This may improve our ability to understand the pathophysiology of osteoporosis and other bone disorders, and to estimate bone biomechanical properties in terms of fracture resistance given that the mechanical competence of Tb bone is a function of its apparent density and 3D distribution. During aging and osteoporosis, Tb plates are perforated and connecting rods are dissolved, with a continuous shift from one structural type to the other, which cannot be evaluated by 2D histological sections. There is debate among histomorphometrists about whether Tb thinning, or rather Tb disappearance occurs with aging and/or menopause based on 2D sections using the parallel plate model. We examined 39 paired iliac crest bone biopsies (78 specimens). For 20 TransM women, the 1st biopsy was from normal, premenopausal women, mean age 49 (±SD, ±3) years (yrs), and the 2nd biopsy from the same group of women, but 1 yr postmenopausal, occurring 5 (±2) yrs) after the 1st biopsy. From 19 PostM women with osteoporotic vertebral fractures and the hip/lumbar BMD at least 1 SD below the mean value in normal young women, the 1st biopsy was taken at age 68 (±7) yrs, 20 (±9) yrs since menopause, while the 2nd was 19 (±5) months later. The specimens were scanned using a Scanco micro CT with isotropic resolution of 17 μ. 3D Tb structural parameters were directly measured without stereological model assumption. Structure model index 0 represents an ideal plate structure and 3 represents rod structure. There was a significant change between paired bone biopsies in 3D Tb bone volume fraction (TransM versus PostM, -4.0%/yr vs. -3.3%/yr), Tb number (-1.2%/yr vs. -0.1%/yr), Tb thickness (-2.7%/yr vs.-0.5%/yr), Tb separation (2.4%/yr vs. 0.5%/yr), structure model index (8.0%/yr vs. 4.6%/yr), degree of anisotropy (-0.6%/yr vs. -0.1%/yr), and connectivity density (-1.1%/yr vs. -9.2%/yr). The percentage change was greater in 3D Tb thickness than in Tb number and Tb separation. Thus, 3D Tb microstructure rapidly deteriorates in the iliac crest, which is more pronounced in TransM women than in PostM women with osteoporotic fracture, while loss in Tb connectivity density is much greater in PostM women than in TransM women. Tb thinning does occur and trabeculae dramatically shift from a plate-like structural type to a rod-like pattern, and become more isotropic.

Disclosures: J.J. Zhao, None.


Performance of In Vivo Micro-CT Analysis of Mouse Lumbar Vertebral and Knee Trabecular Bone Architecture.P. L. Salmon, E. Buelens*, A. Sasov*. Skyscan N.V., Aartselaar, Belgium.

The knee and lumbar vertebra are commonly studied trabecular bone sites in mice. There are obvious advantages to in vivo microCT analysis of trabecular bone, such as the possibility of sequential analysis of a bone site in a single animal. However there are several factors limiting the quality of microCT images of trabecular bone obtainable in vivo. Xray image contrast is reduced by the presence of surrounding soft tissue causing additional attenuation. The source and detector geometry imposed by an immobile subject limits pixel sizes attainable compared to ex vivo microCT systems. The need to rotate the source and detector array, and small animal movements during scanning, further limit image resolution. Bone morphometric parameters measured by microCT are presented for knee and vertebra both in vivo and ex vivo, using the Skyscan 1076 and 1072 scanners respectively. Measurements of aluminium phantoms are also presented. Sensitivity of morphometric parameter measurement to factors such as pixel size and thickness of surrounding x-ray absorbing material is assessed. Different histomorphometric parameters show different sensitivity to varying image resolution and contrast. Basic structural parameters such as bone volume and surface are relatively less sensitive. However parameters indicating connectedness or dissociation of structures, such as trabecular pattern factor and Euler connectivity, are acutely sensitive to image resolution. We demonstrate that despite the above mentioned constraints on in vivo microCT image resolution, useful and representative architectural parameters of mouse trabecular bone can be obtained by microCT in vivo, with pixel size of 8.9 micron and 10 percent MTF resolution below 15 micron.

Disclosures: P.L. Salmon, Skyscan N.V. 3.


Sex and Strain-Dependent Variation in Vertebral Cortical and Trabecular Bone Traits in Inbred Mice.M. L. Bouxsein1, M. Russ*1, M. Shea2, E. S. Orwoll2, R. F. Klein2. 1Orthopedic Biomechanics, Beth Israel Deaconess Med Ctr, Boston, MA, USA, 2Oregon Health & Sciences University, Portland, OR, USA.

Bone density and structure are strongly influenced by genetic factors. To date, most genetic studies have assessed BMD, bone size or strength characteristics that reflect either cortical bone, or a combination of cortical and trabecular bone, with few focusing exclusively on genetic determinants of trabecular bone density. We previously evaluated female mice from the C57Bl/6J-C3H/HeJ F2 intercross to show that vertebral trabecular bone traits are highly heritable, and we identified several QTL associated with these traits. In this study we used μCT to evaluate sex- and strain-dependent differences in vertebral trabecular and cortical bone traits in 4 mo-old male (M) and female (F) mice from 8 inbred strains: 129S1, Castaneus (Cast), BALB/cByJ (Balb/c), C57Bl/6J (B6), C3H/HeJ, DBA/2J (D2), A/J and AKR/J (n=13--20/gr). Narrow-sense heritability estimates for weight-corrected BV/TV, trabecular number (TbN) and separation (TbSp) were high in both M and F (H2 = 0.74 to 0.83), but somewhat lower for trabecular thickness (TbTh, H2 = 0.33 for M, 0.53 for F). Effects of strain, sex, and the interaction of strain x sex were highly significant for all vertebral traits (p<0.0001 for all). For example, trabecular BV/TV was highest in 129S1 (33.1±1.3%), intermediate in A/J, AKR, Balb/c, B6 and DBA (22 to 28%) and lowest in Cast (16.5±0.6%)(p<0.0001 for strain). Interestingly, in 6/8 strains, TbN was higher in M than F (p<0.0001), whereas TbTh was consistently higher in F (7/8 strains, p<0.0001). Connectivity varied over 3-fold between strains, and was on average, 21% higher in M (6/8 strains, p<0.0001). Trabecular bone surface to volume ratio was 50% greater in Cast than in AKR and Balb/c (p<0.0001), with M having, on average, 14% higher values than F (7/8 strains, p<0.0001). The thickness of the anterior cortex was highest in AKR (150±2.3 μm) and lowest in Cast (59±0.9 μm), with F having consistently higher values than M (7/8 strains, p<0.0001). Across strains there was no correlation between cortical thickness and trabecular traits. These data confirm strong genetic regulation of vertebral bone density and architecture in inbred mouse strains, with pronounced sex-specific patterns for trabecular microarchitecture. Additional studies of the genetic and sex-specific factors regulating vertebral morphology may provide key information regarding the etiology of vertebral fragility fractures.

Disclosures: M.L. Bouxsein, None.


Effects of Physical Activity and Lifestyle on Evolution of Hip Bending Resistance in Men and Women over 65: The Population-Based EPIC-Norfolk Cohort Study.S. Kaptoge1, N. Dalzell*1, R. Jakes*1, N. Wareham*1, K. Khaw*1, T. Beck2, J. Reeve1. 1Cambridge University, Cambridge, United Kingdom, 2Johns Hopkins University School of Medicine, Baltimore, MD, USA.

That a bone's fracture resistance is more directly related to its resistance to bending than to its areal bone mineral density (BMD) follows from the mechanical principles developed by Galileo and Euler. BMD is used in the investigation of hip fracture without regard to its relationship to bending resistance but little is known of the determinants of changing bending resistance in the proximal femur.

We analyzed data from years at recruitment to test the effects of aging, anthropometry and physical activity on section modulus (SM), a measure of bending resistance, and BMD. All subjects (680M, 666F, 67--76y) had hip Hologic 1000W DXA scans taken at baseline and at 2.9 years (476M, 446F) and 5.4 years (343M, 314F) later. Hip structural analysis was used to calculate BMD and SM on 3 regions; narrow neck (NN), intertrochanter (IT) and shaft (S). Linear mixed modeling was used to test effects of the predictors on both outcomes while adjusting for correlation between regions. Interactions with region and age were tested to determine if the effect of a predictor was similar in all regions and if it modified the effect of aging.

There was significant between-region correlation of outcomes in both genders (coefficients: 0.68 -- 0.84 for BMD, 0.46 -- 0.62 for SM). Hypothesis tests accounting for this between-region correlation indicated that the effects of most predictors varied significantly between regions. SM in women increased with increasing age, weight, height and grip strength and all had a significant interaction with region (P < 0.003). Weekly recreation time (WRT) and lifetime physical activity score were positively associated with SM and the coefficients were similar in the 3 regions (P < 0.015). SM was lower in women with history of any fracture and differences varied by region (P<0.0001). An interaction between age and weight indicated that low body weight was associated with lower SM that increased faster with age (P = 0.001). The principal discordance seen in contrasting BMD with SM was that BMD declined instead of rose with age, due to subperiosteal expansion. In men SM increased with aging, weight, height and lifetime physical and sporting activity scores; the size of effect differed by region (P < 0.029). WRT had a positive effect on SM that was similar in the 3 regions (P=0.011).

We conclude that there are important effects of aging, physical activity and anthropometric variables on the evolution of hip bending resistance in men and women. The extent to which BMD can be validly used as a surrogate for SM in the epidemiology of hip fractures remains to be determined.

Disclosures: S. Kaptoge, None.


Impact of Spinal Mobility on Quality of Life in Patients with Postmenopausal Osteoporosis.N. Miyakoshi1, E. Itoi*1, M. Kobayashi*1, H. Kodama*2. 1Orthopedic Surgery, Akita University School of Medicine, Akita, Japan, 2South Akita Orthopedic Clinic, Akita, Japan.

Previous studies have shown that progression of spinal osteoporosis with vertebral fractures results in a progressive decline in the quality of life (QOL). It has also been reported that patients with osteoporotic spinal deformities have a markedly limited range of motion (ROM) in lumbar extension. However, it has not been documented whether or not spinal mobility in patients with osteoporotic spinal deformities affects the QOL. The objective of the study was to evaluate the impact of spinal mobility on the QOL in patients with spinal osteoporosis. A total of 157 postmenopausal women with osteoporosis aged over 60 years was divided into five groups according to their spinal deformities: round back (RB, n=41), hollow round back (HRB, n=33), whole kyphosis (WK, n=40), lower acute kyphosis (LAK, n=18), and normal posture (NP, n=25). The QOL was evaluated using the Japanese Osteoporosis QOL Questionnaire (JOQOL) proposed by the Japanese Society for Bone and Mineral Research. This questionnaire contains six domains with higher scores indicating higher levels of QOL. The number of vertebral fractures, angles of thoracic kyphosis and lumbar lordosis, and spinal ROM during maximum flexion and extension were measured with radiographs. The total QOL score in RB, HRB, WK, and LAK groups was significantly lower than that in NP group (p<0.05). Among the groups with spinal deformities, WK group showed the lowest QOL score (p<0.05). All the groups with spinal deformities, but not NP group, showed significant positive correlations between the QOL and spinal ROM (0.52r>0.75, p<0.05). In total of 157 patients, the total QOL score showed significant correlations with age, number of vertebral fractures, lumbar lordosis angle, and spinal ROM. Multiple regression analysis revealed that the spinal ROM best correlated with the total QOL score. We conclude that the QOL in patients with osteoporosis is impaired not only by the spinal deformities, but also by the spinal mobility.

Disclosures: N. Miyakoshi, None.


Binge-Like Alcohol Exposure of Adult Male Rats Alters Bone Collagen Degradation and Bone Mineral Density in Femur and Lumbar Spine: Effects Abolished by Risedronate.J. J. Callaci*, D. Juknelis*, N. Frost*, F. H. Wezeman. Orthopaedic Surgery, Loyola University Medical Center, Maywood, IL, USA.

Chronic alcohol consumption reduces bone mass and strength, increasing fracture risk for alcohol abusers. Mechanisms underlying this vulnerability involve modulation of bone remodeling. Direct effects of alcohol on bone formation are documented, those on bone resorption are less well studied. The skeletal effects of exposure to high blood alcohol concentrations (BAC's) attained during binge drinking have not been studied to date. In this report we examine the effects of repeated high dose alcohol treatment on bone collagen degradation, a marker of bone resorption and bone mineral density in an adult rat model of binge-like alcohol exposure. Intraperitoneal (IP) injections were used to administer a 20% (vol/vol) ethanol/saline solution (3g/kg, 1X/day) to rats on four consecutive days for 1, 2 or 3 weeks. Effects of treatment with the anti-resorptive agent risedronate (35 mg/kg, 1X/ week) were also evaluated. Peak BAC's averaged 308.5 + 12 mg/dL, with an average BAC of 258.6 + 28.7 mg/dL at time of euthanasia. No significant effects of treatment were observed after 1 or 2 weeks of binge alcohol exposure. After 3 weeks of binge-like alcohol treatment, total serum deoxypyridinoline (Dpd) a crosslink of bone type I collagen released during the resorption process was significantly increased (205%, p < 0.05) over control levels. Bone mineral density (BMD) in cancellous bone of the distal femur and lumbar spine were significantly decreased (34 and 21% respectively, (p < 0.01) also after 3 weeks of alcohol binge treatment. Significant effects of risedronate treatment were noted in reversing both ethanol-induced Dpd increases (p < 0.01) and BMD decreases (p < 0.001) of binge-treated rats to control levels. These findings suggest that BAC's attained during alcoholic binge drinking may effect the skeleton in part by stimulating bone resorption, an effect that is mitigated by risedronate.

Disclosures: J.J. Callaci, None.


Factors Responsible for Pain in Osteoporosis and Degenerative Joint Disease Based on Electroalgometry Using Fall of Skin Impedance in Response to Exercise Load.T. Fujita1, M. Ohue*1, S. Nakamura*1, Y. Fujii2, A. Miyauchi3, Y. Takagi3. 1Katsuragi Hospital, Kishiwada, Osaka, Japan, 2Calcium Research Institute, Kishiwada, Osaka, Japan, 3National Sanatorium Hyogo Chuo Hospital, Hyogo, Japan.

Accuracy and efficiency of electroalgometry (EAM) measuring skin impedance was improved by using portable Prep Check Electrode Impedance Meter Model EIM 105-T1 (General Devices, NJ) and Vitrode L (Nihon Kohden, Tokyo). Measurement range was 100 to 2000 k ohms with an accuracy of ± 3 ohms using 9μA, 5Hz test current. Seasonal variation was minor and insignificant in an air-conditioned test room. Basal impedance tended to rise with age in both males and females probably reflecting drying and coarsening of the skin, but the response to exercise load was independent of the basal value. Responses as pain expressed by visual rating scale (VRS) and fall of skin impedance measured by EAM were significantly correlated on knee extension and flexion (knee pain), spine extension and flexion (back pain) and walking (overall pain). Back pain expressed in VRS and EAM was significantly correlated with relative cortical volume in radius measured by pQCT P=0.0007 and 0.0368) but not with lumbar bone mineral density (LBMD) measured by DXA, radial trabecular BMD measured by pQCT, degree of spondylotic changes in X-ray and intra-individual coefficient of variation of LBMD used as a measure of spondylotic deformity. Spinal compression fracture was significantly correlated with back pain expressed in EAM (p= 0.0214) but not subjective pain expressed in VRS. Back pain induced by spinal extension and flexion appears to be mainly influenced by vertebral instability dependent on the strength of cortical bone, the major supporter of the skeletal resistance to external force. Back and joint pain in osteoporosis and osteoarthritis improved in response to bisphosphonates, active absorbable algal calcium (AAA Ca) and collagen, probably through improvement of spinal stability by inhibition of osteoclastic and chondroclastic resorption and collagenase activity, according to the sensitive and objective pain measurement by elctroalgometry utilizing the fall of skin impedance as an index.

Disclosures: T. Fujita, None.


Osteoporosis in Melanin Concentrating Hormone Receptor 1 (MCHR1) Deficient Mice.M. Bohlooly-Y*1, M. Mahlapuu*1, H. Andersén*1, A. Åstrand*1, S. Hjorth*1, L. Svensson*1, J. Törnell*1, M. R. Snaith*1, D. G. Morgan*1, C. Ohlsson2. 1AstraZeneca R&D Mölndal, Mölndal, Sweden, 2Centre for Bone Research at the Sahlgrenska Academy (CBS), Department of Internal Medicine, Göteborg University, Göteborg, Sweden.

Osteoporosis is a metabolic bone disease characterized by low bone mass, leading to an increase in fracture risk. However, the mechanisms that lead to osteoporosis are still poorly understood. Recent studies indicate that the hypothalamus plays an important role in the regulation of bone mass. Melanin concentrating hormone (MCH) is highly expressed in the hypothalamus and has been implicated in the regulation of energy homeostasis (MCH). We investigated if MCH receptor 1 (MCHR1) -signalling is involved in bone homeostasis. Here we present data demonstrating that MCHR1-signalling is involved in a tonic stimulation of bone mass. Mice with an inactivated MCHR1 had osteoporosis, caused by a clear reduction in the cortical bone mass due to decreased cortical bone thickness. Serum levels of c-telopeptide, a marker of bone resorption, were increased in MCHR1−/− mice, demonstrating that the MCHR1−/− mice have a high bone turnover osteoporosis. We conclude that the MCHR1 is a novel drug target for osteoporosis.

original image

Disclosures: C. Ohlsson, None.


Six Minute Walk Test: A New Powerful Predictor of Functional Dysfunction in Osteoporotic Patients with and without Vertebral Fractures.J. T. Lin*1, S. Backus*2, E. Myers*3, M. Peterson*4, J. M. Lane5. 1Physiatry/Metabolic Bone, Hospital for Special Surgery, New York, NY, USA, 2Rehabilitation Medicine, Hospital for Special Surgery, New York, NY, USA, 3Biomechanics, Hospital for Special Surgery, New York, NY, USA, 4Statistics, Hospital for Special Surgery, New York, NY, USA, 5Orthopedics/Metabolic Bone, Hospital for Special Surgery, New York, NY, USA.

Osteoporotic patients with vertebral compression fractures (VCF) are known to have increased morbidity and mortality. While it is recognized that VCF impair function, specific functional deficits have not been clearly identified. The purpose of our study was to identify functional deficits using kinesiologic tests performed in the motion analysis laboratory. 89 osteoporotic patients both with and without VCF underwent functional evaluation in the motion analysis laboratory, including timed up and go, functional reach, six minute walk, and bilateral single limb testing. Age of patients and number of compression fractures was recorded. Patients without VCF (n=41) versus patients with VCF (n=48) had an average age of 74.58 ± 8.86 vs. 73.39 ± 6.99 years, functional reach of 11.68 ± 2.94 vs. 10.81 ± 2.60 inches, six minute walk of 388.03 ± 127.99 vs. 315.20 ± 140.89 meters, average right single limb stance of 16.12 ± 19.26 vs. 15.27 ± 18.14 seconds, and average left single limb stance of 16.10 ± 18.93 vs. 13.40 ± 16.44 seconds. Patients with VCF versus patients without VCF were found to have statistically significant differences in the ability to perform six minute walk (Levene's equality of variances = 0.332, 2-tailed t test p=0.013). Analysis of our data shows trends suggesting that while all of the kinesiologic tests used may be useful in identifying functional deficits, the 6 minute walk is clearly efficacious. Previous authors have identified other balance instruments such as the get up and go and functional reach tests as useful predictors of functional limitation. Based on our data, we propose that the six minute walk test be used to identify dysfunction in osteoporotic patients. The six minute walk, a test of endurance and balance, may be a better predictor of the ability to perform instrumental activities of daily living such as banking and grocery shopping. Our data did not show direct correlations between number of VCF and functional limitations, suggesting that other factors, such as pain and kyphosis, may play important roles in dysfunction.

Disclosures: J.T. Lin, None.


The Effect of Age on the Expression of Nerve-Related Genes During the Healing of Femoral Fractures in the Rat.M. H. Meyer, W. Etienne*, R. A. Meyer. Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC, USA.

Bone formation to bridge the fracture gap following femoral fracture slows with age in both humans and rats for unknown reasons. Abnormalities in the innervation of the fracture site will slow skeletal healing clinically. We explored whether abnormal nerve behavior in the older rats might contribute to this slowing of skeletal repair. Simple, transverse, mid-shaft, femoral fractures with intramedullary rod fixation were induced in female Sprague-Dawley rats at 6, 26 and 52 weeks of age. At 0, 0.4, 1, 2, 4, and 6 weeks after fracture a bony segment, one-third the length of the femur, centered on the fracture site, including the external callus, cortical bone, and marrow elements, was harvested from each subject. Total RNA was extracted from each segment. RNA was pooled between two rats of the same age and time after fracture. cRNA was prepared and hybridized to 18 Affymetrix U34A microarrays (1/age/time point). Of the 8,700 genes on each array, an average of 3,300 were scored as present. 240 genes were related to neural function. The mRNA expression of most of these responded to fracture. About 70 were increased by fracture, such as galanin (J03624), pleiotrophin (AI102795), and glia maturation factor B (Z11558). 52 decreased after fracture at all three ages, such as synuclein (AF007758), neurogranin (L09119), and neuropeptide Y (M15880). There was some differential effect with age. A few genes, 12, had a stronger response to fracture in young rats than in older rats, such as PCTAIRE 2 (AB005540) and 3 (AB005541) and synaptic scaffolding molecule (AF034863). More genes, 42, had a greater rise after fracture in the older rats than in the younger rats, such as glial-derived neurotrophic factor (L15305), thrombospondin 4 (X89963), and postsynaptic density protein (PSD-95) (M96853). In conclusion, mRNA of genes related to neuronal function is found in bone and in the fracture callus. Most of these genes respond to fracture with altered mRNA gene expression. Differential expression with age may reflect altered nerve cell function at the fracture site that may be related to the slowing of fracture healing.

Disclosures: M.H. Meyer, None.


The Effect of Age on the Expression of Mitochondrial Genes During the Healing of Femoral Fractures in the Rat.R. A. Meyer, M. H. Meyer. Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC, USA.

In both humans and rats, bone formation to bridge the fracture gap after femoral fracture slows with age for unknown reasons. Cellular energy production is important to fracture repair. We had noted earlier transient decreases in the expression of mitochondrial genes following femoral fractures in the rat (JBMR 17 (Suppl. 1): S326, 2002). In this study, we have explored these genes in greater detail. In particular, we asked whether abnormal mitochondrial gene expression might contribute to the slowing of skeletal repair in the older rats. Simple, transverse, mid-shaft, femoral fractures with intramedullary rod fixation were induced in female Sprague-Dawley rats at 6 (young), 26 (adult) and 52 (old) weeks of age. At 0, 0.4, 1, 2, 4, and 6 weeks after fracture a bony segment, one-third the length of the femur, centered on the fracture site, including the external callus, cortical bone, and marrow elements, was harvested from each subject. Total RNA was extracted from each segment. RNA was pooled between two rats of the same age and time after fracture. cRNA was prepared and hybridized to 18 Affymetrix U34A microarrays (1/age/time point). Of the 8,700 genes on each array, an average of 3,300 were scored as present. 74 genes related to mitochondrial function were studied. The mRNA expression of these genes fell into one of three patterns. First, genes located within the mitochondrial DNA showed no decline in gene expression with age in unfractured bone. Fracture induced a moderate decrease in gene expression with recovery to baseline activity at 4 weeks after fracture in young and adult, but no recovery in the oldest rats. Examples are ND1 (M35826) and ND5 (S46798). Second, some autosomal genes for mitochondrial function showed reduced activity in unfractured bone in the oldest rats. Fracture led to a profound transient decrease in mRNA levels with recovery by week 2 in young and adult rats but no change in expression from their low basal values in the oldest rats following fracture. Examples include ATP synthase (D13123) and adenylate kinase 1 (D13376). Third, other autosomal genes for mitochondrial function also had a decrease in mRNA levels with age in unfractured bone. But, for all three ages, fracture caused a decrease in gene activity with subsequent recovery to baseline activity which was slower in the oldest rats. Examples include rhodanese (X56228) and creatine kinase (X59736). In conclusion, mRNA of mitochondria-related genes decrease after fracture. The prolonged down-regulation of some of these genes in the oldest rats may be related to the slowing of fracture healing.

Disclosures: R.A. Meyer, None.


Beta-blocker Use, BMD and Fractures in the Study of Osteoporotic Fractures (SOF).I. R. Reid1, G. D. Gamble*1, A. B. Grey1, D. C. Bauer2. 1Department of Medicine, University of Auckland, Auckland, New Zealand, 2University of California, San Francisco, San Francisco, CA, USA.

The central nervous system has been demonstrated to regulate bone mass in mice, and recent work suggests that this is mediated by beta-adrenoreceptors on osteoblasts (Takeda et al, Cell 111:305, 2002). Beta-blockers have been shown to increase bone mass in mice. Since these agents are widely used therapeutically, it is possible that they may influence fracture epidemiology in humans, and they are a potential therapy for osteoporosis. We have studied their association with BMD and fracture rates in SOF.

Beta-blocker use was recorded at the 4th visit (94--96), and this information was available in 8412 women, of whom 1099 were users. Users had significantly higher weight, less smoking, more thiazide use, more HRT use, less glucocorticoid use, more statin use, and more hypertension than non-users. Total hip BMD at the 4th visit was higher in the beta-blocker users (0.746 vs 0.735 g/cm2, P=0.02), but adjustment for weight alone, or together with these other variables, eliminated this difference (P=0.62). There was no effect of beta-blocker use on loss of hip BMD over a mean follow-up of 4 years (0.69% per yr in users vs. 0.62% in non-users, P=0.25). Os calcis BMD at visit 4 was also higher in those taking beta-blockers (0.385 vs 0.375 g/cm2, P=0.005). Once again, weight-adjustment eliminated this difference (P=0.14).

The prevalences of hip, wrist or any fracture (since age 50 and prior to visit 4) were similar in users and non-users. However, over a mean follow-up of 7yr after visit 4, the incidence of hip fractures was 5.8% and 7.8% for users(n=1028) and non-users(n=6577), respectively, (P=0.03). Adjustment for weight and other factors previously shown to influence hip fracture incidence in this cohort, eliminated this effect (OR 0.95, 95%CI 0.64--1.40). There were 353 incident wrist fractures, but no effect of beta-blocker use was found (adjusted OR 1.30, 95%CI 0.91--1.84). The same pattern was observed for non-vertebral fractures, which were not different between-groups after adjustment for weight.

Thus, there are not major associations between beta-blocker use and either BMD or fracture rates in this cohort. The differences between these findings and those in mice may be related to differences in study design, in doses or durations of drug use, or in selectivity of beta-blockers studied. It is concluded that the use of these drugs is not associated with a reduced risk of osteoporosis in older women.

Disclosures: I.R. Reid, None.


Status of 25(OH)D in Korean Postmenopausal Women and its Relations to the Bone Metabolism.Y. Rhee*1, Y. Kim*1, Y. Won2, S. Lim1. 1Internal Medicine, College of Medicine, Yonsei University, Seoul, Republic of Korea, 2Internal Medicine, College of Medicine, Kwandong University, Seoul, Republic of Korea.

Active vitamin D is one of the key bone-related hormone. Vitamin D deficiency causes osteomalacia, and even its insufficiency would bring irreversible bone loss and increased risk of fracture. We evaluated the concentration of 25-hydroxyvitamin D (25(OH)D) and parathyroid hormone(PTH) level in 172 Koreans postmenopausal women who visited our hospital during October 2001 to March 2002. For further analysis of the factors affecting the vitamin D status and relations with bone mass, Among them, 83 women were studied; demographic characteristics, bone mineral density(BMD), and the bone markers. The mean serum 25(OH)D level was 14.0 ng/ml(34.8 nM). The prevalence of vitamin D insufficient patients defined as 25(OH)D less than 12 ng/ml was 23.8% which is quite high prevalence concerning that of the other countries. Serum 25(OH)D was inversely related to serum PTH level (r= -0.187, p=0.016) and also negatively associated with aging(r= -0.143, p=0.06). Eighty three postmenopausal women had taken the DXA(Hologic QDR 4500A), then they were divided into 3 groups; vitamin D sufficient group(S)[25(OH)D>12 ng/ml], vitamin D insufficient group(I)[12>25(OH)D>6ng/ml], vitamin D deficient group(D)[25(OH)D<6ng/ml]. In the D group, women were older and had significantly lower BMD at the lumbar vertebrae, the femoral neck, femoral trochanter and total hip(p<0.05) than the S group. There were more patients with vertebral fracture in the D group than in I and S group(75% vs. 18.9%, 16.0%, p=0.046). Ten patients with low bone mass had been treated with alfacalcidol for 1 year and BMD increased 5.8%(p=0.04) at the lumbar spine and 2.0%(p=0,04) at the total hip respectively, We found out that the status of vitamin D expressed in 25(OH)D in Koreans postmenopausal women were much worse and were affecting bone negatively. However, we also had the positive result with the 1 year administration of active vitamin D against further bone loss. Therefore we need to be alarmed at this finding since this surely will affect the bone health especially in the elderly and to consider the adequate replacement of vitamin D in these patients.

Disclosures: Y. Rhee, None.


Trabecularization of Femoral Neck Cortex through Fenestration of Osteonal Walls: Association with Endocortical Thinning and Hip Fracture.J. Power*, N. Loveridge, A. Lyon*, J. Reeve. University of Cambridge, Cambridge, United Kingdom.

Femoral neck fractures are associated with increased intra- and endo-cortical remodelling and reduced wall thickness of endocortical packets. Furthermore, giant cortical canals develop in association with clustered intracortical remodelling. We hypothesised that these two characteristic phenomena of disordered remodelling may be driven by the same patho-physiological processes (eg. secondary hyperparathyroidism or mechanical underloading). If this were true they should be statistically associated.

Femoral neck fracture biopsies (female, n=12, mean age±SE = 81.3±1.3) and age/gender matched post-mortem controls (n=12; 81.4±1.8) were fixed and embedded in PMMA. Sections were stained with solchrome cyanine R for quantification of osteoid bearing canals and the extent of endocortical osteoid surface (%OS/BS). Osteonal wall thickness (W. Th.) and endocortical bone packet W.Th. were measured under polarised light. Eroded canals and endocortical surfaces (%ES/BS) were quantified on Goldner's stained sections.

There was a significant correlation between cortical and endocortical %OS/BS over the whole biopsy (P=0.041; Spearman-Rho test) and in the anterior (P=0.026), inferior (P<0.001) and posterior (P=0.047) regional quadrants. For %ES/BS, only in the inferior region was there a significant correlation (P=0.031) between compartments. Endocortical W.Th. data was compared with cortical osteon W.Th. where the canals were catagorised into simple (canal surrounded by a single undisrupted cement line) and composite systems in which the osteon is comprised of at least 2 separate bone packets. Endocortical mean W.Th. (31 microns ± 0.4) was identical to that in composite osteons (31 microns ± 0.9); and this was markedly lower (P<0.0001) than seen in simple osteons (46 microns ± 1.8), however there was no significant case/control difference within the regression model (P=0.33).

In conclusion, remodelling indices significantly associated between the intra- and endo-cortical envelopes are consistent with both being controlled by common regulatory processes. Furthermore, W.Th. of endocortical bone packets was the same as that in composite osteons. These results suggest the following scheme for cortical thinning: remodelling, regulatory osteonal canals merge to form giant canals through osteoclastic fenestration of their walls. Bone formation in these canals is reduced to the level in the endosteum. As the composite canals enlarge and the marrow compartment advances, trabeculae develop out of the residue of cortical bone. Bending resistance declines, while at the same time risk of femoral neck buckling during a fall onto the trochanter increases.

Disclosures: J. Power, None.


The Effects of Hindlimb Unloading and Alcohol on Cancellous Bone Histomorphometry and Gene Expression.T. E. Hefferan, G. L. Evans*, M. Zhang*, A. M. Kennedy*, R. T. Turner. Orthopedic Research, Mayo Clinic, Rochester, MN, USA.

Alcoholism, a known risk factor for osteoporosis, can occur in combination with other risk factors. In this study we examined the effect of disuse and alcohol consumption on cancellous bone. The hindlimb unloading model was used to study 6 month-old intact male Fisher 344 rats for a period of 4 weeks. The treatments were baseline, control or alcohol diet; normal weight bearing, control or alcohol diet; and hindlimb unloaded, control or alcohol diet, with alcohol contributing 35% of the calories. Histomorphometry and molecular biology measurements were done on the tibia and femur metaphyses respectively. A two-way ANOVA was used to determine the effects of unloading and alcohol, as well as the interaction between treatments. Unloading resulted in a significant decrease in bone volume, while double-labeled surface was significantly decreased with unloading and alcohol consumption. Trabecular thickness and number were significantly decreased with unloading, while trabecular separation was increased. Mineral apposition rate was significantly decreased in the unloaded animals with significant interaction between unloading and alcohol. Indices of bone formation, mineralizing surface/bone surface, bone formation rate/bone surface and volume were significantly reduced with both unloading and alcohol treatments. There was a significant interaction between unloading and alcohol. Northern blot analysis and ribonuclease protection assay were used to measure mRNA levels of matrix proteins and cytokines, IL-1β, IL-6, IFN-γ, TGF-βs, and TNF-α. Type I collagen and osteonectin gene expression was significantly decreased with unloading, 4.5 fold and 3 fold. Osteocalcin gene expression was significantly decreased 4 fold with unloading and 2 fold with alcohol and there was a significant interaction between the two variables. IL-1β was significantly increased 0.8 fold with unloading and decreased 1.25 fold with alcohol treatment. In summary, histomorphometric and gene expression analysis show significant detrimental skeletal changes with unloading and alcohol consumption. Specifically, reductions in indices of bone formation were noted with both treatments. However, the detrimental effects of alcohol and unloading were not additive suggesting that they share, in part, common cellular pathways.

Disclosures: T.E. Hefferan, None.


Endothelial Nitric Oxide Synthase Expression Decreases in the Growth Plate but Increases in the Metaphyses of Bones from Lactating Rats in Association with Reduced Endochondral Growth and Increased Resorption and Osteoid Deposition, J. I. Aguirre1, S. U. Igal*1, A. Larsen*1, A. Quiroga*1, N. Lausada*1, M. Petruccelli*1, C. Perfumo*1, S. Wimalawansa2. 1Facultad de Ciencias Veterinarias, Univ. Nacional de La Plata, La Plata, Argentina, 2Endocrinology, Robert Wood Johnson Medical School, New Brunswick, NJ, USA.

Lactation is a physiological condition in which a significant reduction in bone mineral density and loss of cancellous and cortical bone mass are observed. Like postmenopause or after ovariectomy, it represents an estrogen-deficient state due to the absence of estrous cycle. Previous studies demonstrated that estrogen regulates endothelial nitric oxide synthase (eNOS) expression and that nitric oxide is one of the local mediators by which estrogen regulates bone cell activity. Based on this, we hypothesized that the reduction in bone mass that occurs during lactation is mediated, at least in part, by a reduction in eNOS expression in the bone microenvironment as a result of estrogen deficiency. To examine this possibility, we studied bone metabolism in lactating rats by histomorphometry of the distal femur, proximal tibia and the first lumbar vertebra metaphyses and by using biochemical serum markers of bone turnover. In addition, we analyzed the pattern of eNOS expression by immunohistochemistry. Osteocalcin and crosslaps were significantly increased in the serum of lactating rats compared to age-matched, postpartum and non-lactating rats controls. Moreover, lactating rats showed a reduction in endochondral growth (EG), a decrease in bone volume (BV/TV), trabecular thickness (Tb.Th.) and mineralized surface (MS/BS) and an increase in osteoid surface (OS/BS) and eroded surface (ES/BS). In contrast, in ovariectomized (OVX) rats, EG was normal but the MS/BS was increased compared to sham operated rats. Interestingly, eNOS expression dramatically decreased in hypertrophic chondrocytes of the growth plates of lactating rats compared to their respective controls, but remained unchanged in OVX rats compared to sham. On the other hand, the immunoreactivity of eNOS increased particularly in osteoblasts in the trabecular surface of the secondary spongiosa in lactating rats. Taken together these data indicate that the estrogen deficiency state during lactation causes a reduction in bone growth, an increase in osteoclast resorption and in osteoid deposition without mineralization; and that these changes are associated with decreased eNOS expression in the chondrocytes of the growth plates and increased expression in osteoblasts in bone metaphyses suggesting a critical role of eNOS in lactation-induced bone loss.

Disclosures: J.I. Aguirre, None.


Long-Term Treatment with Risedronate Preserves Bone Quality.E. Paschalis1, R. J. Phipps2. 1Hospital for Special Surgery, New York, NY, USA, 2Procter & Gamble Pharmaceuticals, Mason, OH, USA.

It is now well established that bone quality, in addition to bone microarchitecture and bone mineral density (BMD), is an important determinant of bone strength and fracture risk. Treatment of postmenopausal osteoporotic subjects with risedronate reduces vertebral and nonvertebral fractures while concomitantly preserving bone microarchitecture and increasing BMD. In this analysis we compared the effects of up to 5-yr treatment with placebo or risedronate on two parameters of bone quality, mineral crystallinity and collagen cross-link ratio (pyr/deH-DHLNL), via Fourier transform infrared microscopic imaging (FTIRI).

Paired iliac crest biopsies were obtained from postmenopausal osteoporotic subjects at baseline and after 3-yr treatment with placebo (n=8) or risedronate (5 mg/day po; n=11). Biopsies were also obtained after 5-yr treatment with risedronate from 8 of these 11 subjects. Biopsies were embedded in methylmethacrylate, and the trabecular bone region was analyzed by FTIRI in ∼4 um thick sections for mineral crystallinity (bone mineral crystallite size in the crystallographic c-axis) and collagen cross-link ratio. Analysis was focused on trabeculae devoid of resorbing surfaces. Three images per section were acquired (each image 400 × 400 um2 area or >2000 pixels with a spatial resolution of 7 um). Crystallinity and cross-link ratio (mean ± standard deviation) were compared before and after treatment (*P<0.05).

Subjects treated for 3-yr with placebo had statistically significant increases in both mineral crystallinity and collagen cross-link ratio, a pattern consistent with untreated osteoporosis. In contrast, 3-yr and 5-yr treatment with risedronate preserved mineral crystallinity and collagen cross-link ratio of trabecular bone.

This lack of an increase in both mineral crystallinity and collagen cross-link ratio coupled with increased BMD and preservation of microarchitecture suggests that risedronate suppresses osteoclastic activity relatively more than osteoblastic activity. These results contrast to previously reported increases in mineral crystallinity seen with other antiresorptive therapies, including ibandronate and estrogen. 

Table  .  
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Disclosures: E. Paschalis, None.


LS BMD Increase Accounts for only a Fraction of the Vertebral Fracture Reduction at 1 Year: Results from the VERT and HIP Trials of Risedronate.N. B. Watts1, T. D. Johnson*2, Z. Li*2, C. Kasibhatla2. 1University of Cincinnati, Cincinnati, OH, USA, 2Procter & Gamble Pharmaceuticals, Mason, OH, USA.

The relationship between BMD increase and vertebral fracture risk reduction is not linear. The proportion of risk reduction explained by BMD increase varies amongst different studies. The proportion of vertebral fracture risk explained by LS BMD increase at 1 year has been investigated for osteoporotic women taking risedronate and the results are reported here.

The analysis included patients from the VERT and HIP studies, who were enrolled either on the basis of low LS BMD (T-score < -2.0) and at least one prevalent vertebral fracture (VERT-NA) or two prevalent vertebral fractures (VERT-MN) or FN T-score < -4 or -3 with at least one nonskeletal risk factor for hip fracture (HIP studies). Patients were randomized to receive either placebo or risedronate 2.5 mg or 5 mg daily. In addition, patients also received vitamin D, if baseline levels were low, and1000 mg/day calcium supplementation. Cox regression analysis was used to estimate the overall treatment effect and the treatment effect explained by LS BMD at 1 year. Only patients who had a post-baseline BMD measurement prior to 1 year and a known fracture status were included in the analysis. There were 70 patients who had an incident vertebral fracture, out of a total of 1739. The overall risk reduction over 1 year was 60% (CI: 54--65). The proportion of fracture risk reduction explained by LS BMD increase over 1 year was estimated to be 7%.In conclusion, the increase in lumbar spine LS BMD only weakly contributes to vertebral fracture risk reduction at 1 year.

Disclosures: N.B. Watts, None.


Only a Small Proportion of Non-Vertebral Fracture Risk Reduction is Explained by BMD Increases.N. B. Watts1, D. Felsenberg2, T. D. Johnson*3, Z. Li*3, R. Eastell4. 1University of Cincinnati, Cincinnati, OH, USA, 2Department of Radiology, University Hospital Benjamin Franklin, Berlin, Germany, 3Procter & Gamble Pharmaceuticals, Mason, OH, USA, 4University of Sheffield, Sheffield, United Kingdom.

The objective of the present study was to determine the contribution of BMD to osteoporosis-related nonvertebral fracture risk reduction in postmenopausal osteoporotic women. The analysis included patients from the two risedronate VERT studies who were enrolled in the trials on the basis of two prevalent vertebral fractures or low LS BMD (T-score < -2.0) and at least one prevalent vertebral fracture. Patients were randomized to receive either risedronate 5 mg or placebo daily. Patients also received vitamin-D, if baseline levels were low and 1000 mg/day calcium supplementation. Risedronate 5 mg daily reduced the risk of nonvertebral fractures by 35% over 3 years (95% CI=11%, 52%). The mean BMD percent increases over placebo at endpoint were 4.6% at lumbar spine and 2.6% at femoral neck. There was a non-linear relationship between BMD increases and nonvertebral fracture risk reductions. The proportion of nonvertebral fracture risk reduction explained by the BMD increases over 3 years (5mg vs. placebo) was estimated to be 12.2% (95% CI=5.7%, 26.1%) for lumbar spine and 5.5% (95% CI=2.6%, 11.9%) for femoral neck. The relationship between BMD increases and nonvertebral fracture risk reduction is non-linear. BMD increases observed during risedronate therapy only explain a small portion of the reduction in risk of non-vertebral fractures.

Disclosures: N.B. Watts, None.


Anti-Fracture Efficacy of Risedronate Is Greater than Nasal Calcitonin and Alendronate in an Observational Setting.N. B. Watts1, K. Worley*2, J. Doyle*3, R. Sheer*2, M. Steinbuch*2. 1University of Cincinnati, Cincinnati, OH, USA, 2Procter & Gamble Pharmaceuticals, Mason, OH, USA, 3Aventis Pharmceuticals, Bridgewater, NJ, USA.

To compare the anti-fracture efficacy of risedronate with other osteoporosis therapies in an observational setting, we used an administrative claims database to conduct a cohort study of patients age ⩾ 45 years initiating bisphosphonate or nasal calcitonin therapy (93% women, mean age 69 years). Patients with prescriptions in the prior 6 months for a bisphosphonate, nasal calcitonin, or raloxifene were excluded. All risk estimates were adjusted for age, sex, estrogen use, prior fragility fractures, and a general morbidity indicator (number of concomitant medications). The incidence of nonvertebral fractures (clavicle, humerus, wrist, pelvis, hip and leg) was compared for patients receiving risedronate, alendronate, and nasal calcitonin.

The first analysis consisted of six-month fracture assessment in 7081 individuals with a new “index” prescription for risedronate (5 mg daily or 30 mg weekly), alendronate (5/10 mg daily, 35/70 mg weekly) or nasal calcitonin between July 2000 and December 2001. A second analysis examined 12-month fracture incidence in 5024 patients with an index prescription between July 2000 and June 2001. Compared with patients receiving nasal calci-tonin, patients receiving risedronate showed statistically significant reductions in risk of nonvertebral fractures at 6 and 12 months (see table). Compared with patients receiving alendronate, risedronate-treated patients had a lower risk of nonvertebral fractures at 6 months that attained borderline statistical significance, and a significant reduction in non-vertebral fractures at 12 months (see table). 

Table  .  
  1. p=0.067, *p<0.05, **p<0.01

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In conclusion, in a large administrative claims database, patients initiating therapy with risedronate had a significantly lower risk of nonvertebral fractures compared with both alendronate and nasal calcitonin during the first 12 months of therapy. This study demonstrates substantial and rapid anti-fracture efficacy for risedronate in an observational setting, consistent with the fracture reductions observed at 12 months in clinical trials.

Disclosures: N.B. Watts, None.


Economic Evaluation of Gastrointestinal Events in Osteoporotic Patients Receiving Bisphosphonate Therapy in a Managed Care Setting.N. N. Borisov*1, J. J. Doyle*2, C. P. Brezovic*1, R. L. Sheer*1. 1Procter & Gamble Pharmaceuticals, Mason, OH, USA, 2Aventis Pharmaceuticals, Bridgewater, NJ, USA.

The objective of this study was to compare resource utilization and associated direct medical costs of GI-related events for both alendronate and risedronate patients utilizing an integrated administrative, medical and pharmacy claims database.

A retrospective cohort study was conducted among 4259 women and men (aged 65+) with a new prescription for risedronate (5mg/day), or alendronate (5mg/day, 10mg/day, 35mg/ week or 70mg/week) between November 1, 2000 and February 28, 2002 (16-month capture period). GI-related medical resource utilization and direct medical costs were assessed for a 4-month period following initiation of the bisphosphonate therapy. GI-related events and/or medications were assessed to select only patients with no GI events in the 6-month pre-treatment period. Utilized GI-related resources (pharmacy, outpatient, and inpatient) were valued using 2002 US dollars.

There were 623 (15%) patients treated with risedronate, and 3636 (85%) patients treated with alendronate. The mean age in both cohorts was 75 years and 94% were women. In the first four months after initiating bisphosphonate therapy, the average GI-related direct medical per-member-per-month (PMPM) cost was significantly lower for risedronate patients compared to alendronate patients ($2.15 vs. $6.00, p = 0.0001). Risedronate patients had almost three times fewer inpatient visits (per 100 patients per month) than alendronate patients (0.24 vs. 0.60, p = 0.0157). Also, risedronate patients averaged half the number (per 100 patients per month) of outpatient visits incurred by alendronate patients (0.88 vs. 1.53, p = 0.0273). In a patient population aged 65+ years receiving osteoporosis treatment, risedronate was associated with markedly lower GI-related medical costs compared to alendronate. The main source of the GI related cost difference between risedronate and alendronate was inpatient care utilization, suggesting that alendronate patients were experiencing clinically more severe GI events.

Disclosures: N.N. Borisov, None.


An Observational Study of the Incidence of GI Events Among Patients Receiving Treament with a Bisphosphonate.K. Worley*1, J. J. Doyle*2, R. L. Sheer*1, M. Steinbuch*1. 1Procter & Gamble Pharmaceuticals, Mason, OH, USA, 2Aventis Pharmaceuticals, Bridgewater, NJ, USA.

The purpose of the present study is to evaluate potential differences in the occurrence of gastrointestinal (GI) events between patients taking risedronate and alendronate, two oral bisphosphonates for the prevention and/or treatment of osteoporosis, in an observational setting.

A retrospective cohort study was conducted among 835 risedronate patients and 4828 alendronate patients, aged 65 years and older, in the Protocare Sciences administrative claims database. Women and men initiating therapy with daily risedronate (5 mg), daily alendronate (5/ 10 mg), or weekly alendronate (35/70 mg) between November, 2000, and February, 2002, were included in the analysis. In order to evaluate only newly starting bisphosphonate patients, individuals were excluded from the analysis based on one or more prescriptions for risedronate or alendronate in the 6 months prior to the index prescription. A GI event was defined as a GI-related primary ICD-9 diagnosis code (e.g., esophagitis, heartburn, gastric ulcer). The incidence of GI events during the 4 months after initiation of therapy was compared for alendronate and risedronate patients. The occurrence of GI events in the 6 months prior to therapy was also evaluated for the two groups so as to capture any potential differences in GI risk prior to initiation of treatment.

Alendronate patients exhibited a 44% higher risk of incurring a GI event compared to risedronate patients during the treatment period. This difference in risk was statistically significant (p = 0.01), adjusting for age, sex, and GI-related events in the 6 months prior to initiating treatment. The two dosing regimens of alendronate were combined in the analysis since the weekly users did not notably differ from the daily users with respect to incidence of GI events (RR = 1.08). Patients initiating alendronate were 20% less likely to have a history of GI events compared to those initiating risedronate (RR = 0.80, p < 0.05), suggesting a possible preference by physicians for prescribing risedronate among high-risk GI patients.

This study shows that among patients aged 65 years of age and older, alendronate users are at a significantly higher risk of GI events compared to risedronate patients during the first 4 months of therapy. The elevated risk cannot be attributed to age, sex, or preexisting GI problems

Disclosures: K. Worley, None.


Bone Histology and Histomorphometry in a 2-Year Risedronate Study Comparing Daily and Weekly Dosing.R. R. Recker1, E. W. Sod2, Z. Li*2, A. A. Chines2. 1Creighton University, Omaha, NE, USA, 2Procter & Gamble Pharmaceuticals, Mason, OH, USA.

The risedronate once-a-week efficacy study compared daily 5 mg to 35 and 50 mg once-a-week treatments in postmenopausal osteoporotic women. The primary endpoint of the non-inferiority study was lumbar spine BMD percent change at 1 year with safety evaluated over 2 years. Both weekly doses were non-inferior to 5 mg daily in lumbar spine BMD. Iliac crest biopsies were obtained in a subset of patients at baseline (n=109) and year 2 (n=86). Histology and histomorphometry data were collected from the biopsy samples as part of the safety evaluation. 193 of the biopsy samples were suitable for evaluation.

No histological abnormalities were observed in any of the risedronate-treated groups, suggesting normal bone quality even at the highest dose of 50 mg once- a-week. All endpoint biopsies had double tetracycline labeled surfaces.

Bone structural parameters were generally unchanged and similar among the treatment groups. Bone turnover by histomorphometry was significantly and similarly reduced in all groups by approximately 65%. This was consistent with observed decreases in the resorption marker NTx (50--60% reduction). All groups showed increased formation periods (FP) with significant changes in the 5 mg daily and 35 mg weekly groups. Consistent with previous 5 mg/d data, median mineralizing surface was approximately 1.5% in all groups after 2 years of therapy. Median change in mineral apposition rate (MAR) was positive in all groups and reached statistical significance in the weekly dose groups. Osteoid measures (surface, thickness, and volume) decreased in all treatment groups. Although wall thickness was unchanged in all groups, increased MAR and FP is suggestive of an anabolic effect of risedronate.

This is the first study demonstrating daily and weekly risedronate regimens produce similar results at the bone tissue, BMU, and cellular levels. The histology observations and histomorphometry data support the safety of the daily and once-weekly doses tested. These bone biopsy results, in combination with the non-inferiority of lumbar spine BMD in both weekly regimens, supports the selection of 35 mg once-a-week as an alternative to 5 mg daily risedronate.

Disclosures: R.R. Recker, None.


A New Dosing Concept for Bisphosphonate Therapy: Rationale and Design for the Monthly Oral iBandronate in LadiEs (MOBILE) Study.R. R. Recker1, J. Y. Reginster2, P. D. Delmas3, K. Coutant*4, B. Bonvoisin4. 1Creighton University, Omaha, NE, USA, 2University of Liège, Liège, Belgium, 3Claude Bernard University and INSERM Research Unit 403, Lyon, France, 4F. Hoffmann-La Roche Ltd, Basel, Switzerland.

Due to stringent dosing recommendations, oral daily and weekly bisphosphonate regimens may not be fully acceptable to some patients, potentially compromising adherence and therapeutic outcomes. Simplified, less frequent regimens may help optimize patient management. Ibandronate is a highly potent, nitrogen-containing bisphosphonate with proven fracture efficacy when administered as an oral daily (2.5mg; vertebral fracture risk reduction: 62%) or novel oral intermittent (20mg every other day for 12 doses every 3 months; vertebral fracture risk reduction: 50%) regimen. An oral monthly formulation of ibandronate is currently undergoing clinical assessment and is expected to provide an optimal combination of efficacy, tolerability and patient convenience. A randomized, double-blind, parallel-group study (Monthly Oral iBandronate In LadiEs: MOBILE) is exploring the non-inferiority of once-monthly oral ibandronate (100mg or 150mg) to the proven oral daily ibandronate (2.5mg) regimen in terms of lumbar spine (L2--L4) BMD change in post-menopausal women (aged 55--80 years, TSM ⩾5 years) with osteoporosis (lumbar spine BMD [L2--L4] T-score <−-2.5 and ⩾-5.0). Approximately 1,600 patients were randomized to one of four treatment groups for 24 months: 2.5mg oral daily ibandronate; 100mg oral monthly ibandronate (given on a single day); 100mg oral monthly ibandronate (given as separate 50mg doses on two consecutive days); 150mg oral monthly ibandronate (given on a single day). All patients receive the same medication schedule, with placebo replacing active medication as required for blinding. Patients receive oral daily calcium (500--1500mg) and vitamin D (400IU). The primary endpoint is the relative change in lumbar spine (L2--L4) BMD after 12 months. Secondary endpoints include the relative change in lumbar spine (L2--L4) BMD after 24 months, relative change in hip BMD (all sites) after12 and 24 months and biochemical markers of bone turnover after 3, 6, 12 and 24 months. Adverse events, including clinical vertebral and non-vertebral fractures, are monitored throughout the study. Non-inferiority analysis is an accepted and widely used methodology for demonstrating therapeutic equivalence between alternative regimens (e.g. weekly vs daily alendronate, weekly vs daily risedronate). Thus, in the MOBILE study, likely vertebral fracture efficacy will be demonstrated if the study s