Axial and Appendicular Bone Properties in Ambulatory Children with Cerebral Palsy.Tishya Wren*1, David Lee1, Robert M. Kay1, Frederick J. Dorey1, Vicente Gilsanz2. 1Childrens Hospital Los Angeles, USA, 2Children's Hospital of Los Angeles, USA

Children with cerebral palsy (CP) have deficient bone growth and an increased propensity for non-traumatic fractures. However, most studies of bone in children with CP have been limited to individuals with moderate to severe involvement (GMFCS levels 3-5). Higher functioning children with CP (GMFCS 1-2) may also be at risk for deficient bone acquisition due to muscle weakness and mobility limitations. The purpose of this study was to examine the relationship of axial and appendicular bone properties in ambulatory children with CP to functional (GMFCS) level.

Quantitative computed tomography (QCT) measurements were compared among 37 children with CP (N = 12, 5, 18, 2 for GMFCS levels 1, 2, 3, 4; age 9.3 ± 1.5 years; 18 male) and 37 controls with the same age and sex distribution. Vertebral (L3) properties were compared between CP and control and across GMFCS levels. Tibia properties were compared only across GMFCS levels because tibia data were not available for the controls. The primary method of analysis was linear regression. Simple and multivariable regression produced similar results, and only the multivariable results are presented here. The final multivariable model included height, weight, sex as covariates since height, weight, and their respective percentiles decreased with increasing disease severity (p<0.03).

There was no difference in vertebral cancellous bone density based on diagnosis (95% CI for coefficient: -15 to 7; P=0.49) or GMFCS level (CI: -12 to 8; P=0.68). The control group had larger vertebrae (CI: -81 to 9; P=0.02) than the children with CP, primarily due to smaller vertebral size in GMFCS levels 3 and 4 (CI: -98 to -7; P=0.02). In the tibia, geometric properties of the diaphysis decreased with increasing GMFCS level (CI: 35 to -1 for CSA, -25 to -6 for CBA; p<0.05). Cancellous density of the metaphysis also decreased with increasing GMFCS level (CI: -16 to -2 for entire metaphysis, -35 to -6 for slice; p<0.02) although values were similar between GMFCS levels 1 and 2.

This study is among the first to examine bone in higher functioning children with CP. The results suggest that children with CP of all levels may have reduced bone mass in their tibias, while spine deficits differentially affect more involved children. Since the lifespan of persons with CP is increasing and even small bone deficits may manifest as osteoporosis later in life, it is important to study bone acquisition in all children with CP.

Disclosures: Tishya Wren, None.


Effect of Vitamin D2 and D3 Supplementation in Healthy Adolescents from a Risk Region of Vitamin D Deficiency in Argentina.Cristina Tau*1, Edit Scaiola2, Juliana Castagneto1, Marina Rodriguez3, Carina De Roccis4, Zulma Pellisa1. 1Metabolismo Cálcico y Óseo, Endocrinología, Hospital de Pediatría J.P. Garrahan, Buenos Aires, Argentina, 2Servicio de Pediatria, Hospital Regional de Ushuaia, Tierra del Fuego, Argentina, 3Servicio de Clíínica Médica, Hospital Regional de Ushuaia, Tierra del Fuego, Argentina, 4Laboratorio, Hospital Regional de Ushuaia, Tierra del Fuego, Argentina

In order to improve vitamin D status of healthy adolescents from Ushuaia (55° S), in the south of Argentina, three periodic vitamin D2 or vitamin D3 supplementations of 100,000 IU were administered every three months: at the beginning of Winter (March), during winter (June), and in spring (September). Serum 25-hydroxyvitamin D (25OHD) was measured before the study, a month after the first supplementation, and two months after receiving the third supplementation (September 2008, April, and November 2009). After informed consent was signed by the parents, we studied 34 healthy adolescents, age (mean ± SD) 18.5 ± 2 years old (range 14.7 to 21.6), 21 girls and 13 boys. An inquiry was made in reference to sun exposure, calcium intake by dairy products. Weight, height, and type of skin of each individual were considered. None had received previous vitamin D. Serum calcium(SCa), phosphate(SP), and alkaline phosphatase(AP) were measured at the beginning of the study. Weight was 0.16 ± 0.74 (Z-Score), height was 0.2 ± 0.81. Seventeen adolescents received vitamin D2 or ergocalciferol and 17 received vitamin D3 or cholecalciferol. SCa was 9.35 ± 0.37 mg/dl, SP was 4.3 ± 0.73 mg/dl, AP: 91 ± 63 mg/dl. Basal levels of serum 25OHD were 13 ± 6.3 ng/ml (range 2-26) in september/spring 2008 (D2 group: 14.4 ± 6.4, D3 group: 11.8 ± 6.1). One month after the first supplementation, 25OHD increased significantly in both groups: 29 ± 7 range 16-44 ng/ml (p< 0.01), but was higher in the D3 than in the D2 group (31.6 ± 5.4 vs 25.7 ± 7.3 ng/ml, p<0.012). At the end of the study, 14 months after the beginning of the study, 25OHD decreased significantly: 22 ± 5.3 range 12-32 ng/ml (p<0.018). However, although 25OHD decreased in both groups when compared with the one-month peak, they did not differ between them (D3: 23.9 ± 5.1 and D2: 20.2 ± 5ng/ml). None of the adolescents had vitamin D intoxication (>50 ng/ml). Values less than 15 ng/ml were seen at the end of the study in 2 individuals who received D2. Calcium intake was below FDA recommendations: 570 ± 320 mg/day. These results indicate that to prevent vitamin D deficiency in risk zones, three supplementations of 100,000 IU of vitamin D during fall, winter and spring, would be adequate and safe to increase 25OHD levels. Morever, vitamin D2 or D3 are efficient to prevent vitamin D deficiency.

Disclosures: Cristina Tau, None.


Increased Bone Turnover in Preterm Infants Assessed by Serial Measurements of Urinary Osteocalcin and C-terminal Telopeptides of Type I Collagen (α-CTX-I and β-CTX-I).Kaisa Ivaska*1, Leena Kilpelainen2, Mikko Hakulinen3, Tiina Lyyra-Laitinen3, Esko Vanninen3, Natalia Habilainen-Kirillov1, Tanja Kuiri-Hanninen2, Ulla Sankilampi2, Kalervo Vaananen1, Leo Dunkel2. 1University of Turku, Department of Cell Biology & Anatomy, Finland, 2Kuopio University Hospital, University of Eastern Finland, Department of Pediatrics, Finland, 3Kuopio University Hospital, Department of Clinical Physiology & Nuclear Medicine, Finland

Moderate prematurity and rapid postnatal growth in preterm infants may have an impact on bone metabolism and development. The purpose of the present study was to evaluate bone metabolism in preterm infants during the first six months of life and compare them with full term infants.

Bone turnover was analyzed in 67 preterm infants (gestational age 24.7-36.7 weeks, median 32.9) and in 58 full term infants (gestational age 37.0-42.1 weeks, median 39.7) at postnatal day 7 (D7) and thereafter monthly until the age of six months (M1-M6). Bone turnover was assessed by measuring urinary osteocalcin midfragments (U-OC) and urinary concentrations of C-terminal telopeptides of type I collagen (α-CTX-I and β-CTX-I, indicative for resorption of native and aged type I collagen, respectively). All results were corrected for urinary creatinine. Bone mineral content was assessed by dual energy x-ray absorptiometry at the corrected age of 14 months.

U-OC was significantly higher (p<0.001) in preterm group compared with the full term group at all time points analyzed (D7-M6). Similarly, α-CTX-I concentration was significantly higher in preterm group from time point M2 to M6 (all p<0.001). β-CTX-I concentrations were similar in preterm and full term infants. In preterm infants, median U-OC concentration increased by two fold by M2 (p<0.001) and then started to decline, reaching the initial level by M6. Similar postnatal increase in median α-CTX-I concentration was seen in both preterm and full term groups by the age of one month. Bone mineral content at the age of 14 months did not differ between the two groups and there were no gender differences.

In conclusion, urinary OC and α-CTX-I concentrations reflect bone metabolism in infants and are suitable markers for serial monitoring of bone turnover in early postnatal life. Bone turnover, as assessed by urinary OC or α-CTX-I, was faster in preterm infants than in full term infants. Increased bone turnover in preterm infants resulted in normal bone mineral content by the age of 14 months.

Disclosures: Kaisa Ivaska, None.


Measuring Trabecular Density in the Peripheral Pediatric Skeleton: How Well Does pQCT Compare to QCT?Babette Zemel*1, Keenan Brown2, Soroosh Mahboubi3, Rohama Khadija4, Krista Whitehead3, Mary Leonard1. 1Children's Hospital of Philadelphia, USA, 2Mindways Software, USA, 3The Children's Hospital of Philadelphia, USA, 4University of Texas, USA

Appendicular sites are preferred for QCT measures of trabecular density (TrabDen) in children due to lower radiation exposure compared with spine scans. However, open growth plates complicate reference line placement, and differences in TrabDen distribution along the metaphysis and changes in bone dimensions during growth present additional challenges. This study compared distal tibia TrabDen measured at a single 2.3 mm slice by peripheral QCT (pQCT, Stratec XCT 2000) vs. QCT TrabDen averaged across the entire distal metaphysis (Siemens Somatom Sensation, Mindways QCT Pro Software) taken at two study visits, 12 ± 1 months apart.

Subjects (n=40, ages 9 to 21y) were participants in an ongoing blinded randomized control trial to treat low TrabDen in youth with Crohn disease. Tibia length was measured anthropometrically and from the QCT scan. Metaphyseal length (Met Length) was defined using density values along a central ellipse as the distance from the distal peak (just proximal to the growth plate) to the zero density point where trabecular bone was no longer detectable (see Figure). QCT TrabDen was calculated using a threshold of 450mg/cm3 and averaged across the MetLength. pQCT TrabDen was measured at 3% from the proximal edge of the distal physis (threshold 600 mg/cm3). Correlation analysis and paired t-tests were used for cross-sectional and longitudinal comparisons.

TrabDen by pQCT vs. QCT at baseline (r=0.76) and 12 months (r= 0.78) showed excellent agreement. Baseline vs. 12 month correlations for TrabDen were high and not significantly different for pQCT (r=0.68) and QCT (r=0.79), respectively. MetLength was consistent over time (r=0.77) and varied modestly as a function of tibia length and sex (p for interaction=0.03, overall R2=0.20). pQCT TrabDen (but not QCT TrabDen) was significantly associated with MetLength (r=0.33 & 0.42, for baseline and follow-up, respectively). pQCT Trabden increased significantly over 12 months (p<0.01) but not QCT TrabDen.

These findings demonstrate good agreement between a single slice 3% distal tibia pQCT TrabDen measurement and QCT TrabDen averaged across the entire metaphysis at multiple time points, and stability in both measures over time. However, longitudinal changes in pQCT TrabDen may be related to bone growth and re-distribution of trabecular bone along the MetLength. Further research is needed to optimize the use of pQCT in the assessment of TrabDen for bone health in children.

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Figure Figure. BMD Gradient Along the Distal Tibia Using A central Ellipse Male Subject, Age 11.7 years at baseline

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Disclosures: Babette Zemel, None.


See Friday Plenary number FR0005.


Systemic Biomarker Profiling of Metabolic and Dysplastic Skeletal Diseases Using Multiplex Serum Protein Analyses.Michael Whyte*1, Deborah Wenkert1, Denise C. Dwyer2, David L. Lacey2, Marina Stolina2. 1Shriners Hospital for Children, USA, 2Amgen Inc, USA

Understanding the etiology and pathogenesis of dysplastic and metabolic bone diseases has come from both genetic and biochemical investigation. Here, we describe our approach to systemic biomarker profiling using multiplex serum protein analyses to study age- and disease-mediated changes in healthy controls and patients as well as family members with these disorders.

The Research Center at Shriners Hospital for Children, St. Louis, Missouri, serves as a national referral center for inpatient diagnosis, treatment, and investigation of pediatric dysplastic, nutritional, and metabolic bone disorders where collection of fasting blood is routine. At Amgen (Thousand Oaks, California), Luminex-based, micro-bead, multiplex kits are used for osteopontin, osteocalcin, ligand of receptor activator NF-κB (RANKL), osteoprotegerin (OPG), fibroblast growth factor 23 (FGF23), matrix metalloproteinases (MMP 1, 3, 7, 8, and 13), and cytokine quantitation. ELISA kits are used for prostaglandin E2 (PGE2), bone-specific alkaline phosphatase (BAP), tartrate-resistant acid phosphatase (TRACP 5B), and collagen formation/degradation markers. Serum levels of sclerostin (SOST) and dickkopf (DKK1) were evaluated by custom ELISAs (developed at Amgen, Inc).

All sera were obtained after donors fasted at least 4 hours. To establish reference ranges, we studied 9 healthy children and 18 healthy adults (ages 6-60 yrs). MMPs (1, 3, 7, 8, and 13) correlated positively with age (R2 0.26 - 0.64, p<0.05), whereas osteocalcin, BAP, c-terminal propeptide of collagen type I (CICP), and TRACP 5B correlated negatively (R2 0.23 - 0.71, p<0.05). The patients represented 30 established and 14 unique diagnoses. Affected family members (dominant disorders) and obligate carriers (recessive disorders) were also studied, reflecting a total of 110 individuals. Our analyses revealed that serum concentrations of FGF23 were significantly increased in x-linked hypophosphatemia (XLH), TRACP 5B in osteopetrosis, and RANKL in juvenile Paget's disease, whereas decreases in BAP were documented for hypophosphatasia, validating our array strategy.

Hence, our multiplex serum protein analyses could reveal unique markers for specific bone disorders, and enhance understanding for a wide range of skeletal diseases.

Disclosures: Michael Whyte, Amgen, Incorporated, 3

This study received funding from: Amgen, Incorporated


To Asses the Incidence of Low Vitamin D Levels in Children with Osteogenesis Imperfecta and to Determine its Effect on Bone Healing and Number of Fractures.Antony Kallur*, Richard Kruse, Lauren Davey, Michael Bober. A.I. DuPont Hospital for Children, USA

Purpose: The prevalence of Vitamin D insufficency inchildren with OI is unknown. The aims of this study are to determine the correlation between low vitamin D level on bone quality and number of fractures. Low vitamin D levels in children with Osteogenesis Imperfecta negatively affects bone quality and increases fracture risk, requiring routine monitoring of vitamin D levels and supplementation.

Methods: We retrospectively reveiwed the medical records of patients on our database with diagnosis of Osteogenesis Imperfecta. All the patients with Vitamin D levels done were included in the study. We reveiwed the database and extracted the first ever value of Vit D assayed.

The treatment, number of fractures, surgeries and whether Bisphonates were used and complications were analysed.

Results: We had 61 patients who met our inclusion criteria. The average levels were 32.4ng/ml with 30 patients (50%) with levels less then 30ng/ml. 9 of the 61 patients had levels less then 20ng/ml (15%). The values ranged from 61ng/ml to 14ng/ml with a standard deviation of 11.66.

Conclusions: Children with O.I. have significant incidence of Vitamin D insufficency and defficency futher putting them at riskl for fractures. This has to be addressed when assessing and treating patients with OI.

Disclosures: Antony Kallur, None.


Effects of Repetitive Loading on the Growth-Induced Changes in Bone Mass and Cortical Bone Geometry: A 12-Month Longitudinal Study in Pre- and Post-Menarcheal Tennis Players.Gaele Ducher*1, Shona Bass2, Leanne Saxon3, Robin Daly4. 1Penn State University, USA, 2Deakin University, Australia, 3Royal Veterinary College, United Kingdom, 4The University of Melbourne, Western Hospital, Australia

Pre- and early-puberty may be the most opportune time to strengthen the skeleton, but there is little longitudinal data to support this claim. The aim was to study longitudinal growth and exercise-induced skeletal benefits relative to menarche in young female tennis players. Competitive players (n=45) aged 10-17 yrs were followed over 12 months. At baseline, 13 players were pre-menarcheal, of which 10 had menarche during follow-up (PRE/PERI). The remaining 32 players were post-menarcheal at baseline (POST). All players started playing tennis before menarche (mean 6.9+ 1.9 yrs); PRE/PERI had similar training volume than POST (11 ± 5vs. 12 ± 6 hrs/wk) but shorter playing history (5.7 ± 2.0 vs 7.5 ± 2.0 yrs, p<0.05). The osteogenic response to loading was studied by comparing the playing and nonplaying humeri for DXA BMC and MRI total bone area (ToA), medullary area (MedA), cortical area (CoA) and muscle area (MCSA) at the mid and distal humerus. Growth effects: Over 12 months, growth-induced gains (nonplaying arm) in BMC, ToA and CoA at the mid and distal humerus were greater in PRE/PERI (9-19%, p<0.001) than in POST (2-7%, p<0.05-0.001). There were no within group changes in MedA, with the exception that mid humerus MedA decreased in POST. Exercise effects: At baseline, BMC, ToA, CoA and MCSA were 7-19% greater in the playing vs. nonplaying arm in both PRE/PERI and POST (p<0.001); MedA was smaller in the playing vs. nonplaying arm in POST only (p<0.05). Over 12 months, these side-to-side differences increased further for mid ToA and CoA and distal ToA in PRE/PERI, and BMC, distal ToA and CoA in POST (p<0.05). When comparing the annual gains in the playing arm after accounting for the corresponding changes in the nonplaying arm, the increases in mid ToA, CoA, and MCSA were greater in PRE/PERI than POST (p<0.05). At both skeletal sites, the smaller the side-to-side differences in BMC and CoA at baseline, the larger the exercise benefits at 12 months (r=-0.36 to -0.48, p< 0.01). Training parameters and exercise-induced changes in MCSA were not predictive of the exercise benefits in BMC, ToA or CoA. In conclusion, both pre- and post-menarcheal tennis players showed significant exercise-induced skeletal benefits within a year, but these benefits tended to be greater in pre-menarcheal girls. Accelerated rates of growth and smaller pre-existing bone asymmetries due to past training were associated with greater skeletal responsiveness to loading.

Disclosures: Gaele Ducher, None.

This study received funding from: National Health and Medical Research Council, Australia


See Friday Plenary number FR0009.


See Friday Plenary number FR0010.


Maternal Perinatal Diet Induces Developmental Programming of Bone Architecture.Maureen Devlin*1, Corinna Grasemann2, Alison M. Cloutier1, Mark R. Palmert2, Mary Bouxsein1. 1Beth Israel Deaconess Medical Center, USA, 2The Hospital for Sick Children, Canada

The discovery of complex interactions of bone, fat, and brain suggests that osteoporosis and obesity may share common origins. Prior work has shown maternal diet in gestation and lactation triggers developmental programming that raises metabolic disease risk, but potential skeletal effects are unknown. We hypothesize that maternal diet induces perinatal programming that alters postnatal skeletal acquisition, bone mass and strength. Given recent evidence for central hypothalamic mediation of skeletal homeostasis, we predict that such programming alters central regulation of bone mass. Methods: To test this hypothesis and establish a contribution of developmental programming to skeletal acquisition, we compared postnatal skeletal acquisition in pups from mothers fed high fat (HF, 45% kCal/fat) or normal (N, 18% kCal/fat) diet. Female (F) C57Bl/6J mice were fed N or HF diet ad lib. from 6 wks prior to breeding through gestation and lactation. At 3 wks of age we weaned F pups from HF mothers (HF-N) and N mothers (N-N) onto N diet ad lib. Outcomes at 14 and 26 wks of age included body mass, body length, %body fat, total body bone mineral density (BMD, g/cm2) via pDXA, cortical (Ct) and trabecular (Tb) architecture at the midshaft and distal femur via μCT, and glucose tolerance. Results: At 14 and 26 wks of age, body mass, body length, %body fat and glucose tolerance did not differ in F HF-N vs. N-N. Total body BMD increased by 50% more in N-N vs. HF-N from 14-26 wks of age (+12% vs. +8%), suggesting impaired skeletal acquisition. Thus HF-N had 3% lower BMD at 26 wks and 5-9% lower BMC at 14 and 26 wks vs. N-N (p<0.05 for all). In the midshaft femur, Ct.Th was 6% lower in HF-N vs. N-N at 14 wks of age (p<0.03), but Ct.BA/TA (%) did not differ between HF-N and N-N at either timepoint. Most provocatively, in the distal femur, Tb.BV/TV (%) was 19-26% higher in HF-N vs. N-N F at 14 and 26 wks (p<0.05 for both). Conclusion: These data reveal complex effects of perinatal diet on postnatal skeletal acquisition. Female HF-N pups show lower bone acquisition from 14-26 wks of age, with lower Ct.Th at 14 wks of age, but higher distal femur Tb. bone volume vs. N-N at both timepoints. We propose that maternal HF diet may impair postnatal BMC acquisition in pups, but also slow age-related Tb. bone loss. Our data support the hypothesis that maternal diet alters postnatal skeletal homeostasis, and highlight the need for further work to identify the mechanisms involved.

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Distal femur BV/TV (%)

Disclosures: Maureen Devlin, None.


See Friday Plenary number FR0012.


Physical Activity is Associated with Increased Volumetric Bone Density and Bone Strength in Early Childhood.Zoe Cole*1, Nick Harvey2, Miranda Kim2, Keith Godfrey2, Hazel Inskip2, Nick Wareham3, Ulf Ekelund3, Elaine Dennison2, Cyrus Cooper4. 1MRC Epidemiology Resource Centre, Southampton General Hospital, United Kingdom, 2MRC Epidemiology Resource Centre, United Kingdom, 3MRC Epidemiology Unit, United Kingdom, 4University of Southampton, United Kingdom

Introduction: Both femoral geometry and bone mass have been shown to independently to predict both hip strength and fracture risk in adults. Whereas intrauterine and early postnatal life has been shown to influence bone mass, the relationship between hip geometry and strength is poorly understood. We therefore used a large prospective cohort study to explore the relationships between growth and lifestyle in early life and hip geometry, strength and volumetric density at 6 years old.

Methods: Children were recruited at 6 years old from the Southampton Women's Survey. Their mothers diet lifestyle and anthropometry had previously been characterised before and during pregnancy. The children underwent measurement of bone mass by DXA (Hologic), including hip structure analysis (HSA), and by pQCT at the tibia (Stratec). Physical activity (PA) was assessed by accelerometry (Actiheart, Cambridge Neurotechnology Ltd, Cambridge, UK) for 7 continuous days. Diet was assessed using a validated food frequency questionnaire and detailed anthropometric data was also collected.

Results: There were 215 children with PA data who underwent a DXA scan, of these 49 children also underwent pQCT assessment. Mean daily time spent in vigorous activity (VPA) was positively associated increased measures of volumetric BMD for whole body (r=0.32, p<0.001), lumbar spine (r=0.16, p=0.02) and hip (r=0.15, p=0.03). In the subset that underwent pQCT, VPA was positively related to cortical volumetric bone mineral density (r=0.29, p=0.05). Increased daily time in VPA was also positively associated with femoral neck and intertrochanteric section modulus (r=0.23, p=0.001; r=0.23, p=0.001 respectively), cross sectional area (CSA) (r=0.26, p=0.0002; r=0.24, p=0.0009) and cortical thickness (r=0.17, p=0.02; r=0.19, p=0.009). These relationships were independent of maternal and childhood dietary, lifestyle and anthropometric factors. Similar associations for VPA with section modulus (r=0.17, p=0.02) and CSA (r=0.16, p=0.02) were observed at the femoral shaft.

Conclusions: Higher levels of vigorous physical activity in childhood are associated with increased femoral neck strength, both in terms of geometric shape and volumetric mineral density. This work supports the notion that increasing physical activity in childhood is likely to be a potential public health strategy to improve childhood skeletal development and reduce the burden of osteoporotic fracture in later life.

Disclosures: Zoe Cole, None.


Skeletal Deficits and Recovery in Methylphenidate Treated Rats.Michelle Mary*1, Haden Janda1, Christine Snearly1, Winfred Abrams2, Nora Volkow3, Gene-Jack Wang4, Lisa Robinson4, Mala Ananth4, Tim Wigal5, James Swanson5, Panayotis Thanos4, Michael Hadjiargyrou6, David Komatsu1. 1InMotion Orthopaedic Research Center, USA, 2University of Tennessee, USA, 3Laboratory of Neuroimaging, NIAAA, NIH, USA, 4Behavior Neuropharmacology & Neuroimaging Lab, Medical Dept. Brookhaven National Laboratory, USA, 5Child Development Center, University California Irvine, USA, 6State University of New York at Stony Brook, USA

Up to 9% of American youths are diagnosed with Attention Deficit Hyperactivity Disorder (ADHD), 90% of which are treated with methylphenidate (MP). This study examined the effects of MP in adolescent rats to test the hypotheses that long-term treatment is detrimental to skeletal development and cessation of treatment leads to recovery of normal skeletal characteristics.

Four-week old male Sprague-Dawley rats were randomized to 3 groups (n=24/grp): low-dose (MP low), high-dose (MP high), and vehicle (Veh). MP was administered daily via drinking water with MP high and MP low rats receiving 30 or 4 mg/kg, respectively, for 1 hour followed by 60 or 10 mg/kg, respectively, for 1 hour. Veh rats received water with no MP. After 3 months, half of the rats (n=12/grp) were sacrificed. The remaining rats (Recovery) were taken off treatment and sacrificed 5 weeks later. Femora and tibiae were harvested for: DXA determination of BMD, BMC, and area; caliper measurements of length, anterior-posterior (AP) and mediallateral (ML) diameter; and 3-point bending tests for energy to failure (EF), stiffness (SF), and ultimate force (UF), as well as histologic and gene expression analyses. Serum was also collected for biomarker analyses. One-way ANOVA and Dunnett's tests were used to identify significant differences (p<0.05).

Weight gain during treatment in MP high rats was 15% less than Veh rats. Femoral and tibial BMD and BMC were lower in MP high compared to Veh (femur: 6% & 9%; tibia: 5% & 9%, respectively). In addition, femoral AP diameter was smaller in MP low (5%) and MP high (9%) as compared to Veh. Biomechanically, EF was lower in MP low (25%) and MP high (31%) compared to Veh, and UF was 14% lower in MP high. In the Recovery rats, none of these parameters were significantly different. Serum ALP was 31% higher in MP high rats compared to Veh, and similarly elevated in Recovery rats.

In summary, MP treatment resulted in less weight gain and weaker, less mineralized, smaller bones, particularly at high doses. However, these effects were ameliorated within 5 weeks. Higher serum ALP in MP high rats suggests increased osteoblast activity, but bone specific ALP remains to be quantified. Also, ongoing experiments to assess osteoclast activity, histology, and gene expression need to be completed to determine how MP impairs skeletal development. Clinically, these data suggest that adolescents treated with high dose MP may have weaker bones and an increased fracture risk.

Disclosures: Michelle Mary, None.


The Lifetime OsteoGenic Exercise Score: A Method to Estimate Bone Status in Young Adult Women.Tara Dahn, Jo Welch*. Dalhousie University, Canada

A simple tool to predict bone status would have both clinical and public health applications. Furthermore, if bone status was known to women when they were young adults, early adoption of lifestyle choices that favor better bone health could minimize future age-related bone deterioration. The purpose of the study was to develop a tool based on exercise history to predict the category of bone status in young adult women. For this study, 80 non-smoking women, aged 18-25, mean height, 67 cm, mean weight 63 kg, were recruited. They completed a simple questionnaire that included their exercise history and age at menarche. A Lunar Achilles InSight bone ultrasonometer was used to measure Stiffness Index (SI) at the calcaneus. An equation that could quantify impact loading from exercise was then developed. The Lifetime OsteoGenic Exercise Score (LOGES) uses the following formula to score each exercise reported in an exercise history questionnaire: LOGES = (AR2*D*F*M)/[(YS/2)+1], where AR = activity rating; activities were divided into 5 categories based on published effects on bone, D = duration of participation (y), F = frequency of participation (hr/wk), M = developmental period multiplier, based on whether activity occurred before, during or after puberty, and YS = years since cessation of activity (y). The score for each exercise activity was then summed to provide a total score for each subject. Subjects were ranked by their total scores. The list of ranked subjects was then partitioned into five equal groups, based on activity levels, which resulted in mean SI per group of 99.8, 105.9, 108.2, 110.5, and 114.3. The relationship between group mean activity level and SI was strong (R2 = 0.96). To test the reliability of the scoring system, 8 people unfamiliar with exercise and bone health were provided with 5 minutes of verbal explanation and a short written description of the scoring system. Each novice rater then scored the same 5 randomly selected subjects from their initial questionnaires. The Pearson's correlation coefficient for inter-rater reliability was R = 0.996. In conclusion, the LOGES system enabled the classification of young women into categories of bone status based on exercise and pubertal history. This system of estimating bone status is reliable and offers a method to screen young women, who rarely receive bone tests, for bone status.

Disclosures: Jo Welch, None.


IBD Causes Greater Bone Loss in Male Compared to Female Mice.Regina Irwin, Laura McCabe*. Michigan State University, USA

Inflammatory bowel disease (IBD) is the most common chronic gastrointestinal disease in the United States. Nearly 1.4 million males and females are affected with one of the two major forms of IBD which are Crohn's disease and ulcerative colitis. One complication of IBD is bone loss. Purpose: To examine the influence of gender on IBD induced bone loss. Method: Helicobacter hepaticus was used to induced IBD in male compared to female IL-10 knockout mice. This model effectively simulates Crohn's disease. Mice, at 14 weeks of age, were gavaged with bacteria and after 6 weeks bone parameters were examined. Results: Both sexes experienced significant decreases in bone parameters, however, the losses were less in females. Specifically, IBD decreased bone mineral content and denisty by 26% in males, but only by 15% in females. Similarly, IBD caused a decrease in bone volume fraction and trabecular thickness, by 37% and 19%, in males and only by 16% and 10% in females. In contrast, osteocalcin expression in bone was reduced to a similar extent (50%) in both males and females. Histomorphometry demonstrated similar changes in osteoblast and osteoclast numbers in male and female mouse bones. Conclusion: Our findings suggest that the female skeleton is partially protected from IBD induced bone loss. A potential beneficial role for estrogen is currently being investigated.

Disclosures: Laura McCabe, None.


PTH Increases and Serum NTX is Associated with Maternal Bone Loss in Pregnant Adolescents.Bridget Essley*1, Allison McIntyre2, Beth Cooper2, Thomas McNanley2, Tera Kent1, Frank Witter3, Z. Leah Harris3, Kimberly O'Brien1. 1Cornell University, USA, 2University of Rochester Medical Center, USA, 3Johns Hopkins University, USA

Bone turnover increases during pregnancy and a net loss of bone can occur over the course of pregnancy. Pregnant adolescents may be at increased risk for bone loss as the calcium (Ca) demands of the developing fetus are added to the demands of adolescent growth. Pregnant teens are also at increased risk for insufficient Ca intakes and vitamin D insufficiency. To assess the relationships between vitamin D status, parathyroid hormone (PTH), and Ca intake on maternal bone health across gestation, pregnant adolescents (≤ 18 y) were recruited at ≥ 12 weeks of gestation and followed at 3 points across pregnancy (at approximately 16, 25 and 34 wks gestation). At each visit an ultrasound of the calcaneus was performed and a 24-hr dietary recall and FFQ were obtained. Blood samples were collected at mid-gestation (25.3 ± 3.4 weeks) and maternal and cord blood were obtained at delivery (39.2 ± 2.8 weeks). 25(OH)D, intact PTH, osteocalcin (OC: a marker of bone formation) and N-telopeptide (NTX: a marker of bone resorption) were measured in all blood samples. Teens were 17.1 years of age at entry into the study (n=141). Vitamin D insufficiency (25(OH)D ≤ 20 ng/mL) was found in 49% of teens at mid-gestation, 48% at delivery, and 46% of neonates were D insufficient at birth. In adolescents, 25(OH)D was inversely related to PTH at mid-gestation (p=0.034, n=93) and delivery (p=0.066, n=76). PTH increased across gestation (p<0.05, n=76) and was elevated (PTH ≥ 46 pg/mL) in 13% of teens at midgestation (n=93) and 32% of teens at delivery (n=81). Ca intake averaged 874 ± 379 mg/day (recall) and 975 ± 484 mg/day (FFQ) and was not related to PTH or changes in maternal calcaneus measures. Markers of bone turnover increased across gestation and the increases in OC and NTX per week were positively correlated (p=0.002, n=57). The average change in ultrasound bone measures were negative (n=120) indicative of maternal bone loss across gestation. The increase in NTX per week was positively associated with changes in bone ultrasound measures (SOS, QUI, BMD; p<0.05, n=94). This population exhibited a high prevalence of vitamin D insufficiency, net loss of maternal bone across gestation and unexpected increases in PTH across gestation. Calcitriol analyses are underway and a model will be constructed to elucidate the main determinants of maternal bone loss and fetal bone growth in this population of pregnant adolescents.

Disclosures: Bridget Essley, None.


Acute Mandibular Lesion in a 12 year old Girl with McCune Albright Syndrome and Fibrous Dysplasia.Nancy Dunbar*1, David Ebb2, Ksenia Tonyushkina3, Thomas Carpenter4, Leonard Kaban2. 1Baystate Medical Center, USA, 2MassGeneral Hospital for Children, USA, 3Baystate Children's Hospital, USA, 4Yale University School of Medicine, USA

McCune Albright Syndrome (MAS) is caused by activating, somatic mutations of GNAS, resulting in fibrous dysplasia (FD) of bone, café-au-lait skin markings and precocious puberty. Only two mutations have been reported in MAS (commonly, at R201 and also at Q227). No genotype/phenotype correlations are known. Fibrous dysplasia in MAS can be severe, causing significant disfigurement, pain and restriction of function. Uncommonly, aggressive cystic lesions arise, usually in the craniofacial area, and are associated with disfigurement and pain. These lesions have been shown to produce excessive amounts of FGF23.

We report a 12 year old girl with MAS, severe polyostotic fibrous dysplasia, and a 5×5cm mandibular lesion, which had acutely expanded in one week. The superior aspect of the lesion was bony to palpation but ballotable inferiorly; the overlying skin was yellowed. The lesion stabilized after 10 further days and the pain remitted. Radiographic studies revealed an expansile lesion with fluid-fluid levels eroding through the inferior cortex of the mandible (see images below). Widespread fibrous dysplasia occurred throughout the mandible and skull base. Bone pain had been treated during the previous year with intravenous pamidronate at 3-month intervals, last administered 10 weeks prior to the observation of the lesion. Of interest, an acutely expanding lesion involving her right orbit led to surgical excision in Kyrgystan at age 7.

During the preceding several months, serum alkaline phosphatase activity increased from 513 to 686 IU/L, circulating PTH increased from 31 to 60 pg/ml, and circulating 1,25(OH) decreased from 64 to 24 pg/ml. Serum 25-OHD, calcium and phosphorus levels were stable within the normal range. Surgical enucleation of the mandibular lesion was performed. Gross inspection was consistent with an aneurysmal bone cyst. Histopathology and pre/post-operative serum FGF23 levels are pending. Treatment with interferon to stabilize the vasculature is under consideration pending the histopathology.

This case presents the clinical course and therapeutic response to an aggressive cystic lesion in the mandible of a 12-year-old girl with severe MAS/FD presumed to be an aneurysmal bone cyst. The role of FGF23 in the pathogenesis of acute cystic lesions in FD, and the role of interferon in their treatment are currently unknown.

Disclosures: Nancy Dunbar, None.


Bone Health in Children with Neurofibromatosis Type 1 - a High Incidence of Abnormal Vertebral Morphology and Altered Skeletal Geometry.Peter Simm*1, Julie Briody2, Aaron Schindeler2, Christopher Cowell2, David Little2, Kathryn North2, Craig Munns2. 1Royal Children's Hospital, Melbourne, Australia, 2The Children's Hospital At Westmead, Australia

There is increasing evidence that some young people with Neurofibromatosis Type 1 (NF1) have an abnormal bony phenotype, however this is yet to be fully characterized. We performed a bone health assessment on 23 patients with NF1 (11 female) aged between 5 and 16 years (mean age 9.5 yrs).

Results showed that this is a highly heterogenous group, however there is a subgroup who have an abnormal bone phenotype. Dual energy absorptiometry (DXA) scanning showed a mean lumbar spine bone mineral density (BMD) Z score of 0.1 SD (corrected for height), with a wide spread (+/-1.43).

Peripheral quantitative computerised tomography (pQCT) showed a distinct phenotype, with the mean results showing a low trabecular BMD (-0.89 SD, +/-0.61, p <0.001) at the 4% site and reduced bone area (-0.73 SD, +/-1.31, p = 0.02), cortical thickness (-0.51 SD, +/- 0.81, p = 0.009) and a trend towards reduced stress strain index (-0.38 SD, +/- 1.15, p = 0.14), but normal cortical BMD (0.65 SD, +/-1.23, p = 0.03), at the 66% site of the tibia. Muscle cross sectional area was also reduced (-0.63 SD, +/- 1.06, p = 0.01) which may be contributing to the observed skeletal geometry.

Of the 21 patients who had spinal Xrays, 7 (33%) had significant anterior wedge deformity by Genant criteria with greater than 15% loss of anterior height compared with posterior height, predominantly in the mid thoracic region. A further 4 patients had abnormal shaped vertebrae without meeting the Genant criteria. Therefore over 50% of our patients showed vertebral body changes. The significance of these findings is unclear as there was no correlation between low BMD on DXA scan and vertebral changes. Whether the vertebral changes reflect bone weakness and fragility, or rather dysplasia, remains to be determined.

Metacarpal index was reduced (1.91, N >2.0), with 8 pts (35%) having a score less than 1.75. 4 patients had a vitamin D level of <50 nmol/L, despite no patients having established risk factors for Vitamin D deficiency. Only 3 patients reported a previous fracture. The subjects had mean height Z score of -0.4 SD but had increased body mass index Z score (0.4 SD).

Our results confirm that there is a subset of patients with NF1 who have an abnormal bone phenotype. We plan to follow our cohort longitudinally to obtain data on prospective incidence of fracture and to follow the vertebral body changes in the more severely affected individuals.

Disclosures: Peter Simm, Pfizer, 2

This study received funding from: Pfizer


Impact of Weight Gain on Bone Mineral Content in Adolescents with Type 1 Diabetes.Sowmya Krishnan*1, Brianna Bright2, Mary Murray3, Kenneth Copeland2, David Fields2. 1The University of Oklahoma Health Sciences Center, USA, 2University of Oklahoma health science center, USA, 3University of Utah, USA

Background: Lower spine and whole body bone mineral content (BMC) has been reported in adolescents with type 1 diabetes, while overweight status in adolescents without diabetes has been shown to be associated with higher BMC. It is not known if being overweight is protective for the bones of adolescents with type 1 diabetes. The following is a cross sectional study examining the influence of weight status on BMC in overweight and normal weight adolescents with type 1 diabetes.

Methods: A total of 31 adolescents with type 1 diabetes (HbA1c range 6.5% to 10.7% with a mean of 8.5%) participated in the study. All were Tanner stage 3 or above. 16 were overweight (mean BMI = 28.3 ± 3.6) and 15 were normal weight (mean BMI = 20.6 ± 2.0). All adolescents underwent a whole body DXA scan (Hologic QDR 4500, Waltham, MA). Additionally 21 wore a step activity monitor and 18 completed a detailed 4-day food recall.

Results: Adolescents with type 1 diabetes who were overweight had higher total BMC and subtotal BMC than adolescents with type 1 diabetes who were normal weight, though this difference did not reach statistical significance (p=0.0692 and 0.0594 respectively). As expected, both total BMC and subtotal BMC correlated significantly with age (r=0.55, p=0.0014 and r=0.50, p=0.0038 respectively). BMC measures (both total and subtotal) did not significantly correlate with any other variables, including physical activity measures (total steps/day, sedentary time/day) or vitamin D and calcium intake.

Conclusion: Overweight adolescents with type 1 diabetes had higher total and subtotal BMC values compared to normal weight adolescents with type 1 diabetes, indicating a similar relationship of BMC to overweight and normal weight status as that reported in non-diabetic adolescents. These observations pertain to adolescents with relatively well-controlled type 1 diabetes; whether they can be generalized more broadly to all diabetic patients regardless of glycemic control remains to be demonstrated.

Disclosures: Sowmya Krishnan, Novo Nordisk, 2

This study received funding from: Novo Nordisk


See Friday Plenary number FR0021.


Paternal Deletion of the GNAS Imprinted Locus in a Girl Presenting with AHO, Severe Obesity and ACTH-independent Adrenal Hyperplasia.Caroline Kannengiesser1, Stéphanie Maupetit-Méhouas2, Sabine Rouleau3, Régis Coutant3, Agnes Linglart4, Caroline Silve*5. 1Assistance Publique-Hôpitaux de Paris; Université Paris 7. Hôpital Bichat Claude Bernard, Service de Biochimie hormonale et génétique, France, 2INSERM U986, France, 3Department of Pediatric Endocrinology, University Hospital, France, 4INSERM U986; Assistance Publique-Hôpitaux de Paris. Endocrinologie-diabétologie pédiatrique et Centre de référence des maladies rares du métabolisme du calcium et du phosphore, Hôpital St-Vincent de Paul, France, 5INSERM Unit 986, France

Diseases due to GNAS genomic or epigenomic alterations include: 1. pseudohypoparathyroidism (PHP) 1a characterized by Albright Hereditary Osteodystrophy (AHO) with multi-hormonal resistance due to maternally inherited Gsa loss of function (LOF) mutations; 2. pseudoPHP, characterized by AHO without hormonal resistance due to paternally inherited Gsa LOF mutations; 3. PHP1b usually presenting with selective PTH resistance without AHO due to maternal epimutations of the GNAS differentially methylated regions (DMRs); and 4. McCune-Albright syndrome characterized by hyperplasia and increased function of endocrine glands, including the adrenal, due to somatic Gsa gain of function mutations. Patients with Gsa LOF mutations usually present with ectopic mineralization, which are more severe when the mutation are paternally inherited. We investigated a girl who presented with AHO, no ectopic ossification, severe obesity (as in PHP1a), no hormonal resistance (as in PPHP), bilateral adrenal hyperplasia and increased function (as in McCune Albright) as indicated by hirsutism, amenorrhea, acne, elevated circulating androgens (testosterone, SDHA) and elevated cortisol cycle albeit normal urinary cortisol and no Cushing syndrome. She was the only child born to healthy parents. Systematic sequencing of the GNAS coding exons 1 to 13 did not show any mutation nor heterozygous polymorphism. Methylation analysis of the GNAS DMRs using enzymatic digestion after bisulfite treatment or methylquantification showed a maternal pattern of methylation of the entire GNAS locus. Genome wide CGH-array analysis (Nimblegen 12*385K) demonstrated the presence of a ∼1.575 Mb genetic deletion encompassing the entire GNAS locus. The deletion was confirmed by allelic quantification of genomic DNA; microsatellites analysis of the parents and proband DNAs demonstrated the paternal origin of the deletion. In summary we describe a new case of constitutional paternal deletion of chromosome 20 encompassing the entire GNAS locus and associated with a paradoxical phenotype. While the short stature and the bone features defining AHO are likely due to Gsa haploinsufficiency, the mechanisms leading to the severe obesity and adrenal gland bilateral hyperplasia need to be elucidated. Because a defect in none of the genes included in the deleted genetic interval are likely to be associated with the phenotype, it is tempting to speculate that the absence of XLS and/or AB and AS transcript expression due to the paternal deletion contributes to the phenotype.

Disclosures: Caroline Silve, None.


Evidence of Metabolic Bone Disease in Young Infants with Multiple Fractures Misdiagnosed as Child Abuse. Chuck Hyman1Marvin Miller2, David Ayoub*3. 1Pediatrician, USA, 2Wright State University Boonshoft School of Medicine, USA, 3Clinical Radiologists, SC, USA

Purpose: Assess the presence of clinical and evidence of metabolic bone disease in young infants presenting with multiple unexplained fractures (MUFs) undergoing an investigation for child abuse.

Methods: We have evaluated the radiographs and pertinent medical records of 40 infants with MUFs and their mothers for signs and risk factors of impaired bone metabolism.

Results: The average age of presentation was 12.6 ± 9.0 weeks (range 2-52). Twelve of thirteen (92.3%) mothers tested had subnormal 25OH vitamin D. Ten were deficient (<20 ng/ml) and two within the insufficient (<30 ng/ml) range. Ten of seventeen (58.8%) infants had subnormal 25OH vitamin D. Most infants with normal vitamin D levels were tested many weeks after presentation and had been supplemented. Twenty-seven mothers (68%) had 2 or more risk factors for VDD. There was evidence of decreased fetal bone loading in 17/40 (43%) pregnancies. Twenty percent of women had gestational diabetes. Acid lowering drugs which can decrease calcium absorption were used in at least 16 (40%) mothers while pregnant and in 11 (27.5%) of infants.

There were 14.1 ± 9.0 fractures, including classic metaphyseal lesions (CMLs), per infant (range 3-47). One hundred eighty-nine CMLs were encountered in 37 infants (5.1 per infant; range 0-15). Epiphyseal separations were suspected in 5.8% of CMLs. All other CMLs were clinically silent and healed without callus or periosteal reaction. Multiple rib fractures were seen in 33 infants (7.8 per infant; range 2-20). Infants had at least one non-CML fracture at these sites: skull (20.0%), which was always linear and usually diastatic, clavicle (32.5%), thoracolumbar spine (17.5%), appendicular diaphysis (40.0%) or metaphysis (15.0%), scapula (5.0%), and small bones of hands/feet (20.0%). All infants displayed multiple radiographic signs of metabolic bone disease (Table 1).

Conclusions: This study reports a large series of infants with MUFs who had clinical and radiographic evidence of metabolic bone disease likely of multifactorial etiology overlooked during an investigation for child abuse. While metabolic bone disease is a known cause of increased bone fragility and MUFs, its differentiation from inflicted or accidental injury remains challenging. Careful review of the radiographs with attention to the unappreciated signs or risk factors that impair fetal and infant bone mineralization is critical to avoid an erroneous diagnosis of child abuse and its consequences.

Table Table 1. Radiographic signs* in 40 infants with MUFs
  1. *per infant basis

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Disclosures: David Ayoub, None.


See Friday Plenary number FR0024.


New Case of Infantile Generalized Arterial Calcification: Evolution from Prenatal Diagnosis to 12 Months of Age under Bisphosphonates' Therapy.Thomas Edouard*1, Nathalie Alos2, Joaquim Miro1, Chantal Lapierre1, Jacques Michaud1, Gilles Chabot3. 1CHU Sainte-Justine - Université Montréal, Canada, 2CHU Sainte Justine, Canada, 3Hopital Saint Justine, Canada

Background: Generalized arterial calcification of infancy (GACI, OMIM 208000) is a rare autosomal recessive disease characterized by extensive calcification of large- and medium-sized muscular arteries. The diagnosis is prenatal or in early infancy, with variable clinical presentations including heart failure, hypertension or failure to thrive. This disease is linked to mutations in the ENPP1 gene which encodes an enzyme that generates inorganic pyrophosphate (PPi), a potent inhibitor of hydroxyapatite crystal formation. Treatment with bisphosphonates, which are synthetic PPi analogues, has been proposed as a means to reduce arterial calcifications in GACI patients. GACI has been reported to be frequently lethal, and the efficiency of any therapy, including bisphosphonates, remains unknown.

Case report: We report on a girl who was diagnosed prenatally at 32 weeks gestation with GACI when a routine fetal ultrasonographic examination revealed arterial calcification and pericardial effusion. The parents were healthy and non consanguineous without relevant family history. The newborn baby was born by vaginal delivery at 364/7 weeks gestation with normal birth weight and length but no pulses were palpable. Secondary to pericardial effusion, he developed mild respiratory distress in the first hour of her life and required pericardial fluid drainage and respiratory support for 6 days. Ultrasonographic and tomodensitometric evaluations confirmed diffuse arterial calcification that involved the coronary, main and branch pulmonary and aorta arteries. Mutational screening demonstrated mutations in ENNPP1 gene, which correspond to c.583T/C.

Evolution: Intravenous disodium pamidronate (3 infusions at days 8, 15, 18 of 0.25mg/kg, 0.50 mg/kg and 0.50mg/kg respectively) was changed to oral disodium etidronate (starting dose of 20 mg/kg daily: 50 mg die) at 3 weeks of age. Follow-up included 3-monthly clinical reviews and assessments of mineral homeostasis, 6-monthly DEXA, ultrasonography and tomodensitometric studies, and annual x-rays of the wrists and knees. Progressive resolution of arterial calcifications was seen by 3 months of age and etidronate dosage was kept at 50 mg daily. At 12 months, under 6mg/kg of etidronate, growth and development, serum mineral homeostasis and areal bone mineral densitometry of the lumbar spine remained normal. X-rays of the wrist and knees didn't show any sign of rickets.

Disclosures: Thomas Edouard, None.


The Use of Intravenous Bisphosphonate Therapy to Treat Vertebral Fractures due to Osteoporosis among Boys with Duchenne Muscular Dystrophy.Anne Marie Sbrocchi*1, Frank Rauch2, Pierre Jacob1, MaryAnn Matzinger1, Leanne M. Ward1. 1University of Ottawa, Canada, 2McGill University, Canada

Purpose: Boys with Duchenne Muscular Dystrophy (DMD) are at risk for vertebral fractures. Bisphosphonates have been used to treat the spine fragility; however, detailed analyses of the response to therapy are lacking. The objective of this study was to assess the efficacy and safety of IV bisphosphonate treatment for painful vertebral fractures among boys with DMD.

Methods: This was a one-year, retrospective observational study of 7 boys (age 7.9-14.0 years) with DMD who had received either IV pamidronate (9 mg/kg/year) or zoledronic acid (0.1 mg/kg/year) to treat painful vertebral fractures. The co-primary outcomes were back pain status and change in vertebral morphometry (with categorization as per the Genant score) at 12 months post-bisphosphonate initiation. Changes in vertebral heights were deemed to be statistically significant if they exceeded the Least Significant Change at the 99% confidence level. Secondary outcomes included development of new vertebral fractures, changes in lumbar spine areal bone mineral density (BMD), and adverse events.

Results: A description of the cohort at baseline and 12 months is presented in the Table. Pubertal development was Tanner stage 1 (n=6), and Tanner stage 2 (n=1). All but one boy had received glucocorticoids prior to treatment initiation (average cumulative steroid exposure per patient 16,058 mg/m2 in prednisone equivalents), and only one was fully ambulatory. There were 27 fracture events noted in the 7 patients at baseline; 3 fractures in 1 patient, 2-4 in 2 patients and >5 in 2 patients. Of the 27 fracture events, 14 improved, 11 stabilized and 2 deteriorated (30% to 37% and 15% to 32% loss in vertebral height). All but one Grade 2 (moderate) and Grade 3 (severe) vertebral fractures improved (n=11) or stabilized (n=6) at 12 months. There were 3 new Grade 0.5 fractures in 2 boys apparent after 1 year of therapy. Back pain either resolved completely (n=4) or improved (n=3). The mean ± SD spine BMD also increased (see Table). First-dose side effects were present in 4 patients and included fever and malaise (N=4), and hypocalcemia (N=2).

Conclusion: In boys with spinal osteoporosis and DMD, IV bisphosponate therapy administered over 12 months was associated with improvements not only in spine BMD, but also in the more clinically relevant back pain and vertebral morphometry. The therapy was generally well-tolerated.

Table  .  
  1. Median (min, max) values reported unless otherwise specified.

  2. VF = Vertebral fracture, VH_Vertebral height, BMD = Bone mineral density; a=areal, v-volumetric.

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Table. Clinical Parameters Pre- and 12 Months Post-Treatment

Disclosures: Anne Marie Sbrocchi, None.


In vivo Microstructural Quantification of Finger Joints using High-Resolution pQCT.Christoph Kolling1, Kathryn S Stok*2, Thomas L Mueller2, Ralph Muller2, Jorg Goldhahn2. 1Schulthess Clinic, Switzerland, 2ETH Zurich, Switzerland

Fingers are a primary target of destructive processes like rheumatoid arthritis (RA) leading to bony erosions, destruction of the subchondral bone and local osteopenia, and results in loss of hand function. Evaluation of treatment success is limited to measures such as counting of swollen joints or scoring of X-rays. In contrast, highresolution peripheral quantitative computed tomography (HR-pQCT) can detect small changes in bone microarchitecture and is established for analysis of peripheral joints in vivo (distal tibia and radius). High-resolution structural measurements of finger joints, however, have not been performed to-date. The goal of this study was to quantify structural parameters in finger bones from a group of healthy volunteers, and to correlate this with established parameters at the ipsilateral radius.

A group of 19 healthy volunteers (aged 39 to 69) underwent clinical assessment to ensure the absence of any systematic disease. HR-pQCT measurements (Scanco Medical AG) in conjunction with a fixation device (Pearltec AG) were performed at the metacarpal-phalangeal (MCP) and proximal interphalangeal (PIP) joints of the right hand (Fig 1a) with an isotropic nominal resolution of 82 μm. Two volumes of interest were defined; the trabecular bone of the epiphyseal region, and the cortical shell surrounding it (Fig 1b). Morphometric parameters were computed, and Wilcoxon Rank-Sum and Spearman correlation coefficient were used to test statistical relationships.

Significant differences were seen for all parameters between fingers and between joint parts for average bone density, D100, and cortical thickness, Ct.Th. In the MCP, D100 and Ct.Th increased in the distal region. From finger 2 to 3 to 4, D100 decreased in the MCP and increased in the PIP. Strong correlations were observed in finger joints between the proximal and distal aspects, and strong correlations were also found between the fingers themselves. Finally, the ipsilateral radius correlated weakest to finger 2, and strongest to fingers 3 and 4, especially in the PIP distal region.

In conclusion, the data show distinct structural relations between joint parts, between joints of the same finger, and within the same region of different fingers. These findings indicate that HR-pQCT has the potential for analysing structural changes in small finger bones with the onset of RA. The healthy cohort will now be used as a normative database for examining subchondral bone changes with the onset of RA.

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Figure Figure 1 (a).  

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Figure1. (a) Measurement regions for HR-pQCT scans, and (b) Specific joint volumes of interest.

Disclosures: Kathryn S Stok, None.


See Friday Plenary number FR0028.


Agreement between pQCT and DXA-derived indices of bone geometry, density and theoretical strength in females of varying age, maturity and physical activity.Portia Flowers*1, Jodi Dowthwaite2, Rebecca Hickman1, Tamara Scerpella3. 1SUNY Upstate Medical University, USA, 2State University of New York, Upstate Medical University, USA, 3State University of New York Upstate Medical University, USA

Purpose: DXA is commonly used to assess bone but does not specifically measure cross-sectional geometry, compartmental density or skeletal strength. Indices of bone geometry and strength can be derived from DXA output (Sievanen 1996). Previously, we used pQCT as a standard to validate these DXA-derived indices in post-menarcheal females (Dowthwaite 2009). In the current study, we validate these indices across a broader maturational range.

Methods: Contemporaneous pQCT and DXA scans of the distal radius were performed in 97 females, aged 8.0 to 22.8 yrs, mean=15.1 yrs (premenarche n=42; postmenarche n=55). Ultradistal (UD) and 1/3 DXA indices were compared to 4% and 33% pQCT results, using Pearson correlations and mean differences (Bland-Altman plots). The original Sievanen formulae were modified to improve inter-method agreement for UD output.

Results: DXA and pQCT indices were significantly positively correlated with stronger correlations at 1/3 (R=0.85-0.93, p<0.001) than at UD (R=0.64-0.97, p<0.001). At UD, all DXA indices exceeded pQCT measures except periosteal cross-sectional area (CSA). At 1/3, DXA indices exceeded pQCT measures for all CSA indices. DXA indices were lower than pQCT results for bone mineral apparent density (BMAD), cortical thickness, section modulus, and fall strength ratio. Inter-method agreement was only affected by maturity level for UD BMAD and fall strength; DXA vs. pQCT differences were positive premenarche and negative postmenarche.

Conclusion: Across a broad age and maturity range, DXA-derivations for the distal radius yield useful indices of bone geometry, density and strength, providing a valuable supplement to traditional DXA assessments of the distal radius during growth. 


Sievanen, H., P. Dannus, V. Nieminen, A. Heinonen, P. Oja, I. Vuori. 1996 Estimation of various mechanical Characteristics of human bones using dual energy x-ray absorptiometry: methodology and precision. Bone 18:17S - 27S.

Dowthwaite, J.N., R.M. Hickman, J.A. Kanaley, R.J. Ploutz-Snyder, J.A. Spadaro, T.A. Scerpella. 2009 Distal Radius Strength: A Comparison of DXA-Derived vs pQCT-Measured Parameters in Adolescent Females. J CLin Densitom 12: 42 - 53.

Table Table 1.  
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Disclosures: Portia Flowers, None.


See Friday Plenary number FR0030.


See Friday Plenary number FR0031.


Comparison between Clinical Diagnostic Tools and QCT-based Finite Element Models for Predicting Human Vertebral Apparent Strength In Vitro.Enrico Dall'Ara*1, Dieter Pahr1, Peter Varga1, Franz Kainberger2, Philippe Zysset1. 1Vienna University of Technology, Austria, 2Medical University of Vienna, Austria

Vertebral fracture is a common medical problem in osteoporotic individuals. Bone Mineral Density (BMD) is nowadays the gold standard to evaluate fracture risk in vivo. Quantitative computer tomography (QCT)-based finite element analysis (FE) may be an attractive method to predict vertebral strength. The aim of this study was to compare the ability of FE and clinical diagnostic tools to predict vertebral strength in vitro using an improved testing protocol.

Soft tissues and endplates were removed from 37 vertebral bodies (level T12-L5, gender 7M-3F, age 44-82). The obtained sections were scanned with QCT (0.39×0.39×0.45 mm3) and the images calibrated with a phantom. Bone mineral content (BMC), total BMD (vBMD), areal BMD from lateral (aBMD-lat) and anterior-posterior (aBMD-ap) projections were evaluated. Wedge shape fractures were then induced in each section with a novel testing setup [1]. FE models were generated from QCT images coarsened to 1.33 mm3 by converting voxels directly to hexahedral elements. The boundary conditions were well controlled during the test and accurately reproduced in the models. For both experiments and nonlinear FE, both strength (defined as maximal load: Exp_FM and FE_FM) and apparent strength (defined as maximal load divided by mean cross section area: Exp_App_FM and FE_App_FM) were computed. Density-based (vBMD) and mass-based (BMC) measurements were used to predict Exp_App_FM and Exp_FM, respectively. FE and mixed variables (aBMD-ap and aBMD-lat) were used to predict both.

FE was found to better predict Exp_App_FM and Exp_FM (R2=0.80 and R2=0.79) than diagnostic tools (Exp_App_FM: R2=0.74 for vBMD, R2=0.41 for aBMD-lat and R2=0.48 for aBMD-ap; Exp_FM: R2=0.70 for BMC, R2=0.67 for aBMD-lat and R2=0.63 aBMD-ap). In particular, prediction of Exp_App_FM with FE was quantitatively correct without fitting and significantly better than the ones found for aBMD-ap and aBMD-lat (Fischer's z-test: p<0.01 for both).

Due to the normalization with area that accounts for bone dimension, Exp_App_FM may be the most relevant parameter for vertebral fracture risk. It should also be emphasized that our aBMD measurements were free of the artifacts that are usually generated by the posterior elements and iliac crest during standard DEXA. Nevertheless, it remains to be tested if significantly improved FE prediction of vertebral strength enhances significantly the determination of vertebral fracture risk.

[1] Dall'Ara et al., Bone, 44: P325, 2009

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Exp_App_FM predictions with aBMD-ap and FE_App_FM. Red dashed line represents 1:1 relation.

Disclosures: Enrico Dall'Ara, None.


CT-based Structural Rigidity Analysis Can Alter the Course of Treatment in Patients with Skeletal Metastasis.Brian Snyder*1, Ara Nazarian2, Vahid Entezari3, John Hipp4, Nathan Calderon5, Richard Terek6, Edward Cheng7, Albert Aboulafia8, Megan Anderosn3, Mark Gebhardt3, Timothy Damron9. 1Children's Hospital BostonHarvard Medical School, USA, 2Beth Israel Deaconess Medical Center, USA, 3BIDMC, USA, 4Baylor College of Medicine, USA, 5UT Austin, USA, 6Brown University, USA, 7University of Minnesota, USA, 8Sinai Hospital, USA, 9State University of New York Upstate Medical University, USA

The skeleton is the 3rd most common site of metastatic cancer, and a third to half of all cancers metastasize to bone. Clinicians make subjective assessments regarding fracture risk using clinical guidelines now recognized to be inaccurate. We have developed and validated a CT-based structural rigidity analysis (CTRA) method to monitor the fracture risk associated with metastatic lesions. We hypothesize that introduction of CTRA to the physicians' decision making process will alter the management of patients with skeletal metastasis. The aim of this study was to evaluate whether patient's treatment based on clinical guidelines was changed by CTRA.

X-rays and CT scans of the involved bones were obtained on all patients with a hydroxyapatite phantom included in the image to convert the X-ray attenuation for each pixel to bone mineral density. The elastic and shear moduli of elasticity were calculated from bone mineral density using empirically derived constitutive relationships. The cross-section through the affected bone that has the largest reduction in rigidity is the weakest and assumed to govern failure of the entire bone. The orthopaedic oncologist was asked to select between: Observation, chemo/radiation, non-invasive surgical stabilization and tntercalary allograft/prosthetic replacement based on the fracture risk assessment using Mirels' score and then again after reviewing the CTRA report. Treatment plans based initially on clinical guidelines and then again after CTRA were compared to identify cases where the treatment plan was changed as a result of CTRA.

Forty seven patients with 56 metastatic lesions to the appendicular skeleton were enrolled into the study. Based on Mirels' criteria (score ≥9), 43 (78.2%) lesions needed prophylactic fixation, while CTRA-based fracture threshold identified 28 (50.9%) lesions at risk for fracture and suggested prophylactic surgery. Treatment based on CTRA would have changed the initial treatment plan for 19 (34.5%) lesions. In 43 lesions, the clinician did not deviate from the original treatment plan, although in 11 of them CTRA indicated a different course of treatment, which of those 3 out of 11 (27.3%) were at high risk for fracture.

The results of the study suggest that CTRA has the potential to alter treatment plan based on clinical guidelines in 35% of the time. CTRA was most valuable in cases where bone destruction was moderate or osteoporosis was coexistent.

Disclosures: Brian Snyder, None.


See Friday Plenary number FR0034.


Effects of Rosiglitazone on Bone: Assessing QCT Parameters in a Mechanistic Study in Postmenopausal Women with type 2 Diabetes Mellitus.John Bilezikian*1, Barbara Kravitz2, E. Michael Lewiecki3, Colin Miller4, Allison R. Northcutt5, Gitanjali Paul5, Alexander R. Cobitz5, Antonio Nino6, Lorraine Fitzpatrick7. 1Columbia University College of Physicians & Surgeons, USA, 2GlaxosmithKline Pharmaceuticals, USA, 3University of New Mexico School of Medicine, USA, 4BioClinica, Inc. (formerly Bio-Imaging Technologies, Inc.), USA, 5CVM/MDC, GlaxoSmithKline, USA, 6GSK, USA, 7GlaxoSmithKline, USA

Abstract: This mechanism-of-action study assessed the effect of rosiglitazone (RSG) on bone mass and structure through imaging modalities including areal bone mineral density (BMD) and hip structural analysis (HSA) with dual-energy X-ray absorptiometry (DXA) of the lumbar spine and hip, high resolution magnetic resonance imaging (hr MRI) of the wrist, digitized hip radiography and volumetric BMD of the lumbar spine and hip with quantitive computerized tomography (QCT). Serum biomarkers of bone formation and resorption, calcium homeostasis and selected sex hormone changes over time were assessed. Parameters of glucose control included measurement of hemoglobin A1C (HbA1C), fasting plasma glucose (FPG) and serum insulin. Here we present the effect of RSG on volumetric BMD at the hip and lumbar spine as measured by QCT (

Methods: This was a Phase IV, 52 week, double-blind, multicenter study. A total of 226 postmenopausal women were randomized in a 1:1 ratio of RSG or metformin (MET). At baseline mean age was 63.8 ± 6.5 years while mean years post-menopause was 16.9 ± 8.4. Median duration of diabetes was 3.5 (1.8 -7.8) years. Other baseline characteristics were: mean body mass index (BMI) 31.4 ± 5.9 kg/m2; mean FPG and HbA1c were 111.7 ± 23.4 mg/dl and 6.4 ± 0.65% respectively. Femoral neck BMD was 0.83 g/cm2 ± 0.141, at total hip 0.98 g/cm2 ± 0.123, and at total spine 1.05 g/cm2 ± 0.161. Femoral neck T-score was -0.95 ± 0.908, total hip -0.02 ± 0.970, and total spine was -0.55 ± 1.247.

Using spiral multi-detector scanners, a subset of 79 subjects underwent QCT scans of the lumbar spine and hip at baseline and after 52 weeks of treatment. All scans were centrally evaluated and read. The change in integral, cortical and trabecular BMD at the lumbar spine and hip from baseline to week 52 was estimated within and between the two treatment groups.

Conclusion: Volumetric trabecular, cortical and integral BMD at the hip and the lumbar spine may provide insight on the effect of RSG on bone and may help explain the increased risk of fracture reported in long-term controlled clinical trials.

Disclosures: John Bilezikian, GlaxoSmithKline, 5

This study received funding from: GlaxoSmithKline


Effects of Rosiglitazone on Bone: Understanding its Effects through Assessment of Bone Structure using Digitized x-rays in a Mechanistic Study of Postmenopausal Women with type 2 Diabetes Mellitus.Antonio J. Nino*1, Claude Arnaud2, Rene Vargas-Voracek3, Cesar Bogado4, Barbara Kravitz5, Allison R. Northcutt1, Gitanjali Paul1, Alexander R. Cobitz1, Lorraine Fitzpatrick6. 1CVM/MDC, GlaxoSmithKline, USA, 2University of California at San Francisco, USA, 3Imaging Therapeutics, USA, 4Instituto De Investigaciones Metabolicas (IDIM), Argentina, 5GlaxosmithKline Pharmaceuticals, USA, 6GlaxoSmithKline, USA

Abstract: The study objective was to characterize the effects of rosiglitazone (RSG) on bone mass and structure in postmenopausal women with type 2 diabetes mellitus through different imaging modalities including dual-energy X-Ray absorptiometry (DXA) of the lumbar spine and hip region, hip structural analysis (HSA), high resolution magnetic resonance imaging (hr MRI) of the wrist, quantitative computerized tomography (QCT) of the lumbar spine and structural assessment using hip radiographs (ImaTx). Serum biomarkers of bone formation and resorption, calcium homeostasis and selected sex hormone changes over time were assessed. Parameters of glucose control included measurement of:hemoglobin A1C (HbA1C), fasting plasma glucose (FPG), and serum insulin. Here we present findings from assessment of hip radiographs.

Methods: This was a phase IV, double-blind, multi-center study ( NCT00679939) of 52 weeks duration. A total of 226 postmenopausal women were randomized to RSG or metformin (MET) in a 1:1 ratio. At baseline mean age was 63.8 ± 6.5 years while mean years post-menopause was 16.9 ± 8.4. Median duration of diabetes was 3.5 (1.8 -7.8) years. Other baseline characteristics were: mean body mass index (BMI) 31.4 ± 5.9 kg/m2; mean FPG and HbA1c were 111.7 ± 23.4mg/dl and 6.4 ± 0.65% respectively. Femoral neck BMD was 0.83 g/cm2 ± 0.141, at total hip 0.98 g/cm2 ± 0.123, and at lumbar spine 1.05 g/cm2 ± 0.161. Femoral neck T-score was -0.95 ± 0.908, total hip -0.02 ± 0.970, and lumbar spine is -0.55 ± 1.247.

All subjects underwent hip radiographs at Baseline and Week 52. All radiographs were read centrally. A total of 30 femoral geometry and cortical macro-anatomical-parameters were measured including hip axis length, neck-shaft angle, neck cortical thickness and shaft cortical thickness.

Conclusion: Trends and differences, observed between relevant group comparisons of bone structure measurements from hip radiographs of postmenopausal women followed during the study, may provide insight on the effect of RSG on bone and its relation to the risk of bone fractures.

Disclosures: Antonio J. Nino, GlaxoSmithKline, 3

This study received funding from: GlaxoSmithKline


See Friday Plenary number FR0037.


In Vivo Evaluation of an Anatomical Subject-specific FE-model to Predict Hip Fracture Load.Erwan Jolivet*1, Marie-Astrid DUCOBU2, Valérie Bousson3, Klaus Engelke4, Jean-Denis Laredo5, David Mitton2, Wafa Skalli6. 1Arts et métiers ParisTech, France, 2Arts et Metiers ParisTech, CNRS, LBM, France, 3Hôpital Lariboisière, France, 4University of Erlangen, Germany, 5Service de Radiologie ostéo-articulaire, Hôpital Lariboisière, Assistance Publique des Hôpitaux de Paris et Faculté de Médecine Diderot, Université Paris 7, France, 6Arts et Metiers ParisTech, CNRS, France

Finite element models have proved their ability to predict bone strength better than DXA and several author have developed and validated in vitro subject specific finite element models. However there is still no validation on in vivo data. Using a previously validated finite-element model of the proximal femur, this study investigated first whether contra lateral femur strength could be an appropriate indicator for in-vivo model evaluation and secondly if such finite element model is able to discriminate fractured patients from controls.

The model was applied to 22 patients, 11 controls and 11 with a hip fracture. All subjects were women with age ranged from 71 to 96 years and data were collected during the 3D-QCT European project.

The CTscan acquisition and reconstruction parameters were 1 mm contiguous slices, B40s convolution kernel and 0.293 mm in-plane pixel size. First, periosteal and endosteal surfaces were semi-automatically segmented using MIAF software (Kang et al. 2004) and hexahedral patient-specific mesh was automatically generated. Bone was considered as a heterogeneous isotropic material with a Poisson's ratio of 0.4. A Young's modulus and an ultimate stress were attributed to each element based on Hounsfield Units (Duchemin et al. 2008) and a stress based criterion was defined to assess fracture load. For simulation, the boundary conditions reproduced a stance configuration.

To validate the hypothesis of using the contra lateral femur for patient analysis, both right and left femurs in the control groups were modelled. No significant difference of the fracture load was observed using a paired t-test (p<0.05).

Considering three range of the fracture load (low < 5000 N, middle 5000-7000 N and high fracture load > 7000 N), 55%of the patients were classified in the low level group and only 18% in the high level group. On the contrary, 14% of the control subjects were in the low level group and 55% were classified in the high level group.

These results are promising as for the identification of subjects with high risk of hip fracture. Taking into account clinical parameters, such as defined by Frax index, should still improve the accuracy to go towards fracture risk estimation.

Duchemin, L., et al. Med Eng Phys 30(3): 321-8, 2008.

Duchemin, L., et al. Comput Methods Biomech Biomed Engin 11(2): 105-11, 2008.

Kang, Y., et al. Med Image Anal 8(1): 35-46, 2004.

Disclosures: Erwan Jolivet, None.


See Friday Plenary number FR0039.


Lean mass predicts bone mineral density and estimates of hip strength in middle-aged individuals with non-insulin-requiring type 2 diabetes mellitus.Kendall Moseley*1, Devon Dobrosielski2, Kerry Stewart2, Deborah Sellmeyer2, Suzanne Jan De Beur3. 1The Johns Hopkins Hospital, USA, 2The Johns Hopkins Bayview Medical Center, USA, 3Johns Hopkins University, USA

Purpose: Individuals with type 2 diabetes mellitus (T2DM) typically have higher bone mineral density (BMD), but higher fracture risk, than non-diabetics. Our aim was to examine the independent contributions of fat and lean mass to BMD and estimates of hip strength in those with T2DM.

Methods: Subjects for this cross-sectional analysis were men (n=78) and women (n=56) aged 56 ± 6 years with uncomplicated, non-insulin-requiring T2DM. Total body fat and lean mass as well as total body, hip, and lumbar spine BMD were measured with dual energy x-ray absorptiometry (DXA). Abdominal visceral, subcutaneous (SQ) and total abdominal fat were measured with magnetic resonance imaging. Averaged bilateral femoral neck strength measures including section modulus (SM), cross sectional area (CSA) and buckling ratio (BR) were estimated from DXA using validated hip structure analysis (HSA) formulae.

Results: Participants had normal BMD at all sites and were overweight to morbidly obese (BMI 29 to 41 kg/m2) with well controlled T2DM (HbA1C in women 6.6 ± 1.2%, men 6.7 ± 1.6%). In bivariate analysis, higher lean mass was associated with higher total body, femoral neck and hip BMD and higher SM and CSA across both sexes (r=0.36-0.55). In men only, lean mass was inversely correlated with BR (r=-0.27, p<0.05). Increased total body fat mass and abdominal SQ fat correlated with increased total body and hip BMD as well as increased SM and CSA in women (r=0.34-0.57). In men, these fat measures correlated with only total body BMD and no estimates of hip strength. In multiple regression analyses, lean mass remained a significant predictor of total body, hip and femur BMD in men and women, while fat mass predicted only total body BMD (p< 0.05). In a separate model including both sexes, lean mass significantly contributed to all three measures of hip strength.

Conclusions: In middle-aged men and women with non-insulin-requiring T2DM, lean mass was a significant predictor of BMD at the total body, hip and femoral neck. Lean mass predicted measures of hip strength including SM, CSA and BR. Fat mass may additionally contribute to BMD and HSA in women. These data suggest that maintenance of lean mass may preserve BMD and maintain hip integrity in those with T2DM who lose weight to improve insulin sensitivity. Strengthening exercises may be an intervention for hip fracture prevention in T2DM.

Disclosures: Kendall Moseley, None.


See Friday Plenary number FR0041.


See Friday Plenary number FR0042.


Predicting Subchondral Bone Structural Properties Using a Depth-Specific CT Topographic Mapping Technique in Normal and Osteoarthritic Proximal Tibiae.James Johnston*1, Saija Kontulainen1, Bassam Masri2, David Wilson3. 1University of Saskatchewan, Canada, 2University of British Columbai, Canada, 3University of British Columbia, Canada

Altered subchondral bone mechanical properties are undoubtedly involved in the initiation and/or progression of osteoarthritis (OA). To better understand the role of bone in OA we developed a 3D in vivo imaging tool: computed tomography topographic mapping of subchondral density (CT-TOMASD). CT-TOMASD employs quantitative CT (QCT) and surface projection image processing to characterize and map subchondral bone mineral density in relation to depth from the subchondral surface. The main objective of this study was to determine if CT-TOMASD could predict the structural stiffness of proximal tibial subchondral bone. A secondary objective involved assessing whether factors such as compartment (medial/lateral), age or cartilage degeneration (an indicator of OA disease) affected these predictions.

Fourteen proximal tibial compartments (4 medial, 10 lateral) from 10 donors (10M; age: 73+/-11 years) were CT scanned (0.5mm isotropic voxel) with a QCT bone density phantom. CT-TOMASD averaged subchondral bone density to depths of 1.5, 2.5, 5 and 10mm from the subchondral surface (Fig. 1). We performed indentation testing (3.5mm diameter flat indentor, 2mm/minute rate, 0.5mm displacement, 250N max load) directly at the subchondral surface (4-6 sites per compartment, 68 sites in total). Prior to indentation, cartilage degeneration was graded using ICRS scoring. Test sites were classified as normal (ICRS<2; n=39) or OA (ICRS>=2; n=29). We related structural stiffness to CT-TOMASD measures using power-law regression models (SPSS). We adjusted the models for compartment, age and cartilage status to test effect on model prediction. If no effect was observed, we tested the coincidence of separate regression models using F tests.

CT-TOMASD measures across the 0-1.5mm layer explained 54% of the variance in stiffness (p=0.000) (Fig. 2). After adjustments, the model explained 61% of the variance in stiffness (p=0.004). Density was the primary predictor (beta=0.77; p<0.001) while side (beta=0.24; p=0.005) improved model prediction. Age (beta=0.01; p=0.859) did not improve model prediction nor did cartilage status (beta=0.13; p=0.087). F tests revealed that separate normal and OA regression models exhibited similar stiffness-density relationships. CT-TOMASD measures to depths of 2.5, 5 and 10mm from the subchondral surface explained less variation in stiffness than across the 0-1.5mm layer. CT-TOMASD has potential to predict subchondral bone stiffness in vivo.

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Figure Figure 2. Stiffness-density plots for medial and lateral plateaus

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Figure Figure 1. CT-TOMASD images subchondral density from OA and normal knees

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Disclosures: James Johnston, None.


Predicting Trabecular Bone Elastic Properties from MRI-derived Measures of Bone Volume Fraction and Fabric.Michael Wald*1, Jeremy Magland1, Chamith Rajapakse2, Ed Guo3, Felix Werner Wehrli1. 1University of Pennsylvania Medical Center, USA, 2University of Pennsylvania School of Medicine, USA, 3Department of Biomedical Engineering, Columbia University, USA

Significant effort has been expended on predicting bone strength through in vivo assessment of its structure. Even though bone volume fraction (BVF) is predictive of bone strength, the variation in mechanical properties with test direction requires inclusion of measures of structural orientation. Here, we examine whether the inclusion of trabecular bone (TB) fabric can improve the BVF-based prediction of the elastic constants derived from linear μFE analysis of μMR images acquired with resolution and signal-to-noise (SNR) characteristics achievable in vivo.

Thirty demarrowed, cadaveric human distal tibia specimens were fixed in 1 mM aqueous Gd-DTPA/10% formalin solution. Images with a 160 μm isotropic voxel size were acquired using a two-channel phased-array ankle coil and a custom-designed 3D spin-echo sequence at 1.5T field strength. Micro-MR datasets were processed to yield BVF images with pure bone and pure marrow corresponding to intensities of 100 and 0, respectively. Two 7.5×7.5×7.5mm3 sub-regions of TB were extracted from the posterior (P) and lateral (L) portions of the tibia where the TB orientation is known to be different (Figure a). Each sub-region was subjected to orientation analysis via the mean-intercept-length (MIL) algorithm where angularly-sampled MIL measurements were fit to an ellipsoid with axes λ1, λ2, λ3. Six stress/strain simulations were performed via μFE analysis where each element was assigned a modulus proportional to its BVF with pure bone having 15GPa. The nine orthotropic elastic constants (three Young's moduli - Eii (i=1, 2, 3), three shear moduli - Gij (j=1, 2, 3), and three Poisson's ratios - vij) were fit to a model [Cowin, 1985] of the normalized fabric eigenvalues λ1, λ2, λ3 and power-law functions of BVF where the exponent a was allowed to vary from 1 to 3.

Each sub-region's Eii were best predicted by separate power-law functions of BVF (R2s=0.92-0.98, Figures b and c - P and L sub-regions, respectively). Adding fabric into the model predicted the pooled data for both analysis regions and all testing directions with high accuracy (R2=0.986, Figure d). Model agreement for pooled Gij and vij were also good when fabric information was considered (R2s=0.99 and 0.68). Non-pooled models of BVF explained variations in Gij, but not in vij (P: R2s=0.92-0.97 and 0.08-0.51, L: R2s=0.92-0.96 and 0.04-0.42). Inclusion of fabric information established a general model between BVF and elastic constants independent of subregion and test direction. The data emphasize (1) the importance of fabric as a predictor of bone mechanical properties, and (2) that these relationships can be assessed in the limited resolution and SNR regime of μMRI. 

Disclosures: Michael Wald, None.


Quantitative Characterization of Motion Artifact in HR-pQCT Images of the Distal Radius and Tibia.Miki Sode*1, Andrew Burghardt2, Jean-Baptiste Pialat3, Thomas Link2, Sharmila Majumdar2. 1University of California San Francisco & Berkeley, USA, 2University of California, San Francisco, USA, 3INSERM U831, Université de Lyon & Hospices Civils de Lyon, France

Motion artifacts are commonly seen in in vivo high-resolution peripheral quantitative computer tomography (HR-pQCT) images, especially at the radius. Compromised image quality confounds the accuracy of cortical and trabecular densitometric and structure indices. It often leads to repeating the acquisition, thereby exposing the subject to more radiation. Criteria for accepting/rejecting an image are highly subjective. Therefore, the objective of this study was to develop a technique to measure motion and estimate error.

A total of 54 sets of HR-pQCT images of the distal radius (N=33) and tibia (N=21) acquired for various studies conducted in our laboratory were reviewed. Each set consisted of 2 acquisitions of the same site before and after repositioning. At least one image was graded 1 (no visible motion) according to the manufacturer-suggested quality grading system. The cortical and trabecular densitometric and structure indices were calculated for each image, and the % difference between paired was calculated. The amount of motion during a single acquisition was quantitatively measured in the following manner. First, the raw cone beam projection images were parallelized and corrected for dark and flat field intensity. Next the projected bone region at 0° and at 180° flipped with respect to the detector center were compared using i) normalized mutual information (MI), ii) sum of squared intensity difference (SSD), iii) entropy (E), iv) ratio image uniformity (RIU), and v) maximum normalized cross correlation (NXC). The mean % difference between paired acquisitions was calculated for each measure. A linear regression analysis was performed for the correlation (Spearman's ρ) between the % differences in each index and the similarity measure. Radius and tibia data were pooled.

The % differences in E, RIU, and NXC positively and significantly correlated with the % differences in cortical and trabecular densitometric and structure indices (ρ=0.27-0.58; all p≤0.04). Cortical area and thickness as well as trabecular bone volume fraction, -number and -spacing had strong correlations with these similarity measures (all ρ≥0.36 and p≤0.007) (Figure 1).

The result of this study provides a basis for quantitative characterization of motion artifact.

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Figure Figure 1.  

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Disclosures: Miki Sode, None.


Radiation Therapy Causes a Rapid Loss of Bone Mineral Density of Thoracic Vertebra in Women and Men Lung Cancer Patients.Michael Lawrence*1, Jeffrey Willey2, Mert Saynak1, Ted Bateman3, Lawrence Marks1. 1University Of North Carolina, USA, 2Clemson University, USA, 3Univesity of North Carolina, USA

Cancer patients receiving radiation therapy (RT) for pelvic tumors are at greater risk of hip fractures. The mechanisms are unknown, but recent preclinical studies have identified a rapid activation of osteoclasts. We hypothesize that RT also causes atrophy of thoracic vertebra (ThV) during lung cancer treatment. A retrospective analysis of 25 lung cancer patients was performed. Change in volumetric bone mineral density (vBMD) was examined using pre- and 6 months post-RT thoracic CT scans. Ten women (mean age of 61 years) and 15 men (72 years) were studied; no patients received chemotherapy or antiresorptive treatment.

Patients typically received a total RT dose of ∼66 Gy in 2.0Gy/day fractions to the tumor. The trabecular bone of 6 vertebral bodies were contoured using UNC's RT treatment planning system (PlanUNC); the average CT density was calculated for pre- and post-RT contours. The mean dose for each ThV was determined by PlanUNC, permitting assessment of the dose-dependent nature of vBMD changes. All density changes were normalized to tissue changes well outside the treatment beam. Significance was determined by paired t-test.

The average loss of ThV vBMD for each patient was -21% (p<0.001), with no difference in the degree of loss for women (-21%, p<0.001) and men (-21%, p<0.001). Linear regression analysis showed no correlation with patient age and vBMD decline. By calculating RT dose to each vertebra within a patient, we were able to examine the relative radiation sensitivity of ThV trabecular bone. Significant loss of vBMD occurred across dose ranges: 45+ Gy (-24%, p<0.001); 35-45 Gy (-23%, p<0.001); 25-35 Gy (-15%, NS); 15-20 Gy (-16%, p=0.047); and 5-10 Gy (-20%, p=0.003). No differences were observed between doses using a 1-way-ANOVA.

Lung cancer patients receiving RT lost ThV vBMD in incidentally irradiated bone at a rapid rate of ∼3% per month. Trabecular bone within the ThV appears to be a radiation-sensitive tissue, occurring with only a total dose of 5-15 Gy. This rate of TrV bone loss confirms other clinical data from pelvic tumor RT. This rate of loss also conforms to preclinical reports showing early osteoclast activation from RT. Past studies showing bone loss or fractures from RT have only examined women. In this study, men and women were equally susceptible to bone loss. Future studies need to examine subsequent recovery of vBMD and strength. Antiresorptive therapies may be appropriate to prevent this RT-induced bone loss.

Disclosures: Michael Lawrence, None.


Relationship Between Age, Mineral Characteristics, Mineralization, Micro-hardness, And Microcracks In Human Vertebral Trabecular Bone.Delphine Farlay*1, Yohann Bala2, Brigitte Burt-Pichat3, Roland Chapurlat3, Georges Boivin4, Helene Follet5. 1University of Lyon, France, 2Université De Lyon, France, 3INSERM U831, Université de Lyon, France, 4INSERM, France, 5INSERM, Université De Lyon, USA

Relationship between mineralization and microcracks has been mainly studied on the same Bone Structural Units in cortical bone (1). Our purposes were to analyze at tissue level the contribution of bone mineralization and mineral characteristics in the development of microcracks in trabecular bone. Human vertebrae trabecular cores (L2, Ø 8.2mm, height 10mm) from 53 cadavers (54-95 yrs) were embedded in PMMA for analysis of pre-existing microcracks by bulk staining in xylenol (2), Crack length (Cr.Le) and crack density (Cr.Dn) were measured. Fourier Transform Infrared Microspectroscopy was performed on 2μm-thick sections (mean of 20 areas by sample, 30*100μm each). Mineral maturity (MM) was the ratio between apatitic/non apatitic phosphates, and mineralization index (MI) the ratio between mineral/organic matrix. Crystallinity index (CI) reflected size/perfection of bone crystals (3-4). Quantitative microradiography performed on 100μm-thick sections allowed the measurement of the degree of mineralization (DMB), and microhardness (Hv) was calculated on the surfaced block (5). Normality of the distributions was tested, and logarithmic transformation was performed for Cr.Le and Cr.Dn. Relationships between variables were tested using Pearsons' correlations. A positive correlation between age of donor and CI was found. DMB, Hv and MM were positively associated with MI. Interestingly, a positive correlation was found between Cr.Le and DMB, MI, MM, but not with CI (Table). The more mature the mineral was, the higher were the degree of mineralization and the crystallinity, and the harder was bone tissue. Moreover, the length of microcracks increased with the mineralization (degree and maturity), whereas crystals size/perfection had no influence. Thus, propagation of microcracks was more influenced by the age of mineral than by crystals dimension and composition. Finally, the density of microcracks was not influenced by mineralization, suggesting that their propagation and density are not linked. In conclusion, in vertebral bone, crystallinity (crystal size/perfection) increased with age of donor, and could explain partly bone fragility.

1 Wasserman et al Eur J Morph 2005, 42: 43.

2 Arlot et al J Bone Miner Res 2008, 23: 1613.

3 Farlay et al J Bone Miner Metab 2010 Epub

4 Bala et al Bone 2010, 46: 1204.

5 Boivin et al Bone 2008, 43: 532

Table Table. Pearsons' coefficient correlations
  1. *p<0.05; **:p<0.005

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Disclosures: Delphine Farlay, None.


See Friday Plenary number FR0048.


See Friday Plenary number FR0049.


See Friday Plenary number FR0050.


The Effects of Simulated Microgravity and Return to Weightbearing on Densitometric and Mechanical Properties of the Femoral Neck in the Adult Rat HU Animal Model.Joshua Kupke, Scott Morgan*, Yasaman Shirazi-Fard, Joshua Davis, Joseph Marchetti, Alyssa McCue, Susan Bloomfield, Harry Hogan. Texas A&M University, USA

Microgravity-induced bone loss is one of the primary concerns for astronaut health on long duration spaceflights. However, bone mineral density (BMD) does not always provide accurate predictions of changes in bone quality and strength. Thus, bone strength indices based on CT scans have been devised using various combinations of both geometric and densitometric properties The objective of the current study was to characterize the effects of microgravity, and recovery, on the femoral neck (FN) using the adult hindlimb unloaded (HU) rat model. An additional important goal was to compare calculated strength indices to mechanical testing results at the FN.

Adult male Sprague-Dawley rats (6-mo) were grouped into baseline (BL), weightbearing cage control (CC), and hindlimb unloaded (HU). HU animals were further divided into sub-groups (n=15 each): HU euthanized after 28d of suspension, and HU euthanized after 28, 56, and 84 days of recovery. CC groups were euthanized at the same four end points. The excised right femur was scanned ex vivo at the FN using peripheral quantitative computed tomography (pQCT). Mechanical testing was performed with the femur oriented vertically and quasi-static loading applied to the femoral head in a proximal-to-distal direction. This conventional approach creates a combination of compression, bending, and shear in the FN.

HU exposure gave rise to 6.3% lower bone mineral content (BMC) compared to BL and 7.8% lower total volumetric (integral) bone mineral density (vBMD) at the FN. The most dramatic effect was for the neck compression strength index (NCSI), defined as vBMD2 times the minimum cross-sectional area. The mean NCSI was 13.4% lower for HU compared to BL. By contrast, the neck bending strength index (NBSI) was only 3.9% lower. As an estimator of bone strength, NCSI corresponds more closely to the actual maximum breaking force for the femoral neck, which was 17.1% lower after 28d HU. Considering recovery after return to weightbearing, BMC was only 2.5% lower than BL after 28d of recovery and returned to the same level as BL after 56d. After 28d of recovery, vBMD was 5.1% lower than BL but was higher after 56d. The NCSI remained 10.5% and 5.8% lower after 28d and 56d recovery, respectively. However, the measured breaking force returned to BL levels after only 28d of recovery. Thus, the NCSI is a reasonable predictor of strength for the initial exposure to HU, but underestimates actual strength values during recovery.

Disclosures: Scott Morgan, None.


See Friday Plenary number FR0052.


See Friday Plenary number FR0053.


Mechanically-induced Signaling Events in Loaded Bones In-vivo Include Activation of ERK1/2 During Functional Adaptation.Jason Bleedorn*1, Susannah Sample2, Troy Hornberger3, Peter Muir4. 1University of Wisconsin-Madison, USA, 2UW-Madison School of Veterinary Medicine, USA, 3School of Veterinary Medicine - UW Madison, USA, 4University of Wisconsin, Madison, USA

Purpose: Mechanical stimuli play an important role in the regulation of bone mass. Failure of the skeleton to maintain bone mass contributes significantly to patient morbidity and mortality in conditions such as osteoporosis. The mechanisms involved in converting biophysical signals into the molecular events that regulate functional adaptation have not been defined. The present study was designed to determine activation of mechanically-induced signaling events in bone in an in-vivo bone loading model.

Methods: 22 male rats aged 10-12 weeks were used. 8 rats were used to determine the timeline of signaling events after mechanical loading. 4 rats were used as controls, 2 of which received insulin treatment (5 IU/kg Humulin R) as positive control, and 2 which were sham loaded. The remaining 4 rats had their right ulna loaded at 4Hz for 1,500 cycles at -3,750μe (-18N). Individual rats were euthanatized and both right and left ulnas were collected at 0, 15, 30 and 60 minutes after in vivo loading. The remaining 14 rats were used for further investigation of specific time points after bone loading using the same loading protocol. Right and left ulnae were collected at 15 minutes (n=4 loaded, n=3 sham) and at 60 minutes (n=4 loaded, n=3 sham) after right ulna loading. Bones were immediately frozen in liquid nitrogen, ground to a powder with a pestle and mortar, and the proteins extracted. Phosphorylation of p70S6k, PKB, p38, JNK and ERK was examined using Western blot analysis.

Results: Mechanical loading resulted in a cascade of protein phosphorylation evident at various time points in the first phase of the study. Activation of p70S6k(389) PKB(308) PKB(473), JNK1/2 and ERL1/2 were detected in the loaded right ulna after mechanical loading. Activation of p38 was not detected at the time points examined. In the second phase of the study, activation of the ERK signaling pathway (p-ERK1/2) was consistently (n=4) seen in loaded bone at 15 minutes after mechanical loading.

Conclusion: These results confirm that it is possible to detect mechanically-induced signaling events in loaded bone during functional adaptation in-vivo. Load-induced bone formation is associated with activation of p70S6k, PKB, ERK1/2 and JNK1/2 at distinct time points in loaded bones. Further work is needed to determine whether additional mechanically-induced signaling events are evident to further characterize the molecular mechanisms that regulate functional adaptation of the skeleton.

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Signaling time line for p70S6k(389) after mechanical loading of the right ulna

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ERK signaling at 15 minutes following mechanical and sham loading of the right ulna

Disclosures: Jason Bleedorn, None.


See Friday Plenary number FR0055.


Are Exercise-induced Gains in Lumbar Spine vBMD Driven by Changes in Back Extensor and Psoas Muscle Size in Older Men? An 18-month Randomised Controlled Trial.Christine Bailey*1, Sonja Kukuljan2, Riku Nikander3, Caryl Nowson2, Robin Daly3. 1The University of Melbourne, Australia, 2School of Exercise & Nutrition Sciences, Deakin University, Melbourne, Australia., Australia, 3The University of Melbourne, Western Hospital, Australia

Since muscle and bone are inextricably linked, it is often assumed that changes in muscle size and strength should affect bone strength predictably and correspondingly. For older adults, progressive resistance training (PRT) can markedly improve muscle mass and strength, but its effects on LS BMD are mixed. In addition, from the limited Ex trials that have reported a positive effect on LS BMD, few have examined whether the changes in bone are predominantly mediated by gains in back muscle CSA. The aims of this study were to examine: 1) the effects of a multi-component Ex program incorporating PRT and weight-bearing impact Ex on erector spinae (ErSp) and psoas muscle CSA, and CT-measured lumbar spine vBMD in older men; 2) whether any exercise-induced changes in bone were mediated by changes in muscle CSA; and 3) the effects of training load on muscle and bone. Men (n=180) aged 61 ± 7 years (± SD) were randomised to an Ex or non-Ex group with or without additional Ca-Vit D3. The Ex consisted of high intensity (60-85% 1RM) PRT and weight-bearing impact Ex performed 3 d/wk for 18 mo. QCT was used to assess L1-L3 total and trabecular vBMD (TrvBMD), and muscle CSA. Ex led to a significant 2.3% net gain in TrvBMD and 1.6-4.9% net increase in ErSp and psoas muscle CSA relative to no-Ex (all p< 0.05). The Ex-induced gains in ErSp, psoas and total back muscle CSA were correlated with the changes in TrvBMD (r=0.26-0.33, p<0.05-<0.001). Similar significant correlations were observed in the no-Ex group (r=0.33-0.41, p<0.01-< 0.001). However, further analysis revealed that there were no differences in the slopes nor intercepts for the changes in muscle and bone between the Ex and no-Ex group. This suggests that the Ex-induced gains in vBMD were not mediated by the changes in muscle size. When we examined the influence of training load on the skeletal changes, we found that the average RT load and number of impacts per session were related to the changes in TrvBMD (r=0.38 and 0.43, respectively, p< 0.001). When both parameters were entered into the regression model, only the number of impacts per session remained significantly related to the changes in TrvBMD (p<0.05). In conclusion, an 18-month Ex program consisting of PRT and impact training was effective for improving both back muscle size and lumbar spine BMD, but these skeletal changes appeared to be related to the number of impacts per session and not the gains in muscle size.

Disclosures: Christine Bailey, None.


Effects of Pulsed Electromagnetic Field Stimulation on Gene Expression Related to Bone Formation in Spontaneously Hypertensive Rats.Hiroaki Takekura1, Hiroyuki Tamaki*2, Hideaki Onishi2, Tomie Nishizawa3, Kengo Yotani1, Atsumu Yuki1, Futoshi Ogita1, Hikari Kirimoto2, Kounosuke Tomori1. 1National Institute of Fitness & Sports, Japan, 2Niigata University of Health & Welfare, Japan, 3Chukyo Women's University Junior College, Japan

Pulsed magnetic stimulation (PMS) has been used to treat bone disorders and reportedly modifies differentiation, proliferation and mineralization of osteoblasts in vitro. The spontaneously hypertensive rat (SHR) displays a disorder characterized by cancellous bone deficit in the skeleton. We examined the temporal effects of repetitive PMSs (rPMS) on bone formation in vivo for SHR tibiae and femora and investigated magnetic field mapping in bone during PMS. The magnetic sensor was placed in the distal metaphysis of the femur to determine maximum magnetic field strengths during PMS. Measurements were made at 9 positions (X-Y axis) and 9 distances (Z axis) from the sensor during PMS supplied at 10 stimulation intensities in increments of 10% of the maximal output of the stimulator. One hindlimb of male SHR and normal rats at 6 and 33 weeks old was held on the table in the prone position and the PMS probe was placed 10-20 mm from the thigh and leg. A stimulation intensity of 60-80% was used for rPMS at a frequency of 0.5-1 Hz, for 3-5 min. After sacrifice, tibiae were analyzed by quantitative histomorphometry and femora were used for analysis of bone morphogenetic protein (BMP)-2, transforming growth factor (TGF)-β2, osteocalcin (OC), and collagen (Col)-I mRNA expressions by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Magnetic field strength in the bone during PMS showed a nonlinear hyperbolic decrease and a linear increase depending on probe distance and stimulation intensity, respectively, within the range of 250-450 mT at a distance of 10-20 mm and 60-80% stimulation intensity. Lower trabecular bone volume (BV/TV) was observed in old SHR compared to normal old rats. RT-PCR showed that rPMS stimulated mRNA levels of BMP2 and TGF-β2 after 24-h of treatment compared to the contralateral hindlimb. These findings suggest that temporary local rPMS on the bone provides potent stimulation of bone formation in vivo for SHR by promoting BMP-2, and TGF-β2 production.

Disclosures: Hiroyuki Tamaki, None.


Loaded Physical Activity Predicts Bone Strength, but not bone microstructure, at the Distal Radius in Adolescents.Heather McKay*, Danmei Liu, Deetria Egeli, Melonie Burrows. University of British Columbia, Canada

Physical activity (PA) has a positive effect on bone mineral content and bone geometry in male and female adolescents across a range of skeletal sites. There is also evidence for the beneficial effects of PA on bone strength and bone microstructure at the distal tibia, an inherently loaded site, in adolescents during growth. However, no studies have examined the relationship between PA, bone strength and bone microstructure in adolescents at inherently unloaded sites. Our aims in this cross-sectional study were to evaluate the independent contribution of PA to bone strength and bone microstructure in adolescent males and females at the radius. We assessed the distal radius using XtremeCT (Scanco Medical) at the 7% site in 113 males (Age 17.4 ± 1.6yrs; Height 175.6 ± 7.3cm; Weight 69.0 ± 15.4kg), and 97 females (Age 17.9 ± 1.8yrs; Height 162.8 ± 6.9cm; Weight 59.5 ± 11.3kg). We assessed PA (loaded and unloaded) using the modified PA Questionnaire for Adolescents [1]. Our primary outcome was bone strength index (BSI, mg2/mm4; ToA (mm2) × ToD2(mg/cm3) [2]. We also assessed cortical bone density (CoD, mg/cm3), cortical thickness (Cort.Th, mm), trabecular bone density (TrD, mg/cm3) and trabecular number (Tb.N, 1/mm). We used multiple regression (STATA, Version 10.1) to evaluate the independent contribution of PA (PA, hours per week) to bone outcomes after adjusting for maturation (Tanner), height (cm), weight (kg), radius length and calcium intake (mg/day). Loaded PA was a significant predictor of BSI, ToA and TrD in males (P<0.001; Table 1). Loaded PA was a significant predictor of BSI only in females (P<0.05; Table 1). There was no influence of loaded PA on bone microstructure in either sex (P<0.05). There was no influence of unloaded PA on BSI or bone microstructure in either sex (P<0.05). Our results suggest that whilst loaded PA has a positive effect on bone strength at the radius in both sexes, it has no significant effect on bone microstructure. These findings are at odds with the reported beneficial effects of PA on bone microstructure at the distal tibia. Data are required to further assess the influence of PA on inherently loaded and unloaded skeletal sites across growth.

1. Crocker PR, Bailey DA, Faulkner RA et al.(1997) Measuring general levels of physical activity. Medicine and Science in Sports and Exercise 29:1344-1349

2. Martin RB (1991) Determinants of the mechanical properties of bones. J Biomech 24 Suppl 1:79-88.

Table Table 1. Multiple regression results for HR-pQCT variables at the distal radius (7% site) for male and female participants.
  1. Note: Only models and variables with significance p<0.05, are shown β = estimated regression coefficient; SE = standard error.* R2 change significant at p<0.05; **R2 change significant at p<0.001.

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Disclosures: Heather McKay, None.


Maturity-specific Differences Attributed to Gymnastic Loading at the Distal Radius.Jodi Dowthwaite*1, Portia Flowers2, Rebecca Hickman2, Paula Rosenbaum2, Tamara Scerpella3. 1State University of New York, Upstate Medical University, USA, 2SUNY Upstate Medical University, USA, 3State University of New York Upstate Medical University, USA

Purpose: Loading exposure during growth is linked to high bone mineral indices, but few studies address maturity and tissue-specific adaptations in bone geometry and density. The current study tests for an interaction between gymnastic loading and menarche status.

Methods: Contemporaneous pQCT (pQ) and DXA (DX) scans were performed at the radial metaphysis (4%, Ultradistal (UD)) and diaphysis (33%, 1/3) in 113 females aged 8-25 yrs (mean 14.9 yrs). Premenarcheal (PRE) subjects included 24 nongymnasts (NON), 30 gymnasts (GYM, exposure >4 h/wk). Post-menarcheal subjects (POST) included 5 gymnasts and 26 exgymnasts (EX/GYM exposure >2yrs at >6h/wk); all others were defined as NON (n=28). Two factor analysis of covariance evaluated gymnastic exposure and menarche status, adjusting for age and height.

Results: EX/GYM and NON differed in height and % body fat (NON>EX/GYM)(p<0.02); POST exceeded PRE means for all but % body fat (p<0.05). At both sites, DX area, aBMD, all BMC, all periosteal and cortical areas were greater in EX/GYM than NON (p<0.05), as was 4% trabecular vBMD. However, diaphyseal cortical vBMD was higher in NON than EX/GYM (p<0.05). Interactions were influential at both sites. EX/GYM emphasized greater periosteal and trabecular/intramedullary dimensions in late puberty, whereas POST NON emphasized only thickened cortices, yielding higher total vBMD/BMAD than EX/GYM (p<0.05). Fewer interactions were detected by DXA.

Conclusion: Distal radius differences attributed to gymnastic loading during growth are maturity-specific. POST EX/GYM advantages emphasize bone geometry rather than density; this may reflect pubertal loading and/or training cessation (84% ex). High adjusted bone outcomes in POST NON suggest pubertal acceleration of cortical growth. Generally, DXA and pQCT results agreed. However, pQCT detected more significant interactions, potentially reflecting greater sensitivity, accuracy or positional influence. 

Table Table.  
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Disclosures: Jodi Dowthwaite, None.


Normal Masticatory Function Protects the Rat Mandibular Bone from Estrogen Deficiency-related Bone Loss.Anestis Mavropoulos1, René Rizzoli2, Stavros Kiliaridis1, Patrick Ammann*2. 1University of Geneva, Switzerland, 2University Hospital, Geneva, Switzerland

Purpose. In a previous investigation we showed that mandibular alveolar (trabecular) bone appears to be less sensitive to estrogen-related osteoporosis than the proximal tibia spongiosa. We hypothesized that the heavy mechanical loading during mastication may protect the alveolar bone from the detrimental effects of estrogen deficiency observed in other skeletal sites. In this study we tested this hypothesis by comparing the effect of ovariectomy on the mandibular alveolar bone of Sprague-Dawley rats fed either a normal (hard) or a soft diet. Methods. Forty six-month-old female Sprague-Dawley rats underwent transabdominal ovariectomy (OVX) or sham operation (SHAM). Half of the animals received their food in the usual form of pellets (hard food), while the other half received a soft, porridge-like, isocaloric food. The experiment lasted 16 weeks. Micro-computed tomographic histomorphometry was used in order to evaluate the effect of estrogen deficiency on the mandibular alveolar bone. Results. Both experimental manipulations (OVX, soft diet) had a significantly negative impact on the micro-architecture of the mandibular alveolar bone. Soft diet led to a reduction of BV/TV (p=0.02), and trabecular thickness (p=0.03) and number (p<0.03). The conjunction of soft diet and OVX led to a significant reduction of BV/TV (p<0.001), and trabecular thickness (p<0.001), number (p<0.001), and separation (p<0.001). Ovariectomy led to 21.2% reduction of BV/TV in rats fed the soft diet (p<0.001), whereas a non significant change (-8.2%) was observed in those receiving the hard diet. Conclusions. Estrogen-deficiency seems to only minimally influence rat mandibular alveolar bone under normal masticatory mechanical loading, at least for the duration of this experiment. Soft diet and the ensuing hypofunction of the masticatory system significantly accentuated the negative effects of estrogen deficiency on the mandibular alveolar bone. 

Disclosures: Patrick Ammann, None.


Site and Frequency Specific Effects of Whole Body Vibration on Axial and Appendicular Structural Bone Parameters in Aged Rats.Marion Pasqualini*1, Norbert Laroche1, Arnaud Vanden Bossche1, Laurence Vico2. 1Inserm U890 - Lyon University, France, 2University of St-Etienne, France

Mechanical stimulation of bone by Whole Body Vibration (WBV) was shown to have osteogenic effects. Most studies of WBV in rodents were done in growing, or gonadectomized animals. There is a lack of reports of WBV in aged adult animals and thus no preclinical evidence to support WBV in the prevention of bone loss. The goal of this study was to test the skeletal effects of different WBV frequencies, applied with the same acceleration, in aged rats. Our hypothesis was that high frequencies (90Hz) are more anabolic than low frequencies (8Hz). We exposed 48 male Wistar, 10-month rats to 4 weeks of vertical vibrations for 10 min/day, 5 days/week at 0.5g at frequencies of 8Hz, 52Hz or 90Hz. Trained rats stood free on the vibrating platform, control rats stood on the unmoving platform. Proximal tibia metaphyses were scanned in vivo by micro-CT (VIVA CT40, Scanco Medical) at the initiation and the end of the training period. At sacrifice, we measured trabecular and cortical bone parameters in the distal femur and L2 vertebral body. Post-hoc multiple comparisons were done when ANOVA results were significant. Rats vibrated at 8 and 52Hz lost body weight, while weight increased under a 90Hz training. Longitudinal follow up of the trabecular proximal tibia showed no significant effect of WBV. At sacrifice however, the trabecular compartment of the distal femur showed larger trabeculae (+11%, n=12, p<.0001) and an increased degree of anisotropy (+29%, n=12, p<.0001) at 90Hz vs controls. In contrast, these parameters were decreased at 52 Hz and even more at 8Hz vs 90Hz. Bone volume, trabecular number and connection density were all increased at 90Hz as compared to 52 and 8Hz. At 90Hz vs control, the cortical thickness is increased (+11%, p<0.01) and the porosity is reduced (-50%, p<0.05). In the L2 vertebra, all trabecular parameters were improved at 90Hz vs controls, bone volume and density connection were also increased at 52Hz, while no effect was observed at 8Hz. We showed that 0.5g WBV at 8Hz and 52Hz is either deleterious or inefficient in long bones. The anabolic effects of the 90Hz regimen observed in the vertebrae suggest that bone volume would also increase in the femur and tibia after a longer training period. The cortical effects also suggest improved biomechanical properties. This study showed that in WBV, in addition to the acceleration level, frequency is an important parameter with site-specific effects. 

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Figure Figure 1.  

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Disclosures: Marion Pasqualini, None.


A New Urinary Bio Marker of MMP-derived Cartilage Degradation.Anne-Christine Bay-Jensen1, Jianxia Wang2, Inger Byrjalsen3, Yi Li4, Qinlong Zheng4, Kim Henriksen5, Per Qvist5, Morten Karsdal*5. 1Nordic Bioscience, Denmark, 2Nordic Bioscience China, Denmark, 3CCBR-Synarc, Denmark, 4Nordic Bioscience Beijing, China, 5Nordic Bioscience A/S, Denmark

Joint degenerative disease is characterized as progressive damage of the joint tissue, which includes cartilage erosion, osteophyte formation, subchondral sclerosis, and synovial alterations. The turnover of cartilage is normally maintained by a balance between catabolic and anabolic processes. However, in the case of pathological matrix destruction, the rate of cartilage degradation exceeds the rate of formation, resulting in a net loss of cartilage matrix. The specific products of tissue formation and degradation can be measured in serum and urine, as markers of pathology. A monoclonal antibody was raised against a MMP9-derived neoepitope in type II collagen, which was identified mass spectrometry (N-terminal-GRDGAAG1053). An ELISA assay, called CIIM, was developed and optimized. The level of CIIM fragments was measured in urine samples from a normal population of men and women (N=145, age range 22 - 84 years). Immunohistochemistry was done to validate the presence of the neoepitope OA cartilage samples from patients undergoing knee replacement. The technical performance of the assay was accepted, giving inter- and intra-assay CV below 6%. We verified that the neoepitope was actually MMP-dependent by adding MMP-inhibitor (GM6001) to cartilage cultures and measuring the release of the fragment to the medium. GM6001 could inhibit the release of CIIM from catabolic active cartilage when compared to catabolic active control. Next, we measured the level of CIIM in urine of a normal population and found that there was a significant difference between men (84.8 pg/mmol creatinine, n=82) and women (126.4 pg/mmol creatinine n= 74, p<0.01). We also found that post-menopausal women (153.2 pg/mmol creatinine, n=47) had a higher level than pre-menopausal women (83.0 pg/mmol creatinine, n=27, p<0.01). We found the neoepitope to be immunolocalized to OA pathological features of the cartilage: 1) to eroded surface of the cartilage; 2) to the calcified cartilage; 3) to lesions. We developed a new biomarker assay that measures type II collagen degradation by MMP. We believed that this new markers could be a new and valuable tool for measuring cartilage breakdown observed in joint degenerative disease such as OA.

Disclosures: Morten Karsdal, Nordic Bioscience, 3


See Friday Plenary number FR0063.


Exercise May Prevent Regional Bone Loss Induced By Monosodium Iodoacetate Osteoarthritis Model.Arnaud Boudenot*1, stephane pallu2, Eric Dolleans2, Eric Lespessailles3, Christelle Jaffre2, Claude Laurent Benhamou4. 1Inserm Unit U658Hopital Porte MadeleineOrléansFrance, France, 2INSERM UNIT U 658, France, 3Centre Hospitalier Regional, France, 4INSERM Orléans-France, France

Knee injuries (i.e. anterior cruciate ligament tear or menisectomy surgery) are frequent in contact sports and may lead to early knee osteoarthritis. Monosodium iodoacetate (MIA) injection into joints is a validated animal model of induced osteoarthritis. This study aims to demonstrate the possible protective effect of exercise (treadmill running) on the subchondral bone changes at the proximal tibia (PT) and distal femur (DF) in the MIA-induced osteoarthritis model.

Male Wistar rats aged of 13 weeks (n=47) were divided into two groups: exercise (Ex) (n=24) or non exercise (NEx) (n=23). Ex rats were subjected to intermittent progressive training one hour per day, 5 days per week for 10 weeks. At the end of the training period, each group was divided into two subgroups and underwent either MIA injection (1mg/100μL NaCl) or NaCl (100μL) into the right knee joint leading to 4 groups: 1) Ex-NaCl, 2) Ex-MIA, 3) NEx-NaCl and 4) NEx-MIA. Bone Mineral Density (BMD) was evaluated on specific regions of interest (PT and DF) at baseline, after training (10 weeks) and at sacrifice (14 weeks). All data are expressed as mean ± SD. Mann-Whitney U test was used to determine the statistical significance of the differences between groups, p values lesser than 0.05 were considered significant *.

Before the MIA or NaCl injection we did not observed any significant differences between groups in BMD at the PT or DF subregions. Mean PT-BMD in the Ex-MIA group before injection was 0.429 ± 0.030 and 0.435 ± 0.021 g/cm2 at sacrifice (p = NS). In contrast, in the NEx-MIA group we observed significantly lower values of mean TP-BMD at sacrifice than before injection (0.409 ± 0.022 vs 0.429 ± 0.025 g/cm2; p=0.03). At sacrifice, we did not observed significant differences in BMD at DF between groups. In contrast PT-BMD of the injected knees was significantly lower in the NEx-MIA group compared to the other groups (Fig.).

These results suggest that physical activity could prevent the MIA deleterious effects on the BMD at proximal tibia in this rat model of osteoarthritis.

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Figure Fig. Proximal tibia BMD differences between groups at sacrifice

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Proximal tibia BMD differences between groups at sacrifice

Disclosures: Arnaud Boudenot, None.


Preservation Effects on Bone Tissue Mechanical Properties.Sasha Rosen*1, Bryan Kaye2, Omar Ahmady2, Connor Randall3, Paul Hansma1. 1University of California, Santa Barbara, USA, 2UCSB Hansma Lab, USA, 3UCSB, USA

Recently, The Reference Point Indentation (RPI) instrument has been shown to distinguish patients with and without fractures(1). Patients with fractures tended to have larger values of Total Indentation Distance (TID) and Indentation Distance Increase (IDI)(1-3). For laboratory work on cadaver bone, the bone must typically be preserved. Freezing is a commonly preferred method of bone preservation, as it prevents lysis and, thus, helps maintain the in vivo spatial arrangement of the bone proteins(4). Another accepted approach is via chemical (namely ethanol) fixation. The advantages of chemical storage are primarily twofold; tissue decay is reduced and cell structure is preserved. In this study, the effects of these preservation techniques were evaluated using the Reference Point Indentation (RPI) instrument which performs Bone Microindentation Testing (BMT)(1). Specifically, we studied the dependence of Indentation Distance Increase (IDI) and Total Indentation Distance (TID) on preservation technique.

The effects of preservation techniques were investigated on bovine femur. Baked and control samples of bovine femur were used to contrast weakened (baked) versus strong (control) organic extracellular matrix. Samples were frozen at -20°C. IDI and TID did not change after freezing baked bone. However, a 15% increase (p<0.001) in IDI was noted after the initial freeze cycle of control bone, but there was no difference in TID. Subsequent freeze-thaw cycles did not affect IDI or TID in both, baked and control bone. This leads us to postulate that a bone with less organic matrix will have a smaller degradation due to the first freeze-thaw cycle. Furthermore, subsequent freeze-thaw cycles no longer diminish the bone. Samples were stored in 70% ethanol at 5°C and 25°C for 40 days. The IDI of the samples that were stored at 5°C increased by 18% (p<0.001), compared to a 35% increase (p<0.001) in IDI of the samples stored in 25°C. Ethanol fixation in bone and its corresponding degradation of the extracellular matrix is likely responsible for the deterioration in the mechanical properties. After exploring these common preservation techniques, we were able to determine statistically significant changes in the mechanical properties that were determined with the RPI instrument, revealing that these preservation techniques cannot maintain the mechanical properties investigated in this study.

Disclosures: Sasha Rosen, None.

This study received funding from: National Institutes of Health


Assessment of Bone Mineralization in Rats Treated with Sclerostin Antibody.Lindsey Edwards*1, Amarjit Virdi2, Kotaro Sena2, D. Rick Sumner2. 1Rush University, USA, 2Rush University Medical Center, USA

Sclerostin antibody is being developed for the treatment of postmenopausal osteoporosis because of its robust efficacy in increasing bone volume. Generally, sclerostin, the protein product of the SOST gene, maintains bone surfaces quiescent. Neutralizing sclerostin with an antibody up-regulates Wnt signaling and activates quiescent surfaces into bone forming surfaces. While studies show that antibody treatment in animals and humans increases bone volume, little data has been published describing the quality of the newly formed bone. We hypothesize that treatment with sclerostin antibody will lead to a short-term depression in overall bone mineralization because of the anticipated increase in bone volume and the lag time associated with full mineralization. In the present experiment, the femurs of 10 Sprague Dawley rats that had received titanium implants in their contralateral limbs were used. The rats were treated with saline or sclerostin antibody (Scl-Ab III: 25mg/kg, twice weekly) for 8 weeks. Positive effects of sclerostin antibody treatment on implant fixation were reported previously. In the present investigation, the intact femurs were harvested, formalin-fixed and scanned using micro-computed tomography (microCT) to assess bone volume and mineral density. The bones were then embedded in polymethyl methacrylate, sectioned, polished, carbon-coated and imaged using quantitative backscatter electron imaging (qBEI) as a second assessment of mineral density. Cortical bone area increased by 29% (p <0.001) and trabecular BV/TV increased by 3-fold (p<0.001) in the sclerostin antibody-treated group. The mineral density of the cortical and trabecular bone, whether assayed by microCT or qBEI showed no statistical difference between groups (Table 1). The skewness, kurtosis and variance of the mineral frequency distributions were similar in each group. Thus, in addition to increasing bone volume, sclerostin antibody may also increase the rate of mineralization.

Table Table 1. Assessment of mean bone tissue mineralization at S weeks (n = 5). None of the differences between groups were significant, in contrast to the expectation that, sclerostin antibody treatment would lead to an overall pattern of depressed mineralization.
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Disclosures: Lindsey Edwards, None.

This study received funding from: Amgen


Runx2 Overexpression in Hypertrophic Chondrocytes Delayed Chondrocyte Maturation and Endochondral Ossification.Ming Ding*1, Qiping Zheng2. 1Rush University, USA, 2Rush University Medical Center, USA

Runx2 Overexpression in Hypertrophic Chondrocytes Delayed Chondrocyte Maturation and Endochondral Ossification

Ming Ding1, Sam Abbassi1, Jun Li2, Yaojuan Lu1, Rick D. Sumner1, Vale'rie Geoffroy3, Feifei Li1, Anna. Plaas2, and Qiping. Zheng1,*

Runx2, a member of the Runt domain protein family, has been demonstrated to be an essential transcription factor for osteoblast differentiation as well as a critical regulator for chondrocyte maturation. The type X collagen gene (Col10a1) is a specific molecular marker of hypertrophic chondrocytes during endochondral bone formation. It has been shown that type X collagen plays a critical role during skeletal development and maintenance by impacting the supporting properties of the growth plate and the mineralization process. Recently, a growing body of evidence suggests that Runx2 regulates type X collagen gene expression during chondrocyte maturation in different species. To investigate how Runx2 regulates murine Col10a1 gene expression and impacts chondrocyte maturation during skeletal development, we have generated transgenic mice in which Flag-tagged Runx2 cDNA was under the control of the 300-bp cell-specific Col10a1 regulatory element. Real-time PCR using total RNAs prepared from mouse limbs at the E17.5 and P1 stages showed that both Runx2 and Col10a1 mRNA were significantly increased in transgenic mice compared to their wild-type littermate controls. Histological analysis of long bone sections showed elongated hypertrophic zone with disorganized chondrocytes in transgenic mice. Skeletal staining suggests delayed ossification in craniofacial, long bone digits and the tails from embryonic day 14.5 (E14.5) till postnatal day 3 (P3). microCT analysis of the mice at 1 month stage suggests that the transgenic mice have shorter femur length. We have also performed expression analysis of marker genes for chondrocyte differentiation, maturation or apoptosis. The results showed that Sox9, Ihh (Indian Hedgehog), Mmp13 (Collagenase-3), Bcl-2 (the anti-apoptotic marker gene), and Osx (Osterix) were all significantly increased in transgenic mice. No expression level change was observed for Vegf (Vascular endothelial growth factor). Our data suggest that Runx2 regulates cell-specific Col10a1 expression and chondrocyte maturation in vivo. The Runx2 transgenic mice that have enhanced Runx2 and Col10a1 expression may trigger Ihh and Sox9 expression which dominantly arrest chondrocyte maturation. This may lead to matrix environment change and impact chondrocyte apoptosis during skeletal development and maintenance.

Disclosures: Ming Ding, None.


See Friday Plenary number FR0068.


See Friday Plenary number FR0069.


Dual Action of Von Hippel Lindau (VHL) in Limb Bud Mesenchyme.Elisa Araldi*1, Richa Khatri1, Amato Giaccia2, Ernestina Schipani3. 1MGH, USA, 2Stanford University, USA, 3Massachusetts General Hospital & Harvard Medical School, USA

Numerous lines of evidence suggest that the genetic program activated by hypoxia has a critical role in endochondral bone developmentt in vivo. Consistent with its avascularity, the murine fetal growth plate displays a gradient of oxygenation with a central, hypoxic region. Genetic experiments in vivo have shown that the transcription factor Hypoxia-inducible factor-1α (Hif-1α), which is a major mediator of the cellular adaptation to hypoxia, is necessary for chondrocyte survival and for differentiation of mesenchymal precursors into chondrocytes, by triggering a complex homeostatic response that involves, at least in part, the accumulation of a proper extracellular matrix. To expand our understanding of the role of the Hif family of transcription factors (Hifs) in chondrogenesis, in this study we genetically deleted Von Hippel Lindau protein (pVHL), the E3 ubiquitin ligase responsible for degradation of Hifs under normoxia, in whole limb bud mesenchyme, i.e. both in mesenchymal cells that differentiate into chondrocytes and in mesenchymal cells that give origin to the vascularized soft tissue surrounding the avascular cartilaginous primordia. Lack of pVHL in chondrocytes caused a severe shortening and thinning of the mutant cartilaginous elements, which were markedly deformed and hypocellular, and eventually collapsed, possibly as a result of cellular growth arrest and death. Conversely, lack of pVHL in the soft tissue surrounding the chondrocytic growth plates led to formation of foci of ectopic cartilage, to massive fibrosis, and to appearance of fibromyxoid tumor-like structures, which expanded into and replaced the collapsed cartilaginous structures. We are currently investigating the molecular mechanisms of this apparent dual action of pVHL in cartilage and in the surrounding mesenchyme, with particular emphasis on the role of the Hif family of transcription factors. Taken together, our findings indicate that lack of pVHL may have differential biological effects in cells of mesenchymal origin, depending on their phenotypical identity.

Disclosures: Elisa Araldi, None.


Histological, Functional and Contrast-based çCT Evaluation a Novel Osteoarthritis Model in Mice.Zhechao Merry Ruan*1, Frank Gannon1, Michael Heggeness1, Brian Dawson1, Ming-ming Jiang1, Jennifer Black2, Brendan Lee1. 1Baylor College of Medicine, USA, 2Vanderbilt Univeristy, USA

Osteoarthritis (OA) is common clinical condition characterized by degeneration of articular cartilage, limited intraatricular inflammation with synovitis, and changes in peri-articular and subchondral bone. Current well-characterized animal models of OA include spontaneous models in larger animals, genetics models in mice, and surgery models in rats and rabbits. However, none of these models are optimal for genetics and therapeutic studies that are clinically relevant. Patients with anterior cruciate ligament (ACL) tearing usually develop OA within 1 to 2 years after injury. And, the progression of the disease is similar to spontaneous development of OA in elder patients. Therefore, we hypothesize that ACL transected mice will provide an OA model of great genetic and therapeutic relevance. To test this, we developed a microsurgical procedure to transect the ACL in hind limbs of mouse with minimal damage to the surrounding tissues. We developed a scoring system for mouse OA based on the human OA grading system published by Priztker et. al. in 2006. Samples collected 24 hours after surgery confirmed that little damage were caused directly by the surgery to surrounding tissues. 4 weeks after surgery, histological scoring of ACL transected mouse joint samples by blinded observers showed significantly different scores in transected vs. mock surgery groups (N=10). We also observed synovitis, mast cells recruitment, and neovascularization in cartilage in the ACL transected group. To evaluate the motor skills of mice in this model, ACL transected and mock mice were subjected to various behavior tests before surgery and 4 weeks after surgery. While the walking speed and active/rest time remains similar in ACL transected group and mock, the former showed decreased rotarod running time and increased hotplate response time compared to the latter (N=15). We also observed gait changes in the ACL transected group. Finally, we established a method to evaluate mouse cartilage quantitatively and qualitatively by contrast-based micro-computed tomography (μCT). We are testing whether this method could differentiate ACL transected group from mock surgery. This model will facilitate the study of genetic manipulations in cartilage in the pathogenesis and treatment of OA.

Disclosures: Zhechao Merry Ruan, None.


Hypophosphatemia-independent Changes in Extracellular Matrix Proteins in Hyp Mice may Underlie the Articular Cartilage Degeneration of X-linked Hypophosphatemia.Carolyn Macica*1, Ali Nasiri2, Guoying Liang3. 1Yale University School of Medicine, USA, 2Yale Univeristy, USA, 3Yale University, USA

The two major sites of paradoxical hypermineralization in patients with X-linked hypophosphatemia (XLH) occur in cartilaginous tissues; the fibrocartilaginous tendon/ligament insertion sites and at the margins of synovial joints resulting in formation of enthesophytes and osteophytes, respectively. Despite effective treatment of rickets/osteomalacia, this complication dominates the adult clinical picture. We have previously demonstrated that the Hyp mouse, a murine XLH model, develops an enthesopathy at sites most commonly affected in patients, characterized by a significant expansion of mineralizing fibrocartilage (Liang et al., 2009). Osteophyte formation is an adaptive response that occurs secondary to articular cartilage (AC) degeneration. We now identify alterations in the articular surface of Hyp mice (12 wks) that may predispose patients with XLH to AC degeneration.

We identified a significant reduction in the expression of two matrix proteins normally confined to the mineralized zone of AC; the matrix-degrading enzyme, matrix metalloproteinase-13 (MMP13) and osteopontin (OPN), a mineral-binding secreted glycoprotein. This is accompanied by a significant loss of the mineralized hypertrophic zone of AC (von Kossa), despite elevated alkaline phosphatase activity. EPIC-microCT analysis of hexabrix-labeled-AC (6 micron isometric voxel size, threshold applied to include unmineralized AC but exclude mineralized AC/bone) confirmed an increase in unmineralized AC (wt 0.096 ± 0.01 mm vs. Hyp 0.158 ± 0.01 mm, p<0.05). SafO staining revealed a redistribution of proteoglycans from above the tidemark into the entire articular surface. In contrast, secreted osteoblast MMP13 and OPN were unaffected in subchondral and trabecular bone, underscoring potential regulatory differences between mineralizing osteoblasts and chondrocytes in Hyp mice despite an identical metabolic backdrop.

The cellular cues for these changes likely include both mechanical and biochemical factors and may be unique to cartilaginous tissues. We thus measured markers of mineralizing hypertrophic AC of Hyp mice treated with or without oral P/vitamin D to determine the impact of correcting the abnormal phosphorous and mechanical forces of rickets/osteomalacia. We found that while the rickets was effectively treated, the AC abnormalities persisted. These data suggest a local role of biochemical factors unique to XLH in mediating changes to the architecture of the zonal arrangement that defines AC.

Disclosures: Carolyn Macica, None.


See Friday Plenary number FR0073.


Muscular Function and Bone Strength Related Variables after Knee Replacement.Ari Heinonen*1, Timo Rantalainen2, Anu Valtonen3, Tapani Pöyhönen4, Sarianna Sipilá5. 1Department of Health Sciences, University of Jyväskylä, Finland, 2Neuromuscular Research Centre, Department of Biology of Physical Activity, University of Jyväskylä, Finland, 3Rehabilitation & Pain Unit, Kymenlaakso Central Hospital & Department of Health Sciences, University of Jyväskylä, Finland, 4Rehabilitation & Pain Unit, Kymenlaakso Central Hospital, Finland, 5Gerontology Research Centre, Department of Health Siences, University of Jyväskylä, Jyväskylä, Finland

The bone loss after total knee replacement is apparently stress shielding related and may lead to loosening of the prosthesis. In order to provide more information for rehabilitation after knee replacement to prevent this bone loss, a sample of 29 women and 19 men who were 55 to 75 years old and had undergone unilateral knee replacement surgery an average of 10 months (range 4-17) earlier were recruited. Muscular function was assessed with maximal torgue in knee extension and flexion with an isokinetic dynamometer at an angular velocity of 60°/s. A single computed tomography (QCT) slice was obtained from femoral mid-shaft comprising both legs. The Hounsfield units provided by the QCT device were converted to volumetric bone mineral density (vBMD) values by scanning (K2HPO4) phantom liquids and calculating the linear conversion equations. Cortical cross-sectional area (CoA), cortical volumetric BMD (CoD) and density weighted section modulus (SSI) were calculated from the QCT slices for the operated and the non-operated femurs. The bones were compared to each other with ANOVA having the leg as a within subject factor. The operated thigh CoA was 3% smaller (393 + 82 mm2 vs. 405 + 75 mm2; p = 0.003) and CoD 1% lower (991+ 39 mg/ cm3 vs. 999 + 37 mg/ cm3; p < 0.001) than that in the operated thigh, whereas no side-to-side difference was observed in SSI (2560 + 690 mm3 vs. 2600 + 630 mm3; p = 0.120). In the stepwise regression analyses knee extension and flexion torque, age, height, body mass, pain score (VAS, mm) and time since operation explained 44% of the variance in the CoA, 33% in CoD and 52% of the variance in SSI on the operated side. Body mass explained independently 7% (p = 0.020) of the CoA variation, 14 (p < 0.001) of the CoD and 7% (p = 0.015) of the SSI variation. In addition, maximal knee flexion torque turned out to be the strongest independent predictor, explaining 37% (p < 0.001) of CoA variation, 7% (p = 0.043) in CoD and 45% (p<0.001) of the SSI variation. Further, time since operation explained independently 12% (p=0.020) of the CoD variation. In conclusion, the operated thigh had less cortical bone, and muscle force of the operated leg is a strong independent predictor of the femoral mid-shaft bone strength in subjects undergone unilateral knee replacement surgery. This indicates indirectly that after total knee replacement, early and efficient rehabilitation should be emphasized.

Disclosures: Ari Heinonen, None.


See Friday Plenary number FR0075.


The Potential Role of Gas6 in Growth Plate Chondrocytes.Phillip Newton*1, Ann Canfield1, Colin Farquharson2. 1University of Manchester, United Kingdom, 2Roslin Institute & R(D)SVS, University of Edinburgh, United Kingdom

During endochondral ossification mesenchymal cells differentiate into chondrocytes and further differentiate to a state of hypertrophy. In the developing bone, the chondrocytes align to form the highly organized mature growth plate. At each stage the chondrocytes secrete specific extracellular matrix (ECM) components and ultimately this ECM becomes mineralized allowing vascular invasion and the formation of the primary spongiosa. Gas6 has previously been detected in growth plate chondrocytes by microarray analysis and recombinant Gas6 has been shown to influence chondrocyte differentiation in vitro, although a precise function and mechanism is yet to be elucidated. Signaling within the growth plate has been suggested via interactions between Gas6 and its receptors Axl and most recently, Mer. Axl and Gas6 have also been reported in chondrocytes of articular cartilage and their signalling has been shown to prevent ectopic mineralization within the vasculature via anti-apoptotic mechanisms.

To determine the spatial expression of Gas6 in the growth plate, 28-dayold murine tibial growth plates were analysed by immunohistochemistry. Also, an in vitro model using chondrocyte-like ATDC5 cells was used to determine the temporal expression patterns of Gas6, Axl, Mer and chondrocyte markers by Western blot and qPCR during chondrocyte differentiation and ECM mineralization.

Gas6 was detected by immunohistochemistry in the resting, proliferating and hypertrophic zones of the growth plate but expression was concentrated in the proliferating zone. Analysis of ATDC5 cells confirmed that they underwent the expected stages of differentiation as demonstrated by the expression of collagen types II and X; ECM mineralizationbegan atday14ofculture. Gas6mRNAexpressionwasminimalat day 6 of culture and was maximally expressed at day 13 before decreasing. Total Axl protein and mRNA expression both peaked at day 8 and decreased thereafter, whereas phosphorylation of Axl was prolonged throughout culture. Mer mRNA expression was determined from day 6 and peaked at day 8 before decreasing. This data shows the spatial and temporal distribution of Gas6 within the growth plate and in addition, the detection of phosphorylated Axl within differentiating ATDC5 cells. This strongly suggests a role for Gas6 signaling in the growth plate.

The consequences of Gas6 signaling within the growth plate are unknown and further studies are required to determine Gas6 mediated events.

Disclosures: Phillip Newton, None.


The Role for CTGF and Src in Mesenchymal Stem Cell Condensation Induced by TGF-β1.Fabiola Del Carpio-Cano*1, Steven Popoff2, Raul DeLa Cadena3, Fayez Safadi2. 1Temple University, USA, 2Temple University School of Medicine, USA, 3Temple University School of Medicine, Department of Physiology, USA

The aggregation/condensation of mesenchymal cells (MSC) is a critical step for chondrocyte differentiation. TGF-β1 has been shown to promote MSC migration, proliferation, and condensation. Connective tissue growth factor (CTGF) is a matricellular protein expressed during MSC condensation where regulates matrix production. Previous studies have shown that CTGF acts as a downstream mediator of TGF-β1-induced MSC condensation. Silencing of CTGF expression inhibits MSC condensation induced by TGF-β1. In the present study, we further examined the role of CTGF in TGF-β1-induced MSC condensation. We assessed MSC migration, proliferation, condensation, and other markers of chondrogenesis induced by TGF-β1 in C3H10T1/2 cells infected with adenovirus expressing either CTGF-GFP (Ad-CTGF-GFP) or GFP alone (Ad-GFP; control), both in the presence or absence of exogenous TGF-β1 (5ng/ml) treatment. CTGF overexpression inhibited TGF-β1-induced MSC condensation compared to control cultures. Overexpression of CTGF inhibited the migration and proliferation that is normally observed in cells treated with TGF-β1. We also evaluated gene expression for extracellular matrix and chondrogenesis markers (aggrecan, Col 1α2, Col 2α1, fibronectin and Sox-9) by qPCR analysis. The overexpression of CTGF inhibited the expression of all markers compared to the control infected cultures and also prevented up-regulation of markers induced by TGF-β1. Next, we examined whether Src activation plays a role in MSC condensation induced by TGF-β1. Src was activated upon TGF-β1 treatment of MSC cells. In the presence of PP2, a Src family kinase inhibitor, TGF-β1 failed to induce MSC condensation. In the Ad-CTGF-GFP overexpressing cells, Src was constitutively activated at very high levels despite the fact that these cells did not condense or respond to TGF-β1 treatment. These results suggest that although CTGF and Src play essential roles in the induction of MSC condensation when induced by TGF-β1 in a temporal fashion, the continuos overexpression of CTGF has the opposite effect and prevents TGF-β1 induction of MSC condensation.

Disclosures: Fabiola Del Carpio-Cano, None.


See Friday Plenary number FR0078.


See Friday Plenary number FR0079.


RANKL Expression in Mouse and Human T-Lymphocytes is Regulated by a Set of Cell-Type Specific Enhancers Designated the T-Cell Control Region.Kathleen Bishop*, Xiaohua Wang, Heidi Coy, Robert Nerenz, Mark Meyer, Jenny Gumperz, J. Pike. University of Wisconsin-Madison, USA

Chronic activation of the immune system is directly linked to systemic bone loss. Excess bone resorption is likely due to elevated expression of receptor activator of NF-KB ligand (RANKL), a key osteoclastogenic factor synthesized by both osteoblasts and T-lymphocytes. In osteoblasts, RANKL expression is controlled through multiple distal elements marked by increased transcription factor occupancy, histone acetylation and RNA polymerase II recruitment. In T-cells, however, the mechanisms that underlie the expression of RANKL remain undefined. To explore this process, we confirmed that RANKL was induced in both mouse and human primary T-cells and then scanned over 400 kb surrounding the RANKL locus for regulatory enhancers using ChIP-chip analysis for histone H3/H4 acetylation. We identified potential Rankl enhancers at several regions previously characterized in osteoblasts, including the D5 enhancer, and identified a unique set of regulatory regions located over 120 kb upstream of the mouse Rankl TSS which we designated the T-cell control region (TCCR). Further studies showed that both DNA sequences and epigenetic modifications within the TCCR were highly conserved in both mouse and human and that histone H3/H4 acetylation levels were increased at the TCCR following activation. Inhibition of both MEK1/2 and calcineurin by U0126 and cyclosporine-A, respectively, resulted in decreased RANKL expression, suggesting that multiple T-cell signaling pathways are involved. Accordingly, we found that c-FOS, an effector of MEK1/2, was recruited to both the D5 enhancer and the TCCR, suggesting that c-FOS may mediate RANKL upregulation. The human and mouse D5 enhancers and segments of the TCCR mediated robust inducible luciferase activity following activation in Jurkat T-cells that was abrogated following mutation of the putative c-FOS response elements. Importantly, both D5 and segments of the TCCR were shown using 3C analysis to be in direct contact with the Rankl TSS and thus able to influence the gene's promoter directly. Finally, we note that SNPs linked to bone mineral density in both hip and spine are located within the TCCR, providing additional evidence that RANKL expression by T-cells may contribute to both normal bone remodeling as well as disease-related bone loss. We conclude that both the D5 and TCCR regions represent control segments within the RANKL locus that play an integral role in the transcriptional regulation of this gene in T-cells.

Disclosures: Kathleen Bishop, None.


See Friday Plenary number FR0081.


Effects of Inorganic Phosphate on the Dento-alveolar Complex, In Situ and In Vitro.Yuri Yamamoto*1, Sunao Sato2, Brian Foster3, Kenji Takada2, Takashi Takata4, Martha Somerman3, Satoru Toyosawa2. 1Osaka University, Japan, 2Osaka University Graduate School of Dentistry, Japan, 3University of Washington School of Dentistry, USA, 4Hiroshima University Graduate School of Biomedical Sciences, Japan

Objectives: The genes and associated proteins that regulate phosphate/pyrophosphate homeostasis, e.g., Ank or ENPP-1, are associated with protection from ectopic calcification. Hypercementosis has been reported in mice with mutations/knock-out of Ank and ENPP-1, where tissue pyrophosphate (PPi) levels are decreased and phosphate (Pi) to PPi ratio is increased. The aim of this study was to examine the effect of Pi/PPi imbalance on gene expression in cells associated with periodontal tissues, i.e., osteoblasts, cementoblasts and the precursor cell, follicle cells, in vitro.

Material and Methods: Incisors and molars obtained from mice with Ank and ENPP-1 mutations were examined at the light microscopic level. Osteoblasts, cementoblasts and follicle cells, previously isolated from mouse tissues, were cultured under different concentrations of Pi/PPi as a model to mimic the animal conditions for Ank/ ENPP-1 mutations. Changes in expression of specific genes, osteopontin (OPN), type ?collagen (Col?), dentin matrix protein 1 (DMP1), osteocalcin (OCN) and bone morphogenetic protein 2 (BMP-2), were examined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) at designated times from 12 hours to 72 hours and dose of 0, 1, 3, 5, 7mM Pi.

Results: Histological observations indicated that not only the molars but also the incisors from Ank and ENPP-1 mutant mice showed a marked increase in the amount of cementum when compared to wild-type. In ENPP-1 mutant mice, the thickened cementum was positively stained with OPN and DMP1.

BMP-2 was up-regulated 3-fold in dose and time dependent manner in the follicle cells, but wasn't changed in the osteoblasts and the cementoblasts. In contrast, DMP1 was up-regulated by 7mM Pi more than 200-fold in cementoblasts by 48 hrs and osteoblasts by 72 hrs, but was not changed in follicle cells. OCN was downregulated about 0.1 fold in dose and time dependent manner in the cementoblasts and about 0.2 fold in dose and time dependent manner in the follicle cells, but not in osteoblasts. OPN was up-regulated in all three cells.

Conclusion: These results suggest that functional changes in proteins such as ANK and ENPP-1, by affecting Pi/PPi ratio, may in turn affect mineralized tissue formation by altering expression of key genes and associated proteins in osteoblasts and cementoblasts, and further, may impact differentiation potential in follicle cells.

Disclosures: Yuri Yamamoto, None.

This study received funding from: NIH/NIDCR RO1 DE15109 (MJS)


See Friday Plenary number FR0083.


See Friday Plenary number FR0084.


See Friday Plenary number FR0085.


See Friday Plenary number FR0086.


See Friday Plenary number FR0087.


Amino Acids Differentially Regulate Bone Formation and Resorption.Carlos Isales*1, Kehong Ding1, Qing Zhong1, Michael Cain1, Xing-Ming Shi1, Byung Rho Lee1, Richard Robbins1, Mark Hamrick1, Wendy Bollag1, Clare Bergson1, Crystal Perkins1, Justin Cowart1, Monte Hunter1, Brandon Scott1, Norman Chutkan1, Karl Insogna2. 1Medical College of Georgia, USA, 2Yale University School of Medicine, USA

The impact of dietary protein on bone mass has been controversial. Posited mechanisms for dietary protein's influence on the skeleton have focused on nutrient effects (e.g. the need for protein to model and remodel bone) or metabolic effects (e.g. the metabolic acid load imposed by sulfur-containing amino acids). However, recent data from a number of laboratories suggests that an underappreciated mechanism of nutrient action is directly on bone cells, a paradigm in which nutrients act as signaling molecules. In pursuing the possibility of direct nutrient effects on bone cells we examined individual amino-acid effects on osteoprogenitor/bone marrow stromal cells (BMSCs), a key target for bone anabolism and on osteoclasts, a key target for bone catabolism. Using BMSCs isolated from C57Bl6 mice and exposed individually to twenty of the common amino-acids we found that: (1) tyrosine, tryptophan and phenylalanine induced the largest increases in intracellular calcium as measured by single cell calcium measurements with Fura-2; (2) the same AA's that increased intracellular calcium, also led to a time-dependent increase in ERK phosphorylation, peaking at 10 minutes and returning to baseline by 30 minutes. ERK phosphorylation was not inhibited by Rapamycin (mTOR inhibitor) suggesting that the observed effects were not due to AA uptake and metabolism; and (3) tyrosine and tryptophan resulted in the greatest increases in BMSC proliferation (as measured by thymidine uptake). When the effects of all twenty common AAs were examined on primary osteoclasts isolated from C57Bl6 mice we found that: (1) cysteine, serine, methionine and arginine had the largest effect on early markers of osteoclastic differentiation while cysteine, proline, serine and arginine had the largest effects on late markers of differentiation as quantified by real time RT-PCR (vitronectin, MMP9, cathepsin K and calcitonin receptor); and (5) when osteoclastic activity was examined measuring pit resorption with Osteologic discs, the AAs: arginine, serine and proline were found to be most pro-resorptive. In summary our data demonstrate that individual AAs can selectively activate either bone formation or breakdown. Our data also suggests that the observed differences may account for some of the discrepancies in the literature on dietary protein effects on bone since the AA composition may vary from one dietary source to another.

Disclosures: Carlos Isales, None.


Arthroplasty Patients with Diabetes Mellitus Have Higher Bone Pentosidine Levels and Greater Immunohistochemical RAGE and MMP-1 than Patients without Diabetes.Karen King*1, Allison Williams2, Trevor Oren1, Allan Bucknell2. 1University of Colorado School of Medicine, USA, 2VA Eastern Colorado Health Care System, USA

We previously identified a higher rate of total joint replacement surgeries (arthroplasty) in patients diagnosed with diabetes mellitus compared to patients without diabetes mellitus in a Veterans Affairs database.1 The purpose of the present study was to identify an effect, if any, of diabetes on joint tissue metabolism.

With IRB-approval, cartilage and bone tissues were obtained as surgical wastes from total knee arthroplasty surgeries of osteoarthritis patients diagnosed with diabetes (N=10) or not diagnosed with diabetes (N=10). Osteochondral cores (5 mm dia.) were taken from the tibia plateau and either separated to cartilage and bone samples for collagen analysis or fixed whole for histology. Levels of collagen cross-links (HP, LP, and pentosidine) and total collagen were measured using HPLC. Osteochondral thin-sections were probed with monoclonal antibodies for RAGE or MMP-1.

Overall, cartilage had higher pentosidine levels than bone in both groups (P < 0.001). Bone pentosidine levels were higher in the group with diabetes compared to the group without diabetes (9.153 × 10-3 mol pentosidine/mol collagen vs. 6.945 × 10-3, P < 0.05). Cartilage pentosidine levels were higher, but not statistically significant, in the diabetes group (2.624 × 10-2 mol pentosidine/mol collagen vs. 2.167 × 10-2 P=0.075). There were no differences between groups in the levels of HP or LP in bone or cartilage (P > 0.05). Immunostaining for RAGE was greater in the thin-sections obtained from the diabetes group. Immunostaining for MMP-1 was also greater in the diabetes group. Furthermore, the cells positive for RAGE and MMP-1 were located in the same regions, the cartilage superficial and mid zones and at the bone-cartilage interface.

These results support the hypothesis that hyperglycemia leads to increased pentosidine and other advanced glycation end-products in diabetes mellitus patients and that AGE-RAGE signaling causes inflammatory events such as increased MMP activity.2 We propose that this hypothesis is applicable to the joint tissues and may be a factor (along with others such as BMI) in the increased rates of arthroplasty surgeries.

1King et al. Diabetics receive joint replacements more frequently and at a younger age. Orthopaedic Research Society 56th Annual Meeting, New Orleans, 2010. 2Yan et al. Receptor for AGE (RAGE) and its ligands-cast into leading roles in diabetes and the inflammatory response. J Mol Med. 87:235, 2009.

Disclosures: Karen King, Cerapedics, Inc., 5; Cerapedics, Inc., 2; DePuy Orthopaedics, Inc., 2


Delayed Healing and Efficient In Vivo Retroviral Transgene Transduction in a Mouse Segmental Defect Model of Bone Healing.Charles H. Rundle*, Nicoleta L. Popa, Jon Wergedal, Subburaman Mohan, Kin-Hing William Lau. Jerry L. Pettis Memorial VA Medical Center, USA

Approximately 10% of bone fractures display severely impaired healing that is resistant to current therapeutic approaches. Because the closed three-point bending rodent fracture model traditionally used in fracture studies heals well without intervention, its utility for investigations of impaired bone healing is limited. It is imperative to develop a rodent model that can be used to identify therapeutic approaches for severely impaired bone healing. Ideally, this model should: 1) exhibit non-union healing, and 2) permit efficient delivery of molecular therapy to the injury. To determine whether such a model of impaired bone healing is feasible in the mouse, we compared healing and transgene expression in the three-point bending fracture model with a segmental defect model. In each model, the femur was stabilized with an intramedullary pin; a drill was used to produce a 1-2 mm defect around the entire cortical circumference of the segmental defect model. Healing was monitored by radiology and by histology at 7 and 28 days healing. The segmental defect developed a callus of fibrous tissue during healing, but X-ray examination revealed that the hard callus failed progress to bony union (i.e., healing). Histomorphometry of the callus tissues at 7 days healing (N=4 to N=7 mice per group) established that the callus area of the segmental defect was reduced (38.7 ± 10.5 mm2 vs 34.1 ± 4.8 mm2 p<0.05), as was the proportion of cartilage area per callus area (9.2 ± 5.0% vs 3.2 ± 2.4%, p<0.03). There were no significant differences in these two parameters between models at 28 days healing, but the three-point bending fracture model had progressed to bony union, while the segmental defect model retained fibrous tissue within the defect. This model therefore exhibits bone repair with severely delayed union. To determine whether transgenes of potential benefit can be expressed from these tissues in vivo, we compared the closed fracture and segmental defect models (N=3 each) for expression of a β-galactosidase gene delivered in retroviral-based vector. β-galactosidase staining at 7 days healing demonstrated comparable transgene expression between models. Despite the loss of bony tissue in the segmental defect model, direct injection of viral vector to the injury was effective in transducing resident cells. It is therefore a valuable model to assess the therapeutic efficacy of gene therapy strategies to promote the healing of more clinically challenging bone defects.

Disclosures: Charles H. Rundle, None.


Folate Supplementation During Pregnancy and Lactation Improves Bone Health of Female Mouse Offspring at Adulthood.Jovana Kaludjerovic*, Wendy Ward. University of Toronto, Canada

Supplementation with folic acid during the perinatal period is advocated for women of childbearing age as a prevention strategy against neural tube defects. However, the effects of folate supplementation on bone development have not been well characterized. The objective of this study was to determine if exposure to low (0 mg of folate/kg diet), adequate (2 mg of folate/kg diet) and supplemental (8 mg of folate/kg diet) amounts of dietary folate during perinatal life (defined as the period 1 week before conception to 3 weeks post conception) programs bone health at young adulthood in female mice. CD-1 mice (n = 12-16 pups/group) were randomly assigned to receive an amino acid based diet fortified with 0, 2 or 8 mg of folate/kg diet during pregnancy and lactation. At weaning, all pups were switched to an amino acid diet containing 2 mg of folate/kg diet, as this is the basal dietary requirement for rodents, and were studied to young adulthood (4 months of age). Body weight was measured weekly. Bone mineral density (BMD) and biomechanical strength at femur and lumbar vertebrae were measured at 4 months of age. There were no differences in body weight from postnatal day 1 to 10 among groups, but from postnatal day 10 to 120, mice fed a diet fortified with 2 or 8 mg of folate had significantly higher (p<0.05) body weights compared to folate deficient mice who grew at a significantly lower rate. Females fed a diet containing 8 mg of folate/kg diet had significantly higher (p<0.05) BMC and BMD at the femur and lumbar vertebrae (LV1-3) compared to those fed a folate deficient diet. Moreover, peak load of the femur midpoint, femur neck and LV2 was significantly higher (p<0.05) in mice fed 8 mg of folate compared to those fed a diet devoid of folate. In conclusion, exposure to dietary folate (2 or 8 mg/kg diet) during perinatal life results in normal growth and bone development in CD-1 mice. Future research should determine if the higher BMC, BMD and greater bone strength with supplemental levels of folate in utero and during suckling protect against deterioration of bone tissue during aging.

Disclosures: Jovana Kaludjerovic, None.


Large Scale Destabilization of Type I Collagen Triple Helix may Explain Increased Severity of Osteogenesis Imperfecta Caused by Mutations Near the Collagenase Cleavage Site.Elena Makareeva1, Sejin Han1, Juan Carlos Vera1, Nydea Aviles1, Wayne A. Cabral1, Joan Marini2, Robert Visse3, Hideaki Nagase3, Sergey Leikin*1. 1National Institutes of Health, USA, 2National Institute of Child Health & Human Development, USA, 3Imperial College London, United Kingdom

Most cases of severe and lethal osteogenesis imperfecta (OI) are caused by substitutions of obligatory Gly residues in the triple helical region of type I collagen. Years of studies and hundreds of reported substitutions revealed no simple relationship between OI genotypes and phenotypes, probably because many different factors contribute to the disease severity. One such factor appears to be regional variations in structural and functional properties of the collagen triple helix. In the present study we focused on characterizing the region surrounding the collagenase cleavage site, in which virtually all α1(I) and many α2(I) Gly substitutions were found to be lethal. Analysis of structural changes in this region caused by various mutations revealed reversible unfolding of a significant fraction of the triple helix, clearly detectable by circular dichroism and susceptibility to different enzymes. For instance, we observed not only faster cleavage of mutant molecules by collagenases but also their efficient cleavage by a gelatinase (MMP-2) and the catalytic domain of MMP-1, which have only residual collagenase activity. Our earlier findings suggested that procollagen triple helix folding within this region may be challenging for cells even in the absence of mutations. The large scale triple helix destabilization observed in the present study suggests that mutations within this region may be particularly detrimental for procollagen folding, resulting in severe ER stress and malfunction of osteoblasts, potentially explaining the lethal OI phenotype. Further testing of this hypothesis is currently under way.

Disclosures: Sergey Leikin, None.


Micro-Computerized Tomographic Assessment of Zebrafish Skeleton.THEODORE CRAIG*1, Glenda Evans2, Theresa Hefferan1, Michael Yaszemski3, Stephen Ekker4, Rajiv Kumar3. 1Mayo Clinic, USA, 2Orthopedics Research, Department of Orthopedics, Mayo Clinic, USA, 3Mayo Clinic College of Medicine, USA, 4Department of Biochemistry & Molecular Biology, Mayo Clinic, USA

Skeletal development in the zebrafish (Danio rerio) is similar to that observed in mammals. The expression of genes that might play a role in skeletal development can be readily manipulated in zebrafish, and genetic screens can be used to identify novel genes important in skeletal morphogenesis in this organism. Methods to assess bone architecture and bone mineral content in zebrafish are needed. Bone specific dyes such as alizarin red and calcein can be used to assess skeletal morphology but do not yield quantitative data regarding mineralization. Assessment of skeletal structure, development and mineralization using micro-computerized tomography (μCT), a non-invasive method with high spatial resolution, has not been standardized in this organism. Thirty day-old, juvenile, zebrafish were euthanized and fixed in formalin/phosphate buffered saline. Fish were scanned with a Scanco 35 μCT instrument (integration time 300; isotropic resolution of 3.5 μm; Vasquez et al, Anat. Rec. 291:475-487, 2008) to assess skeletal morphology and bone characteristics. The data were analyzed using the Scanco software. The architecture of mandibular and pharyngeal bones and the components of the dorsal skull was clearly visualized. In the vertebrae of the spine, structure and bone density were readily determined. Data from male and female zebrafish at each of the following ages will be presented: 10 day post fertilization (dpf), 20 dpf, 30 dpf, 60 dpf and 90 dpf. For purposes of comparison skeletal architecture in fish stained with 0.1% alizarin red in 3% potassium hydroxide (Quatro and Longaker, Cells Tissues Organs 181:109-118, 2005) and 0.2% calcein in de-ionized water (Du et al, Dev. Biol., 238:239-246, 2001) will be presented.

Results: Micro-computerized tomography assessed the structure and bone mineral density of the zebrafish skeleton with exceptional clarity and accuracy at ∼30 dpf. In the skull, cranial sutures were readily visualized and bone density determinations were readily performed on bones of the skull and vertebrae. Bone structure was visualized as early as 10 dpf. Micro-computerized tomography is more easily performed and more sensitive than the dye based methods.

Conclusions: Micro-computerized tomography can be used to assess zebrafish skeletal development and bone mineral density and should serve as a powerful tool to assess skeletal defects and changes in mineralization.

Disclosures: THEODORE CRAIG, None.

This study received funding from: NIH


Morphological Comparison of the Skulls of Mice Mimicking Human Apert Syndrome Resulting From Gain-of-function Mutation of FGFR2 Ser252Trp and Pro253Arg.Xiaolan Du, Tujun Weng, Qifen He, FengTao Luo, Lin Chen*. State Key Laboratory of Trauma, Burns & Combined Injury, Center of Bone Metabolism & Repair, Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, China

Apert syndrome is caused mainly by two gain-of-function mutations in FGFR2, Ser252Trp and Pro253Arg. Slaney, et al found that patients with Apert syndrome resulting from FGFR2 Pro253Arg mutation have more severe syndactyly and less common cleft palate. Von Gernet found that patients with Apert syndrome resulting FGFR2 Ser252Trp, in general, have more severe craniofacial phenotypes than those of patients with FGFR2 Pro253Arg. There is, however, no well-controlled comparison of the difference in the skull morphology between Apert syndrome resulting from these two FGFR2 mutations. Taking the advantage of the mouse models mimicking these two syndromes, we did this comparison. The three-dimensional coordinate locations of 27 biologically relevant landmarks located on the skulls of mice mimicking human Apert syndrome resulting from FGFR2 Ser252Trp (Fgfr2+/S252W) and Pro253Arg (Fgfr2+/P253R) and their wild-type (WT) littermates (8w) were collected by using three-dimensional coordinate measuring machine. The euclidean distance matrix analysis (EDMA) was used to statistically characterize the three dimensional form of 10 biologically relevant landmark subsets that were designed to represent the skull shapes of the Fgfr2+/S252W and Fgfr2+/P253R mutants. In general, compared with that in WT littermates, Fgfr2+/S252W mutant mice have more shallow and wider orbit, wider anterior neurocrania, more shortened premaxilla and face height than that in Fgfr2+/P253R mice. We further found that there were significant differences in the biologically relevant landmark subsets for maxilla, orbit and anterior neurocrania (p<0.01). Differences were also found in face height and neurocranium (p<0.05). There were, however, no differences in the landmark subsets for the nasal, frontal, midface, smaller midface and vault (P>0.05). Our results indicate that there are subtle differences in the skull morphology between Apert syndrome caused by FGFR2 Ser252Trp and FGFR2 Pro253Arg mutation. Further studies are needed to confirm these findings in detail and in patients with Apert syndrome. This work was supported by the Major State Basic Research Development Program of China (2005CB522604) and National Natural Science Foundation of China (30971607, 30530410)

Disclosures: Lin Chen, None.


See Friday Plenary number FR0095.


See Friday Plenary number FR0096.


See Friday Plenary number FR0097.


Tgf-beta in the Development of the Intervertebral Disc.Rosa Serra1, Philip Sohn2, Megan Cox*2. 1University of Alabama at Birmingham, USA, 2UAB, USA

Transforming growth factor β (Tgf-β) signaling plays an integral part in skeletal development. Conditional deletion of Tgf-β type II receptor (Tgfbr2) from type II Collagen expressing cells has been shown to cause defects in vertebrae and intervertebral disk (IVD) development. To determine how Tgf-β affects differentiation of mesenchymal progenitor cells in the axial skeleton, we performed an RNA microarray analysis on sclerotome cells that had been cultured with or without Tgf-β 1. A separate microarray screen was done using RNA collected from vertebrae and IVD tissue isolated by laser dissection from e13.5 mice. From the microarray data, we determined through hierarchical clustering analysis that Tgfbr2 deleted IVD more closely resembled developing vertebrae than developing IVD. Furthermore, scatterplot analysis was used to show that the genes that are up-regulated by TGF-β are primarily expressed in the IVD, supporting the hypothesis that TGF-β can promote the IVD phenotype at the molecular level. We selected several transcription factors that were regulated by Tgf-β treatment, differentially expressed in the IVD versus the vertebrae, and/or decreased in IVD with Tgfbr2 deletion, for further study: Erg, Nfatc1, Ebf1, and cMaf. Regulation by Tgf-β was confirmed by semi-quantitative RT-PCR on RNA from primary sclerotome cells treated with Tgf-β1. In situ hybridization analysis and database searches were done to determine the expression pattern of genes in vivo. This analysis showed that Erg1 and Nfatc1 are specifically localized to the IVD and Ebf1 and c-maf are preferentially localized to the vertebrae. To study the effects of Tgf-β overexpression in axial skeleton development, we used the chick model system. Tgf-β soaked Affi-gel beads were placed into the lumbar region of the axial skeleton at stage HH 22- HH 25. Within 48h post-implantation, we are able to see an inhibition of cartilage formation as determined by Alcian blue staining in the area surrounding the bead. This suggested that Tgf-β acts to suppress cartilage formation in the sclerotome. Overall, the data support a model in which Tgf-β acts to maintain the IVD and prevent differentiation into cartilage. These experiments help to address fundamental questions about development of the axial skeleton and provide a basis for future studies aimed at disc repair or replacement.

Disclosures: Megan Cox, None.


The Role of Tgfbr2 in Sclerotome Migration During Vertebrae Development.Ying Wang*, Rosa Serra. University of Alabama at Birmingham, USA

The vertebral column develops from somites. During vertebrae development, sclerotome, a population of undifferentiated mesenchymal cells, differentiates and migrates to give rise to distinct parts of the vertebrae and ribs. Spina bifida occulta is one of the most common congenital malformations in humans leading to disability. Our laboratory has shown that deletion of TGFβ type II receptor (Tgfbr2) in Col2a expressing tissue in mice results in alterations in the formation of the vertebrae. The dorsal vertebrae of these mice failed to fuse, which mimics the symptom seen in patients with spina bifida occulta. Since sclerotome cell (SC) migration plays a key role during dorsal structure formation, we hypothesized that signaling through Tgfbr2 mediates the development of the dorsal vertebrae by regulating migration of SC. We first crossed the Col2aCre mice to the Rosa26 reporter strain to track SC migration in vivo. There was no observable difference between control and experimental group at E15.5. However, migration defects were seen in the mutant mice by E17.5, suggesting that TGFβ signaling is required for sclerotome migration during later stages of dorsal vertebrae development. To determine the underlying mechanism of TGFβ regulation of SC migration, we isolated SC from E11.5 embryos and performed migration assay using a chemotaxis chamber. TGFβ greatly induced wild-type cell migration, while migration of cells lacking Tgfbr2 was disrupted. Since platelet-derived growth factor (PDGF) has been shown to play a central role in regulating sclerotome migration, and it is one of the downstream effectors of the TGFβ signaling pathway, we then determined the direct effect of PDGF on SC migration. Both ligands AA and BB induced migration at distinct concentrations. The expression of PDGF-BB was greatly increased in cells treated with TGFβ, while that was decreased in cells lacking Tgfbr2. These data suggest that the action of TGFβ on SC migration may be mediated by the downstream effector PDGF-BB. The changes of PDGF-BB expression in the mutant mice will be examined by in situ hybridization analysis. To identify additional target genes involved in SC migration, we performed a microarray assay comparing untreated and TGFβ treated SC and found that chemokine CCL21 was upregulated by TGFβ. Preliminary studies showed that CCL21 promoted SC migration. Taken together, our data suggest that PDGF-BB may mediate TGFβ induced SC migration along with other chemokines.

Disclosures: Ying Wang, None.


Hypoxia Promotes Myotube Formation and Fusion via AKT-FoxO3a Pathway.Yoshitaka Kawato*1, Makoto Hirao1, Yui Honjo1, Hiroki Oze1, Kenrin Shi1, Akira Myoui2, Hideki Yoshikawa1, Jun Hashimoto1. 1Osaka University Graduate School of Medicine, Japan, 2Osaka University Hospital, Japan

Muscle is a very important organ in musculoskeletal system. There are two major pathways to create adenosine triphosphate (ATP) in skeletal muscle tissue. Change of oxygen levels is one of major determinant for skeletal muscle bioenergetics; oxidative phosphorylation and glycolysis, however it remains unknown that oxygen level also influence on myogenesis. It has been reported that normal O2 levels in skeletal muscle tissue is relatively low (2-5%) (Kunze 1976; Heinrich 1987; Evers 1997). So, we hypothesized that myogenesis might be promoted when low oxygen tension condition. The murine myoblast C2C12 was induced to differentiate using DMEM supplemented with 2% horse serum and cultured under normoxia (20%O2) for 4days. After that, we separated to normoxia and hypoxia (5%O2) and cultured another 6days. We performed immunocytochemistry using mouse monoclonal anti-myosin MF20 primary antibody. Fusion index (the number of nuclei within myotubes/ total number of nuclei) was increased by hypoxia (85.7 ± 3.7%) (mean ± S.D.) compared to normoxia (34.8 ± 2.7%). Hypoxia also increased the percentage of myotubes with over six nuclei (82.4 ± 6.2%) compared to normoxia (37.8 ± 11.2%). Western blot analysis revealed the increase of myosin heavy chain protein expression under hypoxia in day 7 and 10. Next, we checked the influence of p38MAPK and PI3K-AKT pathway on the hypoxia-induced promotion of myogenesis, because these pathways have been reported to play important role in the myogenesis, furthermore to be activated by hypoxia stimulation. SB203580 (p38MAPK inhibitor) blocked myotube formation dose dependently, however, hypoxia-induced influence was clearly still remained. On the other hand, LY294002 (PI3K-AKT inhibitor) abolished the myotube formation induced by hypoxia. Then, we focused on FoxO3a, which is downstream of AKT pathway and an important transcriptional factor for muscle atrophy-related genes. In fact, atrogin-1 expression was down-regulated by hypoxia. Luciferase reporter assay revealed that FoxO3a transcription activity was down-regulated by hypoxia, and phosphorylation of AKT and FoxO3a was clearly up-regulated. Furthermore, overexpression of FoxO3a using constitutively active form FoxO3a (TM-FoxO3a) almost completely blocked hypoxia-induced myotube formation and fusion. Taken together, although further confirmation is required, down-regulation of FoxO3a activity due to phosphorylation of AKT-FoxO3a pathway is an important mechanism in hypoxia-induced myotube formation and fusion.

Disclosures: Yoshitaka Kawato, None.


Impairment of Long Bone Growth and Progressive Establishment of High Trabecular Bone Mass in Mice Lacking Bone Sialoprotein (BSP).Maya Boudiffa1, Ndéyé-Marième Wade-Gueye2, Marco Cardelli3, Norbert Laroche2, Arnaud Vanden-Bossche2, Jane Aubin4, Laurence Vico5, Marie-Helene Lafage-Proust6, Luc Malaval*7. 1IRCM, Canada, 2Université de Lyon, INSERM U890, Université Jean Monnet, 42023, France, 3Dept. of Molecular Genetics, University of Toronto, Canada, 4University of Toronto Faculty of Medicine, Canada, 5University of St-Etienne, France, 6INSERM Unit 890, France, 7INSERM U890-Université de Lyon-Université Jean Monnet, Saint-Etienne, France

Bone sialoprotein (BSP) is strongly expressed by osteoblasts, osteoclasts and hypertrophic chondrocytes, and is particularly abundant in sites of primary bone formation. We previously showed that adult mice with a knockout of the bsp gene (BSP-/-) present with low bone formation and resorption parameters but a higher trabecular bone mass than their wild type counterparts. This raises the question of the mechanisms of trabecular bone accumulation in the mutants. In this study we analysed bone development in BSP-/- mice.

Newborn BSP-/- mice do not show any general skeletal abnormality. However, microtomographic (μCT) analysis reveal a delay in membranous primary ossification, with wider sutures in BSP-/- than in BSP+/+ fetuses, as well as thinner femoral cortical bone (-/- vs +/+, M ± SEM, 31.4 ± 3.3 vs 56.4 ± 5.3μm, N=4, p<0.01) and lower tissue mineral density (367 ± 5.5 vs 400 ± 11.0, N=5, p<0.05). Histomorphometric measurements on newborns show that trabecular bone volume and osteoclast parameters do not differ between genotypes (BV/TV=8.6 ± 0.7 vs 9.9 ± 2.8%, N=10). However, as early as 3 weeks after birth, osteoclast number (not shown) and surface drop in mutant mice of both sexes (males: 8.6 ± 0.9 vs 6.4 ± 0.7%, females: 7.2 ± 1.4 vs 3.6 ± 0.4%, N=5, p<0.001, 2way ANOVA), concomitant with trabecular bone accumulation (m: 15.9 ± 1.0 vs 11.6 ± 1.6%, f: 18.6 ± 1.6 vs 6.8 ± 1.3%, N=6, p<0.001).

Mutant mice are smaller than wild type since birth and throughout life. The growth plates are thinner in newborn BSP-/- (564 ± 18 vs 655 ± 36μm, N=5, p<0.05), with a thinner hypertrophic zone (204 ± 10 vs 218 ± 8μ m, N=5, p<0.05), suggestive of lower activity. At 3 weeks of age, there is no difference in total growth plate thickness, but the proliferating zone of either sex is thinner and the hypertrophic zone thicker in BSP-/- than in BSP+/+ mice (m: 131 ± 14 vs 94 ± 10μm, f: 98 ± 4 vs 84 ± 8μm, N=5, p< 0.05), maybe reflecting a combination of lower proliferation and impaired resorption. No differences in growth plate parameters were observed in older (10, 16, 40, 48 week) mice.

In conclusion, lack of BSP alters long bone growth through early growth plate kinetics, as well as membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth, concomitant to a drop in osteoclast numbers/surfaces. Further studies are needed to clarify the impact of BSP deficiency on growth plate chondrocytes and the compensatory mechanisms that allow normal endochondral development in BSP-/-mice.

Disclosures: Luc Malaval, None.


A Novel Mechanism for Dose-to-Duration Encoding: ATP Concentration Determines the Persistence of Ca2+/NFATc1 Signaling through Distinct P2 Receptor Subtypes in Osteoblasts.Matthew Grol*, Alexey Pereverzev, Stephen Sims, S. Jeffrey Dixon. The University of Western Ontario, Canada

Cellular responses typically vary with the strength of the stimulus. Thus, underlying transduction mechanisms must relay quantitative information about the intensity of the signal. We and others have shown that ATP, released in response to mechanical stimuli, signals through cell-surface P2 receptors expressed in many cell types including osteoblasts. P2Y are G protein-coupled receptors that classically signal through release of calcium from intracellular stores; whereas, P2X are ATP-gated channels that permit influx of calcium from the extracellular milieu. The calcium-regulated transcription factor NFATc1 (which upon activation translocates from the cytoplasm to the nucleus) plays an essential role in the differentiation of osteoblasts; however, mechanisms leading to activation of NFATc1 during osteoblastogenesis are unknown. Our preliminary studies revealed dramatic differences in the duration of NFATc1 signaling encoded by the concentration of extracellular ATP. Thus, our purpose was to investigate the mechanisms underlying this “dose-to-duration” encoding. Live- and fixed-cell confocal microscopy were used to localize enhanced green fluorescent protein-tagged NFATc1 in osteoblasts. Both low (10-100 μM) and high (1-3 mM) concentrations of ATP induced nuclear translocation of NFATc1 that peaked at 15 min. Notably, NFATc1 returned to the cytoplasm 1 h following stimulation with low [ATP], whereas nuclear translocation induced by high [ATP] persisted for 2-3 h in duration. Since ATP activates P2Y at low concentrations and P2X7 receptors at concentrations equal to and exceeding 1 mM, we tested the contribution of these receptor subtypes. UTP (exclusively activates P2Y) mimicked responses observed with low [ATP], whereas BzATP (P2X7 agonist) induced prolonged NFATc1 translocation. Consistent with these findings, fluorescence measurements revealed that ATP or UTP (10-100 μM) cause only transient increases in cytosolic calcium concentration, whereas BzATP and ATP (1-3 mM) elicit more sustained elevations. Taken together, these findings demonstrate for the first time that the presence of multiple receptor subtypes with different affinities increases the range of ATP concentrations for which dose-dependent responses are possible. This phenomenon provides a novel mechanism by which osteoblasts may transduce differences in the intensity of mechanical stimuli over a wide dynamic range, otherwise unattainable through a single subtype of cell-surface receptor.

Disclosures: Matthew Grol, None.


See Friday Plenary number FR0103.


Hyper-occlusal Force Induced the Expression of Type XII Collagen in Periodontal Tissue.Tetsuomi Nemoto1, Kazuko Goto1, Hiroshi Kajiya1, Yutaka Takahashi1, Tsuzuki Takashi*2, Koji Okabe1. 1Fukuoka Dental College, Japan, 2fukuoka dental college, Jpn

It is well known that excessive mechanical force by hyper-occlusion is induced to occlusal trauma. In clinical, traumatic force produce disorganization of cells and fibers, resorption of bone and cementum, resulting in tooth mobile. However, the mechanism in the process remains to be understood. In the present study, to examine morphological and biological mechanisms in occlusal trauma on periodontal ligament tissue we employed in vivo hyper occlusion model rodents.

Five weeks-old wester rats or ddy mice were kept in hyperocclusion for 0, 2, 4, and 7 days with bonding steel wire on the occlusal surface of the upper light molars. To investigate alveolar bone resorption induced by the hyper-occlusion the sections of the lower right first molar were stained with tartrate-resistant acid phosphatase (TRAP) as a maker for osteoclasts. In parallel experiments, the three parts of tooth, buccal cervix, furcation, and lingual cervix were stained with TRAP. To investigate the relationship between hyper-occlusion and regeneration of cell matrix, we examined the effect of hyper-occlusal force on the expression of type I and type XII of collagens using immunohistochemistry and real time PCR methods.

On control animals (day 0), the furcation of teeth and arrangement of collagens fibers were in order. Type I collagen was localized in the extracellular area at furcation in teeth root. The expression and localization of type I collagens were no effects on the hyper-occlusion treatment. On contrast, the expression and localization of type XII collagen was unclear in periodontal ligament tissues.

On day 4 after the hyper-occlusion treatment, a number of TRAP-positive cells significantly increased in the furcation and lingual cervical parts of teeth compared to control animals. The type XII collagens were gradually up-regulated by hyper-occlusion treatment in time-dependent manner and localized in around periodontal ligament cells. Using real time PCR method, type I collagen mRNA expression was not up-regulated in periodontal tissues during hyper-occlusion treatment, while type XII collagen mRNAs significantly up-regulated on day 2 and 4 after hyper-occlusion treatment.

The results indicated the hyper-occlusal force significantly up-regulated the expression of type XII collagen in periodontal tissue, but not type I collagen, suggesting in the regeneration of periodontal tissues for prevention of occlusal trauma.

Disclosures: Tsuzuki Takashhi, None.


See Friday Plenary number FR0105.


Toughness of Human Cortical Bone under Realistic Loading Conditions.Elizabeth Zimmermann*1, Maximilien E. Launey2, Robert O. Ritchie1. 1University of California, Berkeley, USA, 2Lawrence Berkeley National Lab, USA

Cortical bone invariably contains cracks and fracture mechanics provides the best methodology to characterize their resistance to fracture, or toughness. Fracture studies on the behavior of human cortical bone have provided much information on how the hierarchical microstructure of bone is able to resist the initiation and growth of incipient cracks at numerous length scales. In particular, the toughness of bone originates from a combination of plasticity mechanisms below the micron length scale (i.e., collagen uncoiling, fibrillar sliding, etc.) and crack tip shielding mechanisms at the micron scale (i.e., crack deflection, crack bridging, crack twisting, etc.).

In vivo, these cracks are subject to combinations of tension, in-plane shear and out-of-plane shear at the crack tip, which is called mixed-mode loading. To date, measurements of cortical bone toughness have only been performed under tensile loading conditions because they were assumed to be the limiting value.

In this study, human cortical bone samples were taken from three male donors. The hydrated, notched samples were tested under symmetric and asymmetric four-point bend, which allows the relative amount of tension and shear at the crack tip to be tuned. The tests were performed in an environmental scanning electron microscope to simultaneously image crack growth. Further investigations into the mixed-mode crack growth behavior and the effects of out-of-plane shear were performed.

Our results reveal that in the longitudinal orientation (crack is parallel to the osteons), bone is tougher under in-plane shear. However, in the transverse orientation (crack is perpendicular to the osteons), bone is 25% tougher in tension. This result is a consequence of both the bone matrix's ability to control the crack path through the highly mineralized cement lines, which are oriented along the longitudinal orientation, and the applied mechanical loads that drive the crack forward in tension and cause deflections under in-plane shear.

Thus, when assessing the toughness of bone, the mode I value is not always the limiting value; in addition, one orientation does not sample the same intrinsic and extrinsic mechanisms that control crack propagation in bone. Testing methods to assess the resistance to fracture in bone will be discussed in this context, especially with applications to small animal studies, which are mostly done under tensile loading conditions in the transverse orientation.

Disclosures: Elizabeth Zimmermann, None.


Synthesis, Characterization, and Evaluation of Bone Targeting Salmon Calcitonin Analogues in Normal and Osteoporotic Rats.Michael Doschak, Madhuri Newa, Krishna Bhandari*. University of Alberta, Canada

Purpose: To synthesize, characterize & evaluate the efficacy of bone-targeting salmon calcitonin (sCT) analogues.

Introduction: sCT elicits an antiresorptive effect by acting upon its receptors (CTRs) on bone-resorbing osteoclasts (OC). However, antiresorptive therapy utilizing conventional sCT is severely hampered by its short t1/2 (17-57 min) due to rapid systemic clearance & degradation in kidneys, liver & blood resulting in poor & variable bioavailability. Pegylation, the attachment of polyethylene glycol, improves the pharmacokinetic parameters of sCT by increasing its circulation time, & by reducing proteolysis & immunogenicity. However, as CTRs are also widely distributed in kidneys, lung, and other tissues, the competitive uptake of sCT (or pegylated sCT) by non-bone tissue resident CTRs may not always lead to optimal sCT bioavailability at bone resident OC- the desired site of action for osteopenic bone diseases. Thus, we sought to target sCT & pegylated sCT to bone by conjugation to a bisphosphonate (BP) carrier.

Methods: BP conjugates of pegylated & non-pegylated sCT analogues were synthesized, characterized by MALDI-TOF, Tris-Tricine SDS-PAGE & number of BP/sCT, & evaluated for sCT secondary structure by circular dichroism, in vitro bone mineral affinity & specificity by different calcium salt binding affinity assays, cytotoxicity in OC precursor RAW 264.7 cells by MTT assay, continued sCT bioactivity & CTR binding potential by intracellular cAMP stimulation assay in T47D breast cancer cells, sCT antibody binding ability by ELISA on calcium phosphate coated osteologic cell culture plates, & for their duration of action & effect on plasma calcium & phosphate levels in normal (n=20) & osteoporotic (OP) (n=18) rats.

Results: BP & PEG-BP conjugated sCT resulted in a stable & desirable α-helical form without altered receptor & antibody binding specificity. They exhibited significantly greater bone mineral affinity & specificity over unmodified sCT, retained strong sCT bioactivity, were non-toxic & exhibited an improved duration of action & a comparable reduction in serum calcium to that of parent sCT.

Conclusion: We report the synthesis of a new class of antiresorptive bone drug that has not previously been attempted & neither has a bone targeting formulation of the antiresorptive peptide hormone Calcitonin. These compounds hold great promise for clinical utility in the treatment of osteopenic bone disease & other related indications.

Disclosures: Krishna Bhandari, None.


See Friday Plenary number FR0108.


Differential Gene Expression in Osteoblast/Osteocyte Lineage Cells between Hyp Mouse and Wild-type Mouse.Kazuaki Miyagawa*1, Keiichi Ozono2, Kanako Tachikawa1, Yuko Mikuni-Takagaki3, Mikihiko Kogo4, Toshimi Michigami5. 1Department of Bone & Mineral Research, Osaka Medical Center & Research Institute for Maternal & Child Health, Japan, 2Osaka University Graduate School of Medicine, Japan, 3Kanagawa Dental College & Graduate School of Dentistry, Japan, 4Department of 1st Oral Surgery, Osaka University Graduate School of Dentistry, Japan, 5Osaka Medical Center, Research Institute for Maternal & Child Health, Japan

Among the molecules responsible for hereditary hypophosphatemic rickets, FGF23, PHEX and DMP1 are expressed in osteoblast/osteocyte lineage cells. In X-linked hypophosphatemic rickets (XLH) and its murine homolog Hyp caused by mutations in PHEX/Phex gene, serum levels of FGF23 are elevated, although the underlying mechanism is not fully understood. Therefore, in the current study, we isolated primary osteoblasts and osteocytes from Hyp and wild-type mice to analyze the gene expression by real-time PCR. Long bones obtained from female Hyp mice or wild-type mice were minced and subjected to sequential digestion with collagenase and decalcification with EGTA for fractionation of osteoblasts and osteocytes. We confirmed the high levels of serum FGF23 in female Hyp mice. Among the 9 fractions isolated from wild-type bones, alkaline phosphatase was expressed in fractions 3-5 and attenuated in the later fractions, while strong expression of Dmp1 and Sost was detected in fractions 6-9, suggesting that fractions 3-5 and fractions 6-9 from wild-type mice were considered as osteoblast-rich and osteocyte-rich fractions, respectively. Phex and Fgf23 were expressed in both osteoblast-rich and osteocyte-rich fractions, and the expression of Fgf23 was more intense in osteocyte-rich fractions. Then, we analyzed gene expression in osteoblast/osteocyte lineage cells isolated from Hyp bones, which was compared with that in wild-type cells. The expression of Fgf23 was stronger in Hyp cells, and interestingly, it was relatively high even in the early fractions (fractions 3 and 4) in Hyp. As to the expression of Dmp1, it was also higher in Hyp cells, and its level in fraction 5 was almost comparable to that in fractions 6-9. We also found the slight increase in Hyp cells in the levels of mRNA corresponding to 5'portion of Phex gene retained in Hyp allele. Next, since we have previously demonstrated that extracellular Pi triggers signal transduction via type III Na+/Pi cotransporter Pit1, we determined its expression. In all the fractions, it was almost 2-fold stronger in Hyp cells compared to wild-type cells, suggesting the altered response to extracellular Pi. These results indicate that molecules responsible for hereditary hypophosphatemic rickets are differentially expressed in osteoblast/osteocyte lineage cells between Hyp and wild-type mice, which might involve the difference in responsiveness to extracellular Pi as well as that in the composition of extracellular matrix.

Disclosures: Kazuaki Miyagawa, None.


See Friday Plenary number FR0110.


Increased MEPE Protein Expression in Rat Bone Tissue after a Single Bout of Mechanical Loading.Nathalie Bravenboer*1, Maartje Broeders2, Huib Van Essen3, Christianne Reijnders4, Paul Lips1. 1VU University Medical Center, The netherlands, 2dept Endocrinology, VU University Medical Center, Netherlands, 3VU Medical Center, The netherlands, 4Dept Dermatology, VU University Medical center, Netherlands

Skeletal integrity in humans and animals is maintained by daily mechanical loading. It has been widely accepted that osteocytes function as mechanosensors. Many biochemical signaling molecules are involved in the response of osteocytes to mechanical stimulation. One of these molecules is matrix extra cellular phosphogly-coprotein (MEPE). It has been shown that MEPE gene expression was up regulated in response to 4 point bending load. MEPE plays a role in the regulation of bone mineralization, dentin mineralization, renal phosphate handling and vitamin D metabolism. The aim of this study was to detect MEPE protein expression after mechanical loading in order to elucidate the role in the translation of mechanical stimuli into bone formation. The 4-point-bending model of Forwood and Turner (1) was used to induce a single or a repeated period of mechanical loading on the right tibia shaft, whereas the contra-lateral left tibia served as control. Repeated loading consisted of 10 loading periods in 12 days. Rats were sacrificed 6 hours after the last loading period. MEPE was detected by immunohistochemistry. MEPE positive area relative to total cortical area (PosCo %) was measured in longitudinal bone sections from loaded and unloaded tibiae. A single bout of loading resulted in doubling of PosCo in the loaded right tibia (1.17 ± 0.79%) compared to the contra-lateral control (0.60 ± 0.50%, p= 0.0137). After repeated loading PosCo was unchanged in the loaded right tibia (0.61 ± 0.38%) compared to the contra-lateral control tibia (0.61 ± 0.35%). In rats that received no mechanical loading no difference between the right and the left tibia was observed. In conclusion MEPE protein expression is stimulated by mechanical loading, which confirms the data on MEPE gene expression. This indicates MEPE could play an important role in the translation of mechanical stimuli into bone formation.

(1) Forwood MR, Owan I, Takano Y, Turner CH Increased bone formation in rat tibiae after a single short period of dynamic loading in vivo. Am J Physiol. 1996: 270:E419-23.

Disclosures: Nathalie Bravenboer, None.


See Friday Plenary number FR0112.


See Friday Plenary number FR0113.


Activation of Vascular Smooth Muscle Parathyroid Hormone Receptor Inhibits Wnt/β-Catenin Signaling and Aortic Fibrosis and Calcification in Diabetic Arteriosclerosis.Su-Li Cheng*1, Jian-Su Shao1, Linda Halstead2, Oscar Sierra1, Kathryn Distelhorst3, Dwight Towler2. 1Washington University in St. Louis School of Medicine, USA, 2Washington University in St. Louis, USA, 3Wahsington University in St. Louis School of Medicine, USA

Vascular fibrosis and calcification contribute to diabetic arteriosclerosis, impairing Windkessel physiology necessary for distal tissue perfusion. Wnt family members -up-regulated in arteries by the low-grade inflammation of “diabesity” - stimulate type I collagen expression and osteogenic mineralization of mesenchymal cells via β-catenin. Conversely, intermittent administration of parathyroid hormone (PTH) inhibits Wnt/ β-catenin signaling and calcification in aorta of LDLR (low density lipoprotein receptor) -deficient mice fed with high fat diabetogenic diets (HFD). To better understand PTH type 1 receptor (PTH1R) - Wnt/β-catenin interactions in diabetic arteriosclerosis, we generated SM-caPTH1R transgenic mice, a model in which the constitutively active PTH1R variant H223R (caPTH1R) is expressed only in vasculature by employing minimal SM22 promoter. When fed with HFD, SM-caPTH1R+;LDLR+/- mice become obese and diabetic, with no improvements in fasting serum glucose, cholesterol, body weight, body composition, or bone mass vs. LDLR+/- siblings. Moreover, serum PTH and calcium levels were unaltered and urinary calcium clearance was unaffected by the transgene. However, the expression of SM-caPTH1R transgene decreased aortic β-catenin protein accumulation and aortic β-galactosidase activity in TOPGAL+ (TCF/LEF optimal promoter - β-galactosidase reporter); LDLR+/- mice on HFD. Although TNF, Msx2, Wnt7a, and Wnt7b mRNA levels were not altered, Col1A1, alkaline phosphatase, osteopontin, MMP9, Runx2, Osx, and Nox1 were down-regulated in SM-caPTH1R aorta. In contrast, p21 level was increased. Aortic calcification and collagen and Nox1 protein accumulation were also reduced in SM-caPTH1R mice. Moreover, levels of aortic superoxide - a reactive oxygen species upregulated with calcific arteriosclerosis -were concomitantly reduced by caPTH1R. Ex vivo aortic plethysmography revealed that expression of caPTH1R increased aortic compliance. In vitro, the caPTH1R suppressed myofibroblast proliferation, inhibited Wnt3a, Wnt7a, and Wnt7b -induced TopFlash and Col1A1 promoter activity, and decreased MMP9. Matrix mineralization was also reduced in caPTH1R myofibroblasts. In conclusion, cell-autonomous VSMC PTH1R inhibits arteriosclerotic Wnt/β-catenin signaling and reduces vascular oxidative stress, thus limiting type I collagen and calcium accrual in aortas of diabetic LDLR-deficient mice.

Disclosures: Su-Li Cheng, None.


Circulating Sclerostin Levels in Disorders of Parathyroid Function: Primary Hyperparathyroidism (PHPT) and Hypoparathyroidism (HypoPT).Aline Costa*1, Serge Cremers1, Mishaela Rubin1, Elzbieta Dworakowski1, Donald McMahon2, Shonni Silverberg1, John Bilezikian2. 1Columbia University, USA, 2Columbia University College of Physicians & Surgeons, USA

Sclerostin is a protein product of osteocytes that is encoded by the SOST gene. It plays an important regulatory role in anabolic signaling pathways and, unimpeded, sclerostin inhibits the osteoanabolic Wnt signaling pathway. An attractive hypothesis to account for the anabolic actions of PTH is an osteocyte interaction that leads to the inhibition of sclerostin. However, the data supporting this idea comes from animal studies in which SOST gene expression is measured by mRNA, sclerostin-positive osteocytes (by immunocytochemistry), and sclerostin protein (by Western-blot analyses of bone lysates). To test the hypothesis that sclerostin is regulated by PTH in human subjects, we used a new highly specific and sensitive ELISA for sclerostin (TECOmedical, Sissach, Switzerland) to measure circulating sclerostin levels in serum of 25 subjects with PHPT and HypoPT and compared them with historical controls as reported by the manufacturer. The standard detection range is 0.25 to 4.0 ng/mL and sensitivity is 0.15ng/mL; intra-assay CV 1.3-1.6%, inter-assay CV 1.8-2.7%. Thirteen subjects had PHPT (11F/2M, median age 64 yrs) and twelve subjects had HypoPT (7F/5M, median age 49 yrs). All patients had their disease for at least 3 years. Sclerostin levels were markedly higher in hypoparathyroid subjects as compared to those with PHPT (mean ± SD: 1.13 ± 0.37 vs 0.46 ± 0.10ng/mL, p=<0.0001) and to historical controls (mean ± SD: 0.45 ± 0.23ng/mL, p=<0.0001). Sclerostin levels in PHPT patients did not differ from historical controls p=0.68). These data suggest that sclerostin levels can be accurately measured in the circulation of human subjects. The results are consistent with the hypothesis that low PTH levels appear to favor SOST gene expression, and provide support for the hypothesis that PTH inhibits sclerostin production. The finding of normal levels of sclerostin in PHPT suggests that in a chronic state of PTH excess, sclerostin function is not perturbed, or any perturbation is not reflected in the circulation. Studies to track these levels after intervention with PTH (hypoparathyroidism) or removal of PTH (parathyroidectomy in PHPT) are currently ongoing.

Disclosures: Aline Costa, None.


See Friday Plenary number FR0116.


Heterozygosity in the VDR Gene Influences Body Composition more than Bone Mass.Francisco Jose De Paula*1, Sheila Bornstein2, Ingrid Dick-de-Paula3, Phuong Le4, Bahman Rostama4, Clifford Rosen2. 1School of Medicine of Ribeirao Preto - USP, Brazil, 2Maine Medical Center, USA, 3School of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil, 4Maine Medical Center Research Institute, USA

The influence of modest vitamin D deficiency on bone and body composition in animal models is not clear. Vitamin D receptor null mice (VDR) not only have very low bone mass but also have a lean phenotype and increased insulin sensitivity. We hypothesized that mice with loss of one allele of the VDR gene (VDR+/-) might have moderate skeletal and body composition phenotypes. Female VDR+/- mice on a B6 background and B6 controls (+/+) were raised on a regular chow diet until 12 weeks (wk) of age. VDR+/- and VDR+/+ mice were treated either with PTH (sc, bovine PTH 50 μg/kg daily × 4 wk) or vehicle (VH) × 4 wk. The study comprised 4 female groups of mice: VDR+/+ VH (+/+VH, n=5), VDR+/+ PTH (+/+PTH, n=7), VDR+/-VH (+/-VH, n=10) and VDR+/- PTH (+/-PTH, n=11). Basal PTH serum levels were determined by enzyme immunoassay (Immunotopics), bone mass (aBMD) and body composition were determined by PIXIMUS before and after treatment. Bone microstructure (BV/TV) was determined by μCT in the lumbar spine (L5) and femur; MRI assessed percent body fat and femoral fat after treatment. Baseline PTH serum levels were modestly higher in VDR+/- mice (+/+= 67.7 ± 10 vs +/- = 74.8 ± 7 pg/ml). At 12 wk of age femoral aBMD in VDR+/+ did not differ from VDR +/- mice. Four weeks of PTH treatment for both genotypes showed a 12.2% increase in femoral aBMD from baseline (p<0.01 vs baseline, NS by genotype). With PTH treatment, total body aBMD increased slightly more in VDR+/- than VDR+/+ mice (+7 vs +6%, p=NS), whereas strikingly the VDR+/+ gained more fat mass (+0.32 ± 0.20 g) than the VDR+/- group (-0.022 ± 0.18g). A similar trend in fat mass was noted for the PTH treated groups of VDR+/- and VDR+/+ mice (VDR +/+= +0.44 ± 0.17g vs VDR +/-= +0.28 ± 0.16 g). By μCT, L5 Trabecular thickness was higher in +/-PTH than +/-VH (+/-= 0.05676 μm ± 0.0006656 vs +/+= 0.06092 ± 0.001614 μm). Our results suggest that VDR heterozygote mice respond normally to an anabolic stimulus (PTH) and have a very mild skeletal phenotype associated with slight increases in PTH and reduced trabecular bone volume. Remarkably during continued growth at 16 weeks female VDR+/- mice with or without PTH remained leaner than VDR +/+ mice suggesting that the body composition phenotype is more pronounced than the skeletal one. Further studies to delineate the role of vitamin D in energy metabolism and its relationship to skeletal acquisition are needed.

Disclosures: Francisco Jose De Paula, None.


Irradiation Primes the Skeleton for PTH Anabolic Actions.Teresa Wang1, Amy Koh*2, Chad Novince3, Russell Taichman3, Hector Rios2, Laurie McCauley3, Xin Li2. 1U of M, USA, 2University of Michigan, USA, 3University of Michigan, School of Dentistry, USA

PTH is an anabolic agent used clinically to treat osteoporosis and is in investigational use for increasing hematopoietic stem cells (HSCs). Irradiated (IRR) mice have increased PTH anabolic actions compared to non-IRR mice (30% bone area increase). The purpose of this study was to identify mechanisms responsible for enhanced PTH actions in IRR mice. C57B6 mice were IRR (310-325cGy; 2 fractions 3h apart) then administered PTH (0.05mg/g/d) for 21d. Flow cytometric analysis, microarray, real-time PCR gene expression, serum assays, immunohistochemistry and bone histomorphometry were performed along with primary cell isolation and analyses. IRR reduced bone marrow (BM) cellularity with retention of osteoblastic cells lining trabeculae. IRR decreased cell numbers in calvarial cell cultures in vitro suggesting against a cell autonomous benefit of irradiation. BM added to bone marrow stromal cell (BMSC) monolayers restricted mineralization which was less restrictive with IRR vs. non-IRR BM. IRR co-culture increased BMSC numbers, and IRR BM contained more adipocytes than non-IRR BM. FGF2 and IL-6 mRNA was increased in IRR BM in vivo. IRR decreased, and PTH increased periostin mRNA in the bone marrow with greater and more widespread periostin positivity throughout the marrow. PTH significantly increased serum OCN and P1NP in IRR mice despite lower baseline levels versus non-IRR. IRR decreased B220 cells, while the % of LSK cells (lin-, sca-1+, c-kit+) was increased. IRR mice had increased % BrdU positive cells and PTH increased total numbers of BrdU positive cells in IRR mice. Osteoclast number was not altered with IRR but was increased with PTH in both groups. Megakaryocytes (vWF stain) were reduced yet were located more closely to trabecular surfaces with IRR, and PTH treatment resulted in increased megakaryocyte ploidy status. PTH anabolic activity was augmented evidenced by increased bone area, serum OCN and P1NP in IRR mice. These results suggest IRR increased osteoblastic proliferation in an indirect manner and that PTH acted in the IRR mice by increasing bone active cell populations. IRR via increased IL-6 and FGF2 may prime the skeleton for anabolic actions of PTH. Furthermore, reduced cellularity and the retention of MKs, LSKs and/or adipocytes with IRR may increase osteoblast exposure to these cells to support anabolic actions. Alternatively, irradiation may decrease inhibitory cell types that negatively affect anabolic actions of PTH.

Disclosures: Amy Koh, None.


See Friday Plenary number FR0119.


PTH but not 25(OH)D is Directly Associated with Blood Pressure and Inversely Associated with Carotid-femoral Artery Pulse Wave Velocity.Ashley Davidson*1, Kathy Ryan2, Alan Shuldiner3, Elizabeth Streeten1. 1University of Maryland School of Medicine, USA, 2Department of Endocrinology, Diabetes & Nutrition, USA, 3Department of Endocrinology, Diabetes & Metabolism, USA

Background: Both 25-hydroxyvitamin D (25-D) and PTH have been reported to be associated with cardiovascular (CV) phenotypes in some, but not all, studies. Pulse wave velocity (PWV) is a measurement of arterial stiffness and has been shown to be associated with PTH in populations receiving hemodialysis but has not been reported in healthy individuals with normal renal function. The purpose of this study was to investigate the associations of PTH and 25(OH)D with blood pressure and other subclinical vascular disease markers in the Amish, a population with a relatively homogeneous diet, levels of physical activity and low levels of smoking which can be confounders in other populations.

Methods: This observational study included participants in the Amish Family Osteoporosis and Amish Family Calcification Studies. Generally healthy participants with normal serum calcium and creatinine had the following measured: PTH, 25(OH)D, blood pressure, coronary artery calcification (CAC by electron beam computed tomography), carotid intimal medial thickness (cIMT) and PWV [carotid-femoral (C-F) and carotid-radial (C-R)]. Association analyses were performed between both 25(OH)D and PTH and: BP (n=1108), CAC (n = 650), cIMT (n = 220), C-R PWV (n = 257) and C-F PWV (n = 249). The relations of 25(OH)D and PTH levels, adjusted for season, age and sex, with subclinical vascular disease measures were assessed by comparing mean levels by Pearson's correlation.

Results: The mean age was 51.18 ± 13.02 years (range 18-91). 59.1% of the subjects were female (n = 655). The mean 25(OH)D was 22.32 ± 5.87 ng/ml, mean PTH 55.38 ± 14.27 pg/ml, mean systolic BP 119 ± 12.9 and mean diastolic BP 74.18 ± 7.37. A positive association was found between PTH and systolic (p = 0.04) and diastolic (p = 0.0004) BP. No association was shown between 25(OH)D and BP (systolic p=0.11, diastolic p=0.11). PTH and C-F PWV were inversely associated (p =0.03). There was no association of PTH with CAC, cIMT or C-R PWV (p = 0.85, 0.4, 0.54), nor of 25(OH)D with CAC, cIMT, C-F or C-R PWV (p= 0.8. 0.52, 0.05, 0.6). The correlation between PTH and 25(OH)D was -0.30 (p<0.0001).

Conclusions: PTH level was directly associated with systolic and diastolic BP, and inversely associated with C-F PWV. 25(OH)D was not associated with these phenotypes. These results suggest that PTH may play a direct role in cardiovascular health. Alternatively, PTH may be a surrogate marker for 1,25-dihydroxyvitamin D in these associations. ,  

Table  .  
  1. *adjusted for age and sex

  2. † PTH and 25OH Vitamin D adjusted for season

inline image
Table Table 1.  
  1. *adjusted for age and sex

  2. † p-value based on log-transformed values

inline image

Disclosures: Ashley Davidson, None.


See Friday Plenary number FR0121.


See Friday Plenary number FR0122.


Combined In Vivo MicroCT and Near-Infrared Imaging Allows for Quantitative Analyses of the Tumor Microenvironment.Rachelle Johnson*1, Lindsay Johnson2, Don Nolting3, Steve Munoz1, Gregory Mundy1, H. Charles Manning1, Todd Peterson1, Julie Sterling1. 1Vanderbilt University Medical Center, USA, 2Department of Biomedical Engineering, Vanderbilt University, USA, 3Institute of Imaging Science, Vanderbilt University, USA

The majority of breast cancer and prostate cancer patients with metastatic disease will go on to develop bone metastases, which contribute largely to patient morbidity and mortality. Numerous small animal models of cancer metastasis to bone have been developed in order to study tumor-induced bone destruction, but the advancement of imaging modalities utilized for these models has lagged significantly behind clinical imaging, particularly in the bone microenvironment in response to tumor cell establishment and treatment. We hypothesized that a combination of in vivo micro-Computed Tomography (μCT) and Near-Infrared (NIR) imaging can be utilized to perform quantitative longitudinal and end-point analyses of bone volume and monitor proteinlevel changes in the tumor-bone microenvironment. We utilized the MDA-MB-231 intratibial breast cancer model and imaged mice weekly by Faxitron, in vivo μCT, and Maestro (fluorescence, NIR). We found that in vivo μCT can significantly detect individual and group level changes in bone destruction over time in a bisphosphonate drug treatment model without altering tumor cell growth (tumor-bearing v. control limb, p<0.0001; tumor limb treated v. un-treated p<0.0001). This imaging method was validated by ex-vivo μCT and histological analyses at end-point with comparable findings. In addition, we found a significant accumulation of NIR-labeled TGF-β neutralizing antibody in the hind limbs when compared to a NIR-labeled control IgG (p<0.05), and we found significantly more NIR-labeled TGF-β antibody in the tumor-bearing limb when compared to the contra-lateral PBS-injected limb (p<0.05), indicative of increased bone remodeling at sites of tumor metastasis. More importantly, this technique offers the potential for real-time, non-invasive imaging of protein changes at the tumor-bone interface, such as increased secretion of osteolytic factors. Taken together, these data present a comprehensive analysis of the tumor-bone microenvironment, in which imaging techniques provide an accurate method of monitoring changes in bone destruction and protein expression in the vicious cycle of tumor-induced bone disease.

Disclosures: Rachelle Johnson, None.


See Friday Plenary number FR0124.


See Friday Plenary number FR0125.


Influence of Connexin43 Expression on the Metastatic Phenotype and the Bone Impact of Prostate Cancer Cells.Coralie Lamiche*1, Jonathan Clarhaut1, Pierre-olivier Strale1, Sophie Crespin1, Norah Defamie1, Marc Mesnil1, Francoise Debiais2, Laurent Cronier3. 1CNRS - UMR 6187, Institut de Physiologie et Biologie Cellulaires, France, 2Hopital Jean Bernard, France, 3University of Poitiers, France

Prostate cancer (PCa) has the highest incidence in men with bone metastasis occurring in 80% of cases and mainly characterized by osteoblastic lesions. The mechanisms by which prostate cancer cells are induced to metastasize to bone are variable and complex, implicating oncogenes, pro-angiogenic factors and adhesion molecules. Among them, transmembrane proteins named connexins (Cxs) involved in gap junctional intercellular communication (GJIC) are known to permit coordinated cellular activities during development and differentiation processes. PCa cells are deficient in Cx isoforms however recent studies suggest that Cx43 could be implicated in the last stages of the tumorigenic process. Moreover, cumulated data support the involvement of Cx43 in the differentiation process of bone-forming osteoblastic cells and bone turnover. Therefore, we have investigated the role of Cx43 on the metastatic potential of PCa cells and on their impact on the osteoblastic phenotype.

To examine the effects of increased expression of Cx43 gene in PCa cells, we have performed retroviral infection in two well characterized cells lines representing different stages of cancer progression: PC3 (aggressive) and LNCaP (less metastatic). The impact on proliferation and differentiation of osteoblastic (OB) cells was evaluated in vitro by means of cocultures with OB cells from murine calvarias. In LNCaP-Cx43 cells, the Cx43 was mainly observed in plasma membrane and a functional GJIC was demonstrated by gap-FRAP and preloading assays between PCa cells as well as in the heterocellular configuration (between LNCaP and OB cells). In PC3-Cx43 cells, Cx43 was restricted to the cytoplasmic part and no cell-to-cell communication was measured. In addition, phenotypic characterization of both cell types demonstrated significant differences in adhesion, invasion and proliferation. Finally, proliferation and differentiation abilities were significantly modified by cocultures with PCa cells. The increased proliferation rate due to contact with PCa cells was not linked to the Cx43 expression level. In contrast, the impact on OB differentiation potential, revealed by alkaline phosphastase activity and qPCR analyses of differentiation markers (Cbfa-1, OCN, OPN), seems to depend on the Cx43 level.

Our in vitro study demonstrates that Cx43 could influence the metastatic status of prostate cancer cells. In vivo, preliminary data confirm the potential implication of this connexin in the bone impact.

Disclosures: Coralie Lamiche, None.


Inhibition of ATP6V1C1 (a Subunit of the V-ATPase) Expression Decreases 4T1 Mouse Breast Cancer Cell Invasion and Bone Destruction.Shengmei Feng*1, Lianfu Deng2, Guochun Zhu1, Wei Chen3, Yi-Ping Li3. 1The Forsyth Institute, Shanghai Institute of Trauma & Orthopaedics, USA, 2Shanghai Institute of Trauma & Orthopaedics, Ruijin Hospital, China, 3The Forsyth Institute, Harvard School of Dental Medicine, USA

Vacuolar H+-ATPases (V-ATPases) located at the plasma membrane of highly metastatic human breast cancer cells are involved in the acquisition of a more metastatic phenotype, and V-ATPase inhibitors decrease the invasion and migration of highly metastatic cells. Importantly, the ATP6V1C subunit (C) of the V-ATPase complex is primarily responsible for its enzymatic function through the control of a reversible dissociation of the V0 and V1 domains. ATP6V1C1 (C1), an isoform of the C subunit, is highly expressed in oral squamous cell carcinoma. Furthermore, we recently reported that C1 is an essential component of the osteoclast proton pump and that it is important for F-actin ring formation in osteoclasts. Nonetheless, the role and mechanisms of the C subunit in breast cancer growth and metastasis remain unknown. To reveal the role of V-ATPase subunit C1 in breast cancer, we selected single subclones of 4T1 mouse breast cancer cells after transfection with lentivirus (Lenti)-GFP and Lenti-LacZ (control; siRNA targets LacZ) or Lenti-c1s3 (siRNA targets C1). Immunohistochemistry and Western blot showed that C1 expression was significantly reduced in C1-knockdown 4T1 cells, while ATP6V1C2 (C2) was not expressed in 4T1 cells with or without C1-knockdown. Furthermore, acridine orange staining showed that C1-knockdown 4T1 cells have reduced acidification, which is similar to C1-knockdown osteoclasts. Remarkably, C1-knockdown 4T1 breast cancer cells also exhibited decreased proliferation, migration, and invasion through matrigel.

PBS or one of the different 4T1 clones (i.e. 4T1, 4T1-Lacz, 4T1-c1s3-1, or 4T1-c1s3-11) was implanted into the left femur of female BALB/C mice. After 16 days, X-rays and micro-CT scanning revealed that there was reduced bone destruction compared with the control mice in the femurs and tibiae of mice in which C1-knockdown 4T1 cells were implanted. All assays were done in triplicate, and all animals were maintained according to the Guide for the Care and Use of Laboratory Animals. We report here for the first time, that C1 not C2 is an essential subunit of the V-ATPase complex in 4T1 mouse breast cancer cells and that inhibition of C1 expression in 4T1 breast cancer cells decreased their growth and invasion in vitro and in vivo. Though these data suggest that the inhibition of C1 expression can decrease the invasive properties of 4T1 cells, additional studies are needed to define the molecular mechanisms of how C1 influences 4T1 migration.

Disclosures: Shengmei Feng, None.


See Friday Plenary number FR0128.


Sympathetic Nervous System Activation Increases Breast Cancer Metastasis to Bone.J. Campbell*1, Julie Sterling2, Yun Ma2, GR Mundy2, Florent Elefteriou2. 1Vanderbilt Center for Bone Biology, USA, 2Vanderbilt University, USA

Depression, while not associated with breast cancer incidence, is linked to increased recurrence and decreased survival. Increased Sympathetic nervous (SNS) outflow, a hallmark of mental stress, controls multiple processes in the bone, such as hematopoietic stem cell trafficking, osteoblast proliferation and osteoclastogenesis via β-adrenergic receptors (βAR) and cytokines also involved in cancer metastasis, such as SDF-1 and RANKL. These observations suggested a possible involvement of SNS activation in cancer cell osteotropism, tumor burden and the formation of osteolytic lesions. To address this question, we used an established model of lytic bone metastasis in which MDA-MB-231 human breast cancer cells are injected via left cardiac ventricle in athymic nude mice. Isoproterenol (Iso), a (βAR) agonist, was used as a surrogate for SNS activation. Faxitron, 3D-microtomography and histomorphometric measurements were used to quantify number and size of lytic lesions, tumor burden and BV/TV. We observed that Iso treatment before MDA-MB-231 inoculation increased the number of bone lytic lesions and lesion area (p<0.05). Treating with Iso after bone metastasis increased the size of the lytic lesions (p<0.05) but not lesion number. These results suggested that sympathetic activation alters the bone microenvironment to make it favorable for cancer cell homing and growth. Although it did not affect lytic lesion number in a model without chronic depression, βAR blockade by propranolol decreased lesion area (p<0.05). In vitro, direct stimulation of the β2AR in MDA-MB-231 cells produced no effect on migration, cell growth or PTHrP, Rank and CXCR4 expression. In contrast, stimulation of the β2AR in bone marrow stromal cells, calvaria and MC3T3 osteoblasts increased Rankl but not Sdf1 expression (p>.005). The conditioned media of Iso-treated osteoblasts increased MDA-MB-231 cell transwell migration (p<0.005), which could be blocked by OPG but not by the CXCR4 antagonist AMD300. These results suggest that the increase in osteolysis and metastasis observed following Iso treatment in vivo are due to a predominant effect on the bone marrow stroma rather than a direct effect on cancer cells. These findings support the hypothesis that depression, via sympathetic activation, make the bone marrow environment permissive to cancer metastasis, growth, or recurrence, and imply that β-blockers may reduce both bone tumor burden and relapse in breast cancer patients.

Disclosures: J. Campbell, None.


The Effect of Various Vitamin D Supplementation Regimens on 25-OH Vitamin D Levels in Breast Cancer Patients Undergoing Treatment.Alissa Huston*, Luke Peppone, Kristin Skinner, Michelle Janelsins, Gary Morrow. University of Rochester Medical Center, USA

Vitamin D (VID) deficiency is linked to decreased bone mineral density (BMD), increased breast cancer incidence, recurrence, and mortality. Breast cancer treatment, specifically hormonal therapy (HT), reduces BMD and increases fracture risk. Our study aims include: 1) determining the prevalence of VID deficiency among women receiving treatment, 2) evaluating the effect various VID supplementation regimens on deficiency, and 3) determining the association between VID levels and BMD. Serum 25-OH VID levels were obtained from approximately 224 women and BMD from 75 women undergoing treatment (HT, radiation, chemo, or any combination) for non-metastatic breast cancer (ages 24-88). The proportion of patients with VID deficiency was determined for the total sample, and compared across race and stage of disease at baseline. VID and BMD were compared using ANCOVA models. Severely VID deficient patients (<25 ng/mL) were prescribed high-dose VID (≥50,000 IU/week), mildly deficient patients (26-31 ng/mL) were prescribed low-dose VID (≥2,000 IU/day), and patients with normal VID levels (≥32 ng/mL) were not given any prescription. The change in 25-OH VID levels was compared across the 3 supplementation levels. At baseline, 23% of all women were considered VID deficient (<20 ng/mL), while 43% were considered VID insufficient (20-31 ng/mL). Lower baseline 25-OH VID was observed in Non-Caucasian patients (Caucasian: 28.2 ng/mL vs. Non-Caucasian: 19.3 ng/mL; p<0.01) and patients with later-stage disease (Stage I: 29.7 ng/mL vs. Stage III: 22.9 ng/mL; p<0.01). Weekly high-dose VID resulted in a significant increase (+27 ng/mL; p<0.01) in 25-OH VID, while there was no difference between low-dose VID supplementation (+14 ng/mL; p=0.28) and no supplementation (+9 ng/mL). Among women not on bisphosphonates, spinal BMD was lower for those with deficient (<20 ng/mL: 0.99 g/cm2; p=0.01) and insufficient follow-up VID levels (20-31 ng/mL: 1.18 g/cm2; p=0.07) compared to those with normal VID levels (>32 ng/mL:1.26 g/cm2). VID deficiency was extremely prevalent in women being treated for breast cancer, and more common in breast cancer patients who presented with later-stage disease and were Non-Caucasian. Weekly high-dose VID is required to significantly increase 25-OH VID levels, as opposed to conventional low-dose VID. Lower VID levels were also significantly associated with reduced spinal BMD among women not using bisphosphonates.

Disclosures: Alissa Huston, None.


The Histone Deacetylase Inhibitor, Vorinostat, Reduces Metastatic Cancer Cell Growth and Associated Osteolytic Disease, but Promotes Normal Bone Loss.Jitesh Pratap*1, Jacqueline Akech1, John J. Wixted1, Gabriela Szabo1, Robinder J. Dhillon2, Xiaodong Li2, Krystin Bedard1, Sadiq Hussain1, Andre Van Wijnen1, Janet L. Stein1, Gary Stein1, Jennifer Westendorf2, Jane Lian1. 1University of Massachusetts Medical School, USA, 2Mayo Clinic, USA

Vorinostat, an oral histone deacetylase inhibitor with anti-tumor activity, is in clinical trials for hematological and solid tumors that metastasize and compromise bone structure. Consequently, there is a requirement to establish the effects of vorinostat on tumor growth in the bone microenvironment. Metastatic cancer cells were injected into tibias of SCID mice and the effects of vorinostat on tumor growth and osteolytic disease were assessed by radiography, μCT, histological and molecular analyses. Vorinostat-treated and control mice without tumors were also examined. Vorinostat significantly decreased tumor cell proliferation and promoted apoptosis of prostate and breast cancer cells. Tumor growth in bone was reduced ∼33%. Bone osteolysis was significantly reduced in the first weeks of tumor growth, but then increased with continued vorinostat treatment. The non-tumor bearing contra-lateral femurs showed significant bone loss (50% volume density of controls) after four weeks of vorinostat therapy. This same extent of bone loss occurred in vorinostat-treated normal mice (without tumors). We show that vorinostat mediated bone loss is caused by a 2 to 3 fold upregulation of factors promoting bone resorption (PTHrP, IL-8 and osteopontin); factors secreted by tumor cells to induce osteoclast activity. Thus, our studies indicate that vorinostat effectively inhibits tumor growth in bone, but has a negative systemic effect on the integrity of normal trabecular bone and thereby contributes to osteolysis independent of tumor cell activity. Vorinostat treatment reduces tumor growth but can promote osteopenia throughout the skeleton. Our studies indicate therapeutic regimens should be considered that combine vorinostat with anti-resorptive agents to prevent treatment-induced bone loss.

Disclosures: Jitesh Pratap, None.


The Role of Tumor Derived Interleukin 6 in a Murine Model of Breast Cancer Bone Metastasis.Yu Zheng*1, Anastasia Mikuscheva2, Hong Zhou3, Katja Boernert1, Frank Buttgereit2, Colin Dunstan4, Markus Seibel3. 1Bone Research Program, ANZAC Research Institute, University of Sydney, Australia, 2Humboldt University, Germany, 3Bone Research Program, ANZAC Research Institute, The University of Sydney, Australia, 4University of Sydney, Australia

Breast cancer has a high propensity to metastasise to the skeleton. The bone microenvironment plays a central role in metastatic cancer growth as reduced bone resorption inhibits and increased bone turnover accelerates cancer growth in bone1,2. In patients with metastatic breast cancer, high circulating interleukin-6 (IL-6) levels have been associated with disease progression/ poor clinical outcomes. We found in animal studies that increasing or decreasing bone resorption results in corresponding changes in tumor IL-6 expression and tumor proliferation, indicating that IL-6 expression by cancer cells may play a role in sustaining breast cancer growth in bone3. However, it is unclear whether tumor-derived IL-6 affects cancer cell behavior in vitro and in vivo.

To investigate this question, IL-6 expression was silenced in MDA-MB-231 cells via a lentiviral-based expression system driving the production of short hairpin RNA species (shRNAs). Knock-down efficacy was 80% as assessed by real-time RT-PCR (mRNA) and ELISA (secreted protein).

In vitro characterization of control vs. knock-down MDA-MB-231 cells demonstrated that silencing of IL-6 expression significantly reduces invasiveness without affecting proliferation.

In vivo studies: Control and IL-6 knock-down MDA-MB-231 cells (50000 cells per injection) were implanted via intra-tibial inoculation into 4-week-old BALB/c nu/nu female mice (n=7/group) kept on a low (0.1%) calcium diet to induce high bone turnover1,2. Tumor growth was monitored using X-ray imaging on days 10, 17 and 21, and bones were analysed by histology and histomorphometry following sacrifice on day 21 post inoculation. Compared to controls, IL-6 knock-down resulted in significantly smaller osteolytic lesions on day 10, 17 and 21 (p< 0.05, by X-ray analysis), and significantly reduced total tumor area on day 21 (3.20 ± 0.76mm2vs. 5.57 ± 0.98mm2 p<0.05, by histology). Growth of subcutaneously implanted tumors was similar in animals injected with IL-6 silenced or normal MDA-MB-231 cells.

We conclude that IL-6 knock-down in MDA-MB 231 cells reduces cancer cell invasiveness in vitro and intraskeletal tumor growth in vivo, supporting the conecpt that tumor-derived IL-6 has an important role in the biology of metastatic breast cancer and may be a potential therapeutic target.


  • 1
    Zheng et al., Cancer Res 67, 9542 (2007).
  • 2
    Zheng et al., Clin Exp Metastasis 25, 559 (2008).
  • 3
    Zheng et al., J Bone Miner Res 24 (Suppl 1), (2009).

Disclosures: Yu Zheng, None.


See Friday Plenary number FR0133.


A Role for the ARF Tumor Suppressor in Osteoclasts.Crystal Winkeler*1, Monica Croke2, Emanuella Heller1, Michelle Hurchla1, Chan Lee1, Katherine Weilbaecher3, Jason Weber1. 1Washington University in St. Louis, USA, 2Washington University School of Medicine, USA, 3Washington University in St. Louis School of Medicine, USA

In response to oncogenic stress, the ARF tumor suppressor is able to halt both cell proliferation and growth. ARF stabilizes and activates p53 through the inhibition of Mdm2, the E3 ubiquitin ligase for p53. More recently, ARF has been ascribed p53-independent functions through its ability to inhibit ribosome biogenesis. However, there is an inherent difficulty in separating these two functions of ARF by the fact that cell growth is intimately tied to cell proliferation. To resolve this, we have chosen to study the function of ARF in osteoclasts. Osteoclasts provide a unique model in that they are post-mitotic and function independent of p53. Furthermore, understanding the growth control of osteoclasts is important in understanding diseases such as osteoporosis and osteolytic bone metastasis.

To assess osteoclastogenesis and osteoclast function in vitro, BMMs were harvested from wildtype or Arf-/- mice. Osteoclastogenesis was assessed by TRAP-staining under various concentrations of RANKL. Mature wildtype and Arf-/-osteoclasts were used to assess the role of ARF in ribosome biogenesis, actively-translating ribosomes, protein synthesis, and cell growth. Additionally, we crossed Arffl/fl mice with mice containing Cre under the control of the Cathepsin K promoter. Resultant mice were used to assess osteoclast-specific Arf loss in vivo by vivaCT, serum CTX, and histological analysis. Arf loss caused enhanced osteoclastogenesis and increased osteoclast size, resulting in greater osteoclast activity. This hyperactivity was most evident in our novel osteoclast-specific Arf-null mice, which exhibited a decrease in bone volume and density compared to control mice. Thus, we propose a teleological function for ARF, distinct from its classical role as a tumor suppressor, in the regulation of osteoclast activity.

Disclosures: Crystal Winkeler, None.


See Friday Plenary number FR0135.


See Friday Plenary number FR0136.


Controlled Bone Tissue Ablation using Novel Navigational RF Device.Andreas Kurth*1, Dirk Proschek2, Aaron Germain3, Robert Poser3. 1Goethe-Universitat Frankfurt, Germany, 2Dep. Orthopaedic Surgery, University Medical Center, Germany, 3Dfine Inc., USA

Purpose: Bone metastasis is a debilitating complication that result in severe pain, pathologic fractures, dysfunction, cord compression and decreased quality of life. Radiation and surgery are the common local treatment options, along with systemic treatment for the underlying primary cancer. Radiofrequency (RF) ablation has been successfully used in radiation resistant tumors or small localized disease. Clinical advantages of RF ablation in spinal metastasis are more immediate pain relief and less systemic toxicity. However, clinical limitations of RF ablation in treatment of vertebral lesions include lack of minimally invasive (MI) navigation in hard tissue and accurate ablation zones. The present study evaluates a novel RF ablation device for local treatment of bone metastates.

Methods: A 10 gage, bipolar, RF ablation instrument with a navigational articulating tip containing thermocouples and a proprietary RF generator were used. Based on strategically located thermocouples, the software automatically controls ablation zones using tissue (adjacent to ablation zone) temperature control. Sixty ex-vivo ablations were performed in bovine liver at each power settings for a total of 180 ex-vivo ablations. Infrared thermography recorded tissue temperature gradients during ablation at three power settings (5W, 15W, 25W). Using a porcine in-vivo model, similar ablations were performed in both liver (n=20) and cancellous bone (n=8) sites followed by gross examination, histologic processing and histomorphometric analysis.

Results: Discrete thermal profiles measured by IR and reproducible ablation zones (20×8 mm) were identified in all ex-vivo specimens. While the rate of ablation was dependent on the power setting, the size of ablation was extremely consistent across power settings. In-vivo ablation zone dimensions were within those recorded in extensive ex-vivo tests. Histomorphometric confirmation of effective, reproducible, site specific in-vivo ablation of both liver and cancellous bone were confirmed in a porcine model.

Conclusion: Site and size specific ablation zones are extremely important in treating bone metastasis that can be close to neural elements. These data demonstrate the unique ability to create reproducible ablation zones in hard tissue at variable rates, based on real time temperature mapping controlled software.

Disclosures: Andreas Kurth, None.

This study received funding from: Dfne Inc, San Jose, CA, USA


See Friday Plenary number FR0138.


Epigenetic Silencing of the Homeobox-containing Transcription Factor Dlx2 Promotes Chemoresistance in Human Osteosarcoma Cells.Erik Sampson*1, Vinit Amin2, Laurence Donahue2, Edward Schwarz1, Regis O'Keefe1, Randy Rosier2. 1University of Rochester, USA, 2University of Rochester Medical Center, USA

Osteosarcoma (OS) is the most common primary paediatric bone tumor. Greater than ninety percent of patients who present with metastatic OS and thirty to forty percent of patients with nonmetastatic disease will experience relapse. Drug resistance is one underlying mechanism contributing to the failure of chemotherapy to elicit a lasting response in these patients. Expression of the homeobox-containing transcription factor Dlx2 is associated with chemotherapy sensitivity in various cancer cell lines. In addition, several Dlx family members are expressed early during osteogenesis, raising the possibility that loss of Dlx2 expression during osteosarcomagenesis concomitantly renders OS cells insensitive to chemotherapy. Indeed, the expression of Dlx2 protein is decreased in OS cell lines compared to immortalized human osteoblasts. Retrovirus-mediated expression of Dlx2 in OS cell lines did not affect cell proliferation, basal apoptosis levels or in vitro clonogenicity a surrogate assay for in vivo tumorigenicity. However, OS cell lines stably expressing Dlx2 were up to ten-fold more sensitive to doxorubicin, a chemotherapeutic commonly used to treat OS, as determined by flow cytometric analysis of caspase 3 activation. Numerous homeobox genes, including several Dlx family members are potentially silenced in cancer cells via aberrant promoter DNA methylation. Epigenetic mechanisms of gene silencing including DNA methylation, histone deacetylation and histone methylation are emerging targets for novel cancer therapeutic development. The histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA; vorinostat (Zolinza)) is approved by the Food & Drug Administration for the treatment of cutaneous T-cell lymphoma. The expression of Dlx2 message and protein was increased in OS cell lines treated either with SAHA or another HDACi, valproic acid (VA). Pre-treatment with SAHA or VA followed by doxorubicin had a striking, synergistic effect on apoptosis in OS cells. This synergistic increase in cell death was in part rescued by RNA interference with HDACi-mediated Dlx2 induction. Dlx2 induction therefore partially mediates rather than simply correlates with OS chemosensitization by HDACi. While future studies will be required to determine whether Dlx2 is a bona fide OS-related tumor suppressor gene, strategies to increase Dlx2 expression including HDACi or DNA methyltransferase inhibitors may overcome chemotherapy resistance in relapsed OS patients.

Disclosures: Erik Sampson, None.


See Friday Plenary number FR0140.


Identification of Novel Molecule for Melanoma Malignancy -Role of CIZ-.Kentaro Miyai1, Masaki Noda1, Tadayoshi Hayata2, Tomomi Sakuma*3, Yoichi Ezura4. 1Tokyo Medical & Dental University, Japan, 2Medical Reserach Institute, Tokyo Medical & Dental University, Japan, 3Tokyou Medical & Dental University, Japan, 4Tokyo Medical & Dental University, Medical Research Insititute, Japan

Metastasis of tumor to bone is one of the serious complications during tumor treatment. One of the possible key molecules for the establishment of bone metastasis would be those for cell attachment and transcription of the genes. Cas-Interacting-Zinc finger protein, CIZ, posses these two properties as it is localized at the site of focal adhesion plaque and it could trans locate into nuclei. B16, a malignant melanoma cell, are highly metastatic tumor. Therefore, we examined the possible roles of CIZ for the establishment of tumor metastasis of bone. In B16 cells, CIZ protein was expressed and localized in nuclei and cytosol. Cell migration to wild-type (WT) serum was inhibited by knockdown of CIZ, suggesting that CIZ would be one of factor for cell movement in B16. Treatment of BMP, which is involved in both cancer promotion and inhibition, suppressed migration of B16 to WT serum. Treatment with BMP suppressed CIZ expression in B16, suggesting that down regulation of CIZ by BMP may be involved in this cell migration process CIZ knock down revealed that CIZ interferes with the increase of the transcription through BMP response element by BMP treatment in Luciferase assay. Taken together, these results suggest that CIZ and BMP antagonize each other. In the host side, to examine the difference of local proliferation in bone microenvironment between WT and CIZ KO mice, we inoculated B16 into bone. After 5 weeks, CIZ KO mice suppressed cancer cell proliferation. To investigate whether CIZ-deficient microenvironment also affects cancer cell migration, migration assay was performed. B16 migrated to the side of medium containing the serum derived from WT mice, whereas migration of B16 was suppressed when serum from CIZ KO mice was used. And the size of cells that barely migrated to CIZ KO serum was significantly small compared to WT serum. These results suggest that certain factors under the control of CIZ are required for regulation of migration and morphology of B16. To investigate the molecular mechanism underlying this migration defects in CIZ KO serum, we focused on Cxcl12 involved in tumor growth and metastasis. We analyzed expression level of Cxcl12 in bone marrow, but there is no change between WT and CIZ KO mice. These results suggest that CIZ regulates tumor growth and progression in both sides of the cancer cell and host microenvironment. Breakdown of the balance between BMP and CIZ may be involved in tumor progression.

Disclosures: Tomomi Sakuma, None.


Leukemia Blasts Compromise Osteoblast Function in a Mouse Model of Acute Myelogenous Leukemia.Barbara Silva*1, Yoshihiro Yoshikawa2, Charles Duncan2, Linda Johnson3, John Manavalan2, Ellin Berman4, Stavroula Kousteni1. 1Columbia University Medical Center, USA, 2College of Physicians & Surgeons, Columbia University, USA, 3Cornell University & Memorial Sloan-Kettering Cancer Center, USA, 4Memorial Sloan-Kettering Cancer Center, USA

The bone marrow niche has been implicated in the pathogenesis of leukemia, as a permissive microenvironment required for its emergence or progression. Yet, few studies have shown how the malignant cells influence specific cell populations within the bone niche or characterized signals that orchestrate their crosstalk. In this context, we aimed to define specific bone cell populations and signals that are involved in the changes occurring in the niche after the disease is established. We injected WEHI-3b murine myelomonocytic leukemia cells into immunocompetent Balb/C mice, and compared them to a control group that had not been injected with leukemia cells. Fourteen days later, tumor engraftment was present in both femur and spine of the leukemic mice as observed by histopathological analysis. Leukemic mice showed a marked, 68%, and significant decrease in osteoblast numbers, 50% decrease in bone formation rate, and a less pronounced, 14%, but also significant, trabecular bone loss. Osteoclast numbers were not affected. The decrease in osteoblast numbers appeared to be caused by a decrease in differentiation as evidenced by reduced expression of osteoclacin, Runx2, osterix and alkaline phosphatase. A specific subset of non-hematopoietic (CD45- and CD34- negative) bone marrow cells expressing the endothelial cell marker CD105 were affected, as determined by flow cytometry. This was evidenced by a negative correlation between the tumor burden and the CD45-/CD34-/CD105+ subset of bone marrow cells. wInterestingly, gene expression studies and mineralization assays showed that this particular population of CD45-/CD34- cells has the highest osteogenic potential. Additionally, osteoblastic expression of OPG was decreased, whereas RANKL was increased, leading to a marked increase in the RANKL:OPG ratio, probably explaining the maintenance of osteoclast numbers in the presence of reduced osteoblast numbers. Expression of SDF-1, a gene implicated in normal hematopoietic cell homing in the bone marrow niche, was reduced in osteoblasts, which could contribute to derangements in normal hematopoiesis during the course of the disease. In summary, 1) osteoblastogenesis was decreased, 2) osteoblast- osteoclast uncoupling was noted, and 3) a CD45-/CD34-/CD105+ subset of bone marrow cells was identified as the osteogenic population affected by leukemic engraftment. These data demonstrate that leukemia blasts influence osteoblast growth and functional characteristics.

Disclosures: Barbara Silva, None.


Targeting Wnt or BMP Pathways Differentially Modulates Osteoclastinducing Activity of Giant Cell Tumor Stromal Cells: Biologic and Therapeutic Implications.Matthew Steensma*1, Wakenda Tyler2, John Healey3, F. Patrick Ross4, Steven Goldring4, Ed Purdue4. 1Memorial Sloan-Kettering Cancer Center/Hospital For Special Surgery, USA, 2Rochester University, USA, 3Memorial Sloan-Kettering Cancer Center, USA, 4Hospital for Special Surgery, USA

Giant Cell Tumor of Bone (GCT) is an osteolytic tumor comprised of three cell populations: fibroblast-like stromal cells (SC); myeloid mononuclear cells; and multi-nucleated osteoclast (OC)-like giant cells. The SC is thought to represent the neoplastic element based on a sustained, high proliferation rate in culture and the presence of cytogenetic abnormalities. SC's express genetic markers consistent with an arrest in an early stage of osteoblast (OB) differentiation that is associated with high expression levels of the OC-inducing factor, RANKL. We aimed to test the hypothesis that SC's could be reprogrammed to undergo OB differentiation by targeting the Wnt or BMP pathways resulting in modulation of key regulators of OCgenesis, including osteoprotegerin (OPG). 6 GCT specimens were obtained in accordance with an approved IRB protocol. SC's were isolated from collagenase-dispersed tumor cells using negative selection with a CD14 magnetic bead column. Purity was assessed based on morphology, the absence of CD14 expression (FACS and IF microscopy), and the absence of OC-specific genes, including cathepsin K. SC's were cultured for 10-12 d in the presence or absence of BMP-2, SB415286(a specific GSK-3beta inhibitor and Wnt mimetic), or osteogenic medium (OGM). Differential expression of OB differentiation markers was assessed by qPCR. OPG production was determined by ELISA. The OCgenic activity of SC's was assessed using a human monocyte co-culture system. OC formation was quantified by TRAP stain cell counts done in triplicate. SC's exposed to BMP-2, SB415286, or OGM demonstrated >2-fold induction of OB-associated genes, including Runx2 and osterix. Wnt pathway activation or growth in OGM was accompanied by a marked increase in the OPG/RANKL ratio and loss of SC OC-inducing activity. Conversely, treatment with rhBMP-2 resulted in a decreased OPG/RANKL ratio, and enhanced OC formation. The capacity of Wnt and BMP pathway activation and OGM to induce SC's to undergo OB differentiation provides further evidence of their OB lineage. Surprisingly, the induction of OB differentiation by BMP or Wnt pathway activation produced differential effects on OC-inducing activity. This effect may be attributed to the differential effects of the Wnt and BMP pathways on OPG/RANKL production. The ability to manipulate the OCgenic phenotype of SC's provides a potential novel approach for treatment and insight into the mechanisms by which OB lineage cells regulate OCgenesis.

Disclosures: Matthew Steensma, None.


Unraveling the Molecular Connections between Anti-Cancer and Anti-Osteoporosis Drugs Using the Connectivity Map.Jameel Iqbal*1, Xuan Liu1, Ling-Ling Zhu1, Jianhua Li2, Yuanzhen Peng3, Zuhong Lu4, Guangyu Zhu1, Alberta Zallone5, Li Sun6, Mone Zaidi7. 1The Mount Sinai Bone Program & Department of Medicine, Mount Sinai School of Medicine, USA, 2Tount Sinai School of Medicine, USA, 3The Mount Sinai School of Medicine, USA, 4State Key Laboratory of Bioelectronics, Southeast University, Nanjing China, China, 5University of Bari Medical School, Italy, 6Mount Sinai School of Medicine, USA, 7Mount Sinai Medical Center, USA

Cancer is the second leading cause of death, and osteoporosis the leading cause of morbidity in the aging population. There is mounting evidence that bisphosphonates, the mainstay of osteoporosis therapy, have anti-carcinogenic properties. While it is known that bisphosphonates prevent bone metastases for breast, prostate and lung cancers, recent evidence suggests that these drugs can prevent the growth of breast cancer lesions. We therefore investigated the molecular mechanisms underlying the anti-resorptive and putative anti-carcinogenic actions of bisphosphonates. Human osteoclasts were treated with alendronate or risedronate to generate a unique bisphosphonate gene signature by combining affymetrix datasets from the respective drugs. We then interrogated the Connectivity Map (CMAP, Broad Institute), a powerful genetic database comprising gene expression profiles that define functional connections between drug actions, genetic perturbations, and disease states. Non-parametric, rank-based pattern-matching using the Kolmogorov-Smirnov statistic examined shared mechanisms of action to reveal both mimics and anti-mimics of the bisphosphonates. The bisphosphonate gene signature identified two anti-cancer drugs, the EGF signaling inhibitor tyrphostin AG1478 and the PARP inhibitor 1,5-isoquinolinediol. Both these anti-cancer drugs displayed potent anti-resorptive properties in vitro identical to bisphosphonates, thus attesting to their use as potential candidates for osteoporosis therapy. Given the molecular connectivity of bisphosphonates to EGFR inhibitors, we tested the effects of alendronate and risedronate on the proliferation of lung cancer cells harboring EGF receptor mutations. Both bisphosphonates potently inhibited cell proliferation. Because breast, lung, and prostate cancers are all known to have aberrant EGFR signaling, often with a dependence on Akt, we tested whether the bisphosphonates could inhibit EGF-induced Akt activation. We found that both alendronate and risedronate suppressed Akt phosphorylation: this represents a novel mechanism for bisphosphonate action that may be utilized for inhibiting cancer growth, especially for cancers with known EGFR/Akt mutations. In conclusion, CMAP could be a powerful new alternative to drug discovery, particularly as it unravels new actions of ‘old’ drugs. Its use could conceivably reduce the often prohibitive costs and lengthy time intervals that underpin modern drug development.

Disclosures: Jameel Iqbal, None.


Zoledronic Acid Prolongs Time to First Skeletal-Related Event (SRE), Progression-Free Survival (PFS), and Overall Survival (OS) Versus Clodronate in Patients With Newly Diagnosed Multiple Myeloma (MM): Results of the Medical Research Council (MRC) Myeloma IX Trial.W Gregory1, A. Szubert1, R. Owen2, J. Child1, A. Ashcroft3, M. Drayson4, N Navarro Coy1, Faith Davies5, Graham Jackson6, K Cocks1, Gareth Morgan*7, S. Bell1. 1University of Leeds, United Kingdom, 2St. James's University Hospital, United Kingdom, 3Mid-Yorkshire Hospitals NHS Trust, United Kingdom, 4University of Birmingham, United Kingdom, 5Institute of Cancer Research, United Kingdom, 6University of Newcastle, United Kingdom, 7Royal Marsden Hospital, United Kingdom

Purpose: Myeloma cells grow within the bone marrow, and patients (pts) with MM are at high risk for SREs such as pathologic fracture or spinal cord compression. In addition to prolonging OS, an important goal is preventing these potentially debilitating SREs. Bisphosphonates (BPs) are standards of care for reducing the risk of SREs in pts with bone metastases from solid tumors or bone lesions from MM. In addition, several BPs have demonstrated anticancer activity in preclinical models. In particular, zoledronic acid (ZOL) has shown anticancer activity in preclinical models and clinical studies in various cancer types, including MM. In pts with early breast cancer, adding ZOL improved disease-free survival by 36% (P = .01) vs endocrine therapy alone (Gnant et al. N Engl J Med. 2009).

Methods: In the MRC Myeloma IX trial (ISRCTN68454111), pts with newly diagnosed MM were randomized to IV ZOL (4 mg q 21-28 days) or oral clodronate (CLO; 1,600 mg/d) plus antimyeloma therapy. Treatment continued at least until disease progression. All pts provided written informed consent, were evaluated for disease outcomes and safety, and the study was approved by institutional review boards. OS, PFS, and SREs were evaluated in adjusted Cox models. OS was also evaluated in an exploratory Cox model adjusted for time-dependent SRE effects.

Results: A total of 1,960 pts were evaluable, with median follow-up of 3.7 yr. In the ZOL and CLO groups, mean age was 65 yr, IgG was the predominant subtype, and ∼70% of pts had bone lesions. Overall, ZOL significantly reduced the risk of first SRE by 26% vs CLO (hazard ratio = 0.74; 95% CI: 0.62, 0.87; P = .0004). Pts treated with ZOL had a significant 2-mo increase in PFS (19.5 vs 17.5 mo, respectively; P = .0179) and a 5.5-mo increase in OS (50 vs 44.5 mo, respectively; P = .0118) vs CLO. Moreover, ZOL's survival benefit remained statistically significant after adjusting for potential effects of SREs on OS (P = .0178). Both BPs were generally well tolerated. There was no significant between-group difference in the rate of acute renal failure (ZOL, 6.1%; CLO, 5.8%), and the incidence of confirmed osteonecrosis of the jaw was low (ZOL, 3.5%; CLO, 0.3%).

Conclusions: The results of the MM IX Trial demonstrate a beneficial effect of ZOL on OS and PFS, and superior SRE prevention vs CLO, in pts with MM. Moreover, ZOL OS benefits remained significant after readjustment for SREs, supporting potential anticancer activity.

Disclosures: Gareth Morgan, None.

This study received funding from: Novartis, Boehringer Ingelheim, Pharmion, and Celgene for the clinical trial.


See Friday Plenary number FR0146.


Coexpressions of FGF-23 and MEPE in Causative Tumors of Oncogenic Osteomalacia.Yuki Nagata*1, Yasuo Imanishi1, Jun Hashimoto2, Wataru Ando2, Keisuke Kobayashi1, Takafumi Ueda3, Akimitsu Miyauchi4, Hajime Koyano5, Hiroshi Kaji6, Takatoshi Saito7, Koichi Oba8, Yasato Komatsu9, Hitoshi Goto1, Takami Miki10, Masaaki Inaba1, Yoshiki Nishizawa10. 1Osaka City University Graduate School of Medicine, Japan, 2Osaka University Graduate School of Medicine, Japan, 3Osaka National Hospital, Japan, 4Omura municipal Hospital, Japan, 5Juntendo University, School of Medicine, Japan, 6Kobe University, Japan, 7Jikei University School of Medicine, Japan, 8Graduate School of Medical Sciences, Kyushu University, Japan, 9Kyoto City Hospital, Japan, 10Osaka City University Medical School, Japan

Purpose Oncogenic osteomalacia (OOM) is a rare disease characterized by renal phosphate wasting and osteomalacia, and is caused by the secretion of fibroblast growth factor 23 (FGF-23) from causative tumors. Matrix extracellular phosphogly-coprotein (MEPE) as well as its proteolytic ASARM peptide elevate FGF-23 expression in bone. Although the expression of MEPE was reported in some OOM tumors, little is known whether MEPE contributes to the FGF-23 expression in OOM tumors. In this study, we attempt to determine the expressions of FGF-23 and MEPE in OOM tumors.

Subjects and Methods Eleven causative OOM tumors were examined for expressions of FGF-23 and MEPE by quantitative real-time RT-PCR and immunohistochemistry. Two hemangiopericytomas and 4 giant cell tumors, obtained from non-osteomalacic patients, were used as controls, which pathological diagnoses are common among OOM.

Results The expression levels of FGF-23 in OOM tumors were 105 times higher than those in non-OOM tumors. The expression levels of MEPE in OOM tumors were also 105 times higher than those in non-OOM tumors. Immunohistochemical examinations revealed the presence of FGF-23 in all OOM tumors, and that of MEPE in 10 of 11 OOM tumors.

Conclusions The coexpressions of FGF-23 and MEPE in OOM tumors suggest that MEPE or its proteolytic ASARM peptide may contribute to the FGF-23 overexpression in OOM tumors.

Disclosures: Yuki Nagata, None.


Combined Effects Of Exercise And Alcohol On Bone Status In Rats.Delphine Maurel*1, Christelle Jaffre2, Eric Dolleans2, Nathalie Boisseau3, Claude-Laurent Benhamou2. 1Inserm Unit 658, Hopital Porte Madeleine, Orleans, France, 2Inserm Unit U658, France, 3LAPHAP Laboratory, France

Chronic alcoholism is known to alter bone balance by a decrease of bone remodeling, bone mineral density (BMD), bone mineral content (BMC) and bone resistance in rats and in men. Nowadays most of the treatments to manage bone loss are drug treatments. However, positive effects of exercise on bone are well known. To date and to our knowledge, no study has been driven to determine the possible beneficial effects of regular exercise on bone loss induced by chronic alcohol consumption. To address this question, we designed a study in rats to determine the effects of combined alcohol and exercise on bone.

Thirty-six 2-month old male Wistar rats were divided in 3 groups: Control (C), Alcohol (10 g/kg) (A) and Alcohol+Exercise (AE). The rats were alcoholised for 17 weeks, trained for 14 weeks and then sacrificed. BMC and BMD were assessed by DXA (Hologic, Discovery) and the micro architecture of cortical and trabecular bone has been measured by microCT (Skyscan 1072).

Ethanol consumption decreased significantly the BMC (19.68 ± 0.98; 15.61 ± 1.06 and 16.67 ± 1.73 g respectively for C, A and AE; p<0.0001) and BMD (0.205 ± 0.006; 0.189 ± 0.006 and 0.196 ± 0.008 g/cm2; p=0.0005) of the total body and right femur (BMC: 0.88 ± 0.07; 0.70 ± 0.05 and 0.73 ± 0.08 g for C, A, and AE; p<0.0001; BMD: 0.389 ± 0.020; 0.337 ± 0.019 and 0.359 ± 0.019 g/ cm2 for C, A and AE; p<0.0001) compared to Controls. The BV/TV parameter (15.61 ± 5.49; 11.28 ± 3.05 and 16.43 ± 3.86% for C, A, and AE; p=0.02), the trabecular thickness (0.096 ± 0.012; 0.084 ± 0.007 and 0.101 ± 0.009 mm for C, A, and AE; p=0.0002) and the trabecular number (1.61 ± 0.44; 1.34 ± 0.29 and 1.62 ± 0.31 1/mm for C, A and AE; p=0.01) were also decreased by alcohol compared to Controls while the cortical porosity (1.01 ± 0.27; 1.39 ± 0.33 and 1.06 ± 0.31% for C, A and AE; p= 0.015) and pore number (0.24 ± 0.05; 0.34 ± 0.08 and 0.25 ± 0.06 1/μm for C, A and AE; p=0.001) increased.

The Alcohol+Exercise group had significantly higher femoral BMD, BV/TV, and trabecular thickness compared to A group as well as better cortical porosity and pore number. The maximal strain supported by the bone was also significantly higher in the AE group compared to the A group (182.73 ± 35.10 vs 144.65 ± 18.02 N/mm2 for AE and A; p=0.017).

In conclusion, regular exercise partly prevents the bone loss and deterioration of the trabecular, cortical microarchitecture and BMD induced by chronic alcoholism.

Disclosures: Delphine Maurel, None.


Gene by Diet Interactions in the Alox5 Knockout Mice (Alox5-/-) Is Associated with Significant Bone Loss.Phuong Le*1, Masanobu Kawai2, Sheila Bornstein2, Mark Horowitz3, Clifford Rosen2. 1Maine Medical Center Research Institute, USA, 2Maine Medical Center, USA, 3Yale University School of Medicine, USA

Lipoxygenase 5 encoded by Alox5 gene catalyzes the generation of leukotriene B4 from arachidonic acid, and the loss of Alox5 has been postulated to result in the increased enzymatic conversion of prostaglandin (PG) D2 into PGJ2, an endogenous ligand for PPARγ. Polymorphisms in the Alox5 gene and its associated protein have been linked to cardiovascular disease; intriguingly, Alox5 is also a candidate gene regulating peak bone mass in mice. These findings led us to hypothesize that a high fat diet (HFD) in association with deletion of the Alox5 gene could result in a major bone loss in part through activating PPARy expression. To test our hypothesis, we fed 3 week old male C57BL/6J (B6, control) and Alox5-/- mice either a regular chow diet (10% fat/kcal) or a HFD (45% fat/kcal) for 13 weeks post weaning. We then examined skeletal phenotype by DXA, μCT, serum bone turnover markers, histomorphometry, and cell culture. We also examined PPARγ gene expression in 8-12 week old mice by qPCR. After 13 weeks of treatment, whole body and femoral areal bone mineral density (BMD) were significantly higher in Alox5-/- than in B6 (p<0.01). Similar phenotypic differences were noted by μCT in the femur. Alox5-/- mice had higher serum osteocalcin and PINP levels (p<0.001) than B6. In vitro bone marrow stromal cell cultures from Alox5-/- mice demonstrated greater ALP and von Kossa staining positive colonies compared to cells from B6. Osteoclast number was decreased by histomorphometry in Alox5-/- vs. B6 (p=0.06). On the HFD, there were marked strain differences such that B6 showed increased bone mass in the spine and femur. In contrast, Alox5-/- mice on a HFD had lower whole body BMD, femoral and vertebral trabecular BV/TV than Alox5-/- mice on a regular diet, which was accompanied by reduced trabecular number and increased osteoclast number. Serum Trap5b in the Alox5-/- mice was greater on the HFD while P1NP decreased significantly (p=0.04) compared to mice on a regular diet. qPCR results showed marked increase of PPARγ levels in femur from Alox5-/- vs. B6 on HFD. In summary, PPARγ gene associated with diet interactions in Alox5-/- mice causes significant bone loss by suppressed bone formation and increased bone resorption. This line of evidence may provide an insight into the important interaction between dietary fat intake and genetic predisposition to osteoporosis.

Disclosures: Phuong Le, None.


Potent Inhibition of Heterotopic Ossification by a Selective RARy Agonist.Kengo Shimono*, Wei-en Tung, Hsu-Tsai Chi, Chrissy Mundy, Johanna Jasinski, Daine Pilchak, Julie Williams, Motomi Enomoto-Iwamoto, Maurizio Pacifici, Masahiro Iwamoto. Thomas Jefferson University, USA

Heterotopic ossification (HO) consists of formation of ectopic bone masses following trauma and involves recruitment of progenitor cells, differentiation into chondrocytes and replacement by endochondral bone. Available HO treatments such as irradiation are not fully effective and do not target any of the above HO-formation steps. Differentiation of mesenchymal cells into chondrocytes requires decreases in both retinoic acid (RA) signaling and nuclear retinoic acid receptor α (RARα) action. Indeed, we previously showed that treatment with a synthetic RARα agonist inhibits HO formation in a mouse model. Here, we asked whether another family member -RARγ- represents a previously unsuspected and possibly more powerful regulator of chondrogenesis and HO. Micromass cultures of wild type E11.5 mouse embryo limb mesenchymal cells were treated with RA (that activates every RAR isoform) or a selective RARγ agonist (γ-agonist). Numerous cartilage nodules formed in untreated cultures, but few formed in RA-treated cultures and nearly none in γ-agonist-treated cultures. When the micromass cultures were prepared with double RARα/RARβ-null limb bud cells, the same results were obtained. However, when RARγ-null cells were used, inhibition of cartilage nodule formation by RA was only about 25-30%, strongly indicating that RARγ is the major anti-chondrogenic regulator. Next, we tested whether the γ-agonist acts via a blockage of the key pro-chondrogenic BMP/Smad signaling pathway. It did but, surprisingly, it not only inhibited Smad1 nuclear translocation, but also caused rapid Smad1 degradation via proteasome action. To determine γ-agonist effectiveness in vivo, we induced HO formation by implantation of BMP-2-loaded collagen sponges in mouse calf muscles. Large ectopic HO-like cartilaginous masses formed in control mice and were replaced by bone by day 14. However, cartilage and bone formation was nearly prevented in mice receiving γ-agonist (0.4 to 4.0 mg/kg/day) by gavage; inhibition was about 95% measured by BV/TV (p<0.01). There were no appreciable γ-agonist side effects in terms of body weight, liver and kidney function and articular cartilage phenotype. As expected, we observed partial HO inhibition in mice receiving RA (12 mg/kg/day), but this treatment had side effects including severe skin and articular cartilage damage. In sum, our study shows that selective pharmacologic activation of RARγ represents a novel, powerful and seemingly safe treatment against HO.

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Suppression of Heterotopic Ossification By Selective RARγ Agonist

Suppression of Heterotopic Ossification By Selective RARg Agonist

Disclosures: Kengo Shimono, None.

This study received funding from: NIH, US Army


Studies of Type I Collagen Mutations in Type I and IV Osteogenesis Imperfecta (OI) Patients Induced Pluripotent Stem (iPS) Cells.Xiaonan Xin*, Mary Louise Stover, Dr. Yongxing Liu, Dr. Liisa Kuhn, David Rowe, Dr. Alexander Lichtler. University of Connecticut Health Center, USA

Osteogenesis imperfecta (OI) is a heritable bone disease caused by mutations in the type I collagen genes. Severe have defective collagen fibers with inherently unstable structures causing bone fragility. Previously we reported initial derivation of iPS cells from patients with severe and mild IO, with the long term goals of investigating OI pathogenic mechanisms, and testing therapeutic strategies. In the current studies we have further characterized the cells we produced, and we are in the process of investigating the mutant collagen that they produce. The mutations in the two cell lines have been identified; as expected, the Type I OI cells contain a premature termination codon that leads to mRNA instability, and the Type III/IV cells have an alanine substituted for the glycine at amino acid position 491. RNA analysis using an ABI pluripotency low density array PCR plate showed a similar gene expression profile of the iPS cells to H9 hES cells. With the goal of identifying defined, nonhuman factor-free culture conditions for the iPS cells, we have showed that our cells have similar gene expression profiles when cultured in Stemgent NutriStem media compared to standard mouse embryo fibroblast conditioned media. We have developed a method for differentiating our iPS cells into a mesenchymal stem cell-like population that is over 95% CD73+ and CD44+, and negative for hematopoietic cell markers. Low density array PCR analysis demonstrated expression of mesodermal markers in the differentiated cells, and Type I collagen mRNA is also expressed. We are currently assessing the ability of the iPS cells to produce teratomas that contain bone, and we will analyze the mutant collagen produced by the differentiated cells. We are also working to develop methods to induce differentiation of these cells into osteoblasts, and also to test the zinc-finger methodology for mutation correction.

Disclosures: Xiaonan Xin, None.


Suppression of Bone Quality by Diet-induced Obesity Correlates with Increase in Insulin Resistance and Suppression of Immune Cells.Ete M. Chan*1, Elizabeth M. Fievisohn1, Ada H. Tsoi1, Mario E. Botros1, Benjamin Adler2, Danielle E. Green1, Clinton Rubin3. 1Department of Biomedical Engineering, Stony Brook University, USA, 2Stony Brook University, USA, 3State University of New York at Stony Brook, USA

Obesity is a serious condition leading to diseases such as Type II diabetes (T2D) which is known to affect bone quality, but the underlying mechanisms of this relationship are still unclear. To investigate the effect of obesity on bone quality, twenty 7w male C57BL/6J mice were randomly assigned to either a high fat (HF) diet (45% kcal) or a regular chow diet group (RD) (n=10) for ∼6 months following which the trabecular bone compartment in the proximal tibia was evaluated using micro-computed tomography (microCT). Furthermore, to assess characteristics of T2D and any downstream effects resulting from obesity, 1) abdominal adipose tissue load was evaluated with microCT, 2) glucose tolerance test (GTT) was performed, 3) levels of fasting insulin and fasting glucose in blood were measured and 4) proportional changes in cells from myeloid and lymphoid lineages within bone marrow and blood (subgroup, n=4) were evaluated using flow cytometry. The HF diet treatment induced significant changes in the trabecular bone compartment in the HF group (Fig 1): As compared to the RD group, the HF group has 11% higher trabecular thickness Tb.Th (p=.026), but 24% lower trabecular number Tb.N (p=.023) and 39% larger trabecular separation Tb.Sp (p=.05). Accompanying changes in trabecular bone are increases in body mass (42%, p<.001), adipose tissue volume (56%, p<.001) and fasting insulin (354%, p=.03), indicating the prominent role of obesity in inducing insulin resistance. While body mass is positively correlated to the Tb.Th (r=.63, p=.009) and negatively correlated to the Tb.N (r=-.54, p=.032), glucose intolerance as measured by GTT is also negatively correlated to Tb.N (r=-.61, p=.012) and positively correlated to Tb.Sp (r=.60, p=.014). This indicates that glucose intolerance could contribute to the compromised bone quality. Interestingly, the HF group showed a significant reduction in the B cell proportion both in bone marrow (66.9%, p=.011) and blood (36.5%, p=.045) indicating an impairment of hematopoiesis in the bone marrow. We conclude that obesity upregulated T2D and compromised the bone marrow cells, disrupting the ability of the bone to adapt to the increased load bearing challenges.

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Fig 1

Disclosures: Ete M. Chan, None.


See Friday Plenary number FR0153.


See Friday Plenary number FR0154.


See Friday Plenary number FR0155.


Genetic Variation in the LRP4 Gene Influences Bone Mineral Density and Hip Geometry while Missense Mutations Cause Sclerosteosis.Elke Piters*1, Eveline Boudin2, Olivier Leupin3, Fenna de Freitas2, Karen Jennes2, Manuel Bueno-Lozano4, Feliciano J Ramos-Fuentes4, Peter H Itin5, Torben Leo Nielsen6, Marianne Andersen6, Kristian Wraae6, Kim Brixen7, Michaela Kneissel3, Wim Van Hul1. 1University of Antwerp, Belgium, 2Department of Medical Genetics, University of Antwerp, Belgium, 3Novartis Institutes for Biomedical Research, Switzerland, 4Departmento de Pediatría, Universitad de Zaragoza, Spain, 5Department of Dermatology, University Hospital Basel, Switzerland, 6Department of Endocrinology, Odense University Hospital, Denmark, 7Institute for Clinical Research, Denmark

Some of us have recently reported a role for low density lipoprotein-related protein (LRP)-4 in binding to and facilitating sclerostin in its inhibition of both Wnt signaling and bone mineralization (1). In the scope of these findings, we performed mutation analysis of an extensive set of patients with different high bone mass disorders and additionally carried out an association study with a selection of LRP4 polymorphisms in a population-based cohort from the Odense Androgen Study. Mutation analysis revealed the existence of two missense mutations in the LRP4 gene in two unrelated patients of Mediterranean origin that both showed several typical sclerosteosis features such as an increased bone mineral density (BMD), facial asymmetry and syndactyly. The first patient was homozygous for a p.Arg1170Trp mutation while the other showed a heterozygous p.Trp1186Ser substitution. Both the mutations were absent in a large control set of mixed European origin and completely blocked the interaction with sclerostin and the concomitant sclerostin-enhancer action. The mutations occur adjacent to each other at the surface of the third extracellular β-propeller, which corresponds with the assumption that this domain is involved in sclerostin interaction and function. In another effort to implicate LRP4 in human bone disease, we studied 2 non-synonymous coding polymorphisms in the region encoding the third extracellular β-propeller. BMD measurements of the hip and the spine as well as different hip geometry parameters were available for a total of 1404 Danish men from two age groups ([20-29 y]: n= 804; [60-74 y]: n= 600). Using linear regression analysis adjusted for age, height and weight, we were able to replicate previous associations (2, 3) between rs6485702 and BMD at all sites. The most significant association was found with whole body BMD (p= 4.7*10-5). In addition, we found this SNP to be associated with different hip geometry parameters suggesting that LRP4 is able to regulate bone structure in addition to BMD. However, another SNP in the same region (rs2306033) did not show association with any of the studied phenotypes. Ongoing genetic and functional studies with additional SNPs will aid in delineating the region of LRP4 that partly explains the natural variance in BMD. In conclusion, genetic variation in the LRP4 gene determines BMD both in human bone disease and in the general population. This again illustrates the importance of LRP4 in bone homeostasis.

(1) Leupin et al, J. Bone Miner. Res. 24 (S1): 1250, 2009.

(2) Styrkarsdottir et al, N. Engl. J. Med. 358: 2355, 2008.

(3) Styrkarsdottir et al, Nat. Genet. 41: 15, 2009.

Disclosures: Elke Piters, None.


Low Bone Mass and Turnover Phenotype in a Mouse Model of Down Syndrome.Tristan Fowler*, John W. Bracey, Nisreen Akel, Robert A. Skinner, William R. Hogue, Frances L. Swain, Dana Gaddy, Kent D. McKelvey, Galen R. Wenger, Larry Suva. University of Arkansas for Medical Sciences, USA

Down syndrome (DS) is a human aneuploidy resulting from the presence of a third chromosome 21. It occurs in the human population at a rate of approximately 1 per 1,000 live births in all ethnic groups. This condition is associated with a number of physical characteristics and clinical conditions including a degree of mental retardation, thrombocytosis, and a 500-fold increased risk of developing megakaryocytic leukemia. Recently, DS has also been associated clinically with a dramatically increased rate of fracture in adolescent and adult patients. Since individuals with DS are reported to have a survival rate of >50% to age 50, skeletal complications should be considered in these patients. However, the specific effects of trisomy 21 on the skeleton remain poorly defined. To study this question, we determined the skeletal phenotype of the Ts65Dn mouse model of DS. Ts65Dn mice are trisomic for 104 orthologs of human Chr 21 (HSA21) genes and this model is the most widely used and accepted mouse model for DS. The mice are characterized by segmental trisomy for the region of mouse Chr 16 (MMU16) that extends from just proximal to the amyloid precursor protein (App) gene to the distal telomere and contains about 75% of the HSA21-homologous genes present in the mouse. We have discovered that adult Ts65Dn mice display a profound low bone mass phenotype, with significant decreases in bone volume fraction of the tibia and femur. Interestingly, Tb.N. was dramatically decreased whereas Tb.Sp. and Tb.Th are increased compared with euploid control littermates. The low bone mass in Ts65Dn mice is associated with a dramatic reduction in bone biochemical markers, with an approximately 50% decrease in serum P1NP and TRAP5b levels, indicative of a mechanism for low bone mass involving decreased bone turnover. In addition, the effects of trisomy 21 are not limited to trabecular bone, as the cortical geometric parameters of cortical cross sectional area, cortical thickness, total cross sectional area and periosteal perimeter are all significantly decreased in Ts65Dn mice. These dramatic changes in cortical bone were translated into significant decreases in mechanical strength and stiffness of the Ts65Dn femur. Our studies demonstrate the potential of DS mouse models to improve our understanding of chromosome 21 gene dosage effects in the skeleton and provide novel insight into the causes of the clinical observations of the extraordinary bone fragility in Down's patients.

Disclosures: Tristan Fowler, None.


Low Bone Mass in Genetic Hypercalciuric Stone-Forming (GHS) Rats is Associated with Higher RANKL Expression in Bone Marrow Stromal Cells.Hongwei Wang*1, Randal Zhou1, David Bushinsky2, Murray Favus1. 1University of Chicago, USA, 2University of Rochester, USA

GHS rats are a useful model to study human idiopathic hypercalciuria (IH) as both share increased intestinal Ca absorption and bone resorption, decreased renal Ca reabsorption, low bone mass, and Ca nephrolithiasis. We investigated low bone mass in male and female GHS rats and normocalciuric (NC) controls. Rats were fed a diet adequate in Ca (0.8%) diet. MicroCT scanning showed reduced tibial bone density (BD) in GHS vs NC rats. Microarray analysis of GHS rat bone marrow stromal cells (BMSCs) showed reduced expression of genes that enhance bone formation (BMP2, SPP1 and SOST) while gene expressions that stimulate bone resorption (RANKL, RANK, GM-CSF, TNFa and IL1) were increased. Real time PCR confirmed the microarray results. The numbers of osteoclasts (OCs) in primary cultures of GHS rat bone marrow were increased 2-3 fold. GHS rat OCs were larger by volume, and the BMSCs contained greater RANKL expression by immunostaining. In conclusion, the data strongly suggest that GHS rats have low bone mass due to greater expression of genes of the resorptive stimulatory pathway and suppression of genes that modulate the bone formation pathway. The molecular basis for the dysregulated resorption and formation genes remains to be determined.

Disclosures: Hongwei Wang, None.


Mandible Length Quantitative Trait Loci (QTLs) in HcB-8 × HcB-23 Intercross.In Kyu Han1, Gurpreet Sandhu1, Suzanne J. Litscher1, Neema Saless2, Peter Demant3, Robert Blank*1. 1University of Wisconsin, USA, 2University of California, San Francisco, USA, 3Roswell Park Cancer Institute, USA

We previously mapped quantitative trait loci (QTLs) for femoral biomechanical performance and anatomy in a reciprocal intercross of HcB-8 and HcB-23. In that work, we determined that a robust QTL on chromosome 4 acts by altering the proliferative response to mechanical loading and that the additional affected phenotypes are secondary consequences of the modeling difference. Further, we inferred that the gene underlying the chromosome 4 QTL is Ece1, encoding endothelin converting enzyme 1. To further test our model, we hypothesized that there would be a mandibular size phenotype linked to the chromosome 4 QTL, related to the insertion of the masseter muscle. Here we report mapping of QTLs for mandibular length in our cross.

We subdivided the mandible into 3 segments with boundaries defined by the mentum, the posterior margin of the 3rd molar, the coronoid process, and the angle. We measured the projected distance between the landmarks from digital photographs of the hemimandibles, averaging the left and right sides. We performed linkage analysis using R/qtl and QTL Cartographer. Experiment-wide α = 0.05 significance thresholds varied between 2.8 and 3.0.

The entire F2 progeny included 603 animals. QTLs for the mentum-molar segment are located at 67 cM on chromosome 1 with LOD = 3.8 and at 28 cM on chromosome 6 with LOD 7.5. The molar-coronoid segment has QTLs at 41 cM on chromosome 1 with LOD = 3.8 and at 67 cM on chromosome 4 with LOD = 5.7. No QTLs for coronoid-angle length were detected. There were no significant sex × QTL or cross x QTL interactions. Larger subsets of the mandible did not detect additional QTLs. The chromosome 6 QTL accounts for 8% of the mentum-molar length variance. The chromosome 4 QTL accounts for 7% of the F2 molar-coronoid length variance.

The data support our hypothesis that the chromosome 4 QTL mediates response to mechanical loading. There are no muscle of mastication insertions anterior to the molars, while the masseter inserts primarily in the region between the coronoid process and the 3rd molar. The proximal chromosome 1 QTL may also act via modeling. The chromosome 6 QTL for the anterior jaw is distinct from the male-specific QTL we identified for femoral size and mechanical performance. To the best of our knowledge, this is the first instance in which a skeletal QTL's existence has been predicted on the basis of the affected phenotypes at a remote anatomical site.

Disclosures: Robert Blank, None.


Modelling Bone Healing Deficiencies in Type 1 Neurofibromatosis.David Little1, Aaron Schindeler1, Ian Alexander2, Jad El-Hoss*1. 1The Children's Hospital at Westmead, Australia, 2The Children's Medical Research Institute, Australia

Congenital tibial dysplasia (CTD) involves bowing and weakness of the tibia that leads to fracture and poor subsequent bone healing. CTD and scoliosis are key diagnostic features of the autosomal dominant genetic condition Neurofibromatosis Type 1 (NF1). Based on some compelling new evidence that CTD is associated with local double inactivation of NF1, we are developing advanced murine systems to model the genetics of Nf1 inactivation in mice. We have previously shown deficient bone healing in the Nf1+/- mouse. We are now using a Cre-expressing adenovirus (Ad-Cre) to generate local double inactivation of Nf1 in Nf1flox/- and Nf1flox/flox mice. Briefly, Cre-expressing adenovirus will infect local cells and excise crucial exons of the Nf1 gene in Nf1flox/flox and Nf1flox/- mice. These mice were generated by crossing Nf1 null (Nf1+/-) and Nf1 conditional (Nf1flox/flox) strains. In the context of fracture healing we anticipate this will further impair union compared to the traditional Nf1+/- mouse. Cultured bone marrow cells from Nf1flox/+ mice were exposed to Ad-Cre and this led to efficient Nf1 excision as measured by PCR on genomic DNA. Nf1 gene excision was also detected by PCR in a tibial fracture model where the Ad-Cre was locally injected at the time of surgery. The Z/AP reporter mice were examined to histologically track the efficiency and distribution of Cre-mediated recombination. Alkaline phosphatase positive stain confirmed virus mediated recombination of cells lining the fracture site. In Nf1flox/flox mice, fractures treated with Ad-Cre displayed poor fracture healing compared to Ad-GFP treated controls. Ad-Cre treated fractured where characterised by a fibrocartilaginous plug that impaired further fracture healing. These results show that local knockout of Nf1 impairs fracture healing. Furthermore, we have generated a novel model for the local double inactivation of Nf1 in a fracture setting using the Ad-Cre virus system. This new model will prove beneficial to test novel therapeutics and better mirror the clinical condition.

Disclosures: Jad El-Hoss, None.

This study received funding from: National Health and Medical Research Council


See Friday Plenary number FR0161.


New Mouse Model for Type IV Osteogenesis Imperfecta - Cellular and Molecular Changes in Long Bone Precede Post-Pubertal Adaptation.Frieda Chen*1, Ruolin Guo2, Shousaku Ito3, Luisa Moreno4, Esther Rosenthal1, Tanya Zappitelli1, Ralph Zirngibl1, Ann Flenniken5, William Cole6, Marc Grynpas4, Lucy Osborne7, Wolfgang Vogel1, Lee Adamson4, Janet Rossant8, Jane Aubin9. 1University of Toronto, Canada, 2University of Rochester, USA, 3Osaka University Graduate School of Dentistry, Japan, 4Samuel Lunenfeld Research Institute, Canada, 5Toronto Centre for Phenogenomics, Canada, 6University of Alberta Hospital, Canada, 7Toronto General Research Institute, Canada, 8Hospital for Sick Children, Canada, 9University of Toronto Faculty of Medicine, Canada

Mutations in type I collagen or in proteins involved in its processing have been shown to cause the genetic bone disease Osteogenesis Imperfecta (OI). By a genome-wide N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a founder mouse with the classic clinical manifestations of OI. Mapping and sequencing identified a T to C transition in a splice donor of the collagen alpha1 type I (Col1a1) gene resulting in the skipping of exon 9 and an 18 amino acid deletion in the main triple helical domain of Col1a1. Col1a1Jrt/+ mice are smaller in size and have lower bone mineral density (BMD), decreased bone volume/tissue volume, reduced trabecular number and weaker, more brittle bones than their +/+ (wild type (WT)) littermates. Bone fractures occur at multiple sites at all ages, but with a high incidence of fractures of the pelvis and olecranon process of the ulna, in young Col1a1Jrt/+ mice. Biochemical analyses of matrices in dermal fibroblast cultures indicated that total collagen content of the Col1a1Jrt/+ matrices was ∼40% that of WT, with the content of type I collagen only ∼30% of that in WT cultures, suggesting that mutant collagen chains exert a dominant negative effect on type I collagen biosynthesis. Electron microscopic analysis of tendon and bone samples, as well as extracellular matrices deposited by dermal fibroblasts in vitro, indicated that collagen fibrils are markedly smaller in diameter in Col1a1Jrt/+ versus WT mice and cell cultures. In vitro analysis of stromal cells indicated that the size of the osteoprogenitor population is unaffected, but osteoblast differentiation as assessed by marker expression and mineralization are reduced in stromal cell cultures from age-matched young (5-week-old) versus older (20-week-old) Col1a1Jrt/+ versus WT mice. Expression of Col1a1 and Ocn mRNAs was increased in bones of 5 week-old Col1a1Jrt/+ versus WT mice, while that of Opn was decreased and Alp increased in 20 week animals. Thus, Col1a1Jrt/+ mice are a new model of dominantly-inherited type IV OI. Differences observed in the bone phenotype and osteoblasts at different ages suggest that the Col1a1Jrt/+ model will be useful to explore further the mechanisms by which OI bone adapts during growth and adulthood and other aspects of OI pathophysiology and treatment.

Disclosures: Frieda Chen, None.


Superoxide Dismutase Deficiency in Cytoplasm Exacerbated Bone Loss Under Reduced Mechanical Loading.Daichi Morikawa*1, Yoshitomo Saita2, Hidetoshi Nojiri2, Chizuru Tsuda1, Yoshinori Asou3, Kazuo Kaneko2, Takahiko Shimizu1. 1Molecular Gerontology, Tokyo Metropolitan Institute of Gerontology, Japan, 2Department of Orthopaedics, Juntendo Univ., Japan, 3Section of Regenerative Therapeutics for Spine & Spinal Cord, Tokyo Medical & Dental Univ., Japan

Cu/Zn-superoxide dismutase (Sod1) is one of the major antioxidant enzymes in mammals. Sod1-deficient (Sod1-/-) mice has been reported to present a variety of aging-like pathological changes (muscle atrophy, skin thinning, age-related macular degeneration). Last year, we have reported that Sod1-/- mice exhibit low turnover osteopenia via suppressed osteoblastic bone formation.

In order to clarify the cause of osteoblastic dysfunction, we compared gene expression profile between Sod1-/- and Sod1+/+ osteoblasts isolated from neonates using DNA microarray. This analysis identified the down-regulated genes involved in cell adhesion in Sod1-/- osteoblasts. Among them, we confirmed that osteomodulin (Omd), Comp, chadherin 11, and osteopontin were significantly decreased by realtime PCR analyses in Sod1-/- both in vitro (osteoblast) and in vivo (whole bone). These results suggest that Sod1 might be involved in cell adhesion and sensing of mechanical stress in osteoblast, and that oxidative stress could relate to mechanical loading in bone. Therefore, we performed hindlimb unloading (HU) in Sod1-/- mice.

Ten-weeks-old Sod1-/- and Sod1+/+ mice were conducted in HU for 14 days. Bone mineral density by DEXA was decreased by unloading in both Sod1-/- and Sod1+/+ mice compared to loading group. Interestingly, three dimensional micro CT showed that trabecular bone volume (BV/TV) was decreased more than twice in Sod1-/- (55% loss vs. loading) compared to Sod1+/+ (24% loss vs. loading), however, the decrease of cortical bone volume was not significantly different between Sod1-/- (16.3% loss vs. loading) and Sod1+/+ (15.8% loss vs. loading). In bone marrow cell culture after unloading, the development of TRAP positive osteoclast-like cells were increased in unloading group both in Sod1-/- and Sod1+/+ mice and it tended to be accelerated in Sod1-/- mice. Finally, we examined whether hindlimb unloading increased reactive oxygen species (ROS) in bone marrow cells. Interestingly, ROS production was significantly increased after unloading and it peaked one week after unloading, suggesting that reduced mechanical loading increases oxidative stress in bone marrow.

Our data revealed that cytoplasmic oxidative stress could exacerbate trabecular bone loss under reduced mechanical loading.

Disclosures: Daichi Morikawa, None.


Genome Wide Linkage of Osteocalcin to Chromosome 18 in Multi-generational Families of African Ancestry.Allison Kuipers*1, Caren Gundberg2, Amy Dressen3, Candace Kammerer1, Clareann H. Bunker3, Ashely Edwards3, Alan L. Patrick4, Victor W. Wheeler4, Anne Newman3, Joseph Zmuda1. 1University of Pittsburgh Graduate School of Public Health, USA, 2Yale University School of Medicine, USA, 3University of Pittsburgh, USA, 4Tobago Health Studies Office, Trinidad & tobago

Osteocalcin (OC), synthesized by osteoblasts, is an important protein constituent of bone matrix, and is a marker of bone turnover. Emerging evidence suggests that OC may also be a novel hormonal regulator of energy metabolism. We sought to identify genomic regions involved in regulating total and uncarboxylated OC using genome-wide linkage analysis in 458 individuals from seven large multi-generational families of African ancestry residing on the island of Tobago (mean family size, 56; 4,206 relative pairs; mean age, 42.7 years). We genotyped 5361 single nucleotide polymorphisms with minor allele frequency >0.05 and spaced ∼1 centimorgans apart using the Illumina Infinium II Human Linkage 12 panel. We estimated residual heritability (after adjusting for age, sex, height, and weight) and conducted multipoint quantitative trait linkage analyses using pedigree-based maximum likelihood methods. Means (and residual heritabilities) of total OC, uncarboxylated OC and percent uncarboxylated OC were 4.47 μg/L (0.63 ± 0.09), 1.53mg/L (0.83 ± 0.09) and 35.53%(0.41 ± 0.09), respectively. We detected significant evidence of linkage on chromosome 18 (maximum LOD=4.17, 92cM) and suggestive evidence of linkage on chromosomes 9 (maximum LOD=2.16) and 20 (maximum LOD=2.15) for total OC. Suggestive evidence of linkage for percent uncarboxylated OC was observed on chromosome 12 (maximum LOD=2.23). Interestingly, the structural locus for OC resides on chromosome 1, outside of the linked regions. The linked region on chromosome 18 contains 15 known and predicted genes. A potential candidate gene of interest in this region is the melanocortin 4 receptor (MC4R), which regulates appetite and energy expenditure and is expressed in osteoblasts. Further studies of the linked chromosomal regions may reveal novel insight into the regulation of osteocalcin and bone metabolism.

Disclosures: Allison Kuipers, None.


Heritability of Densitometric, Structural and Strength Properties of Bones: Results from the GAO (Genetic Analysis of Osteoporosis) Project.Jorge Malouf*1, Ana Laiz-Alonso1, Ana Marin1, Angel Martinez-Perez2, Leonor Rib2, Raquel Perez2, Alfonso Buil2, Jordi Casademont1, Jordi Farrerons1, Jose Manuel Soria2. 1Hospital de la Santa Creu i Sant Pau, Spain, 2Institut de Recerca (IR-HSCSP), Spain

Osteoporosis is a skeletal disorder characterized by compromised bone mass and strength that increases the risk of fracture. The genetic basis of primary osteoporosis is complex with multiple genes and environmental factors acting jointly to determine risk. However, recent advances in genetic analyses permit the identification of the genetic determinants of complex diseases. Towards this end, we designed a family-based study of the genetics of osteoporosis in a Spanish population, called GAO (Genetic Analysis of Osteoporosis).

A total of 110 individuals in 5 Spanish extended families (at least three-generation families) were included. All of the families were recruited through a proband with osteoporosis and all of them signed an informed consent. After filling a questionnaire, a spine, femur and whole body densitometry was performed using a Discovery densitometer with the APEX 2.3 software, from HOLOGIC® Bedford, Massachusetts, USA. The strength and geometrical properties of the hip were analyzed using the HSA® software included in the Apex 2.3 software.

Maximum likelihood-based covariance decomposition analysis was used to assess heritabilities (h2) and the genetic and environmental correlations (ρG and ρE) between phenotypes. Table 1 shows the h2 found among the phenotypes.

This is the first study that quantifies the genetic component of several important parameters involved in osteoporosis and fracture risk in extended pedigrees. The high heritability of the majority of them indicate that genetic effects account for a significant proportion of the observed phenotypic variation of these parameters. Thus, a strategy based on a family-based study through a genome scanning can be used to identify the specific genes involved in osteoporosis and fracture risk. Additionally, this knowledge will greatly facilitate the search for genes involved in osteoporosis, and ultimately find its way into the clinic.

Table Table 1. Heritability among phenotypes
  1. h2: Heritability: sd: standard deviation: *p-value at significant level <0.05 (using bonferroni correction for multiple testing).

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Disclosures: Jorge Malouf, None.


A Significant Interaction between Simple Nucleotide Polymorphism (SNP) in NFKB and RANK is Associated with Women BMD in the Framingham Osteoporosis Study.Bi Hua Cheng*1, Xing Chen2, YanHua Zhou3, Serkalem Demissie3, Adrienne Cupples3, David Karasik4, Douglas Kiel4, Yi-Hsiang Hsu5. 1Chang Gung Memorial Hospital - Kaohsiung Medical Center, Chang Gung University, USA, 2HSL, USA, 3BUSPH, USA, 4Hebrew SeniorLife, USA, 5Hebrew SeniorLife & Harvard Medical School, USA

The RANK/RANKL/OPG signaling pathway plays a crucial role in signal transduction in osteoblasts and osteoclasts. Previous GWAS have shown that SNPs in TNFRSF11A (RANK), TNFSF11(RANKL), and TNFRSF11B(OPG) genes are associated with BMD. Although NFKB gene is known to mediate osteoclastogenesis via the RANK/RANKL/OPG signaling pathway, its interactions with RANK, RANKL, or OPG genes in relation to BMD have not been examined in human populations. Therefore, we performed a gene-gene interaction study to investigate this relationship. Femoral neck (FN) and L2-L4 lumbar spine (LS) BMD were measured by a Lunar DPX-L. Genotyping was performed using the Affymetrix 550K SNP chips. A total of 1,813 women and 1,375 men (mean age 62.5 yrs) with both phenotypes and genotypes in the Framingham Osteoporosis Study were in the final analysis. Genotyped SNPs were selected with (1) minor allele frequency ≥ 1% and (2) HWE test p-value ≥ 0.0001 and (3) located in the gene and within 20 kb 5' upstream flanking region (38 SNPs in NFKB, 37 in RANK, 61in RANKL, and 58 in OPG). Sex-specific linear mixed-effect models were performed to determine the single SNP-phenotype associations with LSBMD and FNBMD. Covariates adjusted in the models included age, height, BMI, alcohol consumption, smoking status and principal component for their ancestral genetic background. The most significant association (the lowest p-value) with LS BMD were found for NFKB rs7685474, RANK rs2980973, RANKL rs7334307, and OPG rs7014574 in women and NFKB rs17032779, RANK rs17069956, RANKL rs7989838, and OPG rs11992072 in men. No SNP with association p-value less than 0.01 was found for FN BMD in women or men; therefore we did not perform gene-gene interaction for FN BMD. Six pair-wise interactions between the most associated SNPs in these four genes were performed in each gender. Bonferroni correction was used to adjust for multiple testing (α level=0.00416). A significant interaction between NFKB rs7685474 and RANK rs2298073 (p=0.0039) was found in women only at LSBMD. Individuals with polymorphic alleles in the RANK rs2298073(C) and the NFKB rs7685474(G) had a higher BMD compared to individuals without polymorphic alleles in both of the genes or in one of the genes (Fig 1). No significant gene-gene interactions were found in relation to LS BMD in men. This is the first genetic epidemiological study to reveal an interaction between NFKB and the RANK pathway at genetic level with LSBMD in a Caucasian population.

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Figure Fig 1. Interaction between NFKB rs7685474 and RANK rs2980973 in relation to LSBMD in women

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Disclosures: Bi Hua Cheng, None.


Association of LRP4 Polymorphisms to Bone Properties and Fracture in Women.Maria Swanberg*1, Jitender Kumar2, Fiona McGuigan3, Kristina Akesson4. 1Lund University, Sweden, 2Lund University, Sweden, Sweden, 3University of Lund, Malmö Skane University Hospital, Malmö, Sweden, 4Malmo University Hospital, Sweden

LRP4 and LRP5 are structurally similar members of the low density lipoprotein-receptor gene family that may regulate osteogenesis as co-receptors in the Wnt signaling pathway. Experimental data supports a role for LRP4 and -5 in regulating bone mass, growth and turnover. Genetic association to BMD and fracture has been reported for LRP4 and has been confirmed for LRP5 in several cohorts.

In this study, we examined the association of single nucleotide polymorphisms (SNPs) in LRP4 (rs3816614, rs6485702, rs2306033, rs7926667) and LRP5 (rs68242, rs498832, rs373622) to bone properties and fracture risk. Two cohorts living in Malmö, Sweden, were studied with regard to bone mineral density (BMD) and bone architecture; the PEAK-25 cohort representing 1002 young (25 years old) women, and the OPRA cohort representing 1002 elderly (75 years old) women. The OPRA cohort was followed for 5 years and additional phenotypes studied in this cohort were BMD at age 80, bone loss between age 75 and 80, femoral neck (FN) bone geometry and acquired fractures until age 80.

In the OPRA cohort, LRP4 was associated to BMD corrected for smoking and weight for total body and FN at age 75 (rs2306033 p<0.05) and at age 80 (rs2306033 p<0.01; rs3816614 p<0.01) and for lumbar spine at age 80 (rs6485702, p<0.05). Similarly, in the PEAK-25 cohort, LRP4 (rs6485702) was associated to BMD corrected for smoking and weight at the FN (p<0.05), lumbar spine (p<0.05) and total body (p<0.01).

In the OPRA cohort, LRP4 was also associated with the rate of total body bone loss between age 75 and 80 (rs3816614 p<0.05), bone quality measurements taken through ultrasound (rs3816614 p<0.05) and bone geometry for FN width (rs648570 p<0.01) and moment of inertia (rs648570 p<0.05).

Until age 80 in the OPRA cohort, 609 individuals had suffered at least one fracture, including 139 women with hip fractures, while 329 women had not suffered any fracture. LRP4 was associated to any fracture (rs3816614 p<0.05, rs2306033 p<0.01, rs6485702 p<0.05) and to hip fracture specifically (rs2306033 p<0.01), with more carriers of the variant alleles in the no-fracture group.

In conclusion, non-synonymous polymorphisms in the LRP4 gene were consistently associated with various bone properties i.e. BMD, bone geometry, quality, rate of bone loss and, importantly, to fracture in elderly women. In addition, LRP4 was also associated to BMD in young women, thereby having probable effects on bone throughout life.

Disclosures: Maria Swanberg, None.


Association of Non-coding Variants in the 3'-UTR of the Frizzled-1 Gene with Skeletal Geometry Among Afro-Caribbean Men.Yingze Zhang*1, Allison Kuipers2, Cara Nestlerode1, Clareann Bunker1, Victor Wheeler3, Alan Patrick3, Joseph Zmuda2. 1University of Pittsburgh, USA, 2University of Pittsburgh Graduate School of Public Health, USA, 3Tobago Health Studies Office, Trinidad & tobago

The Wnt/β-catenin pathway is important in osteoblast differentiation, proliferation and apoptosis. Frizzled 1 (FZD1) is a member of the Wnt/β-catenin pathway and is a trans-membrane co-receptor with LRP5 for Wnt molecules. We previously demonstrated a novel association between two common single nucleotide polymorphisms (SNP) in the promoter region of frizzled-1 (FZD1) and bone geometric properties.

In the current analysis, we tested whether SNPs in the 39 untranslated region (UTR) of FZD1 are independently associated with measures of bone geometry. Three SNPs (rs2232163, rs3750145 and rs1052015) in the 39-UTR of the FZD1 were analyzed in 1844 Afro-Caribbean men aged 40 years and older. The minor allele frequencies of these 3 SNPs were: 8.6%, 6.6%, and 5.2%, respectively. Haplotype analysis identified 4 haplotypes GTA, ATA, GCC, and GCA with frequencies of 84.5%, 8.6%, 5.3% and 1.5%, respectively. Association analyses of the SNPs and haplotypes with peripheral quantitative computed tomography measures were performed under an additive model adjusting for age, height and body weight and the two promoter SNPs (rs2232157, rs2232158) to test the independent association of the 39-UTR variants. The minor allele of rs2232163 was associated with decreased (P=0.005) whereas the minor allele of rs3750145 was associated with increased (P=0.04) cortical thickness at the radius. The ATA haplotype was associated with decreased (P=0.008) whereas the GCA haplotype was associated with increased (P=0.002) cortical thickness. Interestingly, these 39-UTR variants encompass several predicted microRNA binding sites. Functional analysis of these SNPs and haplotypes will be important to elucidate the significance of these regulatory region variants.

Disclosures: Yingze Zhang, None.


BMD-associated Variation at the Osterix Locus is Correlated with Pediatric BMI in Females.Jianhua Zhao1, Jonathan Bradfield1, Mingyao Li2, Haitao Zhang1, Frank Mentch1, Kai Wang1, Patrick Sleiman1, Cecilia Kim1, Joseph Glessner1, Edward Frackelton1, Rosetta Chiavacci1, Robert Berkowitz1, Babette Zemel3, Hakon Hakonarson1, Struan Grant*3. 1Children's Hospital of Philadelphia, USA, 2University of Pennsylvania, USA, 3Children's Hospital of Philadelphia, USA

Recent genome wide association studies (GWAS) have revealed a number of genetic variants robustly associated with bone mineral density (BMD) and/or osteoporosis. Evidence from epidemiological and clinical studies has shown an association between BMD and body mass index (BMI), presumably as a consequence of bone loading. We investigated the 34 previously published BMD GWAS-derived loci in the context of pediatric BMI by leveraging our existing genome-wide genotyped European American cohort of 1,106 obese children (BMI≥95th percentile) and 5,997 controls (BMI<95th percentile). Evidence of association was only observed at one locus, namely Osterix (SP7), with the G allele of rs2016266 being significantly over-represented among childhood obesity cases (P=2.85×10-3). When restricting these analyses to each gender, we observed strong association between rs2016266 and childhood obesity in females (477 cases and 2,867 controls; P=3.56×10-4). However, no evidence of association was observed among males. Similar observations were made when treating BMI as a quantitative trait in the same cohort. Interestingly, Osterix is the only GWAS locus uncovered to date that has also been previously implicated in the determination of BMD in childhood. In conclusion, these findings indicate that a well established variant at the Osterix locus associated with increased BMD is also associated with childhood obesity primarily in females. These results are in keeping with the bone loading impact on BMD through increased BMI.

Disclosures: Struan Grant, None.


Bone Mineral Density Locus Associated with Coronary Artery Calcification.Laura Yerges-Armstrong*1, Xinggang Liu1, Elizabeth Streeten2, Alan Shuldiner1, Braxton Mitchell3. 1University of Maryland, USA, 2University of Maryland School of Medicine, USA, 3University of Maryland, Baltimore, USA

Epidemiologic and in vitro studies support a relationship between calcification of bone and vascular tissue, although the nature of the relationship is unclear. To identify genes having pleiotropic effects on these two traits, we tested whether SNPs found by previous genome wide association (GWA) studies (Rivadeneira et al.: Nat Gen 2009) to be robustly associated with BMD were also associated with coronary artery calcification (CAC). Our study was carried out in a population of Old Order Amish individuals in whom we had measured BMD at the hip and spine by DXA (N=889) and calcification of the coronary arteries (N=506) by electron beam CT. On average participants were 56 years of age and 47% of the population was male. The BMD loci were extracted from a recent meta-analysis of Caucasian reference data that identified loci for BMD at the lumbar spine (LS) or femoral neck (FN). We created a genotype score for LS (15 loci) and FN (10 loci) by summing the number of copies of the high BMD allele across all loci. As expected, genotype scores were associated with BMD in the Amish (p=7.7×10-7 for FN and p=0.013 for LS) although the scores explained very little of the total variation in BMD (r2=0.03 and 0.01 respectively for LS and FN). We then evaluated associations of the genotype scores with CAC quantity adjusting for the effects of age and gender. FN genotype score was not significantly associated with CAC. In contrast, analysis of the LS genotype score revealed that an increasing number of high BMD alleles was associated with lower CAC quantity (p = 0.046). Further analyses of the individual SNPs identified an association (p=0.02) between a SNP on 13q14 and CAC in our sample. These results extend previous studies suggesting a relationship between cardiovascular disease and lower bone mineral density by showing a common genetic link between the two disorders. These analyses also reveal a specific BMD variant (rs9533090 downstream of AKAP11) that is also associated with cardiovascular health.

Disclosures: Laura Yerges-Armstrong, None.


Cooperative Effect of Serum 25-Hydroxyvitamin D Concentration and a Polymorphism of Transforming Growth Factor β-1 Gene on the Prevalence of Vertebral Fractures in Postmenopausal Osteoporosis.Kazuki Kobayashi*1, Seijiro Mori2, Noriyuki Fuku3, Yuko Chiba2, Fumiaki Tokimura2, Takayuki Hosoi4, Yoshiyuki Kimbara2, Yoshiaki Tamura2, Atsushi Araki2, Masashi Tanaka3, Hideki Ito2. 1Tokyo Metropolitan Geriactic Hospital, Japan, 2Tokyo Metropolitan Geriatric Hospital, Japan, 3Tokyo Metropolitan Institute of Gerontology, Japan, 4National Center for Geriatrics & Gerontology, Japan

Purpose: A T869RC polymorphism of the transforming growth factor β1 (TGF-β1) gene is reported to be associated with genetic susceptibility to both osteoporosis and vertebral fracture. Low serum 25-hydroxyvitamin D [25(OH)D] level is known to be associated with a higher risk for hip fracture. This study aimed to assess a possible cooperative effect of the gene polymorphism and vitamin D status on vertebral fracture risk.

Methods: Prevalence of vertebral fracture in 168 postmenopausal female patients with osteoporosis was analyzed, and its association with the TGF-β1gene polymorphism and serum 25(OH)D concentration was assessed cross-sectionally.

Results: The fracture prevalence increased according to the rank order of the TGF-β1 genotypes CC < CT < TT, as expected. A significant difference was found not only between the CC and TT genotypes (p = 0.005) but also between the CC and CT genotypes (p<0.05) when the patients with serum 25(OH)D of more than the median value [22 ng/mL (55 nmol/L)] were analyzed. On the other hand, when those with serum 25(OH)D of less than the median value were analyzed, a protective effect of the C allele against the fracture was blunted; statistical significance in the difference of the fracture prevalence was lost between the CC genotype and the other genotypes.

Conclusion: These data suggest that vitamin D fulfillment is prerequisite for the TGF-β1 genotype in exerting its full effect on the fracture prevalence. 

Table Table. Effect of Serum 25(OH)D Level on the Relationship between TGF-βl Genotype and Prevalence of Vertebral Fracture
  1. Number (percent) of patients is presented.

  2. *p<0.05 and **p=0.005 versus CC with 25(OH)D ≥ 22 ng/mL.

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Disclosures: Kazuki Kobayashi, None.


Genetic Association Study of Common Mitochondrial DNA Variants in Osteoporosis.Shufeng Lei*1, Yan Guo1, Tielin Yang2, Hui Shen1, Xianghong Xu3, Shanshan Dong1, Yao-zhong Liu1, Yong-Jun Liu1, Hong-Wen Deng4. 1University of Missouri - Kansas City, USA, 2The University of Missouri-Kansas City, USA, 3University of Missouri-Kansas City, School of Medicine, USA, 4University of Missouri, Kansas City Medical School, USA

Many lines of evidence suggest that mitochondrial DNA (mtDNA) variants are involved in the pathogenesis of human complex diseases, especially for age-related disorders. Osteoporosis is a typical age-related complex disease, however, the role of mtDNA variants in the susceptibility of osteoporosis is largely unknown. In this study, we reported a mitochondrial genome-wide association study for bone mineral density (BMD) in Caucasians.

A total of 445 mitochondrial single nucleotide polymorphisms (mtSNPs) were genotyped in a sample of 2,286 unrelated Caucasian subjects (558 males and 1728 females) by using the Affymetrix Genome-Wide Human SNP Array 6.0. SNPs were tested for association with hip BMD by the quantitative association tests implemented in PLINK, while controlling for potential confounding factors, such as age, gender, height and weight.

A SNP within the NADH dehydrogenase 2 gene (ND2), rs28571027 (C/A), was strongly associated with hip BMD (p = 3.63 × 10-4). The C allele was associated with reduced hip BMD values and the effect size (β) was estimated to be ∼0.022. Our results highlighted the importance of mtDNA variants in influencing BMD variation and risk to osteoporosis. Further genetic and molecular functional investigations for the relationship between mtDNA variants and osteoporosis are warranted.

Disclosures: Shufeng Lei, None.


BMP2 Binds Preferentially to BMP Receptors Localized in Caveolae and Initiates Smad Signaling.Oleksandra Moseychuk*1, Beth Bragdon2, Jeremy Bonor1, Anja Nohe1. 1University of Delaware, USA, 2University of MaineUniversity of Delaware, USA

Bone Morphologic Proteins (BMPs) are growth factors that regulate skeletal development and repair. They are used as therapeutics in spinal fusion and fracture repair but little is known about the initiation of signaling on the receptor level. BMP receptors (BMPRs) are found on the plasma membrane, in caveolae and clathrin coated pits (CCPs). Current literature suggests that BMP2 mediated signaling by Smad 1,5 and 8 is initiated on the plasma membrane followed by endocytosis of receptors through CCPs, however our studies demonstrate that ligand binding, BMP receptor type-Ia (BMPRIa) phosphorylation and Smad 1,5 and 8 activation occurs in caveolae within 10 min.

Combination of Atom Force Microscopy (AFM) and advance confocal imaging were used to determine the binding affinity of ligand with receptors on the surface of live cells. Biologically active AFM tip was designed by covalently linking BMP2, to an AFM tip and live C2C12 cells were transfected with Caveolin-1 GFP conjugated plasmid (protein that is responsible for the flask shape structure of caveolae) as a marker to visualize caveolae. Our AFM data together with high resolution confocal images and retraction curve data demonstrated that BMP2 binds predominantly to BMPRs localized in caveolae. Since BMPR phosphorylation is the first step of the signaling cascade, we performed fractionation of the plasma membrane based on the lipid composition to determine the location of phosphorylated form of BMPRIa in C2C12 cells. We observed phosphorylation in the lipid raft fractions, namely caveolae. Additionally, Smad 1, 5, and 8 phosphorylation was probed at different time points during BMP2 stimulation. It showed the initial phosphorylation takes place in caveolae as early as 10 min post stimulation, followed by endocytosis. After 45 min stimulation with BMP2, the phosphorylated form of Smad 1, 5, and 8 is degraded in the lysosome. Taken together, these data show that BMP2 dependent phosphorylation of BMPRIa and activation of regulatory Smads takes place predominantly in caveolae, not CCPs.

Disclosures: Oleksandra Moseychuk, None.


BMP2 Gene as an Organizer Coordinating Osteogenesis and Angiogenesis Postnatally and Roles in Mechanical Properties of Bone.Wuchen Yang*1, Jeffry Nyman2, Yong Cui1, Jelica Gluhak-Heinrich1, Marie Harris1, Ivo Kalajzic3, David Rowe3, Gregory Mundy4, James Edwards2, Barbara Kream3, Alexander Lichtler3, Xiao-Dong Chen1, Yuji Mishina5, Stephen Harris1. 1University of Texas Health Science Center At San Antonio, USA, 2Vanderbilt University Medical Center, USA, 3University of Connecticut Health Center, USA, 4Vanderbilt University, USA, 5University of Michigan, USA

Coordination between osteogenesis and angiogenesis is important for proper bone remodeling and repair postnatally. Our previous study using the 3.6Col1a1-Cre;BMP2 cKO mice showed that the postnatal deletion of the BMP2 gene from collagen producing osteoblasts inhibits latter stages of osteoblast differentiation and causes an intrinsic 80% reduction in the Mesenchymal Stem Cells (MSCs) population, as determined by CFU-F assays. The long bones of the 3.6Col1a1-Cre;BMP2cKO mice appeared to be less red, suggesting a decrease in vascularization. Since perivascular cells are potentially one of the MSCs niches that associated with blood vessels, we further evaluated the bones by CD146 immunohistochemistry (IHC) and confirmed that the perivascular cells are indeed reduced in vivo in the BMP2 cKO relative to control littermates. Using α-SMA-Cherry transgene, in which the perivascular candidate MSCs are labeled red on microvasculature, we confirmed a reduction of this MSCs population in vivo in the BMP2cKO mice. To address whether this reduction of the MSCs population is caused by a reduction of angiogenesis leading to the reduction of MSCs niches, collagen IV IHC was then performed for both femur and tibia. This revealed that blood vessel density is reduced 20 to 50% in the 3.6Col1a1-Cre;BMP2cKO mice at different ages up to 6 month. These results suggest that the angiogenesis is reduced after BMP2 deletion from osteoblasts. To explore the mechanism by which BMP2 in osteoblasts regulates angiogenesis, the expression level of VEGFA, an angiogenesis stimulator that is secreted by mature osteoblasts, was assayed by IHC and VEGFA association with osteoblasts was reduced 50% in the BMP2 cKO animals. Thus, ablation of BMP2 gene from osteoblasts interferes drastically with the late stage of osteoblast maturation that in turn leads to reduction of VEGF production and results in defective angiogenesis and reduced MSCs niche availability. These phenotypes in the BMP2 cKO lead to altered geometric and mechanical properties of the long bones. There was an over 50% reduction in Work-to-fracture in the femur. A greater than 80% reduction in Post Yield toughness in the BMP2 cKO also suggests these bones are much more brittle than control bones, possibly due to the decreased vascularization and reduced MSCs population.

Disclosures: Wuchen Yang, None.


Dullard, a Novel BMP Inhibitor, Targets Smad1 to Suppress BMP-dependent Transcription in Mammalian Osteoblastic Cells.Tadayoshi Hayata*1, Yoichi Ezura2, Makoto Asashima3, Ryuichi Nishinakamura4, Masaki Noda5. 1Medical Reserach Institute, Tokyo Medical & Dental University, Japan, 2Tokyo Medical & Dental University, Medical Research Insititute, Japan, 3University of Tokyo, Japan, 4Kumamoto University, Japan, 5Tokyo Medical & Dental University, Japan

BMP plays crucial roles during osteoblast differentiation and its signaling effect is negatively regulated to maintain the appropriate signaling levels. However, mechanism of signal regulation by BMP inhibitors during osteoblast differentiation has not been fully understood. Dullard encodes a phosphatase preferentially localized in caveosome and has been reported to suppress BMP signaling pathway primarily by promoting degradation of BMP receptor complex via the lipid raft-caveolar pathway and secondarily by dephosphorylation of activated BMPR1. In osteoblasts, Dullard limits BMP responsiveness. With regard to BMP antagonistic activity in “Xenopus animal cap assay”, Dullard does not suppress constitutively active BMPR1a (caALK3) signaling. However, in the current study, we unexpectedly found that Dullard suppresses constitutively active BMPR1 in “mammalian osteoblastic cells”. To refine at what level Dullard can suppress BMP signaling pathway in “mammalian cells”, BMP responsive element (BRE)-luciferase assay was performed using MC3T3-E1 cell line. Overexpression of Dullard inhibits caALK3-induced activation of BRE-reporter independently of its phosphatase activity. Furthermore, overexpression of Dullard inhibits the activity of both constitutively active phospho-mimetic Smad1 (SMAD1-EVE), in which two serine residues at C-terminal phosphorylated by receptor kinase are mutated into glutamic acid, and more potent SMAD1-EVE-MM, in which MAPK phosphorylation sites in the linker region are mutated. These epistatic experiments indicate that Dullard can suppress BMP signaling pathway at or downstream of the Smad1 level. We also tested whether Dullard affects TGF-β signaling pathway. Dullard suppresses TGFβ-induced reporter activity and constitutively active TGFBR1 (caALK5)-induced TGFβ signaling independently of its phosphatase activity. In BMP-induced osteoblast differentiation, siRNA-mediated knockdown of Dullard decreases alkaline phosphatase production as well as expression of marker genes including Id3, Osterix, osteocalcin, and alkaline phosphatase. Although Dullard was previously identified as a modulator of BMP receptor degradation, it plays more extensive role as a general suppressor of BMP/TGF-β signal transduction downstream of type I receptor/SMAD and promotes BMP-induced osteoblast differentiation. In conclusion, we identified Smad1 and TGF-β signaling pathway as novel targets of Dullard.

Disclosures: Tadayoshi Hayata, None.


See Friday Plenary number FR0176.


See Friday Plenary number FR0177.


See Friday Plenary number FR0178.


See Friday Plenary number FR0179.


See Friday Plenary number FR0180.


Delayed Fracture Healing in Protease Activated Receptor-2 Deficient Mice.Kevin O'Neill*1, Christopher Stutz1, Mignemi Nicholas1, Jeffry Nyman2, Gregory Mencio1, William Obremskey1, Conor Lynch1, Jonathan Schoenecker1. 1Vanderbilt University, USA, 2Vanderbilt University Medical Center, USA

Objectives: Extracellular proteases and inflammation are essential elements of fracture repair. Protease activated receptors (PARs) allow proteases to behave like traditional hormones. It is known that PAR-2 provides an important link between extracellular protease activation and regulation of cellular inflammation. Although PAR-2 is expressed by developing osteoblasts, its function in fracture healing is unknown. The purpose of this study was to compare fracture healing between wildtype (WT) and PAR-2 deficient (PAR-2-/-) mice. We hypothesized that PAR-2-/- mice will demonstrate delayed fracture healing relative to WT mice.

Methods: With IUCUC approval, unilateral transverse mid-shaft femur fractures were created in 10 PAR-2-/- and 11 WT mice after intramedullary fixation. Mice were sacrificed at 7 weeks, fractured and contralateral intact femurs were harvested, and axial micro-computed tomography (μCT) images were obtained. Torsional rigidity and ultimate strength were calculated from torque versus angular displacement curves obtained by torsion testing. Bone volume fraction (BV/TV), bone mineral density (BMD), and polar moment of inertia (pMOI) were determined from μCT images.

Results: For WT mice, the torsional rigidity and ultimate strength of fractured femurs were not significantly different from intact femurs. In contrast, for the PAR-2-/- mice, significant differences were noted between the fractured and intact femurs for both torsional rigidity (p=0.04) and ultimate strength (p=0.04). Significant differences were also noted in PAR-2-/- mice between pMOI, BV/TV, and mBMD (all p<0.001) values of fractured compared to intact femurs (Figure 1).

Conclusions: The PAR-2-/- mice demonstrated delayed fracture healing measured by both biomechanical torsion testing and radiographic μCT imaging. Future studies are needed to determine if the delayed healing observed is secondary to a diminished inflammatory response in the fracture environment. Unveiling this mechanism has potential impact on the basic science of fracture healing and the development of novel pharmaceuticals that, through PAR-2, may enhance fracture healing. ,  

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Figure Figure 1 - A.  

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Figure Figure 1 - B.  

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Disclosures: Kevin O'Neill, None.


Effect of Infection on Bone Marrow Stromal Cells (MSCs): a Role for IL-6 in Erythropoiesis.Brian Sworder*1, David Chou2, Michael Grigg2, Yasmine Belkaid2, Pamela Robey3. 1National Institutes of Health, USA, 2NIAID/NIH, USA, 3National Institute of Dental & Craniofacial Research, USA

Inflammation induces and necessitates changes in hematopoiesis (HP). Previous studies have examined the HP alterations that occur in the bone marrow (BM) during an immune response. However, despite the critical role that BM stroma plays in supporting HP, its function during inflammation remains largely unexplored. This study seeks to determine how BM stromal cells (BMSCs) and their influence on HP are altered during an immune response.

Mice were orally infected with the intracellular parasite, Toxoplasma gondii. This infection model causes severe systemic T helper type 1 inflammation and results in anemia and potential death. Eight days after infection, total cell number in the BM fell to half that of naïve mice. Flow cytometry analysis showed significant changes in the percentages of lymphoid, myeloid, and erythroid constituents. Notably, the fraction of nucleated cells contributing to erythropoiesis was reduced 5-fold. Conversely, there was no apparent change in the number of CFU-Fs (a subset of BMSCs) after infection.

A variety of knockout mice (IFNg receptor, iNOS, TCRa, IL-1, TNFa receptor, IFN-a/B receptor, IL-15, and IL-6) were screened to determine which molecules were important in mediating the pathogenic anemia induced by T. gondii infection. Only IL-6 KO mice were found to have better maintenance of erythropoiesis and a significantly larger erythroblast population than WT mice. IL-6 is known to play an important role in anemia associated with inflammation. However, the relative importance of IL-6 production by various cellular sources in mediating anemia is not known. To determine whether IL-6 from BM stroma might contribute, BMSCs were isolated from naïve and infected mice by FACS (VCAM+/CD45-/Ter119-). Strikingly, analysis by ELISA showed a 2-fold increase in IL-6 secretion by BMSCs after T. gondii infection. Using bone marrow transplants and ectopic ossicle models with WT and IL-6 KO cells, experiments are currently underway to more directly determine the contribution of IL-6 from BM stroma to infection-induced anemia.

Our results demonstrate that IL-6 is a key mediator of erythropoietic collapse during T. gondii infection and that BMSCs may be the relevant source of IL-6. This study also suggests that the bone marrow stroma is functionally different during an immune response, and that BMSCs are potential mediators of changes in HP composition as a consequence of inflammation.

Disclosures: Brian Sworder, None.


See Friday Plenary number FR0183.


Human Blood Eosinophils: An Extrarenal Source of Converting Inactive Vitamin D to Its Active Form and Potential Role in Inflammation.Devendra Agrawal*1, Divya Pankajakshan1, J. Christopher Gallagher2. 1Creighton University School of Medicine, USA, 2Creighton University Medical Center, USA

Rationale: Vitamin D, especially its most active metabolite 1,25-dihydroxyvitamin D3 or calcitriol, is essential in regulating calcium homeostasis, immune modulation, cell proliferation and differentiation. Although extra-renal synthesis of calcitriol associated with inflammatory diseases is known to occur in macrophages and epithelial cells, little is known on the existence of enzymes involved in vitamin D synthesis and metabolism in circulating immune cells. In this study, we examined such enzymes in human blood eosinophils and also investigated the effect of calcitriol, administered in vivo or added exogenously, on the expression of intracellular molecules involved in inflammation in purified white blood cells (WBCs).

Methods: Peripheral blood was drawn from healthy volunteers and WBCs were purified (purity >97%; viability >98%) and stimulated in vitro with calcitriol (100 nM) for 24 hrs. We also purified WBCs from the blood of vitamin D-deficient (25-OHD levels <20ng/ml) subjects (n= 78) before and after treatment with 800-4,200 IU/d vitamin D for 1 year. The mRNA expression of importin-alpha3 (KPNA4), CYP27B1 and CYP24A1 was examined by real-time PCR.

Results: The mRNA transcripts for CYP27B1, CYP24A1, cathelicidin, and KPNA4 were found in human blood eosinophils. In vivo administration of vitamin D for 1 yr in vitamin D-deficient subjects significant increased the mRNA transcripts of CYP27B1, CYP24A1 and cathelicidin, but significantly decreased the mRNA transcripts of KPNA4. Under in vitro conditions, stimulation of human WBCs of healthy volunteers with calcitriol decreased mRNA transcripts of KPNA4 with more pronounced reduction (60-70%) in neutrophils and eosinophils. Calcitriol increased the mRNA transcripts of CYP24A1 in human blood eosinophils.

Conclusions: Presence of both CYP27B1 and CYP24A1 in human blood eosinophils suggest their role in converting inactive vitamin D to its active form and decreased expression of KPNA4 in response to vitamin D could be one of the underlying mechanisms for the anti-inflammatory and immunoregulatory effects of vitamin D in inflammatory diseases involving eosinophils.

Disclosures: Devendra Agrawal, None.


Meox2Cre-CSF-1 Knockout Mice: A Novel Osteopetrosis Model.Sherry Abboud Werner*, Kathleen Woodruff, Diane Horn, Marie Harris, Stephen Harris. University of Texas Health Science Center at San Antonio, USA

CSF-1, a key regulator of mononuclear phagocytes, is expressed in most tissues and exerts pleiotropic effects. This is supported by studies in spontaneous mutant op/op B6C3 mice where absence of CSF-1 leads to osteopetrosis as well as defects in fertility, mammary development and neural function. However, the biologic effect of CSF-1 knockout(KO) in specific tissues has not been explored. To determine whether Cre-lox technology provides a useful strategy for producing CSF-1KO mice, a targeting vector for generating a conditional KO allele for CSF-1(deleting exons 4,5,6) was electroporated into ES cells. Heterozygous C57BL/6 mice harboring the floxed construct containing a 5' loxP site in intron 3 of the CSF-1 gene and a 3' loxP site with a FRT-polII-neo-FRT-loxP cassette in intron 6 were established. The neo cassette was removed by crossing CSF-1flox+neo mice with heterozygous beta-actin-Flip recombinase mice and the CSF-1flox-neo (fx) allelle was confirmed by Southern blot. CSF-1fx/CSF-1fx mice were then bred with Meox2Cre mice to produce heterozygous CSF-1KO mice that were subsequently interbred to generate mice homozygous for the KO allele (hCSF-1KO). At 3 weeks, CSF-1 was analyzed; bones were x-rayed and histological sections were examined using histomorphometry. Offspring showed the expected ratio of genotypes. Mice heterozygous for the KO allele resembled wt controls, whereas hCSF-1KO showed an osteopetrotic phenotype similar to op/op mice. hCSF-1KO showed CSF-1 deficiency, failure of tooth eruption, domed skull, stunted growth and reduced weight compared to wt. hCSF-1KO radiographs showed marked skeletal sclerosis with dense radioopaque bone in tibia and femoral metaphysis, vertebrae and pelvis. Compared to wt, hCSF-1KO hindlimbs showed an expanded growth plate with thick irregular calcified cartilage extending into the metaphysis obliterating the marrow cavity. Numerous TRAP-positive osteoclasts were identified in wt, whereas rare small weak TRAP-positive osteoclast-like cells were detected in hCSF-1KO. These findings provide the first evidence that global CSF-1KO can be achieved using a Cre-based system and that the osteopetrotic phenotype is reproducible in C57BL/6. Development of conditional CSF-1KO mice will be crucial for elucidating the mechanisms by which CSF-1 mediates its effect in targeted tissues. Results may lead to novel approaches for improving reproductive defects and regulating osteoclastogenesis in fracture repair and bone metastasis.

Disclosures: Sherry Abboud Werner, None.


See Friday Plenary number FR0186.


Serum Osteocalcin is Negatively Correlated to Insulin and Adiponectin in Hibernating Bears.Rachel Bradford1, Patricia Buckendahl2, Caren Gundberg3, Kim Henriksen4, Michael Vaughan5, Seth Donahue*6. 1Michigan Technological University, USA, 2Rutgers University, USA, 3Yale University School of Medicine, USA, 4Nordic Bioscience A/S, Denmark, 5Virginia Tech, USA, 6Michigan Tecnological University, USA

Hibernating bears do not lose cortical or trabecular bone despite being physically inactive for up to 6 months per year. Histomorphometry shows decreased bone turnover with balanced bone formation and resorption during hibernation. The purpose of hibernation is to conserve metabolic energy when food is unavailable. Fat is the primary energy source during hibernation; bears lose 30-40% of their body mass (almost exclusively fat mass) during hibernation. Bears also experience markedly decreased kidney function and do not urinate or defecate during hibernation, but maintain eucalcemia. The purpose of this study was to quantify seasonal bear serum markers of bone formation (BSAP) and osteoclast number (TRAP), which do not accumulate with decreased kidney function. To assess bone/fat/energy relationships, we also quantified serum osteocalcin (OC), adiponectin, NPY, insulin, and glucose. Serum was collected every 10 days from black bears before, during, and after hibernation. Serum BSAP and TRAP decreased (p<0.0001) during hibernation by 57% and 29% respectively, supporting the histomorphometric findings of decreased bone turnover. Serum total OC, which is also a bone formation marker, increased 59% during hibernation (p< 0.0001). This increase could possibly be explained by the accumulation of OC due to reduced renal function. Serum NPY was not significantly different in hibernating and active bears. Adiponectin decreased 30%, insulin decreased 20%, and glucose decreased 31% (p<0.0009) during hibernation relative to non-hibernation periods. Total OC was negatively correlated (p<0.0001) with adiponectin, unlike the positive association in humans. Insulin was negatively correlated (p=0.008) to total OC only during the pre-hibernation period. We are currently investigating seasonal changes in uncarboxylated OC. Reduced bone turnover contributes to energy conservation in hibernating bears, but the relationships between osteocalcin, adiponectin, insulin, and glucose may be different in bears than other species since bears rely on lipids for metabolic fuel nearly exclusively during hibernation (fasting). Hibernating bears are a unique model for understanding bone/fat/energy relationships and the prevention of disuse osteoporosis (i.e., bone loss due to prolonged physical inactivity); the biological mechanisms may have implications for human medicine.

Disclosures: Seth Donahue, None.


See Friday Plenary number FR0188.


The Effect of Vitamin K Supplementation on Glucose Metabolism.Hyung Jin Choi*1, Juyoun Yu1, Hosanna Choi1, Jee Hyun An1, Yenna Lee1, Hwa Young Cho1, Sun Wook Cho2, Sang Wan Kim3, Chan Soo Shin1. 1Department of Internal Medicine, Seoul National University College of Medicine, South korea, 2University of Michigan School of Dentistry, USA, 3Seoul National University Boramae Hospital, South korea

Vitamin K has been known to regulate glucose metabolism via modulating osteocalcin and/or inflammation pathway. However, the exact mechanisms for this regulation in vivo have not been fully investigated. We have investigated the effects of vitamin K on beta-cell function and insulin sensitivity in healthy young male volunteers using frequently sampled intravenous glucose tolerance test (FSIGT) before and after 4 weeks of vitamin K treatment. Twenty-two healthy young male subjects (age 28.2 ± 4.5; BMI 24.4 ± 2.5) were given menatetrenone (Vitamin K2) 30mg tid for 4 weeks and FSIGT were performed before and after the vitamin K treatment and carboxylated/undercarboxylated osteocalcin, interleukin-6, CRP, and adiponectin concentrations were measured. Vitamin K treatment significantly decreased and increased undercarboxylated and carboxylated osteocalcin, respectively (both, P < 0.05). However, there were no significant changes in the levels of inflammation markers, ie., interleukin-6 and CRP. Vitamin K treatment significantly increased disposition index (DI), which indicates beta-cell function in relation to insulin action, compared with baseline (baseline: 2507 ± 1448, 4 week treatment: 3468 ± 1584; P = 0.003). There were no significant changes in acute insulin response to glucose (AIRg) and insulin sensitivity (SI). Change in undercarboxylated osteocalcin was significantly associated with change in AIRg after adjusting for confounding factors, including baseline AIRg, age, BMI and weight change (P < 0.05). There was no significant change in body weight and adiponectin concentration. Taken together, these results suggest that vitamin K treatment for 4 weeks increased beta-cell function in healthy young men, which appears to be related to the modulation of osteocalcin metabolism rather than systemic inflammation.

Disclosures: Hyung Jin Choi, None.


See Friday Plenary number FR0190.


Igfb2-/- Mice Exhibit Age-related Changes in Skeletal Mass, Body Composition and Metabolic Status.Victoria Demambro*1, Kathryn Shultz2, Harold Coombs2, Jane Maynard2, David Clemmons3, Wesley Beamer2, Clifford Rosen4. 1Maine Medical Research Institute, USA, 2The Jackson Laboratory, USA, 3University of North Carolina, USA, 4Maine Medical Center, USA

We have previously reported on a global Igfbp2 null mouse on a C57BL/6J background in which the male mice at 16 weeks of age had significantly decreased bone volume and turnover and increased adiposity (Endocrinology 149:2051, 2008). To test the effect of IGFBP-2 absence over time, we aged Igfbp2 null (-/-) and control (+/+) males to 24 months of age. Body composition was examined by PIXImus DEXA at 2, 4, 6, 12, 18 and 24 months. Femurs were analyzed by pQCT at 4, 12 and 24 months for vBMD and MicroCT for structural changes at 4 and 12 months. Mice were evaluated for metabolic changes by ITT and GTT and serum insulin at 12 months. The -/- males were found to be heavier with increased fat mass at all time points. Whole body BMC was significantly reduced in -/- mice compared to +/+ control mice through 18 months. Remarkably, between 12 to 24 months, +/+ males had a dramatic loss in weight (-19%), whole body BMC (-13%), and fat mass (-27%), while -/- male mice appeared protected from these age related decreases by showing a significant increase in BMC (+7%) and a trend towards increased fat mass (+5%). Reductions in femoral vBMD (+/+ = -30%, -/-= -18%) and cortical thickness (+/+ = -22%, -/- = -6%) were observed, however to a lesser extent in the -/- femurs. Due to this differential rate of loss by 24 months -/- femurs were significantly increased for both measures compared to +/+ femurs. MicroCT analysis of distal femurs revealed similar decreases in trabecular BV/TV and number from 4 to 12 months of age. Interestingly there was no change in trabecular thickness in the +/+ mice with the mutants exhibiting a 16% increase during this same period. At 12 months of age, -/- mice were found to be hyperinsulinemic in both the fasted and fed states compared to +/+ mice. When glucose challenged, -/- mice exhibited higher glucose values and ITT results showed a decrease in the hypoglycemic response to exogenous insulin. Taken together, these results are indicative of a mild but significant insulin resistance. In summary, Igfbp-2 null males appear to be protected from age related loss of bone with minor reductions in cortical mass and increased trabecular thickness. On the other hand, they seem to be more susceptible to age related increases in obesity and insulin resistance.

Disclosures: Victoria Demambro, None.


See Friday Plenary number FR0192.


The Skeletal Effect of Parathyroid Hormone Treatment after Ovariectomy is Diminished when Circulating Growth Hormone is Elevated.J. Fritton*1, Devendra Bajaj1, Hui Sun2, Yinjgie Wu2, Mary Bouxsein3, Shoshana Yakar2. 1New Jersey Medical School, USA, 2Mount Sinai School of Medicine, USA, 3Beth Israel Deaconess Medical Center, USA

Parathyroid hormone (PTH) is routinely prescribed to postmenopausal women to restore bone and prevent osteoporotic fracture. However, the effects of the PTH therapy are quite variable in both these women and after ovariectomy (OVX) of animals. Both growth hormone (GH) and insulin-like growth factor (IGF-1) are potent regulators of bone remodeling processes. Previous investigations utilizing OVX have revealed that a serum balance in mice that favors GH over IGF-1 diminishes the effects of ablated ovarian function and leads to reduced endosteal resorption and greater bone formation. We utilized the liver IGF-1 deficient (LID) mouse to study the possible additional effects of PTH treatment. OVX surgery was performed on 12-week-old mice followed by 4 weeks of daily (7d/wk) PTH (50 ng/g body weight) intraperitoneal (i.p.) injection. 12-Lim mCT (Scanco) scans were completed for femurs and vertebrae. Whole-bone mechanical properties were quantified for mid-diaphyseal femurs by loading to failure in 4-point bending. Bone formation histomorphometry measurements were made on sections from double-labeled, calcein-injected (i.p.) mice. Contrary to the expected gains with PTH observed in controls, very small effects were found in LID mice with PTH treatment after OVX (Δ cross-sectional moment of inertia of mid-shaft femur: WT - 25%; LID - < 1%, A trabecular thickness: WT - 3%; LID - < 1%). One explanation for this lack of effect is that cancellous and cortical bone structures may reach homeostasis due to the previously reported enhanced expansion of cross-sectional structures in LID mice that exhibit 3-fold greater circulating GH. Our data suggest little synergism of PTH during estrogen deficiency when GH is elevated and offer a possible insight into the variability of response to PTH treatment for bone loss in postmenopausal women.

Disclosures: J. Fritton, None.


See Friday Plenary number FR0194.


See Friday Plenary number FR0195.


See Friday Plenary number FR0196.


Runx2 Stimulation of HSP70/HSPA1B Gene Transcription Decreases Runx2 Protein Stability in Osteoprogenitors.Nadiya M. Teplyuk1, Jennifer Cotton1, Jonathan Gordon1, Viktor I. Teplyuk1, Mario Galindo2, Di Chen3, Suk-Chul Bae4, Jane Lian1, Janet L. Stein1, Gary Stein1, Andre Van Wijnen*1. 1University of Massachusetts Medical School, USA, 2University of Chili, Chile, 3University of Rochester Medical Center, USA, 4Chungbuk National University, South korea

Inactivation, haplo-insufficiency and dosage insufficiency of Runx2 have been linked to skeletal defects during mammalian development and in human skeletal diseases. Reduced levels of Runx2 in osteoblasts are linked to different skeletal phenotypes, including symptoms of Cleidocranial Dysplasia (i.e., craniofacial abnormalities, supernumerary teeth), as well as absence of clavicles and delayed ossification of cranial sutures in mouse models. Elevation of Runx2 levels in transgenic mouse models is associated with an osteoporotic phenotype. Consequently, it is necessary to establish how osteoblasts succeed in continuously calibrating Runx2 levels to maintain a non-pathological state. Here we show that Runx2 transcriptionally stimulates expression of HSP70, which is a key specificity factor of the C-terminal HSP70 Interacting Protein (CHIP), as well as HSP40 and other HSP proteins, in the absence of external stress signals (e.g., heat shock) in osteoprogenitor cells. Expression of a functionally defective Runx2 protein does not alter HSP70 gene expression. Importantly, we find that HSP70 interacts with Runx2 protein by co-immunoprecipitation and that down-regulation of HSP70 by siRNA depletion increases steady-state levels of Runx2, while forced expression of HSP70 decreases Runx2 levels. We propose that Runx2 controls the levels of HSP70 as a component of an auto-inhibitory rheostat that maintains Runx2 proteins levels within a physiological range that is required for normal skeletal development and bone homeostasis. Interestingly, mutations in the chaperonin function of HSPs have been linked to skeletal dysplasias.

Disclosures: Andre Van Wijnen, None.


See Friday Plenary number FR0198.


A Tentative Role of S100A6 in Regulation of Osteoblastogenesis.Weida Lu*1, Yingchun Zhao2, Jing Xiao2, Robert Recker3, Gary Guishan Xiao1. 1Creighton University, USA, 2Creighton University Medical Center Osteoporosis Research Center, USA, 3Creighton University Osteoporosis Research Center, USA

A progressive adipogenesis with age in bone marrow is associated with reduced bone formation and loss of bone mass. In elderly adults and osteoporotic patients at all ages, increase of bone marrow adipose tissue and decrease of matrix structure are often observed. However, the role of bone marrow adipocytes in the aging bone tissues remains unknown. In our previous study, we used two different modes of co-cultures of adipocytes and osteoblasts, which were derived from human mesenchymal stem cells (hMSC), to study the effects of hMSC-derived adipocytes on osteoblastogenesis. We identified S100A6 in osteoblastogenesis, which is regulated by hMSC-derived adipocytes, suggesting the possible role of S100A6 in the regulatory effects of adipocytes on osteoblastogenesis. In this research, we aimed to study the mechanism of the inhibitory effects of adipoctyes on osteoblastogenesis. S100A6 is one of the members in S100 calcium-binding protein family, and is reported to be an oncogenic transcriptional factor playing a major role in cancer metastasis. However, the role of S100A6 in bone formation is unknown. We hypothesized that S100A6 may play a crucial role in regulation of osteoblastogensis by hMSC-derived adipocytes. To confirm the involvement of S100A6 in osteoblastogenesis, we transfected hMSC with siRNA of S100A6 during osteoblastogenesis, and found that the number of pre-osteoblasts decreased in cells transfected with siRNA of S100A6 compared to cells subjected to negative control. The alkaline phosphatase (ALP) positive area and ALP activity also decreased significantly after transfection of siRNA of S100A6. These suggest that S100A6 may be involved in osteoblastogenesis. To further study the mechanism of S100A6 regulating bone formation, we examined expression of bone formation marker such as Runx2, and found that Runx2 expression was significantly reduced in the transfected cells compared to the non-transfected cells. We also found that the reduced osteogenesis activity, measured by ALP positive area, its activity, and Runx2 expression level, was associated with the decreased expression level of β-catenin after transfection with siRNA of S100A6. Data from this study suggest that S100A6 may play an important role in adipocytes regulatory process of osteoblastogenesis through Wnt signaling pathway. This is the first study that tackles the mechanism of S100A6 regulating osteoblastogenesis.

Disclosures: Weida Lu, None.


See Friday Plenary number FR0200.


Bone Anabolic Factors are Specific to Osteoclasts and the Release of These Factors is Altered by Osteoclast Substrate.Catherine Cruger Hansen*1, Anita Neutzsky-Wulff2, Kim Andreassen3, Morten Karsdal1, Kim Henriksen1. 1Nordic Bioscience A/S, Denmark, 2Nordic Bioscience & University of Copenhagen, Denmark, 3Nordic Bioscience, Denmark

Normal bone remodeling is a coupled process in which bone formation by osteoblasts succeeds bone resorption by osteoclasts, ensuring the amount of resorbed bone is replaced by an equal amount of new bone. However, in osteopetrotic patients suffering from Autosomal Dominant Osteopetrosis II (ADOII) and Autosomal Recessive Osteopetrosis (ARO), an uncoupling phenomenon occurs in which bone resorption is decreased while bone formation remains normal or increased. Additionally, the number of non-resorbing osteoclasts is increased; suggesting bone anabolic factors derive from osteoclasts independent of bone resorption. The aim of this study was to investigate the specificity of the osteoclasts ability to induce bone formation.

Human monocytes were isolated from blood and differentiated into mature osteoclasts using M-CSF and RANKL. For some experiments, RANKL was substituted with OPG to prevent osteoclast differentiation. The cells were then cultured on different substrates including decalcified and non-remodeled substrates. Conditioned medium (CM) and corresponding non-conditioned medium (non-CM) were collected during culture. TRAP activity and bone resorption were used as measurements of osteoclast quality. The pre-osteoblastic cell line 2T3 was subsequently treated with 50% of CM or non-CM in addition to relevant controls. Induction of bone nodule formation was assessed by Alizarin Red-S staining / dye extraction.

CM collected from differentiating osteoclasts was shown to induce bone nodule formation 7+ days after initiation of differentiation while CM collected from macrophages did not. This indicates that secretion of bone anabolic factors is specific to osteoclasts. On non-remodeled dentin and decalcified bone, the osteoclasts were comparable to osteoclasts on calcified bone with respect to number and activity, yet the CMs collected from mature osteoclasts cultured on decalcified bone and dentin showed a 90% reduction in anabolic activity compared to calcified bone.

In conclusion, the secretion of bone anabolic factors is specific to osteoclasts. Furthermore, the secretion of bone anabolic factors by mature osteoclasts is attenuated by altered and non-remodeled substrates. Hopefully these findings, along with further investigations, can lead to a better understanding of the uncoupling between bone resorption and bone formation.

Disclosures: Catherine Cruger Hansen, Nordic Bioscience, 3


Bone Formation Is Predicted by Triiodothyronine and Lean Body Mass in Exercising Women with Hypothalamic Amenorrhea.Jennifer Scheid*1, Nancy Williams2, Gaele Ducher1, Mary Jane De Souza3. 1Penn State University, USA, 2The Pennsylvania State University College of Medicine, USA, 3Pennsylvania State University, USA

Decreased bone formation is related to an energy deficiency in exercising women with functional hypothalamic amenorrhea (FHA), but the underlying mechanism is unclear. This impact of energy deficiency on bone formation occurs despite an apparent osteogenic impact of exercise. The purpose of this study was to examine the relative contributions of energy related factors and factors reflective of the exercise stimulus to bone formation in exercising women with FHA. In this cross-sectional study, regularly exercising women were divided according to menstrual status: 1) Ovulatory (ExOV, n= 31), 2) Amenorrheic (ExAMEN, n=21). Menstrual status was assessed by daily urinary reproductive hormones for a menstrual cycle, or 28-day monitoring period if AMEN. Type I procollagen amino-terminal propeptide (PINP), bone-specific alkaline phosphatase (BSAP), ghrelin, triiodothyronine (TT3), peptide YY (PYY), and leptin were measured in the serum. Exercise minutes per week were assessed by exercise logs. Resting metabolic rate (RMR) was assessed by indirect calorimetry and adjusted for lean body mass. Lean body mass and BMD were assessed by DXA. The ExAMEN group had similar age (23.2 ± 0.8yr), weight (57.9 ± 1.1kg), lean body mass (41.7 ± 0.9kg), and exercise minutes (336.4 ± 44.3min) compared to ExOV, but lower BMI (20.3 ± 0.4 vs. 21.5 ± 0.3 kg/m2 p=0.020). ExAMEN also had lower BMD at the lumbar spine and hip (p<0.01). No differences were found in bone formation markers between the two groups. TT3 was lower (p=0.060) and PYY was higher (p<0.001) in the ExAMEN compared to the ExOV group. All other parameters of energy status were not different between the two groups. Independent variables in a regression model predicting PINP included TT3 and lean body mass which accounted for 34.3% of the variance in PINP (R2=0.371, p< 0.001). In the ExAMEN group alone the proportion of variance in PINP explained by TT3 and lean body mass went up to 67.6% (R2=0.710, p<0.001). Adjusted RMR (r=0.303, p=0.038) was the only energy related variable related to BSAP in the whole sample. Energy status, measured by TT3 and adjusted RMR, and lean body mass are independent predictors of bone formation in exercising women with FHA. Combining an exercise program that improves muscle mass and an adequate energy environment could potentially optimize the skeletal benefits in exercising women with FHA.

Disclosure: Supported by US DoD (PR054531) and CIHR.

Disclosures: Jennifer Scheid, None.


See Friday Plenary number FR0203.


See Friday Plenary number FR0204.


Enhancing Effects of HDAC Inhibitors on Osteoblastic Differentiation.Rabia Islam*1, Hyun-Jung Kim2, Won-Joon Yoon3, Hyun-Mo Ryoo3. 1School of Dentistry, Seoul National University, South korea, 2BioRunx Co. Ltd., South korea, 3Seoul National University, South korea

Recently HDACs have been proven to be associated with endochondral bone formation, osteoblast maturation as well as osteoclast survival. In addition to histone proteins, many non-histone proteins including Runx2 are also reported as substrates for HDACs resulting in regulation of cell growth and differentiation. Although HDAC inhibitors (HDI) including SAHA and depsipeptide are recently approved by FDA as anti-cancer therapy and have been extensively studied for cancer treatment, the studies of HDI for bone diseases are rare. The purpose of this study is to observe the effect of HDIs including SAHA and MS-275 on osteoblast differentiation using C2C12 and MC3T3E1 cells in order to give some clues to the therapeutic usage of HDIs in bone diseases. Firstly, treatment with SAHA or MS-275 induced ALP activity and showed synergistic effect with BMP2 in C2C12 cells. To investigate their effects on bone marker gene expressions during osteoblast differentiation, C2C12 and MC3T3E1 cells were treated with SAHA or MS-275 in doses optimized after cytotoxicity and efficacy observation with cytotoxicity assay and ALP staining, respectively, and then qRT-PCR was performed for cells in different stages of differentiation. Both SAHA and MS-275 significantly increased Runx2, Alp, and collagen type(I) gene expression. We also examined Runx2 protein level in MC3T3-E1 cells treated with or without SAHA or MS-275. Both HDIs also increased Runx2 protein expression. Further studies showed that SAHA and MS-275 increased Runx2 stability and acetylation, leading to transcriptional activation of Runx2. Taken together, SAHA and MS-275 has enhancing effect on osteoblast differentiation, indicating that they might be applicable for therapeutic usage in bone diseases.

Disclosures: Rabia Islam, None.


See Friday Plenary number FR0206.


See Friday Plenary number FR0207.


Human Bone Resorption Lacunas Contain Specific Glycan Epitopes.Meeri Keinanen*1, Jarkko Rabina2, Leena Valmu2, Petri Lehenkari1, Juha Tuukkanen1. 1University of Oulu, Finland, 2Finnish Red Cross Blood Service, Finland

The aim of this study was to characterize the glycan epitopes in human bone located at the resorption lacunas. We hypothesize that the glycans have a particular function in the regulation of bone remodeling.

Human bone slices resorbed with human bone marrow -derived osteoclasts were stained with various lectins. The affinity of various fluorescently labeled lectins towards resorption pits was studied before and after cleaving the glycan epitopes by enzymatic treatments. Results were visualized using confocal microscopy.

Resorption lacunas were positively labeled when treated with sialic acid binding lectins. Also, treatment with N-acetylglucosamine-, mannose- and galactose-specific lectins gave positive result in stainings. Treatment of the resorbed human bone slices with two different sialidases and N-glycosidase changed the binding of lectins, which was observed as weaker signals in confocal microscopy.

These observations show that resorption lacunas contain various glycan epitopes that might have an effect on the subsequent cellular attachment and regulation of the bone formation process.

Disclosures: Meeri Keinänen, None.


See Friday Plenary number FR0209.


Osteoblast Targeted Disruption of Kremen Increases Bone Accrual in Mouse.Zhendong Zhong*1, Cassandra R. Zylstra2, Bart Williams2. 1Van Andel Institute, USA, 2Van Andel Research Institute, USA

Kremen proteins are single-pass transmembrane proteins encoded by two genes in mammals. The molecular functions of Kremens are still being elucidated, although some studies have linked its function to regulation of the Wnt/B-catenin signaling pathway. Recently, Ellwanger et al. (Mol. Cell. Biol., 28:4874, 2008) created mice homozygous germline deletions in Kremen1 and Kremen2 and found that mice lacking both genes, both not either one singly, developed increases in bone volume and bone formation. To gain insight what cell type was responsible for this increase in bone mass, we created mice homozygous for a germline inactivation of Kremen1 that also carry an osteoblast-specific deletion in Kremen2. Analysis of these mice reveals depletion of Kremens in osteoblast can significantly increase the whole body bone density, tibia cortical bone thickness and BV/TV ratio. Thus, our data supports the idea that functions of Kremen within the osteoblast are associated with regulation of normal bone mass.

Disclosures: Zhendong Zhong, None.


See Friday Plenary number FR0211.


Recapturing Fracture Repair and Bone Formation using a Periosteal Derived Cell Population.Scott Roberts*1, Eline Desmet1, Jan Schrooten1, Frank Luyten2. 1K.U. Leuven, Belgium, 2University Hospitals KU Leuven, Belgium

Fracture repair involves several key processes including inflammation, callus formation and bone remodeling. It has previously been shown that the integrity of the periosteum, a fibrous tissue which covers all bones, is required for successful bone repair. This is mainly due to populations of osteo/chondro progenitors being resident within the periosteum which mediate bone healing. In this study a population of human periosteal derived cells (hPDCs) was isolated and pooled from six healthy donors by collagenase digestion. These cells were shown, by FACS, to be positive for the mesenchymal stem cell markers CD73, CD90 and CD105 (100%) as well as the endothelial progenitor cell marker CD146 (4.9%) and the hematopoietic progenitor marker CD34 (45%) indicating the heterogeneous nature of this population. To mimic the environment which these cells would be exposed to during fracture healing, five different commercially available orthopedic 3D matrices composed of calcium phosphate particles in an open collagen network (NuOssTM, CopiOsTM, Bio-Oss®, CollagraftTM and Vitoss®) were selected. Cells were successfully seeded onto the matrices with efficiencies of greater than 90%. Gene expression analysis for the osteo/chondro transcription factors Runx2 and Sox9, following cell seeding and 24 hours and 7 days of passive culture in vitro, showed similar levels of expression, indicating the persistence of bone forming progenitors. Bone formation was observed after eight weeks following implantation, subcutaneously in NMRI nu/nu mice, with hPDCs loaded on NuOssTM, Bio-Oss®, CollagraftTM and Vitoss® matrices. NuOssTM provided the most optimal environment for bone formation with 13% bone observed within the total volume of implant, when compared to Bio-Oss® (5.3%), Vitoss® (3.2%) and CollagraftTM (1.9%). Interestingly CopiOsTM combined with hPDC cells did not exhibit any bone formation. The formation of bone marrow sinusoids were observed throughout the NuOssTM/hPDC implant, in contrast to other tested cell/matrix combinations, suggesting bone formation through a chondrogenic route as seen during fracture repair. No bone formation was observed when these matrices were implanted in the absence of cells. This study highlights the importance of optimizing the cell-matrix combination, whilst also considering the host interaction when investigating bone forming potential of progenitor cell populations.

Disclosures: Scott Roberts, None.


See Friday Plenary number FR0213.


Sodium/Proton Exchange is a Major Regulated Mechanism Supporting Bone Mineral Deposition.Li Liu*1, Peter A. Friedman1, Paul H. Schlesinger2, Harry Blair1. 1University of Pittsburgh, USA, 2Washington University, USA

The osteoblast regulates bone deposition in part by secretion of nucleating proteins for hydroxyapatite, but mineralization proceeds when these proteins are absent. Because bone mineral evolves ∼ 1.5 moles of protons per mole of Ca2+ precipitated, we hypothesized that facilitated acid transport supports mineralization. Using genome-wide expression in mineralizing human osteoblasts, we identified candidate H+ transporters; databases on protein interactions were used to identify potential transport systems. Acid transport and expression of proteins was measured in mineralizing osteoblasts. We find that osteoblast H+ transport involves at least two types of membrane transport. To facilitate mineral deposition at the zone of mineral deposition, there is vectorial H+ uptake by vesicles, possibly a subset of matrix vesicles, which also express alkaline phosphatase to elevate phosphate. This transport was demonstrated by weak base accumulation by intact membrane vesicles isolated from mineralizing bone. Transport requires a K+ gradient but it is independent of ATP; H+ uptake is sensitive to Zn2+ but not to H+pump inhibitors; the identity of this H+ transporter is uncertain. Bulk acid flux at the osteoblast basolateral surface proceeds by a second mechanism, vectorial facilitated H+ transport mediated by Na+/H+ exchanger-6 (NHE6). NHE6 mediates cation exchange at high capacity across the gap-junction connected osteoblasts during bone synthesis. Osteoblasts express both NHE6, a specialized NHE, and NHE1, widely expressed. A chaperon protein, RACK-1, trafficks NHE6 to the cell membrane in osteoblasts. Nonmineralizing MG63 osteoblasts express low levels of NHE6 and have low Na+-dependent H+ transport, shown by whole cell pH in response to 40 mM proprionate, while mineralizing osteoblasts exhibit rapid Na+/H+-exchange and high levels of NHE6. When MG63 is transfected with NHE6, Na+/H+-exchange is similar to mineralizing osteoblasts. Knockdown of NHE1 has minimal effects on pH change with acid loading. Osteoblasts also express the PDZ domain-containing Na+/H+ exchange regulatory factor-1 (NHERF-1), a PTH-receptor binding organizing protein that complexes with SLC9-family NHEs including NHE6, and RACK-1, regulating them. In NHERF-1-/- mice an unusual osteomalacia with broadened mineral deposition zones and ∼30% defect in matrix and mineral synthesis occur. We conclude that NHE-dependent H+ transport, mediated mainly by NHE6, supports hydroxyapatite deposition in bone.

Disclosures: Li Liu, None.


Stimulation of Bone Formation in Cortical Bone of the Mice Treated with a Novel Bone Anabolic Peptide with Osteoclastogenesis Inhibitory Activity.Hisataka Yasuda1, Atsushi Inagaki2, Kaoru Mori2, Yuriko Furuya*2. 1Oriental Yeast Company, Limited, Japan, 2Oriental Yeast Co., Ltd., Japan

In spite of making every effort in developing bone anabolic drugs the only clinically available one is PTH. The major difficulty is lack of clarification of mechanisms regulating osteoblast (Ob) differentiation and bone formation. Here we report that a peptide known to abrogate osteoclast (Oc) differentiation in vivo via blocking RANKL-RANK signaling surprisingly exhibited bone anabolic effect in vivo. In the previous study administration of the peptide significantly inhibited bone loss by reducing NOc/BS and OcS/BS in trabecular bone in OVX mice. To investigate the effects of the peptide it was administered subcutaneously to mice three times per day for 5 days at a dose of 10mg/kg. DXA and pQCT analysis showed that the peptide augmented BMD significantly in cortical bone not in trabecular bone. Histomor-phometrical analysis showed that the peptide had little effect on NOc/BS and OcS/BS in distal femoral metaphysis but markedly increased MAR and BFR in femoral diaphysis. Our findings were inconsistent with the previous report that the peptide acted as an inhibitor of bone resorption and suggested that the peptide exerted its activity through unknown mechanisms. To clarify them, we investigated the effects of the peptide on Ob differentiation/mineralization with MC3T3-E1 (E1) cells and those on Oc differentiation with RAW264 cells and sRANKL. The peptide markedly increased ALP activity and decreased TRAP activity in each cell culture in a dose-dependent manner, respectively. In addition, the peptide stimulated mineralization evaluated by alizarin red staining. Increases in ALP activity and mineralization were similarly observed in human mesenchymal cell culture under osteoblastic conditions. Increases in mRNA expression of BMP4, CTGF, IGF1, IGF2, ALP, and osteoclacin were observed in E1 cells treated with the peptide for 96 h in GeneChip analysis. Addition of p38 MAP kinase inhibitor reduced ALP activity in E1 cells treated with the peptide, suggesting a signal through p38 was involved in the mechanisms. Taken together, the peptide abrogated osteoclastogenesis by blocking RANKL-RANK signaling and stimulated Ob differentiation/mineralization at the same concentration with unknown mechanism in vitro. However, in our experimental conditions the peptide exhibited bone anabolic effect dominantly in vivo. Since the peptide was known to bind RANKL, we hypothesized that the peptide shows the bone anabolic activity with reverse signaling through RANKL in Obs.

Disclosures: Yuriko Furuya, Oriental Yeast Co., Ltd., 3

This study received funding from: Oriental Yeast Co., Ltd.


Suppression of NADPH Oxidases Prevents Chronic Ethanol-Induced Bone Loss.Jin-Ran Chen*1, Oxana P. Lazarenko2, Kelly Mercer3, Kartik Shankar2, Michael L. Blackburn2, Thomas M. Badger2, Martin J. Ronis2. 1University of Arkansas for Medical Science, Arkansas Children's Nutrition Center, USA, 2University of Arkansas for Medical Sciences/Arkansas Children's Nutrition Center, USA, 3Arkansas Children's Nutrition Center, USA

Since the molecular mechanisms through which chronic excessive alcohol consumption induces osteopenia and osteoporosis are largely unknown, potential treatments for prevention of alcohol-induced bone loss remain unclear. We have previously demonstrated that, chronic ethanol (EtOH) treatment leads to accumulation of reactive oxygen species (ROS) in osteoblasts dependent on NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox). EtOH-induced ROS production might mediate both inhibition bone formation and increases in bone resorption. Using total enteral nutrition in a cycling female Sprague-Dawley rat model, we found that EtOH infusion for 4 weeks reduced bone mass (p<0.05) assessed by peripheral quantitative computerized tomography (pQCT) analysis. Co-administration of diphenylene iodonium chloride (DPI) a pan Nox inhibitor by daily s.c. injection of 1 mg/kg/d, abolished EtOH-induced bone loss. Static histomorphometric analysis revealed that EtOH effected both osteoblast and osteoclast indices (p<0.05). EtOH decreased bone volume and the number of osteoblasts, whereas it increased osteoclast number, bone surface covered by osteoclasts and the eroded bone surface characteristic of increased osteoclastic activity. DPI was able to normalize both osteoblast and osteoclast indices affected by EtOH. EtOH-induced bone loss was associated with up-regulation of mRNA levels of all three Nox subtypes 1, 2, 4 and RANKL (receptor activator of NF-kB ligand) in bone, and EtOH-induced RANKL promoter activity in vitro ST2 cell culture (p<0.05). To confirm Nox activation is truly associated with EtOH-induced bone loss, we fed a 36% alcohol containing liquid diet to nicotinamide dinucleotide phosphate oxidase-deficient p47phox-/- female mice for 6 weeks. In vivo systemic CT scan analysis revealed that, in wild type animals, EtOH reduced bone mineral density (BMD) compared to a pair fed group (p<0.05). In contrast, EtOH failed to down-regulate BMD in p47phox-/- female mice compared to their pair fed controls. Bone marrow cells taken from p47phox-/- female mice cultured with 50 mM EtOH for 24 h failed to induce RANKL gene expression but EtOH was able to induce RANKL gene expression in cells from wild type animals (P< 0.05). These data suggest that inhibition of Nox expression or activity may be new target for prevention or treatment of chronic EtOH-induced bone loss, and perhaps other conditions resulting in oxidative stress associated bone resorption such as aging. This study was supported by NIH grant RO1 AA18282 to MJR.

Disclosures: Jin-Ran Chen, None.


See Friday Plenary number FR0217.


See Friday Plenary number FR0218.


Duality of TRIP-1 Function in Regulating Osteoblast Activity In Vitro.Diana Metz-Estrella*1, Tzong-jen Sheu2, J. Edward Puzas3. 1University of Rochester School of Medicine & Dentistry, USA, 2Univesity of Rochester School of Medicine & Dentistry, USA, 3University of Rochester School of Medicine, USA

The importance of the “TGFβ receptor interacting protein” (TRIP-1) in bone remodeling was first recognized when it was found to interact with tartrate resistant acid phosphatase (TRAP). It has been suggested that the interaction of these two proteins activate the TGFβ pathway and increase markers of osteoblast differentiation. Interestingly, TRIP-1 may also have another role inside the cell since it shares a perfect homology to a subunit of the eukaryotic initiation factor 3 complex (i.e. eIF3i). Therefore, we believe that during bone remodeling TRIP-1 integrates extracellular signals in osteoblasts and mediates regulation of protein translation. Consequently, this would lead to a control over osteoblast proliferation and differentiation. To evaluate the role of TRIP-1 in osteoblasts we transiently transfected siRNA against TRIP-1 into primary rat calvarial osteoblasts in vitro. These cells were then cultured in osteoblast differentiation media for 5 days. Cells transfected with TRIP-1 siRNA show an 84% decrease in TRIP-1 protein levels at day 3 after transfection compared to cells transfected with a control siRNA. In addition, cells transfected with TRIP-1 siRNA presented reduced alkaline phosphatase staining and exhibited a 60% decrease in alkaline phosphatase activity when compared to control cells. Using qRT-PCR we found that cells transfected with a TRIP-1 siRNA have decreased expression of osteoblast differentiation markers (i.e., collagen I, alkaline phosphatase, osteopontin and osteocalcin). Interestingly, TRIP-1 siRNA visibly reduced cell number in this assay. In relation to this, cyclin D1 protein levels were dramatically decreased in cells transfected with the TRIP-1 siRNA. These data suggested a role for TRIP-1 in cell cycle regulation. To address this possibility, cells transfected with control or TRIP-1 siRNA were labeled with BrdU and sorted by flow cytometry to assay for possible cell cycle arrest. The results suggested that cells transfected with a TRIP-1 siRNA are arrested in the S and G2M phases of the cell cycle. Moreover, luciferase reporter assays showed that knocking down TRIP-1 interferes with the normal signaling of TGFβ and BMP pathways. In conclusion, our data show that TRIP-1 is an essential protein for normal osteoblast differentiation and proliferation and through its interaction with TRAP may be one mechanism by which site-directed bone formation can occur.

Disclosures: Diana Metz-Estrella, None.


See Friday Plenary number FR0220.




Fibroblast Growth Factor Receptor 2 Expression in Pre-osteoblasts Requires the PBAF Chromatin-remodeling Complex.Fuhua Xu*, Stephen Flowers, Elizabeth Moran. Department of Orthopaedics, NJMS-UH Cancer Center, UMDNJ, USA

Unfolding of the gene expression program that converts precursor cells to osteoblasts is critically dependent on the nucleosome remodeling activity of the mammalian SWI/SNF complex. This ATPase-powered complex regulates stage-specific gene expression in all tissues. Understanding how it acts to maintain or advance specific stages of differentiation is basic to understanding molecular mechanisms of tissue development. The complex can be powered by either of two ATPases: BRM or the BRM-related gene product BRG1. We have recently shown that BRM-specific complexes restrain osteoblast differentiation, helping to maintain the committed precursor state until the pre-osteoblasts receive appropriate signals for differentiation (Flowers et al. 2009. J. Biol. Chem. 284:10067-75). In contrast, BRG1-containing SWI/SNF (also called BAF) generally acts to induce osteogenic genes.

BRG1 also participates in a related complex, designated PBAF. BAF and PBAF share most subunits, but can be distinguished by the choice of another component that also occurs as related alternatives: the ARID family subunits. These are large proteins containing interactive domains for associated proteins, as well as the ARID-motif, which specifies a DNA binding domain of loose specificity. SWI/SNF complexes contain ARID1A or ARID1B. PBAF contains ARID2. To determine whether the PBAF complex is required in osteoblast differentiation, we used an shRNA approach to target ARID2 in MC3T3-E1 pre-osteoblasts.

Cells stably depleted of ARID2 are sharply impaired for induction of the osteoblast specific marker alkaline phosphatase, and show delayed progression to a mineralization phenotype. BRG1-depleted cells are more severely impaired, showing essentially complete failure to differentiate. The results indicate that both BAF and PBAF complexes contribute to induction of the osteoblast phenotype.

Gene array analysis suggests that the BRG1-containing nucleosome remodeling complexes also play an important role in precursor cell commitment to the osteoblast lineage. Analysis of osteogenic gene expression in BRG1-depleted MC3T3-E1 cells reveals several genes expressed in pre-osteoblasts whose expression declines without BRG1. These include BMP4 and fibroblast growth factor receptor type-2 (FGFR2). Preliminary analysis of FGFR2 suggests that maintenance of its expression in pre-osteoblasts requires the PBAF complex.

Disclosures: Fuhua Xu, None.


See Friday Plenary number FR0223.


See Friday Plenary number FR0224.


See Friday Plenary number FR0225.


Phosphorylation-Dependent SUMOylation Regulates the Activity of the αNAC Transcriptional Coactivator.Omar Akhouayri1, Rene St-Arnaud*2. 1Shriners Hospital for Childrent, Canada, 2Shriners Hospital for Children & McGill University, Canada

To act as a transcriptional coactivator of c-Jun-dependent osteocalcin gene transcription in osteoblasts, αNAC shuttles between the cytoplasm and the nucleus. It has been shown that the subcellular localization of the protein is regulated through differential phosphorylation. We have identified an additional mode of post-translational modification of αNAC through covalent attachment of the Small Ubiquitin-like MOdifyer, SUMO. Since sumoylation has been shown to differentially impact on the stability, localization, and activity of transcriptional regulators, we have characterized the SUMO acceptor site on the protein and examined which parameters are affected by sumoylation of αNAC. We first used a commercial assay providing the components of the sumoylation cascade to show that recombinant αNAC could be sumoylated in vitro. Immunoprecipitation followed by immunoblotting with antiSUMO antibodies then confirmed that αNAC is conjugated to SUMO1 in cultured osteoblasts and in calvarial tissue. The amino acid sequence of αNAC contains one copy of the composite ‘phospho-sumoyl switch’ motif, ΨKxExxS (where Ψ is a hydrophobic amino acid and K is the site of SUMO conjugation), that couples sequential phosphorylation and sumoylation. We have engineered site-specific mutants in which the putative lysine sumoylation site (K127) and the potential serine phosphoregulation site (S132) were mutated alone or in combination. Mutation K127R inhibited sumoylation, demonstrating that it is the primary SUMO conjugation site. Interestingly, mutation S132A reduced the sumoylation of αNAC, while the phosphomimetic S132D mutation increased it, confirming that the sumoylation of αNAC is regulated through differential phosphorylation of serine 132. The subcellular localization, half-life, or DNA-binding activity of αNAC were not affected by mutation at residues K127 or S132. Non-sumoylatable forms of αNAC (K127R, S132A, and K127R/S132D) were unable to coactivate c-Jun-mediated transcription, while the hyper-sumoylated S132D mutant had increased coactivating potential. Our studies characterized a novel post-translational modification of the αNAC coactivator and identified one of the rare transcriptional regulators whose activity is potentiated, not inhibited, by sumoylation. The characterization of the upstream kinase involved in the regulation of αNAC sumoylation will identify an additional pathway involved in the regulation of osteoblastic gene transcription.

Disclosures: Rene St-Arnaud, None.


Runx2 Is Necessary for Smurf1 Expression in Osteoblastic Cells.Jeong-Hwa Baek1, Gwan-Shik Kim2, Hyun-Mo Ryoo3, Kyung Mi Woo2, Kyunghwa Baek2, Hye Lim lee*2. 1Seoul National University School of Dentistry, South korea, 2Seoul Nat'l University, South korea, 3Seoul National University, School of Dentistry, South korea

Smurf1 is an E3 ubiquitin ligase and involved in degradation of BMP signaling molecules such as Smad1, Smad5 and Runx2. Previously we have reported that TNF-α stimulates Smurf1 expression via JNK activation and subsequent AP-1 binding to Smurf1 promoter in C2C12 cells. However, TNF-α-induced Smurf1 expression was not observed in Runx2-nulled mouse calvarial cells. Therefore, we investigated the role of Runx2 in Smurf1 expression. Runx2 overexpression induced basal Smurf1 expression and rescued TNF-α-mediated induction of Smurf1 in Runx2-nulled cells. In addition, neither Smurf1 nor Runx2 was expressed in C3H10T1/2 cells. However, Smurf1 expression was induced by exogenous Runx2 expression, not by c-Jun overexpression nor by TNF-α in these cells. Runx2 increased Smurf1 reporter activity in a dose-dependent manner. Studies using Smurf1 promoter mutants have shown that among 4 putative Runx2 binding sites (∼2.7 kb) the most proximal one is important to Runx2-mediated Smurf1 expression. Runx2 directly bound to Smurf1 promoter region, and TNF-α enhanced Runx2 binding to Smurf1 promoter region, which was confirmed by ChIP assay. These results suggest that Runx2 is necessary for Smurf1 expression in osteoblastic cells.

Disclosures: Hye Lim lee, None.


See Friday Plenary number FR0228.


The Osteocyte marker, Podoplanin, is Expressed in Transformed Osteoblasts and is Regulated by AP-1.Takeshi Kashima*1, Akiko Kunita2, Masashi Fukayama2, Agamemnon Grigoriadis3. 1Nuffield Orthopaedic Centre, United Kingdom, 2University of Tokyo, Japan, 3King's College London, United Kingdom

Podoplanin (E11 antigen) is a type-I transmembrane sialomucin-like glycoprotein that has been shown to be highly expressed in osteocytes both in vivo and in vitro, but not in active bone forming-osteoblasts or chondrocytes. The regulation of podoplanin expression is not well understood, although it has been reported recently that the c-Fos proto-oncogene directly regulates podoplanin expression in a skin cancer model. In this study, we aim to examine whether or not the AP-1 protein, c-Fos, upregulates podoplanin expression in transformed bone and cartilage cells, using previously isolated c-Fos-inducible MC3T3-E1 osteoblastic cells (AT9.2) and ATDC5 chondrogenic cells (DT12.4) as well as osteosarcoma cells derived from c-Fos transgenic tumors.

Induction of exogenous c-Fos in AT9.2 cells resulted in a stimulation of podoplanin expression as well as TGF-b1, a known c-Fos/AP-1 target gene. Western blot analysis of c-Fos transgenic osteosarcoma cell lines confirmed high podoplanin levels in cells overexpressing c-Fos. Further, immunohistochemical analysis showed that podoplanin was intensely positive in c-Fos osteosarcoma xenograft tumor cells. To clarify the cell type specificity of podoplanin expression, immunohistochemistry was performed in normal bone and primary c-Fos transgenic osteosarcomas. Podoplanin was expressed in osteogenic tumor cells and in cells within the tumor bone matrix resembling osteocytes. All tumor cells strongly expressed c-Fos protein as well as TGF-b1. Interestingly, no podoplanin expression was observed in chondrogenic areas within the tumors, and this was confirmed by the lack of correlation between c-Fos and podoplanin expression in c-Fos-inducible DT12.4 chondrocytes.

These data imply that c-Fos and TGF-b1 play a role in the regulation of podoplanin expression in c-Fos overexpressing / transformed osteoblasts, and suggest that the specificity of podoplanin expression is expanded along the osteogenic lineage in pathologic osteoblasts.

Disclosures: Takeshi Kashima, The Japanese Ministry of Education, Culture, Sports, Science and Technology, 2


The RUNX2 Cistrome Defines a Regulatory Target Genome Responsible for the Osteoblast Phenotype.Mark Meyer*, J. Pike. University of Wisconsin-Madison, USA

RUNX2 is a master regulator of osteoblast differentiation and function whose primary role is to integrate the complex activities of a collection of transcription factors through target genes and multiple signaling pathways. While many osteoblast genes are targets of RUNX2, the RUNX2 regulome in osteoblast precursors has not been assessed. To define this set of RUNX2-responsive genes, we conducted a ChIP coupled to microarray (ChIP-chip) and sequencing (ChIP-seq) analysis of RUNX2 binding activity on a genome-wide level in mouse MC3T3-E1 cells and correlated these sites of RUNX2 action with enhancer-specific histone marks. RUNX2 was found to be pre-bound to approximately 4822 sites on the mouse genome. While 15% of these sites were within 5 kb of promoters, most were located within introns (36%) or at striking distances (41%) from transcriptional start sites of regulated genes. Two or more binding sites were frequently observed at target genes and over 70% contained a RUNX2 response element. The majority of these sites correlated with elevated levels of histone H4 acetylation, a covalent mark of regulatory activity. Interestingly, RUNX2 binding was specifically observed at both RUNX2 promoters, suggesting that RUNX2 auto-regulates its own expression in osteoblasts. RUNX2 binding was also evident at known sites on genes that were previously characterized as RUNX2 targets, including Bglap2, Spp1, Alkp, Col1a1 and Fgf18, thereby validating the many additional sites found to be direct targets of RUNX2 action. RUNX2 was also found at genes involved in cell cycle control, including Ccnd1, Ccnd2 and Cdk1a. Perhaps most interesting was the finding that RUNX2 localized directly to a surprisingly large repertoire of downstream transcription factor genes involved in osteoblast function. These include genes for Sox9 (but not Sox5 or 6), Sp7 and Atf4 (but not Atf1 or 2) but also genes for C/ebpα, β and δ, Runx2 isotype genes 1 and 3, Vdr, c-Fos, c-Jun and Nfatc1. We also conducted genome-wide ChIP-seq analysis for C/EBPβ and the 1,25(OH)2D3-activated VDR/RXR heterodimer, two transcription factors known to interact directly with RUNX2. Interestingly, while C/EBPβ bound to over 6440 sites in the genome, 1704 of those sites were associated with pre-bound RUNX2. A similar relationship was found with VDR/RXR. We conclude that RUNX2, through its actions on multiple target genes, is indeed a master regulator of the osteoblast phenotype.

Disclosures: Mark Meyer, None.


Cell Surface ATP Synthase: A Novel Mechanism for Extracellular ATP synthesis in Osteoblasts.Shyama Majumdar*1, Vimal Gangadharan1, Kirk Czymmek2, Randall Duncan1. 1University of Delaware, USA, 2University of Delaware & Delaware Biotechnology Institute, USA

ATP, the energy currency of a cell, has many functions other than providing cellular energy; including extracellular signaling in a variety of tissues. We, and others, have previously demonstrated that osteoblasts, osteocytes and osteoclasts use vesicular ATP release to respond to numerous stimuli, including mechanical loading. In addition, these cells have significant basal release of ATP. However, the mechanism for basal release, as well as packaging of ATP into vesicles, remains unknown. We hypothesize that F1FoATP synthase is present on the cell membrane of osteoblasts and is functionally active for basal signaling in unstimulated osteoblasts. Furthermore, we hypothesize that ATP synthase is present in cytosolic vesicular membranes to package ATP in preparation for stimulated release. Immunofluorescence, cell surface protein isolation and membrane fractionation techniques were employed to demonstrate the presence of cell surface ATP synthase in MC3T3-E1 pre-osteoblasts as well in primary mouse calvarial osteoblasts. Using a luminescence based ATP assay to measure extracellular ATP synthesized in the presence of varying doses of extracellular ADP, we found that ATP synthase produces ATP in a dose dependent manner and that this synthesis is attenuated when the functional a and β subunits are blocked using blocking antibodies or angiostatin. We have demonstrated that membrane ATP synthase uses a similar hydrogen ion gradient to the mitochondrial ATP synthase by creating a hydrogen ion gradient across the cell membrane. Reducing extracellular pH significantly increased ATP synthesis. To determine the role of membrane ATP synthase during mechanical stimulation, we subjected MC3T3-E1 cells to fluid shear stress (FSS). ATP synthase protein levels increased within 1 to 5 minutes of application of fluid shear stress indicating a mechanism whereby additional ATP synthase is incorporated into the membrane in response to FSS. These data suggest that ATP synthase is present on the cell membrane of osteoblasts and is functionally active. We also predict ATP synthase is present on the vesicular membrane and may be responsible for ATP packaging into vesicles. These data suggest a novel mechanism for the basal release of ATP and the regulation of purinergic signaling in bone cells.

Disclosures: Shyama Majumdar, None.


See Friday Plenary number FR0232.


Heterodimerization of Purinergic ATP Receptors in Osteoblasts.Roy Choi*, Gallant Chan, Karl Tsim. The Hong Kong University of Science & Technology, Hong kong

Purinergic ATP receptor superfamily contains P2Y (G-protien-coupled receptor) and P2X (ligand-gated ion channel) subfamilies. The metabotropic P2Y receptors have been shown to involve numerous physiological processes, such as neurotransmission, neuromodulation, immunomodulation, and bone formation. Among them, activation of P2Y1 receptor (one subtype of P2Y subfamily) indirectly increases osteoclast formation and bone resorption via the stimulation of RANKL in osteoblasts, while application of P2Y2 receptor agonist blocks bone formation process, suggesting that the extracellular nucleotides can function locally as a crucial negative modulator in bone metabolism. In the present study, we focused on the interaction of P2Y1 and P2Y2 receptors in osteoblasts. Specifically, the possibility of forming P2Y1-P2Y2 receptor heterodimer in osteoblasts would be discussed, which could be used to support the significance and physiological function of receptor dimerization within P2Y receptor subfamily.

Acknowledgement: This study was supported by the RGC grant (660409) to RCYC.

Disclosures: Roy Choi, None.


See Friday Plenary number FR0234.


See Friday Plenary number FR0235.


See Friday Plenary number FR0236.


Adipogenic Cells as Primary Target of Strontium?Carole FOURNIER*1, Anthony Perrier2, Laurence Vico3, Alain Guignandon1. 1INSERM U890, France, 2Laboratoire de Biologie du Tissu Osseux, France, 3University of St-Etienne, France

Strontium Ranelate is used to treat osteoporosis and its ability to reduce fracture risks is clearly established. If Strontium (Sr) effects are late and positive on osteoblastogenesis and/or early and negative on adipogenesis is still an open question. Our work hypothesis is that SR inhibits commitment of stromal cells towards adipogenesis by regulating the balance between Runx2 and PPARy2 master genes. We treated multipotent mesenchymal cell (C3H10T1/2) with various doses of Sr (0 to 3mM) for limited durations (max 5 days) in various differentiation media (neutral, osteoblastic, adipogenic, or both). Whatever the conditions, we found that Sr inhibits PPARy2 expression without major Runx2 alteration. After a one-day-culture PPARy2 inhibition was dose-dependent and reached 80% of inhibition at 3mM of Sr in an adipogenic context. This observation may explain why Sr could be particularly efficient in physiological challenges enhancing bone marrow adiposity and/or oxidative stress such as aging, ovariectomy, and immobilisation. In an attempt to mimic such challenges we treated our cells with rosiglitazone (Rosi) a potent PPARy2 co-activator. We confirmed that Sr (3mM) was able to reduce Rosi-induced adipogenic differentiation by downregulating PPARγ2 expression by 50%. We also showed by immunodetection that PPARγ2 located in the nucleus of preadipocytes, remains in the cytoplasm under 3mM of Sr. At day 5, C3H10T1/2 treated with both Rosi and 3 mM Sr showed expression levels of adipogenic genes (PPARγ2 and C/EBP) and osteoblastic genes (Runx2 and PAL) similar to those of uncommitted cells as seen in C3H10T1/2 cultivated in αMEM alone. We also showed that Rosi alone stimulates catalase (a PPARγ-dependent gene reducing oxidative stress) expression by 300%. Further treatment with 3 mM strontium reduces catalase expression to the level of uncommitted cells. In the same culture conditions, we observed that Rosi induces an upregulation of SOD1 and 2 (cytosolic and mitochondrial antioxidant enzymes, respectively) and that further strontium treatment normalizes these alterations. The upregulation of antioxidant enzymes under Rosi likely reflects the important production of reactive oxygen species, which is counteracted by Sr treatment. These complex changes suggest that strontium acts in part through PPARγ2, key controller of the osteoblastic/adipogenic balance in which actors of oxidative stress is involved.

Disclosures: Carole FOURNIER, None.


See Friday Plenary number FR0238.




See Friday Plenary number FR0240.


Differences in Oxygen Consumption Rate of Osteoblast Lineage Cells in Rats Bred for High and Low Aerobic Capacity.Riyad J. Tayim*1, Jacqueline Cole2, Neil R. Halonen1, Grant C. Goulet1, Lauren G. Koch3, Steven L. Britton3, Ronald Zernicke2, Andrea Alford2, Kenneth Kozloff1. 1University of Michigan Department of Orthopaedic Surgery, USA, 2University of Michigan, USA, 3University of Michigan Department of Anesthesiology, USA

Cells of the osteoblast lineage are particularly responsive and sensitive to their local oxygen environment, and their ability to improve their intracellular aerobic metabolism in response to exercise may have important downstream effects on bone cell function and skeletal phenotype. In this study, we tested the influence of inherent aerobic capacity on bone metabolism independent of applied mechanical loading. Using the Koch-Britton selective breeding rat model of high capacity (HCR) and low capacity (LCR) runners, an intrinsic 5-to-7 fold functional genomic difference in aerobic exercise capacity exists between non-trained animals after 20 generations of selection. HCR have consistently demonstrated heightened skeletal mineralization and osteoblast activity correlated with their inherent aerobic capacity. In the present study, we compared cellular oxygen metabolism between HCR and LCR osteoblast lineage cells during osteoblast differentiation in vitro. Bone marrow stromal cells (BMSCs) were harvested from the femora and tibiae of 8.5 month, female, generation 25 rats (N=3), and were cultured under standard conditions for 12 days followed by osteoblast differentiation. The Seahorse Bioscience XF24 Analyzer was used to assess cellular aerobic capacity on Day 0, 3, 7, 10, 14, 21, and 28 of the differentiation time course. Basal respiration and glycolysis were assessed and normalized to cell number through time-resolved measures of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). OCR and OCR/ECAR ratios in both HCR and LCR cells suggested less glycolytic and more aerobic cell metabolism with differentiation and matrix production followed by a return to glycolysis as cells became engulfed in their ECM. HCR cells consumed less oxygen per cell than LCR through Day 14, suggesting a more efficient phenotype. With the addition of oligomycin, HCR cells showed an increase in percent oxygen consumed for ATP production at Day 14 vs. LCR, but this pattern was reversed by Day 28. The HCR/LCR selective breeding rat model allows us to investigate how intrinsic differences in aerobic metabolism result in differences in cellular aerobic metabolism within osteoblast lineage cells. Thus we are able to define cellular parameters that may be responsible for the heightened mineralization and osteoblast activity found in these animals.

Disclosures: Riyad J. Tayim, None.


Generation and Characterization of iPS Cells from CMD Patients and Healthy Controls.I-Ping Chen*1, Xiaonan Xin1, Mary-Louise Stover2, Shuning Zhan2, Jonathan Kantor3, Ernst Reichenberger1, Alexander Lichtler1. 1University of Connecticut Health Center, USA, 2UCHC, USA, 3North Florida Dermatology, USA

Studying rare genetic bone disorders is clinically significant due to the lifetime debilitating impact they have on patients and limited treatment options for these disorders because little of their pathogeneses is known. Research on rare disorders is not only beneficial for future treatment of patients, but contributes to the understanding of important biological mechanisms in bone development and remodeling. Obstacles for research in this field include unavailability of tissue specimens and lack of animal models. Rapid advances in induced pluripotent stem (iPS) cell biology opened new avenues to study bone cells from such patients.

We propose to use patient-specific iPS cells to investigate craniometaphyseal dysplasia (CMD), a rare genetic bone disorder characterized by progressive thickening of craniofacial bones and widening of metaphyses in long bones. Here, we report our first success of generating and characterizing iPS cells from human skin fibroblasts and from SHEDs (stem cells from human exfoliated deciduous teeth) of CMD patients and healthy controls. Methods for generating human iPS cells from fibroblasts and SHEDs are well established. Reprogramming of somatic cells into iPS cells was performed by retroviral transduction of Oct3/4, Sox2, Klf4 and c-Myc with the addition of a vector expressing Lin28, which contains a GFP cassette to monitor transduction efficiency. Both fibroblasts and SHEDs were reprogrammed into iPS cells. Reprogrammed cells had an indistinguishable morphology from H1 and H9 human embryonic stem (hES) cells. These iPS cells expressed hES cell markers SSEA-4, TRA-1-60, TRA-1-81, Oct4 and Nanog, as shown by RT-PCR and immunocytochemistry. The pluripotency of these iPS cells is further supported by in vitro embryoid body (EB) formation. Assays for testing in vivo differentiation capability of these iPS cells by teratoma formation are in progress.

Previously, we have presented the first CMD knock-in mouse model replicating many characteristics of human CMD. We use this mouse model to generate hypotheses for subsequent iPS-based human studies. Ultimately, we plan to investigate the cellular and molecular basis of CMD in the human system, which is only possible with a sufficient supply of human cells. We expect that combining mouse data with findings generated from the use of human iPS cell technology will significantly add to our understanding of CMD pathology and will establish a system to study other craniotubular disorders.

Disclosures: I-Ping Chen, None.


Integrin-linked Kinase Contributes to Mechanical Regulation of GSK3β in Mesenchymal Stem Cells.Jacob Thomas1, Natasha Case*1, Maya Styner1, Buer Sen2, Zhihui Xie1, Janet Rubin1. 1University of North Carolina, Chapel Hill, School of Medicine, USA, 2University of North Carolina At Chapel Hill, USA

GSK3β is emerging as a critical mediator of mechanical responses in mesenchymal stem cells (MSC). Mechanical strain causes inactivation of GSK3β, resulting in stabilization of β-catenin levels and indirectly increasing COX2 expression via NFATc1. Importantly, both β-catenin and COX2 influence MSC differentiation. The signaling molecules required for mechanical inhibition of GSK3β activity via phosphorylation are unknown. AKT, known to decrease GSK3β activity in response to insulin, may also participate in mechanical effects as strain rapidly activates this molecule. Integrin-linked kinase (ILK) can also directly inhibit GSK3β activity, in addition to indirect effects via activation of AKT. Here we wished to define proximal events whereby mechanical stimulation regulates both AKT and GSK3β activity in marrow-derived MSC. We compared strain to insulin, which is known to activate PI3K. Mechanical strain (2%, 0.17Hz, 30 min) caused phosphorylation of AKT at both Thr 308 and Ser 473. In the presence of the PI3K inhibitor LY294002 (20 mM), strain induction of T308∼P was inhibited, but S473∼P was unimpaired. Importantly, mechanical inactivation of GSK3β, measured by Ser9∼P, was not blocked by PI3K inhibition, nor was the effect of strain to induce a 3X increase in COX2 mRNA. This suggested that strain regulation of GSK3β was largely PI3K independent. This contrasts with effects of insulin, which also phosphorylates AKT at both T308 and S473: inhibition of T308∼P with LY294002 almost completely ablated insulin-induced GSK3β phosphorylation. To assess whether ILK was involved in PI3K independent effects of strain, siRNA (100 nM) to ILK was used. When ILK was decreased by ∼70%, strain still induced AKT T308∼P, but phosphorylation of S473 was absent. This may indicate that ILK is largely responsible for strain induced GSK3β inhibition, and may explain the persistence of strain effects in the presence of PI3K inhibition. In further work we hope to confirm that ILK activation is a primary response to strain, and plays a critical role in regulating MSC lineage selection.

Disclosures: Natasha Case, None.


Lamin A/C is Required during Osteoblast Differentiation to Facilitate Nuclear Mobility and Function of Runx2.Gustavo Duque*1, Christopher Vidal1, Lee Wei Li2, Li Sze Yeo3, Diane Fatkin3. 1University of Sydney, Australia, 2University of Sydney, Nepean Clinical School, Australia, 3Molecular Cardiology & Biophysics Division, Victor Chang Cardiac Research Institute, Australia

We have recently demonstrated that inhibition of the lamin A/C (lmna) gene inhibits osteoblastogenesis in vitro and results in severe osteoporosis in vivo. However, the mechanism by which lamin A/C regulates osteoblastogenesis remains unknown. Lamin A/C is a protein of the nuclear envelope, which interacts with multiple nuclear proteins including Runx2, an important transcription factor in osteoblastogenesis. Low levels of lamin A/C are associated with impaired Runx2 transcriptional activation in vitro. Using lmna knockout (lmna-/-) mice, we undertook studies to characterize the changes in protein interactions in the nucleus in the absence of lamin A/C activity and their potential effect on Runx2 activation in vivo. We focused on MAN1, an inner nuclear membrane protein that not only physically interacts with lamin A/C but also has been associated with osteopoikilosis, a rare type of hyperostosis found after loss of function of the MAN1 gene. Antibodies against MAN1 were able to co-immunoprecipiate Runx2 from marrow cells extracts obtained from lmna-/- mice demonstrating that, in the absence of lamin A/C, they reside in the same complex. In contrast, co-immunoprecipitation between MAN1 and Runx2 was not found in bone marrow cells obtained from WT controls. Furthermore, absence of Lamin A/C determined a reduction in Runx2 nuclear complex activity identified by ELISA and co-immunoprecipitation analyses. We further examined the consequences of lamin A/C knockout on in situ nuclear organization of MAN1 and Runx2 in bone marrow cells using three dye staining confocal microscopy. We established that in WT mice lamin A/C and MAN1 expression closely overlap and the subnuclear distribution of Runx2 shows a clearly punctuated pattern. In contrast, in bone marrow cells of lmna-/- mice, Runx2 lacks a punctuated pattern and closely overlaps the distribution of MAN1. In summary, in absence of lamin A/C, MAN1 and Runx 2 are structurally inseparable thus affecting Runx2 function in osteoblastogenesis. We conclude that the role of lamin A/C in osteoblastogenesis is mediated by a novel molecular mechanism in which the presence of lamin A/C is required to prevent the sequestration of Runx2 by MAN1 in the subnuclear region, thus facilitating Runx2 mobility and function.

Disclosures: Gustavo Duque, None.


See Friday Plenary number FR0245.


See Friday Plenary number FR0246.




See Friday Plenary number FR0248.


Revitalization of Bone Allografts by Murine Periosteal Cells Expressing BMP2 and VEGF.Nick van Gastel*1, Maarten Depypere2, Scott Roberts3, Ingrid Stockmans1, Sophie Torrekens1, Jan Schrooten4, Frederik Maes2, Frank Luyten5, Geert Carmeliet6. 1Laboratory of Experimental Medicine & Endocrinology, Katholieke Universiteit Leuven, Belgium, 2Department of Electrical Engineering (ESAT/PSI), Katholieke Universiteit Leuven, Belgium, 3K.U. Leuven, Belgium, 4Department of Metallurgy & Materials Engineering, Katholieke Universiteit Leuven, Belgium, 5University Hospitals KU Leuven, Belgium, 6Katholieke Universiteit Leuven, Belgium

To date, autologous bone transplantation remains the therapy of choice to treat large bone defects. Despite numerous advantages, its use is restricted by major drawbacks, including limited availability and donor site morbidity. Bone allografts lack these shortcomings and provide the strength and flexibility needed in load-bearing applications. However, allografts are not osteogenic, due to their acellular nature, which prevents integration into the host bone. Here, we show that murine periosteal cells, transiently expressing bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) and seeded on a segmental allograft, can substitute for the absent periosteum and improve callus formation and allograft incorporation.

First, a protocol was established to isolate periosteal cells from long bones of adult mice. Flow cytometric analysis revealed a substantial higher number of mesenchymal stem cells (MSCs) in the periosteal population as compared to bone marrow stromal cells. In addition, periosteal cells showed trilineage differentiation potential (osteogenic, chondrogenic and adipogenic) and when implanted ectopically in mice they formed a substantial amount of bone.

To enhance their therapeutic potential, periosteal cells were transduced with adenoviral vectors encoding BMP2, VEGF or GFP. Increased secretion of growth factors in the culture medium persisted for at least 14 days, as demonstrated by ELISA. Cells expressing BMP2 and VEGF were seeded onto 4mm cortical allografts and implanted into femoral defects in mice. Autografts, empty allografts and allografts with GFP-transduced cells served as controls. After 4 weeks, bone healing was analyzed using μCT and histology. Defects treated with empty allografts or grafts with GFP-transduced cells showed little callus formation and little to no remodeling of the graft. Grafts seeded with BMP2- and VEGF-transduced periosteal cells however induced the formation of a large callus, comparable to autografts, accompanied by manifest graft remodeling.

In conclusion, the successful isolation of adult mouse periosteal cells could help to further explore the role of this cell population using the available genetic mouse models. Moreover, establishing an artificial periosteum consisting of periosteal cells expressing BMP2 and VEGF considerably improves allograft incorporation into large bone defects in mice.

Disclosures: Nick van Gastel, None.


Role of GILZ in TNF-α-Mediated Inhibition of Marrow Mesenchymal Stem Cell Osteogenic Differentiation.Xing-Ming Shi, Nianlan Yang, Linlin He*. Medical College of Georgia, USA

Tumor necrosis factor-alpha (TNF-α) is a potent proinflammatory cytokine and it inhibits osteoblast differentiation while stimulating osteoclast differentiation and bone resorption. TNF-α activates MAP kinase pathway leading to inhibition of osterix (Osx) expression. TNF-α also induces the expression of E3 ubiquitin ligase protein Smurf1 and Smurf2 and promotes degradation of Runx2, another key transcription factor regulating osteoblast differentiation and bone formation. We showed previously that overexpression of glucocorticoid (GC)-induced leucine zipper (GILZ) enhances osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We and others also demonstrated that GILZ is a GC effect mediator and it mimics GCs' antiinflammatory actions. In this study, we asked whether GILZ retains its osteogenic activity while mediating GC antiinflammatory action. We infected MSCs with GILZ-expressing retroviruses and exposed the cells to TNF-α in an osteogenic environment. Our results show that overexpression of GILZ can override the inhibitory effect of TNF-α on MSC osteogenic differentiation as determined by Alizarin red staining of mineralized bone nodules. Real-time qRT-PCR and Western blot analyses show that overexpression of GILZ antagonized the inhibitory effect of TNF-α on Osx and Runx2 mRNA and protein expression. Finally, we demonstrate that GILZ antagonizes TNF-α effect on Osx expression by inhibiting TNF-α-induced MAP kinase activation. Together, these results indicate that GILZ is capable of dissociating the desired anti-inflammatory GC effects from its adverse bone effects and suggest that GILZ may have therapeutic potential for preventing bone loss caused by chronic inflammation such as rheumatoid arthritis.

Disclosures: Linlin He, None.


Zolendronate Induce Expression of Runx2 to Promote Osteogenic Commitment of Human Periodontal and Pulp Cells.Harunur Rashid*1, Nachiket Saoji1, Somsak Sittitavornwong1, Haiyan Chen1, Farah Ghori1, Huw Thomas1, Soraya Gutierrez2, Amjad Javed1. 1University of Alabama at Birmingham, USA, 2Universidad de Concepcion, Chile

Bisphosphonates (BP) are widely used to treat osteoporosis and malignant bone metastasis but osteonecrosis of the jaws and musculoskeletal pain has recently emerged as a significant complication in a subset of patients receiving these drugs. BP inhibits osteoclast mediated bone resorption however its action on other skeletal cells remains largely unknown. We established primary PDL and Pulp cells from non-infected, impacted third molars of healthy individuals to explore the impact of PB treatment on cell growth and differentiation. We determined that a 14-fold dilution of clinically used dose of zolendronate is non cytoxic in exvivo cultures. Exposure of PDL and pulp cells to BP caused a significant inhibition of cell proliferation consistent with its action on osteoclast. However, BP strongly enhanced osteogenic differentiation of both PDL and Pulp cells as evidenced by progressive increase in ALP activity, matrix synthesis and mineral deposition. For a molecular understanding of how BP stimulated osteoblast differentiation, we analyzed gene expression profile by microarray analysis. Interestingly, 70% (78 of 112) of the osteogenic genes were significantly induced and 14% were down regulated upon BP treatment. These changes were noted for all the key ECM genes, considered markers of specific stages of osteoblast differentiation (ALP; 19-fold, Col1a; 19-fold, BSP; 11-fold, DSPP 42-fold, OC; 48-fold, SOST; 103-fold). BP also enhanced the expression of osteogenic regulatory molecules (FGF2; 20-fold, BMP7; 43-fold, TGFβ; 8-fold), their receptors and transducers (TGFβR; 102-fold, BMPR; 12-fold, SMAD9; 18-fold). Moreover, expression of essential transcription factors and regulators of osteoblast differentiation were robustly increased (Runx2; 21-fold, Sox9; 41-fold, Msx2; 3-fold). Similar pattern of gene profile was noted in Pulp cells, indicating BP target a common regulatory pathway in tooth derived mesenchymal progenitors. The upregulation of selected osteoblast marker genes was further confirmed by RT-PCR from primary PDL and Pulp cells of three independent individuals. EMSA and Immunofluorescence studies demonstrated that BP enhanced both DNA binding and nuclear accumulation of Runx2 protein. Finally, BP stimulated Runx2 mediated activation of the OC promoter. Taken together, our studies demonstrate that, bisphosphonate activate program of osteoblast differentiation by enhancing functional competency of Runx2 in tooth derived mesenchymal cells.

Disclosures: Harunur Rashid, None.


Glucocorticoids Attenuate Bone Turnover, but do not Appear to Affect Chondrocytes In Vitro.Kim Henriksen*1, Kim Vietz Andreassen1, Morten Karsdal1, Anne-Christine Bay-Jensen2. 1Nordic Bioscience A/S, Denmark, 2Nordic Bioscience, Denmark

Glucocorticoids are known to attenuate bone formation in vivo leading to decreased bone volume and increased risk of fractures, whereas effects on the joint tissue are not known. This study aimed at characterizing the effect of glucocorticoids on osteoclasts and osteoblasts, as well as the closely related chondrocytes. We used CD14+ monocytes cultured in the presence of M-CSF and RANKL to investigate the effect of glucocorticoids on different osteoclastic parameters. We measured TRACP activity and Calcium release as indices of osteoclast number and activity. 2T3 preosteoblastic cells we used to characterize the effects of glucocorticoids on bone formation, either in the presence or absence of BMP-2. Alizarin Red was used to measure nodule formation, ALP activity as an indicator of osteoblastogenesis. Bovine full depth cartilage explants were cultured with or without TNF-a, OSM and IGF-1 to characterize the effects of glucocorticoids on cartilage turnover. Collagen type II degradation was measured by CIIMB, and aggrecan degradation by 373-G1. Safranin O staining was used to investigate proteoglycan content in sections of the cultured explants. In all assays Alamar Blue was used as an indicator of viability. DEX and PRED dose-dependently inhibited osteoclastogenesis by promoting cell death. For mature osteoclasts, low doses of GLUC induced bone resorption short term, whereas high doses and continued exposure led to osteoclast death. In osteoblasts glucocorticoids induced cell death in the non-stimulated cells. For BMP-2 stimulated cells; we found that GLUCs augmented nodule formation, while still reducing cell viability. In cartilage we found that GLUCs were unable to alter cartilage turnover, although at high concentrations a minor reduction in safranin O staining intensity was observed. In summary, using highly robust models of bone and cartilage turnover, we have shown that the effects of glucocorticoids on bone depend very much in the cell targeted, i.e. activated osteoblasts are further activated by GLUC, whereas non-activated cells undergo cell death. In cartilage no apparent effects were observed, indicating that cartilage may not possess glucocorticoid receptors, or that these are not active. We believe that these model systems are highly relevant for the continued development of glucocorticoid analogues without the detrimental effect on bone.

Disclosures: Kim Henriksen, Nordic Bioscience, 3


See Friday Plenary number FR0253.


See Friday Plenary number FR0254.


See Friday Plenary number FR0255.


Bone-wasting Cytokines are Up-regulated in Fragility Fractures: Role of Bone and Bone Marrow Cells.Patrizia D'Amelio*1, Ilaria Roato2, Lucia D'Amico2, Luciana Veneziano3, Elena Suman3, Francesca Sassi3, Giuseppina Bisignano3, Giancarlo Isaia1. 1University of Torino, Italy, 2CeRMS (Centre for Research & Medical Studies), San Giovanni Battista Hospital, Italy, 3Gerontology Section, Department of Surgical & Medical Disciplines, University of Torino, Italy

The roles of bone and bone marrow cells in bone turnover control and the amounts of cytokines they produce are not clear. This study compares cytokines production in patients with a fragility fracture and those with osteoarthritis (OA).

We evaluated 52 femoral heads from women subjected to hip-joint replacement surgery for femoral neck fractures due to low-energy trauma (37), or for OA (15). Total RNA was extracted from both bone and bone marrow, and quantitative PCR was used to identify RANKL, OPG, M-CSF, TGFβ, DKK-1 and SOST expression.

We found an increase of RANKL/OPG in bone marrow from fractured patients (Fig 1 A), as previously reported for the early post-menopause (1), whereas this parameter was similar in the bone (Fig. 1B). It may thus be supposed that bone marrow cells are the main drivers of osteoclast formation and activity. Both RANKL and OPG were up-regulated in bone marrow from the fractured patients. The increase in OPG may be interpreted as an attempt to inhibit RANKL, and as a sign of increased bone turnover.

This pro-osteoclastogenic cytokine profile was accompanied by an increase in the Wnt pathway inhibitor DKK-1 in bone (Fig. 2A) and bone marrow (Fig. 2B), which reduces osteoblast activity in fractured patients. Osteoporosis may perhaps be regarded as both an osteoblast and an osteoclast disease.

TGFβ was slightly (about 1.5%) increased in OA bone (Fig.3 A). The role of TGFβ in controlling bone turnover is not completely clear. As expected, more was produced by bone marrow (Fig. 3 B) with no significant difference between the two sets of patients (data not shown). Since OA chondrocytes produce more TGFβ, the small increase observed in OA bone may be due to their contribution.

We observe no difference in fractured as respect to OA patients in M-CSF and SOST expression.

In conclusion, we have demonstrated the important contribution of bone marrow cells in the regulation of bone turnover. Bone marrow from fractured patients produces more RANKL than that from patients with OA, whereas bone from fractured patients expresses more DKK-1. The cytokine pattern is thus shifted towards osteoclast activation and osteoblast inhibition in patients with a fragility fracture.


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    Eghbali-Fatourechi G, et al. J Clin Invest. 2003; 111:1221-30.
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Figure Figure 1. RANKL/OPG is higher in fractured patients. A. Box and whisker plot represents RANKL/OPG measured in bone marrow in fractured compared to OA patients. The p value indicated was calculated with the Mann-Whitney U test after correction for age B. As in A: RANKL/OPG measured in bone Graphs represent the median, the upper and the lower quartile and the interquartile range calculated for all the data set

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Figure Figure 2. DKK-1 production in bone and bone marrow from fractured and OA patients. A. Box and whisker plot represents DKK-1 measured in bone in fractured as respect to OA patients. The p value indicated was calculated by means of Mann-Whitney U test after correction for age B. As in A: DKK-1 measured in bone marrow. Graphs represent the median, the upper and the lower quartlle and the interquartile range calculated for all the data set

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Figure Figure 3. TGFβ production in bone and bone marrow from fractured and OA patients. A Box and whisker plot represents TGFβ measured in bone in fractured as compared to OA patients The p value indicated was calculated by means of the Mann-Whitney U test after correction for age B. Box and whisker plot represents TGFβ measured in bone marrow and in bone of all the samples. The p value indicated was calculated by means of Wilcoxon's test. Graphs represent the median, the upper and the lower quartile and the interquartile range calculated for all the data set

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Disclosures: Patrizia D'Amelio, None.


Role of T Cells in the Activation of Osteoclastogenesis in Phenylketonuria Patients.Ilaria Roato*1, Lucia D'Amico2, Francesco Porta3, Alessandro Mussa3, Marco Spada4, Riccardo Ferracini5. 1CeRMS, Center for Experimental Research & Medical Studies, A.O.U. San Giovanni Battista, Italy, 2CeRMS, Italy, 3Department of Pediatrics, University of Turin, Italy, 4Department of Pediatrics, University of Torino, Italy, 5Department of Orthopaedics, A.O.U. San Giovanni Battista, Italy

Phenylketonuria (PKU) is a rare inborn error of metabolism commonly complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques. We studied osteoclastogenesis and T cell activation state in 40 PKU patients, considering their bone condition and metabolism in an attempt to elucidate the pathogenesis of bone damage. Peripheral blood mononuclear cell (PBMC) cultures were performed to study osteoclastogenesis, adding or not recombinant human monocyte-colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL). TNF-a, RANKL and OPG were dosed in cell culture supernatants by ELISA. RANKFc and anti-TNF-a were added in some experiments to investigate their ability to inhibit osteoclastogenesis. T cell activation state was analyzed evaluating CD69 and CD25 expression by flow citometry. Bone conditions and the phenylalanine levels in PKU patients were clinically evaluated.

PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. These OCs formed resorbing lacunae on the mineralized supports used to test OC activity. We dosed higher TNF-a levels in PKU patients than in healthy controls. RANKL levels were detected both in PKU patients and in healthy controls cultures, but they were higher in patients than in controls. OPG levels were not statistically different between patients and controls, but the RANKL/OPG ratio was higher in PKU patients, explaining the spontaneous osteoclastogenesis in the unstimulated cultures of patients. The addition of the specific antagonist RANK-Fc inhibited osteoclastogenesis, whereas anti-TNF-a failed to inhibit it. RANKL was produced by T and B cells both in patients and in controls, but T cells from patients resulted more active than ones from controls. Osteoclastogenesis in PKU patients was inversely related to bone condition assessed by Quantitative Ultrasound and directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia.

Spontaneous osteoclastogenesis is present in PKU patients and correlates with hyperphenylalaninemia, suggesting its role in the pathogenesis of bone impairment in PKU. Osteoclastogenesis is dependent on RANKL, which is produced by T and B cells, suggesting a direct involvement of immune system in the bone damage of PKU patients.

Disclosures: Ilaria Roato, None.


See Friday Plenary number FR0258.


Comprehensive Analysis of Epigenetic Role of TGF-beta in RANKL-induced Osteoclastogenesis by ChIP-seq Approach.Tetsuro Yasui*1, Takumi Matsumoto2, Hironari Masuda2, Jun Hirose2, Yasunori Omata2, Yuho Kadono2, Hisataka Yasuda3, Daizo Koinuma2, Shuichi Tsutsumi2, Kozo Nakamura2, Hiroyuki Aburatani2, Sakae Tanaka2. 1University of Tokyo, Japan, 2The University of Tokyo, Japan, 3Oriental Yeast Company, Limited, Japan

Osteoclast differentiation from monocyte-macrophage lineage precursor cells is regulated by two essential cytokines, receptor activator of NF-κ B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). TGF-β is a cytokine with ubiquitous proliferation and differentiation activity in many types of cells, and we recently demonstrated that TGF-β is essential for RANKL-induced osteoclastogenesis. In this study, we analyzed the role of TGF-β in osteoclastogenesis through comprehensive epigenetic approach.

To identify TGF-β-regulated genes in osteoclast precursors (OCPs), chromatin immunoprecipitation using anti-Smad2/3 antibody and massively parallel DNA sequencing was performed (ChIP-seq). 2,786 Smad2/3 binding sites were identified, and 903 genes were extracted as Smad2/3 target genes. Microarray analysis demonstrated that expression of these genes was in fact upregulated by TGF-β treatment.

To further specify the key regulatory genes of osteoclast differentiation, we analyzed histone modification profiles of OCPs. It has been recently recognized that trimethylation of histone H3 lysine 4 (H3K4me3) is associated with active transcription of a gene and trimethylation of histone H3 lysine 27 (H3K27me3) is associated with gene silencing. Dynamic changes in the histone modification pattern of cell lineage-specific genes from H3K4me3/H3K27me3 bivalent to H3K4me3 monovalent are known to be involved in the differentiation of stem cells to terminally differentiated cells. We therefore analyzed histone methylation patterns by ChIP-seq approach using anti-H3K4me3 antibody or anti-H3K27me3 antibody. We found that histone modification pattern was converted from K4/K27 bivalent to K4 monovalent by TGF-β treatment in 85 genes, with 18 of them being Smad2/3 target genes. Expression of these 18 genes was significantly upregulated by TGF-β treatment, while none of them was upregulated more than 2-fold by RANKL treatment. Interestingly, in as many as 6 out of the 18 genes, treatment of OCPs with RANKL for 72 hrs converted histone modification pattern to be H3K27me3 monovalent and gene expression was downregulated less than 1/4 fold. These findings indicate that RANKL epigenetically silences transcription of TGF-β-regulated key osteoclastogenic genes.

Combined with our finding that TGF-β most strongly promoted RANKL-induced osteoclastogenesis when TGF-β was applied to OCPs prior to RANKL stimulation, it is suggested that the role of TGF-β in osteoclastogenesis is to maintain OCPs in undifferentiated state and thus support RANKL-induced osteoclastogenesis.

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Disclosures: Tetsuro Yasui, None.


Elucidating the Mechanism of Impaired Osteoclastogenesis in Cultures of Cells from +/R740S Osteopetrotic Mice.Irina Voronov*1, Noelle Ochotny1, Morris Manolson1, Jane Aubin2. 1University of Toronto, Canada, 2University of Toronto Faculty of Medicine, Canada

Osteoclasts are multinucleated cells responsible for bone resorption. Vacuolar H+-ATPases (V-ATPases), the multimeric enzymes present at the ruffled border of osteoclasts, are necessary for acidification of the resorption lacunae. V-ATPases consist of fourteen subunits, one of which, the “a3” subunit, is highly enriched in osteoclasts. Heterozygous mice with an R740S mutation in “a3” (+/R740S) have defective V-ATPase activity resulting in a higher bone density. Osteoclast number is increased in +/R740S bones, but in cultures of either bone marrow- or spleen-derived +/R740S cells, osteoclastogenesis is decreased compared to wild type (+/+) cells. We hypothesize that “a3” not only is important for acidification, but also plays a role in osteoclast differentiation.

To characterize osteoclastogenesis in vitro, +/R740S and +/+ bone marrow cells were cultured in the presence of RANKL and M-CSF for 6 days; the expression of osteoclast markers was analyzed by RT-PCR and apoptosis was assessed by TUNEL labeling. Gene expression levels of all osteoclast-specific markers, including tartrate resistant acid phosphatase (TRAP), osteoclast associated receptor (OSCAR), dendritic cell specific transmembrane protein (DC-STAMP), and the “d2” subunit of V-ATPase, were decreased in +/R740S cells in vitro, while “a3” expression was not changed compared to +/+ cells. “a3” and “d2” protein expression levels showed a similar pattern as determined by Western blotting. TUNEL assay results demonstrated that there was no difference in the number of apoptotic osteoclasts in +/R740S versus +/+ cell cultures, indicating that decreased osteoclast numbers in +/R740S cell cultures were not due to increased cell death. The expression levels of both the proapoptotic marker Fas and the anti-apoptotic marker Bcl2 were unexpectedly higher in +/R740S versus +/+ cultures. Besides acidification of extracellular spaces, the V-ATPases are known to play an important role in vesicular trafficking. Our results to date indicate that there is no difference in “a” subunit localization between +/R740S and +/+ cells grown on non-resorbing surface as assessed by immunofluorescence. Future experiments will determine whether the R740S mutation affects vesicular trafficking in osteoclasts. These studies will help to elucidate precise roles of the V-ATPase “a3” subunit in osteoclast maturation and activity.

Disclosures: Irina Voronov, None.


See Friday Plenary number FR0261.


Live-Cell Microscopy of Osteoclast Precursor Fusion and Osteoclast Fission.Ineke Jansen*1, Veerle Bloemen2, Jan Stap3, Ton Schoenmaker4, Teun De Vries5, Vincent Everts5. 1ACTA, The netherlands, 2Vrije Universiteit Medical Center, The netherlands, 3Department of Cell Biology & Histology, Netherlands, 4Dept. of Oral Cell Biology & Periodontology, Netherlands, 5ACTA, Vrije Universiteit, The netherlands

Purpose: Osteoclasts are specialized cells with the unique capacity to resorb bone. The formation of these cells is a multistep process in which migration is essential for both the precursors and the osteoclast itself. Yet, surprisingly little is known about the dynamics of cell migration throughout the process of osteoclast formation. In this study we investigated the migratory behavior and cell-cell interaction of osteoclast precursors and mature osteoclasts.

Methods: Human peripheral blood mononuclear cells (PBMCs) were cultured with the osteoclastogenic cytokines macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-KB-ligand (RANKL). The cultures were analyzed by live cell microscopy during 96 hours, both after one week and after two weeks of culture. In addition, we analyzed the migratory behavior of mature osteoclasts isolated from rabbits.

Results: At an early stage the osteoclast precursors were actively migrating over relatively long distances and migrated further away from their initial position compared to the later stage. At the two weeks time point, just prior to fusion, the cells were motile, but did not travel long distances. We next monitored isolated osteoclasts and showed for the first time a unique phenomenon: osteoclasts can undergo fission to generate functional multinucleated compartments as well as compartments that contained apoptotic nuclei.

Conclusions: These findings indicate important differences in the dynamics of cell migration during osteoclastogenesis: First, the osteoclast precursor explores the environment in search for fusion partners whereas in a later stage the osteoclast precursor moves in a much more localized area, possibly preparing for an interaction with neighboring cells. The most intriguing observation of dividing osteoclasts suggest that the osteoclast divides into several multinucleated cells in order to simultaneously control bone resorption at different sites and shed apoptotic nuclei to free the cell from non-functional elements. Our observations provide new information on the cellular behavior of multinucleated cells and open a new prospective for controlling bone resorption.

Disclosures: Ineke Jansen, None.


See Friday Plenary number FR0263.


Osteocyte Apoptosis Directly and Indirectly Regulates Osteoclast Formation In Vitro.Lidan You, Axel Guenther, Saja Al-Dujaili*. University of Toronto, Canada

Introduction: Microdamage is believed to disrupt interstitial fluid flow in bone, thereby reducing solute transport and causing osteocyte apoptosis. Osteocyte apoptosis in microdamage areas was shown to precede increased local osteoclast formation and activity, thereby initiating targeted bone remodeling in vivo. Osteocytes have been demonstrated to release RANKL, M-CSF and VEGF as pro-osteoclastogenic factors in vivo. However, the mechanisms for osteoclast formation and resorption in microdamage areas are unknown. We hypothesize that: a) apoptotic osteocytes localized near microdamage are directly responsible for initiating targeted bone remodeling, and b) apoptotic osteocytes indirectly regulate bone remodeling by sending cues to nearby healthy osteocytes, which then initiate targeted remodeling.

Methods: We serum-starve MLO-Y4 osteocyte-like cells for 24hr to achieve similar apoptosis effect associated with bone microdamage. Conditioned medium was obtained at 2hr and 24hr post-apoptosis to represent early stages in the initiation of bone remodeling in the following groups:

A) Apoptotic osteocytes (i.e. osteocytes at microdamage)

B) Healthy osteocytes treated with apoptosis conditioned medium (i.e. healthy osteocytes sensing apoptosis cues from microdamage)

C) Healthy osteocyte treated with non-apoptosis conditioned medium (i.e. healthy osteocytes in the absence of microdamage)

We measured soluble concentrations of RANKL, VEGF and M-CSF in conditioned medium, and conditioned medium effect on osteoclast formation/size. Discussion:

Our findings indicate that both apoptotic osteocytes and apoptosis-conditioned healthy osteocytes are involved in regulating osteoclast formation, as was demonstrated by elevated soluble RANKL and VEGF concentrations in conditioned medium. Moreover, our results suggest that healthy osteocytes near apoptosis are not only responsible for initiating the resorption response, but also for confining osteoclasts to the apoptosis area, thereby creating a “halo” around the microdamage. These findings are first to provide a mechanism linking osteocyte apoptosis with bone microdamage, and suggest that osteocyte apoptosis both directly and indirectly regulates the initiation of targeted bone remodeling.

Disclosures: Saja Al-Dujaili, None.


Regulation of Osteoclast Formation by Pyruvate Kinase M2.Seong Sik Kim*1, Ryan Ricofort2, Eric Serrano2, Jian Zuo2, Alan Jenkins2, John Neubert2, Mathew Boxer3, Douglas Auld4, Craig Thomas5, Lexie Holliday2. 1Department of Orthodontics, College of Dentistry, Pusan National University, Pusan, Korea., South korea, 2University of Florida College of Dentistry, USA, 3NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, USA, 4NIH Chemical Genomics Center, USA, 5NIH Chemical Genomics Center, National Institutes of Health, USA

Recent studies suggest that a physical link occurs between the vacuolar H+-ATPase (V-ATPase) and several glycolytic enzymes, and that these interactions are required for assembly of the V-ATPase (1). Given that osteoclasts express very high levels of V-ATPase, this predicts that increases in levels of glycolytic enzymes might be expected during osteoclast formation in order to support V-ATPase assembly. This study was initiated to test this prediction. By quantitative immunoblotting we found that aldolase, glyceraldehyde 3-phosphate dehydrogenase and phosphofructokinase all increased by approximately 10-fold relative to the level of actin during osteoclastogenesis. Because high levels of glycolytic enzymes are expressed in cancer cells and this is associated with a switch from utilization of pyruvate kinase M1 (PKM1) to the use of PKM2, we used an anti-PKM2 antibody in immunoblots to demonstrate high level expression of PKM2 in osteoclasts. RT-PCR was used to confirm the expression of PKM2 and PKM1. A small molecule activator of PKM2 (NCGC00185916-01; ref. 2) at a concentration of 1 micromolar increased the number of osteoclasts (tartrate-resistant acid phosphatase expressing multinuclear cells) formed during differentiation of RAW 264.7 cells stimulated by RANKL and mouse marrow osteoclasts stimulated by calcitriol by from 1.5 to 5-fold. Stimulaton was concentration-dependent with highest stimulation achieved at 1 micromolar of the activator. These data suggest that osteoclastogenesis is associated with expression of increased levels of several glycolytic enzymes and with the expression of PKM2. Osteoclast formation was significantly increased by a small molecule activator of PKM2. The unusual expression of glycolytic enzymes and the presence of PKM2 represent potential therapeutic targets for the treatment of osteoclast-mediated disease processes.

1. Lu, M., Ammar, D., Ives, H., Albrecht, F., and Gluck, S. L. (2007) J. Biol. Chem. 282, 24495-24503

2. Boxer, M. B., Jiang, J. K., Vander Heiden, M. G., Shen, M., Skoumbourdis, A. P., Southall, N., Veith, H., Leister, W., Austin, C. P., Park, H. W., Inglese, J., Cantley, L. C., Auld, D. S., and Thomas, C. J. (2010) J. Med. Chem. 53, 1048-1055

Disclosures: Seong Sik Kim, None.


See Friday Plenary number FR0266.


The RANK IVVY535-538 Motif Plays a Critical Role in Tumor Necrosis Factor-α-mediated Osteoclastogenesis by Rendering Osteoclast Genes Responsive to Tumor Necrosis Factor-α.Joel Jules*, Zhenqi Shi, Xu Feng. University of Alabama at Birmingham, USA

Tumor necrosis factor-α (TNF), a proinflammatory cytokine, is implicated in bone loss stemming from various bone disorders, but its precise role in osteoclast (OC) formation remains controversial. While several groups showed that TNF can promote OC formation independent of RANKL, others demonstrated that TNF-mediated OC formation needs permissive levels of RANKL. To address this discrepancy, we independently examined the role of TNF in OC formation in culture dish and bone slices using primary bone marrow macrophages (BMMs). Our data indicate that OC formation in both conditions requires permissive levels of RANKL. Moreover, we show that TNF can stimulate OC formation from BMMs previously exposed to RANKL for as short as 6h. These data indicate that RANKL plays a critical role in TNF-mediated OC formation by priming BMMs into OC lineage. RANKL induces OC formation by activating the expression of numerous genes, including those encoding metallopeptidase 9, carbonic anhydrase 2, cathepsin K and tartrate resistant acid phosphatase. To investigate the molecular basis of the RANKL-mediated lineage commitment, we studied the effect of TNF on the expression of these genes. While TNF alone cannot activate the four genes, TNF is able to do so either in the presence of permissive levels of RANKL or from BMMs previously exposed to RANKL, indicating that the RANKL-mediated lineage commitment involves reprogramming of OC genes into an inducible state. We have previously shown that the RANK IVVY535-538 motif plays a critical role in mediating OC lineage commitment. Hence, we examined whether this motif is involved in reprogramming of OC genes into an inducible state. To this end, we used 2 chimeras: Ch1 & Ch2. Ch1 comprises the human Fas external domain linked to the transmembrane & intracellular domains of normal RANK and Ch2 has inactivating mutations in the IVVY motif. The chimeras were activated by a human Fas activating antibody (Fas-AB), specific to human Fas, without affecting endogenous RANK or Fas. BMMs expressing Ch1 or Ch2 were pretreated with Fas-AB/M-CSF for 18h, followed by TNF/M-CSF for 96h. BMMs expressing Ch1, but not Ch2, formed OCs. More importantly, the expression of the four genes in BMMs expressing Ch1, but not Ch2, was activated by TNF stimulation. Taken together, we conclude that the RANK IVVY motif plays a crucial role in TNF-mediated OC formation by reprogramming of OC genes into an inducible state in which they can be activated by TNF.

Disclosures: Joel Jules, None.


See Friday Plenary number FR0268.


Characterization of the V-ATPase a3-B2 Subunit Interaction.Norbert Kartner*1, Yeqi Yao2, Keying Li3, Morris Manolson1. 1University of Toronto, Canada, 2Faculty of Dentisty, University of Toronto, Canada, 3Faculty of Dentistry, University of Toronto, Canada

V-ATPases are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. V-ATPases are composed of at least 14 subunits, some of which have multiple isoforms. Mammalian cells have 4 isoforms of the a subunit (a1-a4) and two isoforms of the B subunit (B1 and B2). To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation-domain fusion library was constructed from an in vitro osteoclast model, RANKL-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of GAL4 binding-domain fused to the 50 kDa N-terminal cytoplasmic domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts. One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts. Further characterization, using affinity pulldowns and solid-phase binding assays, revealed an interaction of apparent high affinity between NTa3 and C-terminal domains of both B1 and B2 subunits. Dual B-binding domains of equal apparent affinity were observed in NTa3, suggesting a possible model for interaction between these subunits in the V-ATPase complex. There did not appear to be any discrimination between binding of B1 or B2 subunits by the four mouse isoforms of the a subunit; however, the a3-B2 interaction appeared to be favored over a1, a2 and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. We present here a novel model for interaction of the NTa domain with the catalytic headpiece of V-ATPase, which takes into account all known intra-complex interactions with NTa. Further understanding of these interactions could aid in the design of targeted therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis. With this goal in mind, we have used the a3-B2 solid-phase binding assay to screen synthetic chemical compound libraries for inhibitors of the interaction. One inhibitor of the a3-B2 interaction was useful in inhibiting acid secretion by osteoclasts in vitro, suggesting that the a3-B2 interaction is of importance in maintaining V-ATPase proton-translocating activity at the osteoclast ruffled border (see abstract, The benzohydrazide derivative KM91104 inhibits osteoclast mineral resorption at μM concentrations that do not affect osteoclast differentiation or fusion. Crasto et al.).

Disclosures: Norbert Kartner, None.


See Friday Plenary number FR0270.


Dissociation of Bone Resorption and Bone Formation in Adult Mice Transplanted with ocloc Hematopoietic Stem Cells.Christian Thudium*1, Carmen Flores2, Anita Neutzsky-Wulff3, Vicki Jensen4, Geerling Langenbach5, Annemarie Brüel6, Jesper Skovhus Thomsen6, Natalie Sims7, Maria Askmyr8, Thomas John Martin7, Vincent Everts9, Morten Karsdal4, Johan Richter2, Kim Henriksen4. 1Nordic Bioscience, Denmark, 2Molecular Medicine & Gene Therapy, Lund University, Sweden, 3Nordic Bioscience & University of Copenhagen, Denmark, 4Nordic Bioscience A/S, Denmark, 5Dept. Oral Cell Biology, Academic Centre of Dentistry Amsterdam (ACTA), Universiteit van Amsterdam & Vrije Universiteit, Netherlands, 6Department of Connective Tissue Biology, Institute of Anatomy, University of Aarhus, Denmark, 7St. Vincent's Institute of Medical Research, Australia, 8St. Vincent's Institute for Medical Research, Australia, 9ACTA, Vrije Universiteit, The netherlands

Patients and mice with mutations reducing the ability of the osteoclasts to secrete acid have osteopetrosis, characterized by defective bone resorption, increased osteoclast numbers, and interestingly normal or even increased bone formation. However, the developmental nature of these phenotypes limits the general applicability of these findings.

To shed light on bone turnover and coupling in osteopetrosis, independent of bone development, we transplanted three-month old mice with hematopoietic stem cells from wt or oc/oc mice, which have defective acid secretion and bone resorption. Changes in bone turnover and structure were investigated in detail during a three month period, which was followed up by a six month study, to allow time for a more drastic change in bone phenotype.

Adult mice were irradiated and transplanted with fetal liver cells from wt or oc/oc mice (Ly5.2 background) by IV injection into wt mice (Ly5.1 background). Engraftment levels were assessed using flow cytometry of the Ly5.2/Ly5.1 ratio. Serum samples were collected every sixth week for measurement of bone turnover markers (CTX-I, PINP, ALP and TRACP 5b). At 12 and 28 weeks the animals were euthanized and bones were collected for histomorphometry, μCT and mechanical tests.

An engraftment level >95% was obtained. The resorption marker CTX-I was reduced in both 3 and 6 month cohorts throughout the timeline, while TRACP 5b was increased in the oc/oc group compared to wt/wt. The bone formation markers PINP and ALP were elevated, when comparing the oc/oc group to wt/wt. μCT analyses of femurs and vertebrae showed a 50% increased bone volume in trabecular and 20% in cortical compartments of oc/oc mice compared to wt. Bone histomorphometry confirms the increased bone volume found in μCT analysis. Furthermore, mechanical tests showed a 35% increase in bone strength in the femoral neck and diaphysis of oc/oc mice compared to wt, as well as a trend in the vertebrae. Bonestrength and volume continue to increase from 3 to 6 months.

In conclusion, we here present data showing that bone formation is uncoupled from bone resorption in adult mice, when the osteoclasts are unable to acidify the resorption lacunae. The observed increase in non-resorbing osteoclasts strongly suggests that bone formation is controlled by the osteoclasts, but not their resorptive activity, and that this uncoupling leads to increased bone quality.

Disclosures: Christian Thudium, Nordic Bioscience, 3


Expression and Activity of Cholinergic Receptors in Osteoclasts.Arik Bar*1, Alon Bajayo1, malka Attar1, Alberta Zallone2, Itai Bab1. 1The Hebrew University, Israel, 2University of Bari Medical School, Italy

Acetylcholine receptors have been reported in osteoblasts but not in osteoclasts. In the present study we have systematically scanned mouse bone marrow-derived monocytes and osteoclasts cultures for the expression and activity of these receptors. RT-PCR analysis revealed the absence of mRNA transcripts for neither of the five known muscarinic receptors. mRNA expression was found mainly for the nicotinic acetylcholine receptor (nAChR) α2 and β2 subunits. Quantitative mRNA analysis indicated a marked increase in the expression of these subunits during osteoclastogenesis. In addition, weaker signals were noted for the nAChR γ, δ and α10 subunits in monocytes and β1, δ and β4 in osteoclasts. The presence of α2 and β2 subunits in osteoclasts was confirmed by immunocytochemistry and in vivo immunohistochemistry. To assess the effect of nAChR activation on osteoclastogenesis and bone resorption, osteoclastogenic cultures (grown in the presence of M-CSF and RANKL) were challenged with the cholinergic agonists nicotine or carbamylcholine. Three-day cultures showed a dose dependent increase in the number of TRAP-positive multinucleated cells. The maximal increase (60%) was at 10-8-10-6 M agonist concentration. By contrast, four-day cultures showed a decrease (60%) in the number of intact osteoclasts at the same agonist dose range. This decrease was associated with a more than 15-fold increase in the number of apoptotic osteoclasts. Resorption analysis on dentine slices demonstrated no effect on pit number, but a 50% reduction in pit size. Taken together, these data suggest a significant role for nAChR signaling in the inhibition of bone resorption, which results from the early formation of immature osteoclasts and their enhanced death.

Disclosures: Arik Bar, None.


See Friday Plenary number FR0273.


Osteoclast Inhibitory Peptide-1 Binding to the FcγRIIB Modulates ITIM and ITAM Signaling in Preosteoclast Cells.Srinivasan Shanmugarajan*1, Craig C. Beeson2, Sakamuri Reddy1. 1Charles P. Darby Children's Research Institute, USA, 2Medical University of South Carolina, USA

Osteoclast inhibitory peptide-1 (OIP-1/hSca) is an autocrine/paracrine inhibitor of osteoclast differentiation, and mice that over-express OIP-1 in osteoclast lineage cells develop an osteopetrosis bone phenotype. We recently demonstrated that OIP-1 binding to the FcγRIIB inhibits osteoclast differentiation, however the underlying molecular mechanism is unclear. Immunoreceptor tyrosine-based activation motif (ITAM)-bearing common γ subunit of FcRs (FcγRI and FcγRIII) and DAP12 are crucial for osteoclast development. Further, Immunoreceptor tyrosine-based inhibitory motif (ITIM) bearing FcγRIIB adapter proteins known to inhibit ITAM signaling in immune cells. Therefore, we examined the OIP-1 inhibition of FcR signaling during osteoclast differentiation. Total cell lysates obtained from the OIP-1 mice derived preosteoclast cells stimulated with RANKL demonstrated increased levels (4-fold) of (ITIM) phosphorylation of FcγRIIB compared to wild-type (WT) mice. In contrast, OIP-1 mouse derived preosteoclasts cells stimulated with RANKL demonstrated inhibition of ITAM phosphorylation of FcRγ but not DAP12. Also, OIP-1 c-peptide treatment to preosteoclast cells obtained from FcγRII-/- deficient mice showed no significant change in the phosphorylation of ITAM. Moreover, evidence suggesting that the tyrosyl-phosphorylated ITIM has affinity with cytoplasmic SH2 domain-containing phosphatases like SHP1, SHP2 and SHIP proteins. Further studies indicate that these inhibitory proteins dephosphorylate tyrosines in ITAM bearing Fc receptors. Total cell lysates obtained from OIP-1 mice derived preosteoclast cells stimulated with RANKL demonstrated a 3-fold increase in phosphorylation of SHP1 but not SHP2. Also, there is no significant change in the levels of phospho-SH2 inositol 5-phosphatases (pSHIP1 and pSHIP2). Interestingly, preosteoclast cells from OIP-1 mice stimulated with RANKL had a 4.5-fold decrease in the levels of ITAM activating phospho-Syk compared to WT mice. These results suggest that cross-regulation of ITIM and ITAM bearing Fc receptors may play a role in OIP-1 suppression of Syk activation and inhibition of osteoclast differentiation. Thus, OIP-1 may have therapeutic utility for bone diseases with high bone turnover.

Disclosures: Srinivasan Shanmugarajan, None.


Peroxiredoxin II Negatively Regulates LPS-induced Differentiation and Bone Resorption.Mijung Yim1, Hyojung Park*2. 1Sookmyung Women's University, South korea, 2Sookmyung Women's University, South korea

Lipopolysaccharide (LPS) is pathogen that causes inflammatory bone loss. LPS has been known to induce the osteoclast formation from osteoclast precursors, which mediates NF-kB and other signaling molecules. Recent study showed that peroxiredoxin II (Prx II) is an essential negative regulator of LPS-induced inflammatory signaling. Prx II is a member of antioxidant enzyme family and plays a protective role against oxidative damage caused by reactive oxygen species (ROS). In this study, we investigated the role of Prx II in LPS induced-osteoclast formation using Prx II-deficient mice. We cultured wild-type and Prx II-deficient spleen cells with RANKL for 48 hours and used as osteoclast precursors throughout the study. When 2 types of cell were treated with LPS, as compared to wild-type cells, osteoclast formation was enhanced in PrxII-deficient cells. To gain molecular insight, we examined the effect of PrxII-deficiency on signaling pathways and transcription factors. The expression of NFATc1, a master regulator of osteoclast differentiation, was enhanced in Prx II-deficient cells. Although LPS did not show different activation patterns of IkB, ERK 1/2, p38 MAPK pathways between wild-type and PrxII-deficient cells, LPS increased the expression of proinflammatory cytokines, IL-1 and IL-6. Furthermore, NO production and iNOS expression is accelerated in PrxII-deficient cells. Also, ROS production is enhanced in PrxII-deficient cells, interestingly, activation of signal transducers and activators of transcription 3 (STAT3) is promoted in PrxII-deficient cells. These results show that PrxII modulates LPS-induced osteoclast differentiation through controlling ROS-related signaling. Consistent with the in vitro result, PrxII-deficient mice showed increased LPS-challenged bone loss. Taken together, PrxII negatively regulates LPS-induced osteoclast differentiation and bone loss. For further study, we continue to make progress in studies about the precise mechanism of Prx II in LPS-induced osteoclastogenesis.

Disclosures: Hyojung Park, None.


Spontaneous Rythmic Fluctuations of Osteoclasts Intracellular pH.Raif Musa-Aziz, Priscilla Morethson*. University of Sao Paulo, Brazil

Large amounts of acid are secreted into the resorption lacuna by the osteoclasts to promote bone mineral dissolution. This extracellular acidification is mediated by a vectorial proton secretion, one of the key elements of the osteoclast activity (Bruzzaniti A & Baron R, 2006) and may have an impact on osteoclast pH regulation. The vacuolar H+-ATPase is the known mechanism related to the proton secretion; however, other proteins, such as Na+/H+ exchanger, the voltage-gated proton channel and a proton-coupled chloride transporter (CLC-7), are also expressed in osteoclasts and may be implicated in the proton secretion process. The role of the aforementioned mechanisms and their interplay during the osteoclast proton secretion are still poorly understood.

This work was performed to evaluate which are the mechanisms involved with proton transport at the osteoclast plasma membrane and their function in the intracellular pH (pHi) regulation. To address these questions, we worked with freshly isolated osteoclasts from long bones or osteoclast-like (OCL) cells generated from bone marrow precursor cells (using M-CSF and RANK-L) of Wistar rats. The cells were plated on glass or plastic coverslips in α-MEM + 10% FBS, pH 7.4, placed in a 5% CO2 incubator at 37°C. At the time of the experiments, osteoclasts and OCL-cells were identified by their morphology using enhanced contrast microscopy and, for some experiments, a further confirmation of the cell phenotype was done by cytochemistry for TRAP or immunocytochemistry for calcitonin receptor. The pHi was measured using the probe BCECF. The emitted fluorescence ratios (R) after excitation of intracellular BCECF at 490 and 440 nm (R = 490/440) were converted to pHi using the high-K+ nigericin technique. The experiments were performed in the presence of standard HEPES solution free of CO2/HCO3- pH 7.4, 37°C, without perfusion (“basal conditions”).

Under these conditions, our preliminary data show that the osteoclasts exhibit cyclic pHi variations characterized by repeated periods of spontaneous acidification and alkalinization. These cyclic pHi variations seem to be regular, with a frequency of 14.5 ± 1.1 min, n=11, and an amplitude---difference between maximal and minimal pHi---of 1.59 ± 0.02 pH units, n=3.

Our data suggest that, at “basal conditions”, osteoclasts do not maintain a constant pHi but, instead, these cells seem to exhibit an oscillatory pHi pattern that may be independent of any stimulus or acid challenge.

Disclosures: Priscilla Morethson, None.

This study received funding from: CNPq, Fapesp


P62, PKCzeta and NF-kappaB Signaling in Human Osteoclasts: a Link With Paget's Disease of Bone.Estelle Chamoux*1, Martine Bisson2, Laetitia Michou3, Jacques Brown4, Sophie Roux2. 1Centre Hospitalier Universitaire Sherbrooke, Canada, 2University of Sherbrooke, Canada, 3Centre De Recherche Du Chuq-Chul, Canada, 4Laval University, Canada

In Paget's Disease of Bone (PDB), osteoclasts (OCs) are larger, more numerous and more active than healthy ones. PDB has been linked to mutations of the sequestosome1 (SQSTM1) gene encoding the protein p62. The most prevalent p62 P392L mutation promotes an active OC phenotype, with a basal and constitutive activation of PKCζ and NF-κB (Chamoux et al, Mol Endocrinol 2009). However, the mechanisms by which the P392L mutation alters p62-associated pathways remain to be investigated. In the present work, OCs generated from cord blood monocytes were transfected with vectors containing the wild-type or mutated p62 gene. Using immunoprecipitations, we determined that p62 associates time-dependently with IκB and that significant degradation of IκB occurs after RANKL stimulation, an effect enhanced by the over-expression of p62WT but prevented by a preincubation with MG-132, a proteasome inhibitor. However, in cells expressing the p62P392L variant, increased expression of IκB was detected in non-stimulated cells, which remained relatively high in the presence of MG132. In addition, we showed that p62/IκB interactions were less consistent in these cells, thus indicating that the p62 mutation may alter the function of p62 in NF-κB signaling by decreasing its capacity to bind IκB, and thus to shuttle this factor towards the proteasome. In order to reconcile these results with our previous observations showing that p62P392L expression lead to over-active OCs, we evaluated the role of PKCζ as a potential intermediate between p62 and NF-κB. The use of a myristoylated PKCζ inhibitor prior to RANKL stimulation significantly decreased NF-κB activation in non-transfected OCs. Moreover, the PKCζ inhibitor abolished both the baseline and RANKL-induced increase in NF-κB activation observed in OCs expressing p62P392L but had only limited effects in p62WTexpressing cells, and tended to decrease the area of bone resorbed by p62P392L-expressing OCs. Thus, our results suggest that the reduced proteasomal degradation of IκB and the increased PKCζ activation both contribute to the resulting NF-κB activation. While the P392L mutation in p62 renders the proteasomal pathway inefficient, it over-activates the PKCζ pathway leading to uncontrolled NF-κB functions and increased bone resorption. The present study highlights for the first time a double function of p62 and clearly points out the importance of PKCζ in the phenotypic changes specific to the P392L mutation in human OCs.

Disclosures: Estelle Chamoux, None.


See Friday Plenary number FR0278.


See Friday Plenary number FR0279.


The Dynamin GTPase-induced Dephosphorylation of Pyk2 is Mediated by PTP-PEST and Regulates Osteoclast Bone Resorption.Pierre Eleniste*1, Angela Bruzzaniti2. 1Indiana University-Purdue University Indianapolis, USA, 2Indiana University School of Dentistry, USA

Osteoporosis is a bone disease that affects millions of people worldwide and is characterized by low bone mass and structural deterioration of bone tissue, which increases the risk of bone fracture, frailty, morbidity and mortality. Excessive bone loss is caused by the activity of osteoclasts which degrade the bone matrix. The specific aim of this study is to identify and characterize the signaling proteins in osteoclasts that regulate the bone resorbing activity of these cells. The non-receptor tyrosine kinase Pyk2is highly expressed in osteoclasts, and mice lacking Pyk2 have an increase in bone mass due to impairment in the cytoskeletal organization and bone resorbing activity of osteoclasts. Following integrin activation, Pyk2 is activated by phosphorylation at Y402, which is necessary for its full kinase activity, and consequently for osteoclast spreading and bone resorption. We previously reported that the GTPase dynamin regulates osteoclast bone resorption in part by leading to the dephosphorylation of Pyk2 at Y402, thus decreasing Pyk2's kinase activity and its ability to bind to Src and downstream signaling proteins. In the current study we examined the intracellular mechanism by which dynamin leads to the dephosphorylation of Pyk2. We report that Pyk2 associates with dynamin via a unique mechanism involving Pyk2's N-terminal domain, most likely its FERM domain, and dynamin's plextrin homology domain. In addition, Pyk2 dephosphorylation requires dynamin's GTPase activity since expression of specific dynamin mutants that have either reduced affinity for GTP or exhibit defective GTPase activity significantly rescue Pyk2-Y402 phosphorylation. Moreover, we find that Pyk2 phosphorylation is rescued in the presence of chemical inhibitors of the tyrosine phosphatases and we identified PTP-PEST as a major phosphatase involved in the dynamin-mediated dephosphorylation of Pyk2. Together, our studies suggest that dynamin and PTP-PEST associate with Pyk2 and regulate the cyclic phosphorylation and dephosphorylation of Pyk2, which is critical for integrin signaling, podosome turnover and osteoclast attachment, migration and bone resorbing activity. Understanding the role of Pyk2 and dynamin and the intracellular mechanism that regulates osteoclast function may lead to the identification of novel therapeutic targets for the treatment of bone-related diseases such as osteoporosis.

Disclosures: Pierre Eleniste, None.


See Friday Plenary number FR0281.


See Friday Plenary number FR0282.


See Friday Plenary number FR0283.


See Friday Plenary number FR0284.


See Friday Plenary number FR0285.


See Friday Plenary number FR0286.


See Friday Plenary number FR0287.


Evidence that Sclerostin is a Locally Acting Regulator of Osteoblast to Osteocyte Transition and a Master Regulator of Mineralization.Gerald Atkins*1, Asiri Wijenayaka1, Katie Welldon1, Peter Rowe2, David Findlay1, Hui Peng Lim1. 1University of Adelaide, Australia, 2University of Kansas Medical Center, USA

Sclerostin is a product of mature osteocytes embedded in mineralised bone and is a negative regulator of bone mass and osteoblast differentiation. The identity of the cells that respond to sclerostin has not been identified, although the localized expression of sclerostin implies that it may have local paracrine or autocrine activities. We tested the hypothesis that sclerostin would regulate the behaviour of the cells actively involved in mineralisation, the late osteoblast or pre-osteocyte. First, long-term cultures of differentiating human primary osteoblastic cells (HO) were exposed to chronic levels of recombinant sclerostin. Significant inhibition of in vitro mineralization occurred in a dose-dependent manner. Analysis of gene expression revealed that sclerostin decreased the expression of mature osteocyte markers, DMP1 and that of SOST itself, whereas the expression of the pre-osteocyte marker E11 was increased. E11 expression was also increased by sclerostin in the MLO-Y4 osteocyte cell line. To separate possible effects on immature cells, primary HO were cultured for 5 weeks, a process that served to differentiate the cells to a post-proliferative and more uniformly mature, pre-osteocyte stage. Cultures were then exposed acutely to sclerostin and gene expression analysed. These cells were exquisitely sensitive to sclerostin and concentrations of 1 - 50 ng/ml consistently increased the expression of E11, while decreasing the expression of the mature markers DMP1 and SOST. Concomitantly, the expression of MEPE was increased by sclerostin, at both the mRNA and protein levels and PHEX mRNA was decreased. This implies that mature osteocytes, by virtue of their expression of sclerostin, are able to impede differentiation of pre-osteocytes as well as alter levels of key regulators of bone mineralization. Consistent with this, MEPE protein levels were increased by sclerostin and immunostaining revealed that sclerostin promoted an increase in the levels of the MEPE-ASARM peptide, shown previously to bind to and inhibit the growth of nascent bone mineral. Our results suggest that sclerostin acts through regulation of the PHEX/MEPE axis and behaves as a master regulator of physiological bone formation, in a local paracrine fashion. The regulation by sclerostin that we have identified of both pre-osteocyte and osteocyte activity is consistent with its localization in the bone and its established role in the inhibition of bone formation.

Disclosures: Gerald Atkins, None.


See Friday Plenary number FR0289.


See Friday Plenary number FR0290.


Effect of Intermittent PTH (1-34) Treatment on Osteocyte Lacunae in Ovariectomized Rats.Donald Kimmel*1, Susan Candell2, Tiffany Fong3, Jack Coats4, Mohammed Akhter5, Thomas Wronski6. 1Kimmel Consulting Services, USA, 2Xradia Inc., USA, 3Xradia Inc, USA, 4Xradia, USA, 5Creighton University Osteoporosis Research Center, USA, 6University of Florida, USA

Parathyroid hormone (PTH) increases serum calcium by several mechanisms including osteocyte (Ocy) activity that may release mineral from bone and increase Ocy lacunar (Ocy.La) size. The purpose of this work is to quantify Ocy.La density and volume in mineralized bone tissue of PTH-treated rats by 3D Xray microscopic imaging.

Female rats aged 13wks were ovariectomized (OVX) (N=24). After 6wks, rats were treated subcutaneously for 16wks with 0 [Veh] or 0.05 mg/kg PTH (1-34) (Bachem) 5d/wk. Tibiae and L2 vertebrae (L2V) were fixed in 10% PO4-buffered formalin for 24hrs, then stored in 70% ethanol. Histomorphometric studies showed typical PTH-related increases in proximal tibial cancellous bone volume and bone formation rate. L2V (4/grp) were trimmed of posterior and transverse processes, leaving whole L2 body (L2VB) samples measuring 12mm diameter X 8mm long. These were scanned (5μm pixel resolution [PR]; 125μm3 voxel resolution [VR]) with a 3D Xray microscope (XCT-200). 3D images were reconstructed and a 1.5mm diameter subregion (0.5-1.3mm distal to the cranial growth plate) rich in trabecular bone was identified and re-scanned (0.9μm PR; 0.729μm3 VR). 3D images were reconstructed and multiple 0.9μm PR 2D slices from each rat were reviewed (Figures). While Ocy.La were more visible in some (Fig 2) than others (Fig 1), automated quantitation of Ocy.La density and volume by segmentation software could not be completed due to insufficient resolution. Blind-coded, randomized 2D slices from all rats were ranked [0-3 semiquantitative scale (Figs 1 and 2)] independently by all authors for Ocy.La visibility. Combined scores (mean ± SD) were respectively, 0.63 ± 0.46 [Veh] and 2.23 ± 0.50 [PTH] (P<.001, Mann-Whitney U).

The semi-quantitative data indicate that each author considered Ocy.La more visible in PTH than Veh rats, perhaps suggesting that Ocy.La are larger in PTH rats than in Veh rats. We also conclude that while images acquired at 0.9μm PR seem acceptable for qualitatively evaluating Ocy.La, images with better than 0.9μm PR are required to facilitate automated analysis of Ocy.La density and volume by segmentation software. Trabecular bone rich specimens from all rats, cut from plastic-embedded L2VBs and measuring 2mmX2mmX8mm, are being re-scanned at 0.125μm3 VR (0.5μm PR), a resolution that was previously proven sufficient to allow automated analysis of Ocy.La properties in human bone.

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Figure Fig. 1. 2D Xray microscopic image of rat vertebral trabecular bone. Few Ocy.La are visible (Rank=0)

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Figure Fig. 2. Many Ocy.La are visible (black dots) (Rank=3)

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Disclosures: Donald Kimmel, Xradia, Inc., 5

This study received funding from: Xradia Inc.


Association of Changes in Serum Levels of Intact Parathyroid Hormone with Changes in Biochemical Markers of Bone Turnover and Bone Mineral Density: A 10-year Follow-up of the Taiji Cohort.Noriko Yoshimura*1, Shigeyuki Muraki1, Hiroyuki Oka1, Hiroshi Kawaguchi2, Kozo Nakamura3, Toru Akune1. 1University of Tokyo, Japan, 2University of Tokyo, Faculty of Medicine, Japan, 3The University of Tokyo, Japan

The aim of this 10-year prospective cohort study is to evaluate changes in the serum levels of intact parathyroid hormone (iPTH) in the general population, and to assess the association of these changes with changes in biochemical bone-turnover markers (BTMs) and bone mineral density (BMD). We randomly selected 400 individuals (aged 40-79 years; 50 men and women in each of 4 age groups) from a list of registered residents of Taiji in 1993 (baseline). The BMD and various serum markers, namely, iPTH, total osteocalcin (OC), beta-C-terminal cross-linking telopeptide of type I collagen (beta-CTX), and N-terminal cross-linking telopeptide of type I collagen (NTX), were measured in the lumbar spine and femoral neck of the participants, at baseline and in the 10-year follow-up survey. The blood and BMD examinations could be performed at both time points in 317 of the 400 subjects (79.3%; 149 men and 168 women). The mean (standard deviation) serum levels of iPTH at baseline were 26.9 (11.0) pg/mL in men and 28.8 (12.6) pg/mL in women. Six individuals (1 man and 5 women) whose iPTH levels were higher than 65 pg/mL (65.02-79.1 pg/mL) at baseline were included in the follow-up analysis. After 10 years, the mean iPTH levels were 32.1 (12.3) pg/mL in men and 32.3 (12.9) pg/mL in women. The serum iPTH levels tended to increase over 10 years in both genders (p < 0.0001 in men, p = 0.059 in women). Multivariate regression analysis after adjustment for age, body mass index (kg/m2), and menstrual status in females (0: normal menstruation, 1: irregular, 2: menopause) at baseline revealed that the changes in the serum iPTH levels were significantly positively associated with the changes in the levels of total OC (men: β = 0.25, p = 0.002; women: β = 0.19, p = 0.03), beta-CTX (men: β = 0.44, p = 0.000; women: β = 0.27, p = 0.002), and NTX in women (β = 0.22, p = 0.01). Multivariate regression analysis after adjustment for the above-mentioned confounders revealed a significant inverse relationship between changes in the serum iPTH levels and changes in the BMD of the femoral neck in women (β = -0.19, p = 0.02). An increase in the serum iPTH levels was found to be related to an increase in all BTM levels in both genders, with the exception of NTX in men. These findings confirmed that the serum iPTH levels were associated with bone metabolism, while they had a limited effect on changes of BMD over 10 years.

Disclosures: Noriko Yoshimura, None.


Is Bone Turnover Adequately Suppressed in Osteoporotic Patients, Treated with Bisphosphonates, in Daily Practice?Danielle Eekman*1, Ben A.C. Dijkmans2, Willem Lems3, Annemieke C. Heijboer2, Irene E.M. Bultink2. 1VU University Medical Center, The netherlands, 2VU University Medical Center, Netherlands, 3Vrije Universiteit Medical Centre, The netherlands

Purpose: In patients with osteoporosis persistence is generally poor. It has been suggested that monitoring therapy by measurement of bone turnover markers (BTM) after 3 months of anti-resorptive therapy might improve persistence.

Methods: We investigated serum bone marker levels in two groups. The first group consisted of patients newly diagnosed with osteoporosis and starting treatment with bisphosphonates. We observed which proportion shows a decrease of BTM of ≥ 30% (the least significant change). Serum levels of procollagen type 1 N-terminal propeptide (P1NP) and C-terminal crosslinking telopeptide (CTX) were determined before the start of treatment and after > 2 months of treatment. Secondly, P1NP and CTX levels were measured cross sectionally in a group of patients who were already treated with bisphosphonates for ≥ 3 months. We observed which proportion reached the biological goal of therapy, BTM in the lower half of the normal premenopausal range. We also measured P1NP and CTX in a reference population of 34 healthy premenopausal women.

Results: In the first group 32 patients were included (23 women) mean age 66. During a mean treatment period of 4 months the P1NP and CTX level decreased significantly with 50% and 63% respectively. In 26 patients (81%) levels of both markers decreased with ≥ 30%. In 6 patients bone turnover was not adequately decreased, probably related to non-compliance (1), prednisone use (1), alcohol abuse (1), decreased mobility (1), paraproteinemia (1) and low calcium intake (1).

In the second group 96 patients were included, mostly women, aged 67. In 95% the serum P1NP levels and CTX levels were in the lower half of the premenopausal range. In 6 of the 7 patients with a level above the premenopausal range a possible explanation could be found: non-adherence (1), non-compliance (2), immobility (1), rheumatoid arthritis (1) and polymyositis and phenytoin use (1).

Conclusion: Our data illustrate that a decrease in bone turnover greater than the LSC can be observed in the majority (81%) of newly treated patients. Of chronically treated patients 95% have a BTM in the premenopausal range. In most patients an explanation for inadeqauate suppression of bone turnover was available. Monitoring treatment effect with bone turnover markers is in daily practice is feasible, and might be an additive tool in improving compliance.

Disclosures: Danielle Eekman, None.


Is Isolated serum 25-Hydroxyvitamin D Measurement to assess Vitamin D Nutritional Status Clinically Relevant?Nayana Parikh*, Tarlisha Eskridge, Leila Idi, Shijing Qiu, Sudhaker Rao. Henry Ford Hospital, USA

There has been growing interest in assessing vitamin D nutrition (VDN) in patients in general and those with bone & mineral disorders in particular. However, there are no guidelines for screening in the general population. Nevertheless, the number of 25-OHD measurements has increased exponentially in the last 2 years especially the number with isolated 25-OHD measurements. The clinical relevance of such practice is yet to be established. Accordingly, we reviewed serum 25-OHD testing in the lab over 5 years to determine the prevalence of 25-OHD measurements with and without simultaneous measurement of serum PTH levels and the relationship between the two, and develop guidelines for appropriate interpretation of serum 25-OHD levels in clinical practice.

Methods: We included all measurements from 2005 to 2009 for both PTH and 25-OHD. The relevant data are summarized in the Table.

Results: While the number of PTH testing remained relatively constant over the 5 years, the number of 25-OHD measurements increased by 9 folds, mostly in the last 2 years. More importantly, the number of isolated 25-OHD measurements as a fraction of total 25-OHD measurements increased from 28% in 2005 to 80% in 2009, again mostly in the last 2 years. However, the fraction of 25-OHD tests <30 ng/ml, a consensus cut-off for optimal VDN, remained constant throughout the 5 year period. There was the expected significant inverse relationship between 25-OHD and PTH both within each year and for the entire cohort, without significant difference between the 5 yearly slopes.

Conclusions: The exponential increase in 25-OHD measurements suggests increased awareness of VDN among clinicians. However, the relevance of isolated 25-OHD measurement without PTH may overestimate vitamin D depletion (because of seasonal or recent vitamin D deficiency) and the need for pharmacologic vitamin D supplementation. Further studies are needed to determine the clinical implications of an isolated 25-OHD measurement to assess VDN.

Table Table. Testing Trends for 25-OHD & PTH from 2005-2009
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Disclosures: Nayana Parikh, None.


Serum Levels of Cathepsin K and Bone Turnover Markers are Decreased in Patients with Type 2 Diabetes.Pedro Rozas*1, Rebeca Reyes2, Antonia García-Martín3, Maria Dolores Perez-Avilés3, Mariela Vasavsky3, Manuel Munoz-Torres4. 1Hospital General, Spain, 2Bone Metabolic Unit., Spain, 3Bone Metabolic Unit. Endocrinology Division. San Cecilio University Hospital., Spain, 4University Hospital, Spain

Background: Studies of bone resorption in diabetes are limited, and the results are conflicting. Aim: To analyse serum levels of bone turnover markers (BTM), cathepsin K, PTH-i and 25 OH vitamin D in patients with type 2 diabetes mellitus (T2DM) and the relationship with bone mineral density (BMD). To compare BTM and cathepsin K levels between T2DM and controls. Patients and methods: Case-Control study including 133 subjects, 78 patients with T2DM and 55 healthy controls. Lumbar spine and femoral BMD were measured by dual X-Ray absorptiometry (Hologic QDR 4500). We measured: bone alkaline phosphatase (b-ALP) (OCTEIATMIDS Ltd Boldon UK), osteocalcin (OC) (DiaSorin, Stillwater, Minnesota USA; tartrate resistant acid phosphatase (TRAP) (Bone TRAP ® Assay IDS Ltd); CTX (Elecsys β Cross-Laps, Roche Diagnostics SL, Barcelona, Spain); Serum cathepsin K levels by ELISA (Biomedica Medizinprodukte GhbH & Co KG Wien, Austria). PTH-i (Intact PTH, Roche Diagnostics SL); 25 OH vitamin D (25-Hydroxyvitamin D 125I RIA DiaSorin). Results: Mean age was 56,7 ± 6,8 yr (57.8 ± 6.4 and 55,1 ± 7,1 in T2DM and control group respectively; p=0.024). Among the T2DM patients (n=78), 47.2% were females (n=35) and 52.8% males (n=43). Serumlevels of bone resorption markers were lower in T2DM compared with controls (TRAP: T2DM 1.39 ± 0.99 UI/l vs controls 1.85 ± 0.81 UI/l, p<0.05; CTX: T2DM 0.20 ± 0.12 ng/ml vs controls 0.33 ± 0.15 ng/ml, p<0.05). There were no differences in bone formation markers (b-ALP: T2DM 14.83 ± 6.5 ug/L vs controls 12.96 ± 6.73 ug/L, p 0.11; OC: T2DM 1.48 ± 1.25 ng/ml vs controls 1.45 ± 1.2 ng/ml, p 0.91). In the subgroup of patients where cathepsin K serum levels were measured (27 T2DM, 11 controls), T2DM presented lower levels although this difference was not significant (T2DM 4.36 ± 5.46 pmol/l vs controls 6.56 ± 10.13 pmol/l, p 0.39). PTH-i serum levels were lower in T2DM (PTH-i: T2DM 38.35 ± 18.20 pg/ml vs controls 50.22 ± 18.99 pg/ml, p<0.05). T2DM have lower levels of 25 OH vitamin D with respect to controls, although differences were not significant (T2DM 17.81 ± 11.14 ng/ml vs controls 21.30 ± 11.05 ng/ml, p 0.07). In T2DM there was a negative correlation between CTX levels and BMD at different sites (p<0.001). Conclusions: T2DM patients have lower levels of bone resorption markers and PTH-i compared with controls. Cathepsin K serum levels seem to be lower in T2DM, although larger studies are needed to clarify this significance of this finding.

Disclosures: Pedro Rozas, None.


Assessment of Tibial and Radial Peripheral Quantitative Computed Tomography: Precision Measurements.Charles Wisniewski*, Giulia Rinaldi, Nithya Setty, Meryl Leboff. Brigham & Women's Hospital, USA

Purpose: Peripheral Quantitative Computed Tomography (pQCT) measures true volumetric (3-dimensional) bone mineral density (vBMD) at the radius and tibia, with minimal radiation exposure. In addition, this technique discriminates cortical and trabecular compartments and cortical thickness. Assessment of the reproducibility of pQCT depends on the machine, technician and testing procedures. Sources of imprecision not related to the instrument are inexact repositioning of the subject in follow-up scans, anatomic site, reference line placement and movement artifacts. Methods: To determine the reproducibility of pQCT, we performed 3 repeated measurements in normal subjects ages 22-35 years within 2 months at the tibia (N=18) and at the radius (N=20) using pQCT (XCT 3000, Stratec, Germany). To minimize extremity movement and discomfort, customized arm and leg holders were used (Bone Diagnostic Inc, WI). We determined the percent coefficient of variation (%CV; standard deviation divided mean BMD times 100) and the least significant change (LSC; root mean square standard deviation times 2.77 for a 95% confidence level) for total area, total density and cortical thickness at each site (4% and 33% radius and 4% and 38% tibia), for trabecular density at the distal sites and for cortical density at the proximal sites of both limbs. Results: At all the tibial sites and proximal radius, the reproducibility (%CV) ranged from 0.34% to 2.41% (Tables 1 and 2). In contrast, the %CV at the 4% metaphyseal site at the radius was > 5% in total area and density; %CV and LSC measures improved when cross sectional area (CSA) at the 4% radial site was within +10 mm2 on repeated measures for each subject (J Musculoskelet Neuronal Interact. 2009 Jan-Mar;9(1):18-24) (Table 2). Cortical thickness at all sites, except at the radial 4%, showed a %CV below 1% and LSC of 0.082-0.42 (Tables 1 and 2). Conclusions: These data show that pQCT is a precise technique for measurements of vBMD and skeletal compartments of the extremities, but the 4% metaphyseal, radial site has the greatest variability. Thus, with standardized measurement procedures, pQCT is a valuable and precise clinical research tool for assessment of the therapeutic benefits of new treatment interventions on bone.

Table Table 1. Reproducibility at the Tibia
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Table Table 2. Reproducibility at the Radius
  1. * =Cross Sectional Area ± 10 mm2

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Disclosures: Charles Wisniewski, None.


Combination of Bone Mineral Density and Trabecular Bone Score for vertebral fracture prediction in secondary osteoporosis.Sophie Bréban*1, Sami Kolta2, Karine Briot3, Simon Paternotte4, Mirieme Ghazi4, Jacques Fechtenbaum2, Maxime Dougados4, Christian Roux5. 1Cochin HospitalParis Descartes University, France, 2Centre D'Evaluation, Des Maladies Osseuses, France, 3Cochin Hospital, France, 4Paris Descartes University, Cochin Hospital, Rheumatology Department, France, 5Hospital Cochin, France

Rationale and objective: The relationship between decreased BMD and fracture risk is less clear in secondary osteoporosis than in post menopausal osteoporosis. The Trabecular Bone Score (TBS) is determined from a grey-level analysis of Dual X-ray Absorptiometry image and is thought to provide non quantitative information on bone. The aim of this study was to test the combination of TBS and BMD for vertebral fracture risk detection in a rheumatoid arthritis (RA) population treated (CS) or not (NCS) with glucocorticoids. Patients: 140 women aged 55.9 ± 14.0 years, with RA since 15.2 ± 10.2 years; 94 were receiving glucocorticoids (mean dose of 6.7 ± 4.7 mg/day) and 129 a disease modifying drug. Lumbar spine and hip BMD (g/cm2) were assessed by DXA and TBS was applied to anteroposterior image of lumbar spine. Vertebral fractures from T4 to L4 were evaluated using Vertebral Fracture Assessment (VFA) software on DXA device.

Results: Mean spine and hip Tscores were -0.9 ± 1.4 and -1.6 ± 1.0, and -0.8 ± 1.5 and -1.5 ± 1.1, in patients with and without CS respectively. There was no difference in BMD between both groups, but TBS was 1.19 ± 0.11 and 1.23 ± 0.09 in CS and NCS groups respectively (p=0.03). TBS was significantly correlated to spine and hip BMD in all groups (r=0.52 to 0.61; p<0.0003). The vertebral fractures prevalence was 16.7% in the whole population, 23.9% in NCS group and 13.0% in CS group. In the whole population, the Area under the Curve (AUC) in the vertebral fracture risk prediction was higher in the TBS model (0.736) compared to the BMD model (0.670 for spine BMD; 0.705 for hip BMD; 0.708 for femoral neck BMD). We calculated a threshold of TBS (1.173) which corresponds to the best sensitivity (75%) and specificity (66%) according to ROC curves. Among patients without osteoporosis (n=97), 13 had vertebral fractures and 8 of them had a TBS lower than 1.173. Conclusion: Trabecular Bone Score provides additional information compared to BMD alone in vertebral fracture risk assessment, in RA population, with or without glucocorticoids. The bone parameters assessed by TBS need to be identified.

Disclosures: Sophie Bréban, None.


Comparison of a Prototype and Current DXA Whole Body Phantoms Provide Inconsistent Relationships.Cassidy Powers*1, Bo Fan2, Colin Miller3, John Shepherd2. 1Bone & Breast Density Group, University of California San Francisco, USA, 2University of California, San Francisco, USA, 3BioClinica, Inc. (formerly Bio-Imaging Technologies, Inc.), USA

PURPOSE: This study was conducted to determine if available whole body phantoms give consistent cross-calibration and quality control relationships between two DXA systems.

METHODS: A BioClinica whole body phantom prototype, the Hologic Whole Body Phantom (each containing an aluminum skeleton), and the BioImaging Variable Composition Phantom (VCP) were scanned 10 times (BioClinica and Hologic) and 5 times in three configurations (VCP) on the Lunar Prodigy and Hologic Discovery DXA scanners using whole body mode (smartscan off). Analysis was performed using Hologic Apex v3.0 (Hologic, Inc.) and Encore v13.2 (GE Healthcare, Inc.) software. Whole body and subregion ROIs were used on the BioClinica and Hologic phantoms to provide BMD, total mass, fat mass, lean mass, percent fat (pfat), BMC and area values. A centered subregion ROI was used to find pfat values for the VCP. Precision was described using means, SDs and %CVs.

RESULTS: The best precision was found using the Hologic WB phantom on both DXA systems. The total mass was very precise for both the BioClinica (17407.08 ± 42.03g, 16893.05 ± 53.40g) and Hologic (29147.17 ± 28.67g, 28274.97 ± 77.79g) phantoms. The Discovery produced better precision for BMD (0.98 ± 0.01, 1.10 ± 0.01) and area (516.44 ± 10.76, 651.21 ± 9.08) measures for the BioClinica and Hologic phantoms. It also gave better precision for total fat, lean, mass, and pfat for BioClinica (6413.86 ± 70.88, 10484.64 ± 81.01, 17407.08 ± 42.03, 36.84 ± 0.41, respectively). Total BMC (489.55 ± 7.58, 699.88 ± 6.07) was more precise on the Prodigy. The assumed single-point cross calibration between the Discovery and Prodigy was in good agreement for mass, BMD, and BMC, but soft tissue variables differed from 6.7% (lean) to 18.0% (pfat) depending which phantom was used. However, the VCP showed a significant offset (7.4%) between the systems for pfat that could not have been predicted by the other two phantoms. Although small (n≤30) in vivo cross calibration populations would provide some adjudication, the power is generally too poor to discern these calibration differences.

CONCLUSIONS: None of the phantoms tested were shown to be superior to the others. A universal standard of accuracy is greatly needed to determine truth in calibration for whole body DXA bone and soft tissue results. However, the phantoms may be useful for evaluation of consistent calibration on a single instrument. 

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Figure Figure 1. Mean percent fat values of the BioClinica, Hologic and VCP phantoms

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Disclosures: Cassidy Powers, None.


See Friday Plenary number FR0299.


See Friday Plenary number FR0300.


See Friday Plenary number FR0301.


See Friday Plenary number FR0302.


Reducing Unneccessary BMD Testing in Healthy Women at Menopause.Gillian Hawker*1, Susan Jaglal2, Jacob Cancino-Romero1, Arielle Mendel1, Samra mian1, Esther Waugh1, Sophie Jamal3. 1Women's College Hospital, Canada, 2University of Toronto, Canada, 3The University of Toronto, Canada

Purpose: Bone mineral density (BMD) testing rates are highest in women at menopause, when fracture risk is low. No BMD testing guidelines exist for this group. Thus, we determined risk factors for low BMD and their ability to discriminate those with/without low BMD in this group.

Methods: Women aged 40-60 years having their first BMD at an academic centre were assessed for osteoporosis (OP) risk factors and spine and hip BMD by dual x-ray absorptiometry. Risk factors were: age, weight, physical activity, smoking, alcohol, caffeine and calcium intake, age at menarche, parity, breastfeeding and menopausal status, and family/personal history of fracture > age 40. We excluded women with medical conditions linked to OP. Using logistic regression and bootstrap technique, we determined the model that best discriminated (using area under the curve, AUC) women with/without low BMD (any site t-score ≤ -2.0). For ease of use, continuous risk factors were then dichotomized at cut-points that best discriminated women with/without low BMD. We compared the AUC of these models with that of the Osteoporosis Self-Assessment Tool (OST score=[weight(kg)-age(yrs)]x 0.2), developed to identify women 65+ years for BMD testing.

Results: Of 958 healthy women sent for a first BMD test, 944 with complete data were included; 87/944 (9.2%) had low BMD. Low BMD was significantly associated with older age, > years post menopause, lower weight, prior fracture, older age at menarche, and physical inactivity in adolescence. In multivariable analysis, older age, lower weight, > years post menopause, and prior fracture were significant independent predictors of low BMD (AUC 0.75 versus OST AUC 0.70, p=0.01). Dichotomizing weight (≤71/>71 kg), years post menopause (≥2/<2), and age (>52/≤52) gave similar discrimination (AUC=0.745). Using the 4 dichotomous risk factors and setting sensitivity to detect low BMD at 95%, 258 women with normal BMD and 4 with low BMD would not have been recommended for testing. None of the 944 women had OST scores < -3 (high OP risk); 41 with low BMD and 205 without low BMD had OST scores between 1 and -3 (moderate OP risk). Using an OST score ≤1 to recommend testing would identify only 47.1% with low BMD.

Conclusions: In healthy women at menopause <10% had low BMD, supporting the need for guidance about testing. Assessment of 4 risk factors was superior to the OST in discriminating women with versus without low BMD.

Disclosures: Gillian Hawker, None.


The Influence of Exogenous Fat and Water on Lumbar Spine Bone Mineral Density in Healthy Volunteers.KYU-NAM KIM*. Ajou University, South korea

Background Changes in human body composition can affect the accuracy of spine bone mineral density (BMD) measurements. The purpose of this study was to evaluate whether fat and water in the soft tissue of the abdomen influence lumbar spine BMD measurements obtained using dual energy X-ray absorptiometry (DEXA).

Methods Healthy volunteers (10 male, 10 female) had duplicate BMD measurements on the same day before and after placement of the following 3 materials in the abdominal area: lard 900g, 1.5cm thick; oil 1.4 liters in a vinyl bag; and water 1.2 liters in a vinyl bag. Duplicate BMD measurements were also made using the Hologic anthropomorphic spine phantom under the same conditions.

Results In the case of human participants, following the placement of exogenous water to mimic extracellular fluid (ECF), there was a significant decrease in lumbar spine BMD (-0.012 g/cm2, P = 0.006); whereas, placement of exogenous lard and oil to mimic abdominal fat, produced a slight increase in lumbar spine BMD (0.006 g/cm2, P = 0.301; 0.008 g/cm2, P = 0.250, respectively). The average percentage of lumbar spine BMD change with and without exogenous lard, oil, and water increased 0.51% and 0.67% and decreased 1.02%, respectively. Using the phantom, BMD decreased with placement of both lard (-0.002g/cm2, P = 0.699) and water (-0.006 g/cm2, P = 0.153); however, there was no difference in BMD after oil placement (0 g/cm2, P = 0.870). Differences in lumbar BMD between lard and oil, representing solid fat and liquid fat respectively, were not significant (P = 0.607).

Conclusions These results suggest that in cases where changes in fat and ECF volume are similar, ECF exerts a greater influence than fat on DXA lumbar BMD measurements.

Disclosures: KYU-NAM KIM, None.


See Friday Plenary number FR0305.


Which Limb to Scan? Revisiting the Relationship Between Skeletal and Functional Limb Dominance.Benjamin Weeks, Belinda Beck*. Griffith University, Australia

Purpose: The typical referral for clinical or research bone densitometry requests a “non-dominant” limb exam. While hand dominance reliably predicts upper extremity skeletal dominance, our experience suggests that functional dominance does not predict lower limb skeletal dominance. In the absence of a single reliable technique to determine lower extremity functional dominance, the extrapolation of the upper extremity rule is widespread; to the extent that scanning devices such as densitometers are generally designed to facilitate scanning of the left hip rather than the right to accommodate the preponderance of right handed individuals in the population. The aim of the current work was to determine the true nature of the association between functional and skeletal dominance of the lower limb. The ultimate goal is to establish a simple and reliable determinant of lower extremity skeletal dominance. Methods: 100 healthy men and women (age 32.5 ± 10.2 years) were recruited for anthropometry and bilateral hip densitometry (BMD; Norland XR-800), calcaneal quantitative ultrasonometry (BUA; QUS-2, Quidel) and tibial peripheral quantitative computed tomography (cortical and trabecular density and area, cortical width and stress strain indices [SSI]; XCT3000 Stratec). Side dominance questionnaires and physical tasks were completed including the Waterloo Footedness Questionnaire (Revised) (WFQ-R), hop distance test, step test, handedness and footedness questions, and side preference for a number of common postures (e.g. folding arms). Correlation analyses and chi square tests with crosstabs were run on all bone and functional dominance parameters using SPSS (17). Results: Significant negative relationships were found for handedness and WFQ-derived dominance with femoral neck BMD dominance (r = -0.35, p = 0.01 and r = -0.32, p = 0.03 respectively), as well as arm folding dominance with tibial SSI dominance at the 14% site (r = -0.62, p = 0.003). While significance was not reached for other measures, a consistent trend for lower limb skeletal dominance in the functionally non-dominant limb was observed for 70% of all bone parameters. Conclusions: Contrary to conventional thinking, the functionally dominant lower extremity exhibits lower bone mass than the functionally non-dominant lower limb. Findings bring into question the standard practice of scanning left lower extremity regions of right handed individuals when skeletally non-dominant lower limb measures are desired.

Disclosures: Belinda Beck, None.


Bone Density and Bone Size in Older Men and Women Assessed by High Resolution pQCT: Contributions of Growth, Consolidation and Aging.Richard Eastell1, Margaret A. Paggiosi2, Jennifer Walsh*2. 1University of Sheffield, United Kingdom, 2NIHR Bone Biomedical Research Unit, United Kingdom

Bone size and density are major determinants of bone strength. HR-pQCT (Xtreme CT, Scanco) measures size-independent volumetric density (vBMD) and structure. The aims of this study were to 1) identify gender differences in bone density and size in older men and women, 2) determine the contributions of growth, consolidation and aging to bone density and size in older men and women 3) identify site-specific changes in bone density and size with aging. We conducted a cross-sectional observational study of 171 male and female healthy volunteers ages 16-18 (end of growth), 30-32 (peak bone mass) and >70 (aging). We measured BMD at the spine and hip with DXA, and the distal radius and tibia with HR-pQCT. HR-pQCT results were analysed by ANOVA with gender, age group and gender* age group interaction as fixed factors. ANOVA with linear contrast was used to compare groups and differences between groups. The table shows subject characteristics, DXA BMD and selected HR-pQCT results.

Radius total density was similar in older men and women, and was similar in men and women at the end of growth and peak bone mass. Density decreased with aging in men and women (M -18%, W -20%, p<0.001). Radius cortical perimeter was greater in older men than older women, and was also greater in men at the end of growth and peak bone mass (p<0.001). Cortical perimeter increased with aging in men and women (M 10%, W 6%). Cortical perimeter increased from the end of growth to peak bone mass in men, but not women (M 7%, W -1%, p<0.05).

Tibia total density was higher in older men than older women (p<0.001). Density decreased with aging in men and women (M -14%, W -23%, p<0.001). Women had slightly lower peak density, and greater loss with aging. Tibia cortical perimeter was higher in older men than older women, and was also higher in men at the end of growth and peak bone mass (p< 0.001). However, cortical perimeter did not differ by age in men or women.

We conclude: 1) radius vBMD does not differ by gender, 2) older men have larger radius size than women due to greater size at the end of growth and a gender-specific increase between the end of growth and peak bone mass, 3) older women have lower tibia vBMD than men due to lower peak values and greater loss with aging, 4) older men have larger tibia size than women due to greater size at the end of growth, 5) weight-bearing and non-weight-bearing bones differ in density and size variation due to gender and age.

Table Table.  
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Disclosures: Jennifer Walsh, None.


Detection of Vertebral Fractures in Lateral Scout Views from Computed Tomography Using Statistical Models of Shape and Appearance.Jane Haslam1, Yoo Mee Kim2, Douglas Kiel3, Elizabeth Samelson3, Joes Staal4, Peter Steiger*5, Mary Bouxsein2. 1Optasia Medical Ltd, United Kingdom, 2Beth Israel Deaconess Medical Center, USA, 3Hebrew SeniorLife, USA, 4Optasia Medical Ltd., United Kingdom, 5Optasia Medical, USA

Purpose: Vertebral fractures (VFx) are underdiagnosed worldwide. New methods that improve reliable VFx assessment are needed. Thus, the aim of this study was to develop and test algorithms which detect osteoporotic vertebral fractures in lateral CT scout view images.

Methods: Subjects included 50 men and 50 women (aged 50-87 yrs, mean 70 yrs) selected from the Framingham Heart Study Offspring and Third Generation cohorts. Vertebral shape was captured by a non-radiologist on CT lateral scout views from T4 - L4 using a novel semi-automated software tool (SpineAnalyzer, Optasia Medical), which captures vertebral shape with 95 points per vertebra. Scout films were also read by two trained radiologists using Genant SQ scores. Machine learning techniques were used to develop classification algorithms (or classifiers) for automatically detecting vertebral fractures given vertebral shape, for two definitions of VFx: 1) SQ grades 1-3 vs. grade 0, and 2) SQ grades 2-3 vs. grades 0-1. The classifiers were evaluated against consensus SQ readings for each definition of VFx in terms of their equal error rates (EERs), area under the Receiver Operator Characteristic (ROC) curve, and kappa score. Classifier testing was performed using “leave one out” cross-validation in order to give very nearly unbiased performance estimates.

Results: Based on radiologists' consensus SQ readings 977 vertebrae were normal and 44 fractured: 25 grade 1 (mild), 16 grade 2 (moderate) and 3 grade 3 (severe). For the two definitions of VFx, classifiers yielded EERs of 8.7% and 0.7%, areas under the ROC curves of 0.97 and 0.998, and maximum kappa scores of 0.57 (95% CIs: 0.42-0.71) and 0.83 (0.70-0.97) respectively. In comparison, for SQ 1-3 vs. SQ 0, radiologist inter-reader kappa scores were 0.56-0.59. For SQ 2-3 vs. SQ 0-1 radiologist interreader kappa scores were 0.68-0.72.

Conclusions: These initial results indicate the potential feasibility of developing algorithms which allow non-radiologist operators to use vertebral shape measurements, determined using a highly automated algorithm, to distinguish VFx in lateral CT scout view images. The algorithms' agreement with “gold-standard” SQ scores is similar to the agreement between experienced radiologists for the same data-set. These data provide strong rationale to validate the results on a larger data-set.

Disclosures: Peter Steiger, Optasia Medical Ltd., 3; Optasia Medical Ltd., 1


Effects of Antiresorptive Agents on Bone Micro-Architecture Assessed by Trabecular Bone Score in Women Age 50 and Older: The Manitoba Prospective Study.Marc-Antoine Krieg*1, Andrew Goertzen2, William Leslie2, Didier Hans3. 1University Hospital, Switzerland, 2University of Manitoba, Canada, 3Lausanne University Hospital, Switzerland

Antiresorptive agents such as biphosphonates induce a rapid increase of BMD during the 1st year of treatment and a partial maintenance of bone architecture. Trabecular Bone Score (TBS), a new grey-level texture measurement that can be extracted from the DXA image, correlates with 3D parameters of bone microarchitecture.

AIM: To evaluate the longitudinal effect of antiresorptive agents on spine BMD and on site-matched spine micro-architecture as assessed by TBS.

METHODS: From the BMD database for Province of Manitoba, Canada, we selected women age >50 with paired baseline and follow up spine DXA examinations who had not received any prior HRT or other antiresorptive drug. Women were divided in two subgroups: (1) those not receiving any HRT or antiresorptive drug during follow up (=non users) and (2) those receiving non-HRT antiresorptive drug during follow up (=users) with high adherence (medication possession ratio >75%) from a provincial pharmacy database system. Lumbar spine TBS was derived by the Bone Disease Unit, University of Lausanne, for each spine DXA examination using anonymized files (blinded from clinical parameters and outcomes). Effects of antiresorptive treatment for users and non-users on TBS and BMD at baseline and during mean 3.7 years follow-up were compared. Results were expressed % change per year.

RESULTS: 1,150 non-users and 534 users met the inclusion critera. At baseline, users and non-users had a mean age and BMI of [62.2 ± 7.9 vs 66.1 ± 8.0 years] and [26.3 ± 4.7 vs 24.7 ± 4.0 Kg/m2] respectively. Antiresorptive drugs received by users were bisphosphonates (86%), raloxifene (10%) and calcitonin (4%). Significant differences in BMD change and TBS change were seen between users and non-users during follow-up (p<0.0001). Significant decreases in mean BMD and TBS (-0.36 ± 0.05% per year; -0.31 ± 0.06% per year) were seen for non-users compared with baseline (p<0.001). A significant increase in mean BMD was seen for users compared with baseline (+1.86 ± 0.0% per year, p<0.0018). TBS of users also increased compared with baseline (+0.20 ± 0.08% per year, p<0.001), but more slowly than BMD.

CONCLUSION: We observed a significant increase in spine BMD and a positive maintenance of bone micro-architecture from TBS with antiresorptive treatment, whereas the treatment naïve group lost both density and micro-architecture. TBS seems to be responsive to treatment and could be suitable for monitoring microarchitecture.

Disclosures: Marc-Antoine Krieg, None.


See Friday Plenary number FR0310.


Measuring Bone Quality using Peripheral Quantitative Computed Tomography at the Tibia in Individuals with SCI: Reproducibility and Methodological Considerations.Lora Giangregorio1, Deena Lala*1, Kayla Hummel1, Christopher Gordon2, B. Catharine Craven3. 1University of Waterloo, Canada, 2McMaster University, Canada, 3Toronto Rehabilitation Institute, Canada

Background: Measurements of trabecular connectivity and hole size can be obtained at the radius using the Stratec pQCT device despite the limited resolution. There are no reports of the same pQCT-based trabecular structure measurements at the tibia. It would be important to establish whether they could be measured reliably, particularly in individuals with low BMD.

Objective: To investigate the reproducibility of bone structure measurements in individuals with spinal cord injury (SCI) obtained using peripheral quantitative computed tomography (pQCT).

Design: Cross-sectional study

Participants/methods: Eleven individuals with SCI (C5-L1) were recruited to participate; 1 was excluded due to contractures. Mean (± SD) age was 43 (10) years and years post injury was 18 (14). pQCT scans were performed twice on the same day at the ultra-distal tibia, with repositioning between scans. The following bone structure variables were determined using in-house software: average and maximum hole size [mm2]; connectivity index; average cortical thickness [mm]; bone volume to total volume (BVTV) ratio. For each outcome, the root mean squared coefficients of variation (RMSCV) were calculated.

Results: Mean (± SD) and RMSCV (in %) for bone structures variable were: average hole size 9.5 (9.7) mm2, 7.0%; maximum hole size 663.1 (253.0) mm2, 5.4%; connectivity index 0.16 (0.06), 2.9%; cortical thickness 0.94 (0.1) mm, 1.8%; BVTV 0.28 (0.12), 0.7%. Variability observed across participants may be attributable to factors such as age, impairment (AIS) or duration of injury. Potential sources of within subject variability include variations in positioning, or movement during scanning.

Conclusions: Although pQCT is a novel and feasible tool for measuring tibia bone structure, the RMSCV for some variables may necessitate multicentre studies with large sample sizes to determine treatment efficacy.

Disclosures: Deena Lala, Merck Frosst, 2

This study received funding from: Canadian Institutes of Health Research, #86521


Reliability of Semi-Automated Vertebral Morphometry Measurements using Lateral Scoutviews from Computed Tomography.Yoo Mee Kim*1, Serkalem Demissie2, Rahel Eisenberg1, Douglas Kiel3, Mary Bouxsein1. 1Beth Israel Deaconess Medical Center, USA, 2Boston University School of Public Health, USA, 3Hebrew SeniorLife, USA

The presence of vertebral fracture (VFx) is among the strongest risks for future fracture. Despite this, underdiagnosis and undertreatment of VFx is a well-known problem worldwide. New methods are needed to improve the accuracy and efficiency of identifying VFx. Thus, the aim of this study was to determine intra- and interreader reliability of vertebral morphometry measurements performed using a new semi-automated algorithm that is based on shape-based statistical modeling (SpineAnalyzer, Optasia Medical, Cheadle, UK). Methods: 100 subjects (50 men and 50 women, aged 50-87 yrs, mean: 70.3 ± 8.9 yrs) were selected from the Framingham Heart Study Offspring and Third Generation Multi-Detector Computed Tomography Study. Two non-radiologist readers independently assessed vertebral morphometry from the CT lateral scoutviews for T4-L4 at 2 time points. Deformities were classified as mild (≥20%), moderate (≥25%) or severe (≥40%) based on Genant's criteria. Intraclass correlation coefficients (ICCs), root mean squared CV (RMS-CV) and kappa (k) statistics were used to assess reliability. Results: Four subjects were excluded due to poor image quality. Of 1248 individual vertebrae from T4-L4 in 96 subjects, 1246 were analyzed. The time per subject needed to conduct morphometry measurements averaged 5 min 22 sec (range: 3:13 to 9:06 min:sec). Intra- and inter-reader ICCs for anterior, mid and posterior vertebral heights for all vertebral levels combined were excellent, ranging from 0.96 to 0.98. ICCs were also excellent at distinct spinal regions (T4-9, T10-12, and L1-4), ranging from 0.87 to 0.96. Intra- and inter-reader RMS-CV ranged from 2.5 to 3.9% and 3.3 to 4.4%, respectively. Based on morphometry measurements alone, Reader A and B identified 51-52 and 46-59 subjects, with at least one prevalent VFx, respectively. Examining all vertebral levels together, we found good intra- (k =0.59 to 0.69) and inter-reader agreement (k= 0.67) for VFx defined by a deformity of ≥ 20%. Conclusions: This new semi-automated method for assessing vertebral morphometry has excellent intra- and inter-reader reliability for vertebral height measurements on lateral CT scoutviews, and requires less time than conventional 6-point morphometry. Reliability for VFx assessment was comparable to previous reports for SQ grading by radiologists. Altogether, this semi-automated technique using CT lateral scoutviews is a convenient and reproducible method to facilitate assessment of VFx.

Disclosures: Yoo Mee Kim, None.


Vertebral Bone Loss Quantified by Normalised Mean Vertebral Area in Lateral Radiographs.Erik B Dam1, Mads Nielsen2, Martin Lillholm*3. 1Nordic Bioscience & CCBR-Synarc, Denmark, 2University of Copenhagen, Denmark, 3Nordic Bioscience, Denmark

Osteoporosis drug trials often use incident vertebral fractures as markers of disease progression and treatment efficacy. The purpose of this study was to investigate bone loss through a continuous measure of vertebral area as a supplement to established progression markers.

The case-control study population consisted of 126 postmenopausal women; a subset of the community recruited PERF study. Case and control groups were matched with respect to age, height, weight, and BMD. Fracture status at baseline and follow-up (6.3 years) was determined from lateral radiographs by an experienced radiologist using Genant's semi-quantitative method. All subjects were fracture-free at baseline and the 101 controls remained fracture-free. The 25 cases developed at least one incident fracture in the lumbar region. The lumbar vertebrae had the corner and mid points marked using a computer tool. The vertebral bone area was calculated as the area of the hexagon defined by the six points. Radiographic scaling invariance was approximated through normalization with the square of the average width of vertebrae (T12-L5). Width was defined as the distance between the midpoints of the anterior and posterior heights. The normalised average vertebral area of the lumbar region was defined as the mean of the six areas. This area was calculated at baseline and follow-up for all 126 subjects. Results are given as mean ± SEM and significance tested using the Wilcoxon ranksum test for unpaired samples and the signed variant for paired.

The mean vertebral areas for the case group at baseline and follow-up and were 0.83 ± 0.01 and 0.77 ± 0.01, p=1 × 10-5. Similarly for the control group at baseline and follow-up: 0.84 ± 0.01 and 0.82 ± 0.01, p=2 × 10-12. The difference between cases and control was significant at follow-up p=2 × 10-5 whereas the difference between cases and controls at baseline was not p=0.35. The average vertebral bone area loss was significantly larger for the case group, p=7 × 10-12.

Several intuitive results were confirmed: The matched case and control groups had comparable vertebral bone area at baseline. Both groups individually suffered significant bone area loss from baseline to follow-up and the case group with fractures lost significantly more bone area than the fracture-free control group. Most promising is, however, the significant bone area loss in the fracture-free control group - a continuous and significant progression that is not registered by fracture readings.

Disclosures: Martin Lillholm, CCBR-Synarc, 3; CCBR-Synarc, 1

This study received funding from: Nordic Bioscience and CCBR-Synarc


Preliminary Comparison of FRAXTM (excluding BMD) with FRAXTM (including BMD calcaneal QUS T-Score) Screening Tool for Estimating Long-term Fracture Risk.Simon Dyall1, Daphne Bird1, Ian Drysdale*2, Heather Hinkley2. 1British College Osteopathic Medicine, United Kingdom, 2British College of Osteopathic Medicine, United Kingdom

The aims of this study were to compare FRAXTM (excluding BMD) with FRAXTM (including calcaneal QUS T-Score) predictions for fracture risk and assess the ability of each to predict actual fracture over the long-term (10 years). Osteoporosis remains a major public health concern and accurate assessment of the associated bone fracture risk should enable appropriate targeting of vulnerable individuals with interventions intended to reduce this risk. Fracture risk may be predicted using simple non-invasive screening techniques that ensure patient safety at relatively low cost such as quantitative ultrasound (QUS) or the WHO fracture risk assessment tool, FRAXTM, an algorithm of hip and other major osteoporotic fracture risk over 10 years.

180 Caucasian females, aged 40 to 79 at baseline were randomly selected from a cohort of 900 subjects screened using calcaneal QUS (McCue Cubaclinical). Relevant patient details were retrospectively entered into the FRAXTM assessment tool, either with or without BMD calcaneal QUS T-score, and indices of fracture risk were compared. Subjects completed a follow up questionnaire to ascertain details of any fracture and level of trauma within the subsequent 10 years, and fragility fractures were abstracted. Scores were classified into low, medium and high risk based on FRAXTM criteria, and the results analysed by Chi-squared statistic.

The FRAXTM scores were significantly different when calculated with and without BMD (p<0.05), and although not significant the Chi-squared analysis indicated a far closer relationship between fragility fracture and high risk with BMD QUS T-score (P=0.08 vs. P=0.93). Furthermore, ten year follow up (mean 10.1 years) revealed that 26 of the 180 subjects sustained fragility fractures, 9 of which were predicted by FRAXTM (18%) and 13 by FRAXTM (with BMD) (28%). There were two spine fractures which were in the high risk category for both techniques, of the 17 wrist fractures 5 were in the high risk categ ory for FRAXTM and 8 for FRAXTM (with BMD), the hip fracture was predicted by both techniques as was one of the two elbow fractures.

It should be noted that the predictive ability of the techniques may have been confounded by increased interim use osteoporosis treatments taken following the initial screen. However, these preliminary findings suggest that the addition of calcaneal QUS T-score improves the ability to predict fragility fractures in women.

Disclosures: Ian Drysdale, None.


Prognosis of Fracture Risk by Quantitative Ultrasound Measurement and Bone Mineral Density.Nguyen Nguyen1, Jacqueline Center1, John Eisman1, Tuan Nguyen1, Mei Chan*2. 1Garvan Institute of Medical Research, Australia, 2Osteoporosis & Bone Biology, Australia

Quantitative ultrasound measurement (QUS) or bone mineral density (BMD) has been shown to predict fracture risk in women. However, whether a combination of QUS and BMD can improve the predictive value of fracture remains unclear. In this study, we sought to determine whether the combined use of calcaneal QUS and BMD measurements could improve the accuracy in fracture risk prediction, and to develop a nomogram based on the predictive model to predict the 5-year and 10-year fracture risk for individual men and women.

The study was designed as a population-based prospective investigation, which involved 407 women and 421 men aged 62-89 year, who had been followed for a median of 13 years (range 11-15 years) during the period of 1994-2009. BMD was measured at femoral neck by DXA using GE Lunar DPX-L densitometer and BUA was measured at the calcaneus using CUBA sonometer. The Cox's proportional hazards regression model was used to assess the association between fracture risk and the predictive variables. Reclassification analysis was used to compare the prognostic performance of the model including age, BMD and fall with the model including BUA.

During the follow-up period, 18% men (n=77) and 38% women (n = 154) had sustained a fragility fracture. Each standard deviation decrease in BUA was associated with a hazard ratio [HR] of fracture 1.86 (95%CI, 1.57-2.27) in women and 1.50 (95% CI, 1.19-1.94) in men. After adjustment for BMD, BUA remained significantly associated with fracture risk in women and men with reduced magnitude (HR 1.57, 95%CI, 1.26-1.95 in women; HR 1.32, 95% CI, 1.03-1.69 in men). For the model with BUA and BMD combined, the AUC increased from 0.71 to 0.74 in women, and from 0.695 to 0.697 in men. Reclassification analysis also yielded a total net reclassification improvement (NRI) of 9.7% (p = 0.02) and 3% (p = 0.69) for women and men respectively. Overall, 21% of women and 33% of men were reclassified into a different risk category.

Based on the estimated parameters of the final model (i.e. BUA, BMD fall and age), two nomograms were constructed for predicting fracture risk for an individual man and woman. These results suggest that calcaneal QUS was significant and independent predictor of fracture risk in both men and women. The combination of QUS and BMD in form of a nomogram can enhance the accuracy of categorizing individuals according to their risk of fracture, supporting the potential role of QUS in the prognosis of fracture.

Disclosures: Mei Chan, None.


See Friday Plenary number FR0316.


Association Between Fat and Lean Distributions with Bone Mineral Density in KNHANES.Woong-Hwan choi1, Sangmo Hong*2. 1Hanyang university hospital department of internal medicine, South korea, 2Hanyang University, South korea

It is uncertain whether obesity is protective or harmful to bone. There were many limitations due to collinearitis between BMD, Fat mass, muscle mass, and body weight in ths studies for role of fat to BMD.

The aim of this study was to define the role of muscle and fat distributions to BMD.

We analyzed body composition and BMD data of 1172 Male and 1546 female from KNHANES IV. Body composition and BMD were measured by DXA. Metabolic syndrome (Mets) modified to define any two of four factors except waist circumference by IDF definition due to conventional Mets has collinearity with central obesity. The relations of [Appendicular lean/Trunk lean; AM] for muscle, [Appendicular fat /Trunk lean; AF] for subcutaneous fat, [Trunk fat/Trunk Lean; CO] for central obesity, age, body weight (BW) and Mets with sub-total BMD analyzed by multiple regressions. We also generated the Z score of each factors by LMS method with LMS chartmaker pro 2.3.

In men, BMD (R2=0.275) had positive relations with AM (B=0.193) and BW (B=0.006) but had negative correlations with AF (B=-0.229), CO (B=-0.111), and Mets (B=-0.016). BMD (R2=0.258) of ≥50 yr men only showed relation with CO (B=-0.187) and BW (B=0.006). Z score of BMD (BMDz, R2=0.251) showed relations with Z score of AF (AFz, B=-0.027), AMz (0.009), BWz (B=0.059) and Mets (B=-0.025) but Coz had no relation with BMDz. In ≥50yr men, BMDz (R2=0.237) showed negative correlation with COz (B=-0.014) and AFz (B=-0.014), but AMz and Mets had no relation with BMD of ≥50yr men. In women, BMD (R2=0.322) had negative relations with AF (B=-0.095), CO (B=-0.045), age (B=-0.002) and Mets (B=-0.009), and positive relations with AM (0.135) and BW (B=0.005). In ≥50 women, BMD (R2=0.202) only had relation with CO (B=-0.043), BW (B=0.004), age (B=-0.004) and AM (B=1.124) but AF had no relations with BMD. BMDz (R2=0.219) in women had negative relations with AFz (B=-0.128), and COz (B=-0.096), and positive relations with AMz (B=0.135) and BWz (B=0.524). In ≥50yr women, BMDz (R2=0.177) only had relation with COz (B=-0.098), BWz (B=0.457) and AMz (B=0.087) and AFz had no relations with BMDs.

We found that central obesity and subcutaneous fat had negative effects to bone except ≥50 women. And muscle mass had relations with BMD, but its significant was disappeared (in men) or weaken [in women AMz B=0.131(<50yr) ® 0.087(≥50yr)] in older subjects.

Disclosures: Sangmo Hong, None.


Association of calcaneal QUS BMD and DEXA BMD at the femoral neck, lumbar spine and whole body in American Indian Men and Women.Maureen Murtaugh*, Molly McFadden, Khe-ni Ma, Tracy Frech, Laurie Moyer-Mileur, Martha Slattery, Tom Greene. University of Utah, USA

Little information is available about bone density, osetoporosis, and fracture risk in American Indian populations in the United States. The feasibility of dual energy s-ray absorptiometry (DEXA) screening among American Indian populations living in remote areas of reservations is met with challenges including cost, radiation exposure, and proximity to a DEXA table. Quantitative ultrasound (QUS) is attractive because it is portable, non-invasive, and does not involve exposure to radiation. Anecdotal reports from Alaska Native healthcare providers suggested that BMD estimated by QUS had a high false positive rate for osteopenia and osteoporosis determined by DEXA at the hip. Therefore, we explored the association of QUS measures as a predictor of DEXA measured BMD (g/cm2) at the femoral neck, lumbar spine and whole body in American Indians living in the southwest. Participants were recruited randomly from the Education and Research Toward Health (EARTH) study to fill age and gender strata. Bone density of the hip, lumbar spine (L1-L4) and whole body were measured using a Hologic Discovery W DEXA machine following standard protocols in 925 men and women. Bone density at the heel was estimated using a Hologic Sahara QUS device in men and women ranging from 18 to 81. We explored the association of estimates of BMD from QUS as a predictor of femoral neck BMD using linear regression analyses stratified by gender. Calcaneal BMD, estimated using QUS, accounted for approximately 26% of the variation of femoral neck, lumbar spine and whole body BMD in women. In men, 15% of the variation of femoral neck BMD but only 4% of lumbar spine BMD and 8% of whole body BMD was explained by QUS BMD. A joint model including age, BMI and QUS BMD accounted for 51% of the variation in measured femoral neck BMD in women and 37% in men (Table 1). Associations with spine and whole body were lower, particularly among men. Using speed of sound (SOS), broadband ultrasound attenuation (BUA) and quantitative ultrasound index (QUI) in place of QUS BMD, resulted in similar associations with DEXA BMD at the three sites. In separate regression models, mean differences between estimates of BMD from calcaneal QUS vs. DEXA at the femoral neck, spine and whole body were systematically related to BMI, suggesting a BMI-dependent bias (Table 2). Population characteristics may be an important consideration in the utility of calcaneal QUS in estimation of BMD and bone health screening.

Table Table 2. DEXA minus Calcaneal QUS BMD
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Table Table1.. R2 for Models predicting DEXA BMD measurements From Calcaneal QUS BMD, BMI and age
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Disclosures: Maureen Murtaugh, None.


Bone Density Following Long-Duration Spaceflight and Recovery.Shreyasee Amin*1, Sara Achenbach1, Elizabeth Atkinson1, Elisabeth Spector2, HJ Hartnett2, L. Joseph Melton1, Sundeep Khosla3, Jean Sibonga4. 1Mayo Clinic, USA, 2Wyle Integrated Science & Engineering Group, USA, 3College of Medicine, Mayo Clinic, USA, 4NASA-Johnson Space Center, USA

Rapid bone loss in the weight-bearing skeleton is well recognized during long-duration spaceflight, but the implications on long-term bone health remain unclear. How bone mineral density [BMD] in US crew members serving on long-duration missions in space [US crew] compare with what would be expected had they not been exposed to microgravity, is unknown.

We therefore examined the observed changes in BMD (g/cm2) among 28 US crew (immediately post-flight and following ∼12 months recovery) relative to comparable age- and gender-expected changes derived from 348 men (age range at baseline: 22-90 yrs) and 351 women (range: 21-93 yrs) representing an age-stratified, random sample of the adult community population. BMD measurements (Hologic QDR 2000) were made at the total hip, lumbar spine, ultradistal and midshaft radius, and total body (sites also measured in US crew). Men were measured at baseline, 2, and 4 yrs; women were measured at baseline, 1, 2, and 4 yrs. Linear mixed effects models were used to predict follow-up BMD using baseline BMD, age, gender, and follow-up time, adjusting for the fact that most people were measured more than once. Models including body mass index (BMI) or lean mass were also considered. In US crew (24 men, age range at pre-flight scan: 36-53 yrs; 4 women, age: 41-53 yrs), BMD was measured pre-flight, immediately post flight and at ∼12 months post-flight using Hologic QDR 2000, QDR 4500 and Discovery scanners. The majority had pre- and post-flight BMD on similar machines. Immediate post-flight BMD was performed a median of 6 (range: 3-33) days after return, with a median flight duration of 167 (range: 95-215) days. 22 men and 4 women had a scan within 6-18 months post-flight; median days from landing to ∼12 months scan was 376 (range: 184-534) days.

The table shows the predicted and observed BMD and rates of change immediately and ∼12 month post-flight for US crew. Findings were similar using prediction models which included BMI or lean mass. Due to microgravity exposure, the observed immediate post-flight BMD at all sites was significantly lower than predicted. However, at ∼12 months post-flight, BMD at most sites in US crew were still lower than would be expected had they not been exposed to microgravity.

These findings have implications on potential long-term adverse effects on bone health of US crew serving on long-duration spaceflight missions.

Table Table.  
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Disclosures: Shreyasee Amin, None.


See Friday Plenary number FR0320.


See Friday Plenary number FR0321.


See Friday Plenary number FR0322.


25-Hydroxyvitamin D-Levels in Community-Dwelling Postmenopausal Women Differ Between Swiss Mountain and Plain Areas During Winter.Jotinder Schuematschek-Kainth*1, David Kissner1, Albrecht Popp1, Günter Menz2, Raphael Kessler3, Matthias Stahl4, Christoph Senn1, Spiros Arampatzis1, Antonella Berlingieri1, Romain Perrelet1, Kurt Lippuner1. 1Osteoporosis Policlinic, University of Bern, Switzerland, 2Hochgebirgsklinik Davos, Switzerland, 3HFR Tafers, Switzerland, 4Kantonsspital Olten, Switzerland

Background: Adequate vitamin D supply plays a critical role in the maintenance of optimal musculoskeletal health. Since dietary vitamin D sources are scarce, sunlight exposure is an important determinant of Vitamin D status which therefore could differ among geographic regions. Little is known about differences in vitamin D status between plain and mountain areas in Switzerland, a country where 25% of the population lives in mountain sites.

Methods: In 150 randomly selected, community-dwelling women, aged 65-80 years, from 3 different regions, serum levels of 25-hydroxyvitamin D (25[OH]D) were assessed using the DiaSorin LIAISON “25-OH Vitamin D Total Assay” kit (Diasorin, Stillwater, MN, USA). One third of the women were living in a mountain area (Davos, 1600 m.a.s.l., n=50), one third in a plain rural area (Tafers, 651 m.a.s.l., n=50) and one third in a plain urban area (Olten, 396 m.a.s.l., n=50). All blood samples were taken during wintertime (February). Current intake of vitamin D supplements were recorded.

Results: Mean (± SD) 25(OH)D levels were highest in Davos (49.9 ± 24.7 nmol/L), followed by Tafers (43.4 ± 23.6 nmol/L) and Olten (38.9 ± 19.2 nmol/L, p=0.015 vs. Davos). The respective rates of women with vitamin D inadequacy (<75nmol/L), insufficiency (<50 nmol/L) and deficiency (<22.5 nmol/L) were: 90%, 64%, and 20% (overall population), 84%, 56%, and 10% (Davos), 88%, 68%, and 18% (Tafers), and 98%, 68%, and 30% (Olten). The rates of inadequacy and deficiency were significanly higher in Olten than in Davos (p=0.015). One third of the women indicated to take current vitamin D-supplements in all three areas.

Conclusions: In Switzerland during wintertime, the prevalence of vitamin D inadequacy in community-dwelling postmenopausal women was high despite intake of supplements. People living in mountain areas may have some advantage over those in plain areas, possibly due to higher sun exposure.

Disclosures: Jotinder Schuematschek-Kainth, None.


A Bout of Resistance Exercise Increased Osteoprotegerin in Healthy Young Men.Katherine Brooke-Wavell*1, James King1, Stephen Burns2, David Stensel1. 1Loughborough University, United Kingdom, 2National Institute of Education, Singapore

Purpose: The binding of receptor activator nuclear factor κB ligand (RANKL) to its receptor promotes osteoclast differentiation, activation and attachment to bone. The soluble decoy receptor osteoprotegerin (OPG) binds to RANKL, preventing the binding of RANKL to RANK, so reducing or preventing bone resorption. In vitro studies have reported increased production of OPG, and reduced production of RANKL, in response to loading. Increases in OPG have also been reported in humans following endurance running but there is little information as to the effects of resistance exercise. The purpose of the study was thus to determine whether a bout of resistance exercise influences serum OPG.

Methods: Participants were 16 healthy young men, who each completed a resistance exercise trial, and a control trial, in random order. The resistance exercise trial consisted of 3 sets of 12 repetitions of 10 resistance exercises, at 80% of 12-repetition maximum. During the control trial participants undertook sedentary activity in the laboratory. Food intake was matched for 48 hours before, and during the trials. A venous blood sample was collected before exercise and after 2.5 hours (30 minutes after the exercise bout). Changes in plasma volume were estimated from haemoglobin and haematocrit concentrations. OPG was analysed using a commercially available enzyme immunoassay from serum in seven participants and plasma in nine. Comparisons between trials were made with paired t-tests and repeated measures analysis of variance.

Results: Participants' mean +/- SD age was 24.1 +/- 3.2 y, height 1.79 +/- 0.06m and weight 77.6 +/- 9.3 kg. Resting OPG concentration did not differ between control and exercise trials (2.9 +/- 0.7 versus 3.2 +/- 0.9 pmol/l respectively). Changes in plasma volume did not differ between trials (p=0.591). OPG increased by mean (standard error) 7.3 (4.0) % after the exercise trial but decreased by 5.1 (3.6) % after the control trial. This difference between trials was statistically significant (p=0.040).

Conclusion: A bout of resistance exercise of an intensity that may be expected to increase bone mineral density increased serum OPG in healthy young men. The OPG/RANKL/RANK system may be involved in bone response to mechanical loading.

Disclosures: Katherine Brooke-Wavell, None.


Age-related Cortical Bone Loss and Fracture Patterns in the Neolithic Community of Catalhoyuk, Turkey.Sabrina Agarwal1, Bonnie Glencross*2. 1University of California, USA, 2University of Toronto, Canada

The Neolithic (from 10,000 to 5,000 cal BC) is broadly characterized by population increase, sedentism, crowding and the adoption of an agricultural lifestyle. Typically prehistoric populations that follow a change in subsistence and lifestyle, show a decline in health as indicated by disruptions in bone remodeling, low cortical bone mass for age, poor bone quality and fragility fracture. The focus of this study is to examine age and sex-related patterns of cortical bone loss as well as skeletal fragility fracture in a Neolithic archaeological skeletal sample from Çatalhöyük, Turkey. Using metacarpal radiogrammetry, a method with specific advantages for investigating cortical bone loss in archaeological remains, 50 adult metacarpals (f=28 m=22) were examined. Measurements of total bone width (TW) and medullary width (MW) were taken and used to calculate cortical thickness (CT) and cortical index (CI). These parameters were then standardized for comparison with historic and modern European data. Çatalhöyük males and females demonstrate an inverse relationship where CI decreases as age and MW increase. Analyses indicate statistically significant age-related change in MW, CT and CI amongst the oldest females. Further, both Çatalhöyük males and females have significantly more compact bone across the entire adult life course than males and females from a medieval historic sample while maintaining similar amounts of compact bone in comparison to recent Europeans. Despite age-related loss of bone, no typical fragility fractures are observed. We discuss possible growth and lifestyle factors at Çatalhöyük that may have contributed to improved skeletal strength and reduction in the risk of fragility fracture, and the implications for understanding age-related bone loss in modern populations.

Disclosures: Bonnie Glencross, None.


Dietary Patterns and Bone Health in Women.Sarah McNaughton1, Tikky Wattanapenpaiboon1, John Wark*2, Caryl Nowson1. 1Centre for Physical Activity & Nutrition Research, School of Exercise & Nutrition Sciences, Deakin University, Australia, 2Royal Melbourne Hospital, Australia

Increasingly, measures of dietary patterns have been used to capture the complex nature of dietary intake. Dietary patterns have been shown to be associated with health but few studies have investigated the impact of specific dietary patterns on bone health. The aim of this study was to examine the association between dietary patterns and measures of bone health in a sample of Australian women aged 18-65 years (n=527) recruited through the Twin and Sister Bone Research Program at the Royal Melbourne Hospital.

Bone mineral density (BMD; g/cm2) at the lumbar spine (L2-L4) and total hip was measured from site-specific scans and total body bone mineral content (BMC; g) was measured from a total body scan using dual energy X-ray absorptiometry. Diet was assessed using a 4-day food diary. Dietary patterns were identified using principal components analysis (PCA). Scores were calculated based on the amount of each food consumed in the pattern and the weightings determined by the PCA. Participants were categorised into quintiles according to the scores. Analysis was conducted using generalised estimating equation methods and adjusted for clustering associated with twin and sister pairs and covariates.

PCA revealed 5 dietary patterns. Pattern 1 was characterised by high consumption of refined cereals, soft drinks, hot chips, sausages and processed meat, vegetable oils, beer and takeaway foods and low consumption of other vegetables, vegetable dishes, tea, coffee, fruit, wholegrain breads and breakfast cereals. This pattern was significantly inversely associated with total body BMC (adjusted for age, height, energy intake, physical activity, smoking, education and calcium intake). Each quintile increase in consumption of this pattern was associated with a 0.7% lower total body BMC. Pattern 4 (high consumption of legumes, seafood, seeds, nuts, wine, rice and rice dishes, other vegetables and vegetable dishes and low consumption of bacon and ham) was directly associated with BMD at both sites and total BMC in adjusted models. Each quintile increase was associated with an average higher bone density of 0.2% at the hip, 0.3% at the spine and 0.6% with total body BMC. The remaining dietary patterns, including those containing dairy foods, were not consistently associated with BMD or total body BMC.

This study identified specific dietary patterns associated with BMD and total body BMC among women and potential food-based strategies for improving bone health.

Disclosures: John Wark, None.


See Friday Plenary number FR0327.


Evidence for an Association Between Seasonal Fluctuation of 25(OH)D and Serum C-telopeptide (CTx): Preliminary Evidence from the D-FINES study.Andrea Darling*1, Fatma Gossiel2, Rosemary Hannon2, Debra Skene1, Jacqueline Berry3, Richard Eastell2, Susan Lanham-New1. 1University of Surrey, United Kingdom, 2University of Sheffield, United Kingdom, 3University of Manchester, United Kingdom

The purpose of this study was to assess whether there is a difference in bone resorption by degree of seasonal change in 25(OH)D and whether this varies by ethnicity. In the recent D-FINES study, (Vitamin D, Food Intake, Nutrition and Exposure to Sunlight in Southern England, 2006-2007), a subset of n=65 from the 293 participants (South Asian (n 30) and Caucasian (n 35)) had blood taken in four seasons for determination of 25(OH)D and serum c-telopeptide (sCTX). sCTX was measured using an electrochemiluminescent immunoassay (Roche cobas e411). Seasonal fluctuation of 25(OH)D was assessed by calculating differences between the winter (nadir) and summer (peak) 25(OH)D. For ease of interpretation these changes were expressed as positive values. This enabled investigation of the absolute change in 25(OH)D but not its direction. This variable was then split into quartiles within ethnicity. The dependent variables were absolute concentration of sCTX in each season as well as summer to winter change in sCTX. ANCOVA was run with absolute summer and winter 25(OH)D status, age, BMI, socioeconomic status, physical activity, and dietary calcium as covariates. In the Asian group there was no clear trend between degree of seasonal fluctuation and absolute sCTX. Indeed, only the autumn data was statistically significant (F=5.93; p= 0.01) and with no consistent pattern among the quartiles. No data were significant for change in summer to winter sCTX in Asians or Caucasians despite a trend in both ethnic groups for lower sCTX in the middle quartiles relative to the highest and lowest. Last, in Caucasians, there was a non-statistically significant (p>0.05) inverse trend between cycling of 25(OH)D and absolute serum C-telopeptide levels. These data suggest lower bone resorption in all seasons in Caucasians with increased cycling, and a reduction in sCTX between summer and winter in both ethnic groups in the middle quartile relative to the other quartiles. As the values were covariate adjusted, these findings are not likely to be due to other variables. However, it must be borne in mind that these results are only trends, which is likely due to the small numbers of subjects. Further research is required to analyse banked urine samples from the D-FINES study (n 293) which would enable us to see if these results are statistically significant with increased statistical power. The D-FINES study was funded by the UK Food Standards Agency. All views are those of the authors alone

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Serum CTX by quartile of seasonal change in 25(OH)D

Disclosures: Andrea Darling, None.


Middle-aged Men with Dietary Intake of Omega-3 Long Chain Polyunsaturated Fatty Acids above the Median have Higher Bone Mass for Age.Nour Makarem*1, Jason DeGuire1, Catherine Vanstone1, Suzanne Morin2, Hope Weiler1. 1McGill University, Canada, 2McGill University Health Centre, Canada

Background: Dietary eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) are associated with improved bone health in postmenopausal women and in animals, but evidence is scarce in men.

Objective: To determine if the dietary intake of EPA and DHA and their subsequent levels in erythrocytes are associated with higher BMD in healthy middle aged men.

Methods: This analysis represents the first 27 participants in a cross-sectional sample of healthy men between 37 and 53 years. Measures included: anthropometry, dietary intake of EPA and DHA using the validated Harvard/Willett food frequency questionnaire (FFQ), assessment of serum 25-hydroxy vitamin D using an automated immunoassay (Liaison, Diasorin), and assessment of BMD at the whole body, spine, total left hip and femoral neck using DXA. The Paffenbarger physical activity questionnaire was used to capture total and weight-bearing activity. BMD of participants with dietary intake of EPA and DHA below (group 1: G1) and above (group 2: G2) the median was compared using t-tests. The results were then confirmed with a multiple regression analysis using EPA and DHA intake, calcium intake, serum 25-hydroxy vitamin D, height, and BMI as independent variables and whole body BMD and spine, hip, and femoral neck BMD z-scores as the dependent variables.

Results: There were no differences between the groups for age (G1: 43.6 ± 1.3 vs G2: 45.9 ± 1.4 y, P=0.259), BMI (G1: 25.7 ± 1.2 vs G2: 28.1 ± 1.1 kg/m2, P=0.149), serum 25-hydroxy vitamin D (G1: 67.0 ± 6.6 vs G2: 67.0 ± 5.0 nmol/L, P=0.497), or calcium intake (G1: 1255 ± 116 vs G2: 1169 ± 103 mg/d, P=0.584). By design, mean intakes of EPA plus DHA were higher in group 2 (P=0.009). Group 2 had higher BMD for whole body (P=0.019), spine (P=0.012), and femoral neck (P=0.040), but not total hip (P=0.074) plus higher BMD z-scores at the spine (P=0.005), total hip (P=0.033), and femoral neck (P=0.016). Multiple regression analysis confirmed that EPA and DHA intake was the only significant independent predictor of whole body BMD (P= 0.014) and regional BMD z-scores (p<0.024).

Conclusion: These data suggest that men with higher intakes of EPA and DHA have improved bone health. This is consistent with evidence from other population groups. Future investigation using a larger sample will include assessment of EPA and DHA using erythrocyte membrane fatty acid analyses, confirm present results, and provide clarification as to whether benefits are also evident for the hip BMD. 

Table  .  
  1. Data are mean ± SEM

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Group 1 and Group 2 BMD and BMD z-scores

Disclosures: Nour Makarem, None.


Temporal Trends and Determinants of Longitudinal Change in 25-Hydroxyvitamin D Levels in a Population-based Study.Claudie Berger*1, Lisa Langsetmo2, Nancy Kreiger3, Linda Greene-Finestone4, Christopher Kovacs5, Brent Richards6, Nick Hidiroglou7, Kurtis Sarafin7, K. Shawn Davison8, Jonathan Adachi9, Jacques Brown8, David Hanley10, David Goltzman11. 1McGill University, Canada, 2Canandian Multicenter Osteoporosis Study, Canada, 3University of Toronto, Canada, 4Public Health Agency of Canada, Canada, 5Memorial University of Newfoundland, Canada, 6Jewish General Hospital, McGill University, Canada, 7Health Canada, Canada, 8Laval University, Canada, 9St. Joseph's Hospital, Canada, 10University of Calgary, Canada, 11McGill University Health Centre, Canada

Vitamin D plays a clear role in skeletal health and in other diseases. We examined vitamin D intake, sun-exposure, and body mass index (BMI) and subsequent changes in serum 25(OH)D over a five-year period among CaMos participants in 2 centres.

We studied 481 women and 213 men aged 30+ years with available 25(OH)D levels drawn in 2000-02 (year 5) and 2005-07 (year 10). Blood samples were all analyzed in the same laboratory using Diasorin Liaison. Our main outcome was change in serum 25(OH)D levels (nmol/L). Potential predictors were vitamin D intake from supplements and drugs (year 10 and change), BMI (year 10 and change), and sun exposure. We used multiple linear regression adjusting for sex, age, season of blood draw, centre (Quebec City and Calgary), and physical activity. Interactions between change in vitamin D intake and season of blood draw and centre were considered.

Vitamin D supplement intake was 174.3 (SD=279.3) IU/day at year 5 and 336.9 (SD=408.6) IU/day five years later. Average BMI increase was 0.27kg/m2 (95% CI: 0.10; 0.44). More than two-thirds of participants reported direct sunlight exposure (>30 minutes) as never or seldom at both time points, while 7.6% reported regular or frequent exposure. 66% of the participants had both draws done in the same season. Average difference between year 5 and 10 in 25(OH)D levels was 3.24 (1.64; 4.83) nmol/L. Those who had their blood drawn in the same season showed an average increase of 3.6 (1.6; 5.7) nmol/L and those who had them drawn in a different season showed a trend toward an increase with a mean of 2.5 (-0.2; 5.1) nmol/L. At year 5, only 33.3% of men, 32.4% of women, and 32.7% overall had 25(OH)D levels >75 nmol/L. By year 10, 38.4% of men, 42.7% of women and 39.6% overall had levels > 75nmol/L. Increase in vitamin D supplements, older age, and being from Quebec city were among the variables associated with an increase in 25(OH)D levels over time. BMI (year 5 or change) was not associated with changes in 25(OH)D levels.

The results, in a longitudinal population-based cohort, indicate a trend toward increasing 25(OH)D levels over the 5-year period. This trend appears to be due to changes in vitamin D intake but indicate that more than 400 IU per day will be required to increase levels in the population above 75nmol/L.

Disclosures: Claudie Berger, None.


50 Year Predicted Changes in BMD Distribution and Hip Fracture Incidence in Canada.Lisa Langsetmo*1, David Hanley2, Claudie Berger3, Nancy Kreiger4, Christopher Kovacs5, Alexandra Papaioannou6, David Hanley2, K. Shawn Davison7, Jerilynn Prior8, Robert Josse9, David Goltzman10. 1Canandian Multicenter Osteoporosis Study, Canada, 2University of Calgary, Canada, 3McGill University, Canada, 4University of Toronto, Canada, 5Memorial University of Newfoundland, Canada, 6Hamilton Health Sciences, Canada, 7Laval University, Canada, 8University of British Columbia, Canada, 9St. Michael's Hospital, University of Toronto, Canada, 10McGill University Health Centre, Canada

Background: The age distribution in the Canadian population is expected to change over the next 50 years, with a consequent increase in the burden of hip fracture. Our objective was to predict the burden of hip fracture based on estimated distribution of age and bone mineral density (BMD) over the period 2006-2056 assuming a) sex-age-specific fracture rates remain constant and b) sex-age-specific fracture rates change according to changes in sex-age-specific BMD.

Methods: Study participants (2590 men, 5740 women) were members of CaMos, an on-going population-based cohort study of Canadians ages ≥ 25 years. We assessed the distributions of BMD at baseline (1995-1997) and year 10 (2005-2007), as well as the mean 10-year longitudinal rate of change by sex and five-year age categories. We assumed constant peak bone mass and constant sex-age-specific rates of change to forecast the future distribution of BMD. Multiple imputation was used to account for missing data. Our base fracture model assumed the sex-age-specific hip fracture rates remain constant over time. Our second model incorporated the expected changes in BMD together with the established relationship between BMD and hip fractures to estimate a predicted fracture rate. Future population age distributions were based on Statistics Canada forecasts.

Results: For men over 50 years, the mean total hip BMD was 0.997 g/cm2 in 2006, 0.979 g/cm2 with same BMD distribution but 2056 age weights, and 0.963 g/cm2 using the predicted 2056 age and BMD distribution. For women over 50 years, the mean total hip BMD was 0.858 g/cm2 in 2006, 0.833 g/cm2 using the same BMD distribution and 2056 age weights, and 0.880 g/cm2 using the predicted 2056 age and BMD distribution. Assuming constant rates, the crude incidence rate of fracture over 50 years would increase by 153% in men and 133% in women, due to estimated changes in age (see Figure). Assuming the predicted changes in BMD distribution, the crude incidence rate of fracture over 50 years would increase by 177% in men, but would increase by only 54% in women.

Conclusion: A simple model based on age alone predicted decreases in mean BMD in both men and women, as well as dramatic increases in hip fracture incidence between 2006 and 2056. However, the predicted increases in hip fracture due to changes in age distribution may be partially mitigated by secular increases in BMD in women, but not in men.

Disclosures: Lisa Langsetmo, None.


See Friday Plenary number FR0332.


Association Between Beta-blocker use and Fracture Risk: the Dubbo Osteoporosis Epidemiology Study.Shuman Yang*1, Nguyen Nguyen2, Jacqueline Center2, John Eisman2, Tuan Nguyen2. 1Garvan Institute, Australia, 2Garvan Institute of Medical Research, Australia

Mice treated with beta-blockers (BB) had increased bone mass. Low bone mass is a predictor of fracture risk. The present study sought to examine the association between BB use and fracture risk in elderly men and women.

Data from 3536 participants (1293 men) aged 50 years and above in the Dubbo Osteoporosis Epidemiology Study were analyzed. Baseline characteristics of participants were obtained at the initial visit which had taken place between 1989 and 1993. Bone mineral density (BMD) at the lumbar spine and femoral neck was measured by dual energy X-ray absorptiometry (GE-LUNAR Corp, Madison, WI). Two hundred and seventy (21%) men and 451 (20%) women had been on BB, as ascertained by direct interview and verification with medication history. The incidence of fragility fractures was ascertained during the follow-up period (1989-2008). Propensity score analysis was used to adjust for potential differences in covariates between BB users and non-BB users.

During the follow-up period, 237 men and 681 women had sustained a fragility fracture. As expected, men and women with a fracture were older and had lower BMD than those without a fracture. Men who used only beta-blocker had a significantly lower risk of fracture than those not on beta-blocker (odds ratio [OR]: 0.63; 95% CI: 0.43-0.92). A similar trend was observed in women, but it was not statistically significant (0.96; 0.77-1.21). These associations did not change after adjusting for age, BMD and common risk factors. A combination of the use BB and thiazide was non-significantly associated with lower fracture risk in both sexes (0.94; 0.37-2.47 for men and 0.95; 0.61-1.49 for women). Men on BB had higher BMD at the femoral neck (0.96 versus 0.92 g/cm2, p<0.01) after adjustment for propensity score.

These data suggest that use of beta blockers was associated with reduced fracture risk in men, and the association is likely to be mediated by BMD. Given the high prevalence of osteoporosis and hypertension in the general population, this finding raises the possibility that these anti-hypertensive agents may have materially affected osteoporosis in many populations with high incidence of hypertension, such as the US, Europe and Japan.

Disclosures: Shuman Yang, None.


BMD Enhances Clinical Risk Factors in Predicting Ten-Year Risk of Osteoporotic Fractures in Chinese Men: The Hong Kong Osteoporosis Study.Cora Bow*1, Shirley Tsang2, Sze Sze Soong1, Siu Ching Yeung1, Annie Kung3. 1Department of Medicine, The University of Hong Kong, Hong kong, 2University of Hong Kong, Peoples republic of china, 3Department of Medicine, University of Hong Kong, Hong kong

Introduction: Clinical risk factors with or without bone mineral density (BMD) measurements are increasingly recognized as reliable predictors of absolute fracture risk. Clinical risk factors may be population specific. The purpose of this prospective study was to determine the risk factors for osteoporotic fractures and to predict the 10-year risk of fractures in Southern Chinese male population.

Materials and Methods: This is a part of the Hong Kong Osteoporosis Study. 1,525 community-dwelling, treatment-naive Southern Chinese men aged 50 or above were recruited. Baseline demographic characteristics and clinical risk factors were obtained, and BMD at the spine and hip were measured. Subjects were prospectively followed for incident low trauma fractures. Ten-year risks of major osteoporotic fracture and hip fracture were calculated using Cox proportional hazards models.

Results: The mean age of subjects was 68 ± 10 years. After 3.5 ± 3 (1-14) years of follow-up, 36 non-traumatic incident fractures were reported. The incident rates for osteoporotic fractures and hip fractures were 676/100,000 and 132/100,000 person-years respectively. The most significant predictors of osteoporotic fracture were history of fall (odds ratio 14.5) and fragility fracture (odds ratio 4.4). Other predictive factors included outdoor activity <60 minutes per day, body mass index (BMI) < 20 kg/cm2, difficulty bending forward, use of walking aid, and age ≥ 65 years. Each SD reduction in BMD at spine or hip was associated with 1.7 to 2.6-fold increase in fracture risk. Subjects with 5 or more clinical risk factors had an absolute 10-year risk of osteoporotic fracture of 6.2%, which increased to 18.2% if they also had total hip BMD T-score ≤ -2.5. Addition of BMD information (total hip T-score ≤ -2.5) significantly enhanced fracture risk prediction when compared to clinical risk factors only (omnibus test p=0.001). Men with multiple risk factors and low BMD T-scores have a higher absolute fracture risk, while men with no risk factors and normal BMD have a lower fracture risk than that predicted by FRAX.

Conclusions: Clinical risk factors are population specific and the addition of BMD measurement to risk factor assessment improves fracture risk prediction in Southern Chinese men.

Disclosures: Cora Bow, None.


Clinical Utility of Combined Femoral Neck and Lumbar Spine Bone Mineral Density Measurements in the Individualized Prognosis of Fracture.Tuan Nguyen*1, Nguyen Nguyen1, Steven Frost2, Dana Bliuc1, Jacqueline Center1, John Eisman1. 1Garvan Institute of Medical Research, Australia, 2University of Western Sydney, Australia

Femoral neck bone mineral density (FNBMD) has been used as the principal predictor of fracture risk in men and women, even though more than 50% of women and ∼70% of men who fracture do not have low FNBMD. Lumbar spine BMD (LSBMD) has also been used in the prognosis of fracture risk, but there is a concern over its predictive value due to degenerative change that affects spinal BMD measurement. The present study sought to address that question by examining the contribution of FNBMD and LSBMD to the prediction of fracture.

The Dubbo Osteoporosis Epidemiology Study was designed as a community-based prospective study, with 1358 women and 858 men aged 60+ years as at 1989. Baseline measurements included femoral neck bone mineral density (FNBMD and LSBMD), prior fracture, a history of falls and body weight. The Cox's proportional hazards model was used to assess the contributions of BMD and clinical risk factors to the prediction of fracture risk.

During the follow-up period, 426 women and 149 men had sustained a low-trauma fracture. There were 77 first hip fractures in women and 26 in men. For predicting any fracture and hip fracture, LSBMD was not a significant and independent predictor in the presence of FNBMD. The area under the ROC (AUC) of the model including FNBMD, age, prior fracture and falls was 0.75; when LSBMD was added to the model the AUC was still 0.75. For hip fracture, the AUC value of the model with FNBMD and the model combined FNBMD and LSBMD were the same of 0.85.

When the lower value of either LSBMD or FNBMD was used in the model, these results remained unchanged. Further re-classification analysis revealed that while the use of lower LSBMD did not improve the AUC over and above that of FNBMD, value of LSBMD was useful in ruling out fracture (i.e., increase specificity by ∼17% to 38%).

These data suggest that although lumbar spine BMD may not be an independent predictor of fracture risk, its use in conjunction with femoral neck BMD could be useful for ruling out fracture cases, particularly hip fracture. This “ruling-out” utility can be very valuable in the individualization of fracture prognosis.

Disclosures: Tuan Nguyen, None.


Creation of an Electronic Order Set to Improve the Care of Patients with Osteoporotic Fractures.Beatrice Edwards*1, Andrew Bunta2, Nikolas Kazmers3, Kenzie Cameron4, Lidia Andruszyn3, Nicole Dillon3, Gabrielle Edwards3, Allison Hahr5, Mark Williams6. 1Northwestern University Medical School, USA, 2Northwestern University Feinberg School of Medicine, USA, 3Northwestern University, USA, 4Division of Internal Medicine, Northwestern University, USA, 5Division of Endocrinology, Northwestern University, USA, 6Division of Hospital Medicine, Northwestern University, USA

Background: Patients with osteoporotic fractures are rarely assessed and treated for osteoporosis Goal: creation of Information technology based intervention to assist physicians in providing medical care for osteoporosis to individuals hospitalized with fractures.

Methods: Study conducted at Northwestern Memorial Hospital (NMH) Chicago from Jan 2008- Dec 2009. Physician focus groups explored attitudes about osteoporosis and secondary prevention in patients with fractures. Physicians provided feedback on their ideal EMR intervention in order to improve care of patients with fractures. Information technology: based on physician recommendation a Powerchart (Cerner) based order set was created for clinical use. Creation of list of patients hospitalized with fractures was performed via text “parsing”. Parsing is the visual recognition of specific terms in radiologic report. Terms searched included: fractures, compression deformity, and insufficiency fracture. A list of patients with fractures was created. Exclusion: patients under the age of 50 yrs, with metastatic cancer, other metabolic bone disorders, or CKD stage 5. Dissemination: conferences were held for medical staff and trainees on secondary prevention of osteoporosis and review of the order set (4 sessions) Evaluation: Medical records of patients hospitalized in the Medicine service were reviewed for the following 9 months. Monthly treatment rates were calculated.

Results: Four focus groups were conducted with six physicians per group, participants were General Internal Medicine (N= 10) Hospital Medicine (n=14). All were attendings at NMH. Physicians noted that lack of radiologist emphasis on presence of fracture, time constraints, competing demands, need for brief hospitalization, and lack of IT-based reminders limited their ability to provide care for such patients. 278 cases were reviewed, diagnosis and anti-resorptive treatment rate varied from 2-12%, mention of need for outpatient osteoporosis care with PCP varied from 3-14% over the next 12 months. Figure 1 Pelvic fractures were readily identfied and treated (n=14/21, 67%)

Conclusion: The creation of an order set was recommended by clinicians but rarely used in practice. Low level of clinician/radiologist awareness, competing demands, need for brief hospitalizations, limit clinicians' ability to address osteoporosis in practice. Other interventions must be explored in an effort to overcome this gap in medical care.

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Figure Figure 1. Osteoporosis Treatment in Patients with Fragility Fractures

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Disclosures: Beatrice Edwards, Amgen, 5; Procter and Gamble, 2; Eli Lilly, 5; Novartis, 5


Has the Cost Burden of Incident Fractures Changed Over Time? A Longitudinal Analysis from 1996-2008.Colleen Metge*1, Mahmoud Azimee1, Lisa Lix2, Suzanne Morin3, Patricia Caetano4, William Leslie1. 1University of Manitoba, Canada, 2University of Saskatchewan, Canada, 3McGill University Health Centre, Canada, 4Manitoba Health, Canada

Cost-of-illness (COI) analysis is used to evaluate the economic burden of illness in terms of health care resource (HCR) consumption and production losses. Incidence-based COI is useful when considering fracture prevention measures and post-fracture management from a cost distribution perspective.

AIM: To estimate costs over time related to incident fractures in older women and men and to describe the incremental costs of fracture in Canada.

METHODS: We used the Population Health Research Data Repository for Manitoba---a comprehensive collection of databases including physician visits, hospitalization and pharmaceutical prescriptions---to identify incident fractures (Apr 1997-Mar 2007) and HCR costs (Mar 1996-Apr 2008). An incidence approach was used to count fracture numbers by site/mechanism (hip, wrist, spine, humerus, other and trauma) and sex in Manitobans aged 50 y and older; a bottom-up approach was used to estimate costs (quantity x unit costs) in the year pre- and post-fracture. Incremental fracture-related costs were calculated as the difference between the year pre- and post-fracture. Costs were expressed in constant dollars (2006 CDN) and evaluated for linear trends over time.

RESULTS: Over the 10 years, adjusted post-hip fracture costs in women changed minimally for hospitalization ($24,863 to $20,085, -1.6% per y, p=.12) and personal care home residence ($11,980 to $12,485, +1.7% per y, p=.15), significantly for physician costs ($2,149 to $3,447, +6.7% per y, p<.0001) and drug costs ($792 to $1974, +14.3% per y, p<.001); home care costs did not change significantly over time ($3,456 to $3,433, -0.5% per y, p=.55). Total costs in the year post-hip fracture were stable in women (+0.1% per y, p=.87) and men (-0.6% per y, p=.58). Incremental costs (the difference between pre- and post-hip fracture costs) increased significantly for physician services (p<.001) and decreased significantly for drug costs (p<.001) in both women and men. Total incremental cost changes over time were variable and depended on type of fracture and sex (see Table); a significant reduction was seen for wrist fractures in women (-4.4% per y, p<.001) with increases for trauma fractures in women (+7.9% per y, p=.003) and men (+8.4% per y, p=.018).

CONCLUSION: Post-fracture costs have changed over time in a site-dependent manner. Identifying projected cost-of-illness including health care resource use will help to estimate the future cost burden of fracture.

Table Table. Change in incremental total costs over time (1996-2008)
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Disclosures: Colleen Metge, Bayer Inc, 5; sanofi-aventis, 5; Amgen, 2 This study received funding from: Amgen Canada


See Friday Plenary number FR0338.


See Friday Plenary number FR0339.


Ontario Osteoporosis Strategy: Three Year Evaluation of Quality Indicators.Gillian Hawker1, Susan Jaglal*2, Cathy Cameron1, Ruth Croxford3. 1Women's College Hospital, Canada, 2University of Toronto, Canada, 3Institute for Clinical Evaluative Sciences, Canada

Purpose: In February 2005, the Ontario Osteoporosis Strategy was launched to address the gaps in care with the overall goal to reduce fractures, morbidity, mortality and costs from osteoporosis through an integrated and comprehensive approach aimed at health promotion and disease management in the population. The province of Ontario, Canada has 11 million inhabitants. The purpose of this research is to evaluate the impact on health system indicators after the first 3 years of implementation of the strategy. Methods: A literature review followed by a Delphi process was used to identify and refine quality indicators for osteoporosis and postfracture care. Quality indicators using administrative data sources were selected for the three year evaluation due to data availability. Data sources included hospital discharge abstracts and emergency department visits to identify fractures; physician billings for BMD testing and drug plan data which only includes individuals 65 years and older. Time trends for each indicator were calculated from 2002 to 2008. Indicators examine fracture rates, BMD testing, treatment rates and adherence. Results: For the primary indicator, fracture rate there was no significant change in fracture rates between 2002 and 2008. The rate of BMD testing was constant over the time period with a greater proportion of patients at “high risk” being tested. The number of BMD tests was constant at about 500,000 per year until 2008 when it dropped to 480,000. The indicator BMD testing one year after a fracture showed an increasing proportion among those who fractured from 2002 to 2007. In 2005 12.7% of hip fracture patients had a BMD test compared to 16.6% in 2007 compared to 24.9% in 2005 and 26.5% in 2007 for wrist fractures. However, over the time period there was no difference in one year adherence rates among those 66+ years who initiated an osteoporosis medication. Conclusions: In the first three years of implementation of the Ontario Osteoporosis Strategy the emphasis was on improving post-fracture care where fracture clinic coordinators were placed in high volume hospitals. This 3 year evaluation suggests that the strategy is successful in targeting these high risk individuals for osteoporosis care. More time is needed before conclusions can be drawn about the impact on fracture rates.

Disclosures: Susan Jaglal, None.


Questionnaire Survey to Validate an Educational Program Intended for Orthopaedic Surgeons to Improve the Management of Severe Osteoporosis.Irene Cerocchi*1, Giuseppina Resmini2, Antonio Capone3, Giovanni Iolascon4, Alfredo Nardi5, Umberto Tarantino6. 1PTV Foundation - University of Tor Vergata, Italy, 2Treviglio Hospital, Italy, 3University of Cagliari, Italy, 4Second University of Naples, Italy, 5Rovigo General Hospital, Italy, 6Azienda Ospedaliera PTV, Italy

The GOST (Management of Severe Osteoporosis in Traumatology) project was intended for orthopaedic surgeons, whose knowledge about osteoporosis and fragility fractures is known to be often poor and superficial, as widely documented in the literature. During 18 educational events the 510 participants were invited to fill in a questionnaire both at the beginning and at the end of the course, in order to verify if its structure and content had brought an improvement in the understanding of osteoporosis and related arguments. The questionnaire was made of 36 multiple choice questions: 7 about basic science, 12 about the clinical aspects of osteoporosis and metabolic bone diseases, 7 about diagnostic tools, 6 about pharmacological therapy and 4 about surgical treatment. There was an overall improvement of 18,77%, and for each question the percentage of right answers was higher at the end of the course compared to the beginning. The data obtained were also compared based on the subject treated. The best results were achieved in the clinical field (62,76% right answers before versus 84.64% after the meeting, with a 21,88% improvement). The percentage of right answers increased by 20,84% in the diagnosis field, and by 16,83% and 16,81% for the questions about medical treatment and basic science, respectively. The lower percentage of improvement in the surgical field (12,21%) was probably due to the already good knowledge about bone implants and the treatment of osteoporotic fractures, as the courses were attended by orthopaedic surgeons only. Moreover, the finality of the educational events was to focus on the biological, clinical and therapeutical aspects of osteoporosis, that are usually poorly considered by surgeons, who tend to focus on the surgical treatment of the fracture, often forgetting the pathophysiology, characteristics and consequences of poor bone quality. In conclusion, the GOST courses proved to be an useful tool in the improvement of orthopaedic knowledge about effective diagnosis and management of metabolic bone diseases.

Disclosures: Irene Cerocchi, None.


See Friday Plenary number FR0342.


See Friday Plenary number FR0343.


See Friday Plenary number FR0344.


What was the FRAX® Value the Day Before the Fracture?Karine Briot*1, Frederic Sailhan2, Antoine Babinet2, JP Courpied2, Philippe Anract2, Christian Roux1. 1Paris Descartes University, Rheumatology Department, Cochin hospital, France, 2Paris Descartes University, Orthopaedics Department, Cochin hospital, Paris, France, France

Rationale and objectives

FRAX® is a fracture prediction algorithm to determine a patient's absolute fracture risk, and to select patients who should receive the highest priority for treatment. FRAX® calculates the 10-year probability of hip (H-FRAX®) and major osteoporotic (clinical spine, forearm, hip and shoulder) fractures (MO-FRAX®). The aim of this study was to calculate the FRAX® in a population of patients with a recent fracture.

Patients and methods: We calculated the FRAX® with femoral neck BMD for all the patients hospitalized for low trauma hip and major osteoporotic fracture over 1 year from February 2009. Bone mineral density (BMD) was measured by DXA and the number of vertebral fractures (VF) were defined using vertebral fracture assessment (VFA) performed during the same exam. The FRAX® was calculated from the data of the day before the fracture, without taking into account the current fracture. All the patients receiving an anti-osteoporotic treatment were excluded.

Results: 435 patients were hospitalized over 1 year in the Orthopaedic Department for a non traumatic non vertebral fracture. 134 were excluded because of cognitive disorders, poor general health and ongoing antiosteoporotic treatment and 59 because of non major osteoporotic fracture. Assessment was thus performed in 242 patients (75.2 ± 11 years); 134 with hip and 108 with other major osteoporotic fractures (clinical spine fractures excluded). Mean femoral neck T score was -2.3 (± 1.1), and 53% of patients were osteoporotic according to spine and/or hip T scores. Mean MO-FRAX® in patients with incident MO fractures was 13.5 ± 9.5% and 44.6% and 62% had a MO-FRAX® ≤10% and 15% respectively. Mean H-FRAX® in patients with a hip fracture was 7.2 ± 5.8% and 28.2% and 50% had a FRAX® ≤3 and 7% respectively. Prevalence of VF by VFA was 42.2% and 50.7% in patients with MO and hip fracture respectively, and 6.3% of these VF were known. Adding these prevalent fractures in the FRAX® calculation increases it to 11.5% and 22.1% for hip and MO fracture.

Conclusion: this study suggests that following the threshold, 25 to 60% of patients with recent fracture would not have been detected by the FRAX® before their fracture. Adding prevalent vertebral fracture in the calculation increases the scores, and thus VFA tool should be consider for fracture prediction improvement.

Disclosures: Karine Briot, None.

This study received funding from: Novartis Laboratory


Decreased Risk Of Vertebral Fracture Is Associated With Low-Moderate Amount Of Alcohol Intake In A Random Sample Of Mexicans.Laura Paola Bernal-Rosales*1, Patricia Clark2, Juan O Talavera-Piña3, Margarita Deleze2, Fidencio Cons-Molina4. 1Hospital Infantil de México “Federico Gómez”, Mexico, 2Laboratorios Clinicos De Puebla, Mexico, 3Centro Médico Nacional Siglo XXI, Mexico, 4Unidad de Diagnóstico de Osteoporosis, Mexico

Purpose: The present study analyzed the potential risk factors in a random sample of Mexicans with vertebral fractures. Methods: A total of 820 subjects were evaluated (406 women and 414 men) aged 50 and over. The sample was randomized and stratified according to four age groups (50-59, 60-69, 70-79, and those over 80) from both sexes. A questionnaire containing information on demographic characteristics, gynecologic history, lifestyle habits, calcium intake, alcohol intake, and physical activity was applied to all participants. Lateral X-rays of the lumbar and thoracic spine and BMD by DXA were obtained in all cases. The protocol was submitted and approved by the Institutional Review Board and written consent was obtained from all participants once the implications of their participation in the study was explained in detail and before the application of questionnaires and/or interviews. A bivariate analysis was performed to estimate the odds ratio and the 95% confidence interval for every risk factor related with the presence of a vertebral fracture; this was followed by a multivariate analysis where each variable was adjusted by the rest of them in a global index. Results: The average age for study group was 69.3 years. Age, gender, and physical inactivity were related to vertebral fracture as follows: age of 70-79 years (OR=2.40, IC95% 1.04-5.68), age over 80 years (OR=6.83, IC95% 3.15-15.16), sedentary lifestyle (OR=1.63, IC95% 0.97-2.72) and female gender (OR=2.28, IC95% 1.40-3.73). A low and moderate intake of alcohol (1-40 g/day) was protective factor for vertebral fracture (OR=0.51, IC95% 0.30-0.87). In the multivariate analysis, low and moderate consumption of alcohol remained as a protective factor (OR=0.56, IC95% 0.33-0.92). Conclusion: Moderate alcohol consumption (1-40g/day) presented a protective effect for vertebral fracture, and, as other studies have showed, an association between the age and gender was observed as risk factors of vertebral fracture.

Disclosures: Laura Paola Bernal-Rosales, None.


Effects of Cigarette Smoke Cadmium on Urine Calcium Excretion in Postmenopausal Women.Maryka Bhattacharyya, Shahram Bozorgnia*. Medical College of Georgia, USA

Low-level cadmium (Cd) exposure in humans is significantly correlated with decreased bone density and increased susceptibility to fractures. In addition, cigarettes are a major source of Cd exposure, with individual smokers having blood Cd concentrations up to 10-fold above those of non-smokers. As part of a larger project, this study investigates if the Cd component of cigarettes is associated with increased calcium excretion from the kidneys of persons who smoke. Eighteen postmenopausal women who smoke cigarettes and 11 non-smokers were studied. From each participant, 2 blood and 11 urine samples were collected over 7 weeks: the first 3 weeks during which smokers smoked at their customary rate; the next 4 days when all smokers ceased smoking; the next 4 weeks when smokers, to different degrees, returned to their previous smoking status. Blood was collected at the beginning and end of the 7 weeks. A first-of-morning (FOM) urine specimen was collected 1- to 2-times per week during the 7 weeks. Smoking status was documented with a daily diary and verified with a carbon monoxide breath test. Analyses included blood Cd concentration (CdB), urine Cd concentration (CdU), and urine calcium concentration (CaU). Results demonstrate that mean values of CdB and CdU were significantly higher in smokers than non-smokers (3.5-fold and 2.6-fold, respectively, p<0.05). In addition, prior to smoking cessation, Cd exposure did not increase mean levels of Ca in FOM urine, with 57 ± 7, 60 ± 14, and 54 ± 10 mg Ca per sample for high-Cd smokers (CdB, 1.0-2.7 ng Cd/ml, n=9), lower-Cd smokers (CdB, 0.5-0.9 ng Cd/ml, n=8), and non-smokers (CdB, 0.2-0.6 ng Cd/ml, n=11), respectively (mean ± SE). However, effects of smoking cessation, measured longitudinally in each subject, did depend upon Cd exposure. During 4 days of smoking cessation, FOM CaU decreased in lower-Cd smokers (-16 ± 5 mg Ca), showed no change in high-Cd smokers (3 ± 4mg Ca), and increased in non-smokers (32 ± 14 mg Ca). These results indicate that subjects with higher CdB values maintained elevated levels of CaU during the short period of smoking cessation, while CaU values decreased in the lower Cd group. For subjects taking Ca supplements, effects of smoking cessation on CaU were also diminished. Results confirm smoking as a significant source of cadmium exposure and demonstrate that the Cd component of cigarettes may maintain elevated calcium excretion by the kidney, which may play a direct role in osteoporosis.

Disclosures: Shahram Bozorgnia, None.


Should Risk of Osteoporosis Restrict Weight Control for Other Health Reasons Among Postmenopausal Women ? - 10-year Follow-up Study.Joonas Sirola*1, Toni Rikkonen2, Marjo Tuppurainen3, Risto Honkanen4, Heikki Kröger3. 1University of Eastern Finland / Kuopio, Finland, 2University of Eastern Finland, Campus of Kuopio, Finland, 3Kuopio University Hospital, Finland, 4University of Kuorio, Finland

Introduction: High body mass index (BMI) is a risk factor for several morbidities but may be protective against osteoporosis. The aim of the present study was to investigate the effects of overweight and obesity on postmenopausal co-morbidity.

Methods: 1970 Finnish women from the OSTPRE cohort were measured with DXA of femoral neck (FN) and lumbar spine (LS) at baseline (1994) and at 10-year follow-up (2004). Women were categorized according to BMI into normal (under 25 kg/m2), overweight (25-29 kg/m2) and obese (over 30 kg/m2). Weight change (WC) during the follow-up was categorized into tertiles: 1) < 1 kg 2) 1-6.2 kg 3) > 6.2 kg. The definition of osteoporosis in the present study was 10-year follow-up FN or LS T-score under -2.5 SD or under -2.0 SD + low trauma energy follow-up fracture. Information about other co-morbidities was based on self-reports. The end-point morbidities were not allowed to be present at baseline. The risk of each co-morbidity was obtained with multivariate logistic regression, adjusted for age, alcohol intake and smoking.

Results: The incidence of co-morbidities during the follow-up were: hypertension (HT) 25.7%, cardiac insufficiency 6.2%, coronary artery disease (CAD) 10.3%, stroke 5.0%, diabetes mellitus (DM) 7.3%, osteoarthritis (OA) 22.3%, breast cancer (BC) 2.8%, depression 2.8%, osteoporosis (OP) 8.2%, chronic back pain (BP) 17.1% and poor self-rated health (SRH) 4.3%. Obesity / overweight predicted higher 10-year risk of HT (OR=2.6 / OR=1.7, p<0.001 compared to normal BMI), CAD (OR=1.6, p<0.05 / OR=1.2, p=NS), DM (OR=11.7 / OR=5.3, p<0.001), OA (OR=1.4, p<0.05 / OR=1.1, p=NS), BP (OR=1.6, p=0.007 / OR=1.2, p=NS) and poor SRH (OR=2.4, p<0.05 / OR=1.5, p=NS) and lower risk of OP (OR=0.13 / OR=0.41, p<0.001). The combined 10-year risk for any co-morbidity significantly associated with BMI, other than SRH and OP, was 2.2 for obese and 1.7 for overweight women (p=0.001). In comparison to the third WC tertile, the first had 1.8 and 2.6 times (p<0.001) and the second 1.6 and 2.9 times (p<0.05) lower risk of having HT and BC, respectively.

Conclusions: The health related risks of obesity, especially DM, outweigh its protective effects on bone health. Weight loss has no significant effect on OP but may protect from HT and BC. Patients with BMI under 25 kg/m2 should be screened for OP, because the risk is over two times higher than the reduction in combined risk of HT, CAD, DM, BP and OA.

Disclosures: Joonas Sirola, None.


Assessing Bone Health in Adult Premenopausal Females in Karachi - A Preliminary Report from Pakistan.Aysha Khan*, Farhan Dar, Romaina Iqbal, Imran Siddiqui, Farooq Ghani. Aga Khan University, Pakistan

Objective: To assess the bone health status in healthy females by using biochemical markers of bone metabolism Material and Methods: Study Design: A cross- sectional study.

Place and Duration: The study was conducted at Section of Chemical Pathology, Department of Pathology & Microbiology, The Aga Khan University Hospital, Karachi, Pakistan, from November 2007 to June 2008.

Methodology: 174 healthy premenopausal female volunteers were recruited through convenient, non-purposive sampling after informed consent. Females taking vitamin D or calcium supplements or any medicine or disease altering bone turnover were excluded. A questionnaire addressing the demographic details was filled; NTx, 25OHD and iPTH were measured in blood in fasting. NTx value >19 nMBCE/L was considered high bone turnover. 25OHD levels were stratified as deficient <20 ng/ml, 21-29 ng/m insufficient, and >30 ng/ml sufficient. Plasma iPTH level of > 87 pg/ml was considered high. Data was analyzed using the Statistical Package for Social Sciences SPSS (Release 16.0, standard version, copyright SPSS). A p-value of <0.05 was treated as significant.

Results: Mean age and BMI of the participants was 29.06 ± 6.89 (18 - 48 years) and 23.12 ± 4.58 (13.84 -41.2) kg/m2 respectively. 82.8% of the females were identified as D deficient, 16.1% had insufficient levels and 1.1% had optimal levels. High bone turnover as depicted by NTx was seen in 36.8% cases. Secondary hyperparathyroidism was present in 25.9% volunteers while others had blunted PTH response. NTx associated with 25OHD (r = 0.198; P-value=0.009); 25OHD associated with iPTH (r = -0.199; P=0.009). Major determinant of high serum NTx were 25OHD, sun exposure and purdah observation. High bone turnover coexists with both low 25OHD (hypovitaminosis D; <30ng/ml) and optimal 25OHD (>30ng/ml).

Conclusion: Vitamin D deficiency is highly prevalent in adult females. Bone turnover is high regardless of vitamin D status. There is a need to develop strategies to understand further, the bone biomechanics and bone quality for Pakistani females.

Disclosures: Aysha Khan, None.


See Friday Plenary number FR0350.


See Friday Plenary number FR0351.


Comparison Between Logistic Regression and Artificial Neural Networks for Morphometric Vertebral Fractures Risk Assessment: New Data from GISMO Lombardia Database.Cristina Eller Vainicher*1, Ivana Santi2, Marco Massarotti3, Luca Pietrogrande4, Matteo Longhi5, Valter Galmarini6, Giorgio Gandolini7, Maurizio Bevilacqua8, Iacopo Chiodini1, Enzo Grossi9. 1Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinco Milano, Italy, 2Istituto Geriatrico ASP IMMeS e Pio Albergo Trivulzio Milano, Italy, 3IRCCS Humanitas Clinical Institute, Italy, 4Ospedale S. Paolo - Università degli Studi di Milano, Italy, 5IRCCS Galeazzi Milano, Italy, 6AO Fatebenefratelli e Oftalmico Milano, Italy, 7Don C. Gnocchi Foundation IRCCS Milan, Italy, 8Ospedale L. Sacco -Polo Universitario, Italy, 9Centro Diagnostico Italiano, Italy

Background: There is growing interest in the development of algorithms, like FRAXTM, using traditional statistical approach, for predicting osteoporotic fractures. Nevertheless, some studies suggest their poor sensitivity. Artificial Neural Networks (ANNs), computer algorithms inspired by the highly interactive processing of the human brain, could represent an attractive alternative to this approach. So far, no reports have investigated the ability of ANNs in predicting osteoporosis fracture.

Aim: To evaluate the capacity of ANNs, compared with Logistic Regression (LR), to recognise patients with or without morphometric vertebral fractures (MVF+ and MFV- respectively) on the basis of classical bone osteoporotic risk factors and other clinical information.

Methods: We compared the prognostic performance of ANNs with that of LR in predicting MFV in 372 female patients affected with postmenopausal osteoporosis (MVF+ n=176; MVF- n=196), described by 42 independent variables. ANNs where allowed to choose the relevant input data automatically (Twist system-Semeion).

Results: 18 variables were selected, as most relevant, among 42 by TWIST system. Using this set of variables the mean sensitivity of LR and ANNs in test data sets was 35.8% and 72.47% respectively, the mean specificity was 76.53% and 78.50% respectively, the overall accuracy was 56.17% and 75.48% respectively.

Age, BMI, years since menopause, smoking habit, hypertension, dislipydemia, calcium intake, bone mineral density, family history of femoral fracture, resulted to be the most relevant variables for the best performing model.

Conclusions: ANNs showed a better performance than LR in predicting the presence of MVF, in particular ANNs showed a higher sensitivity in respect with LR. These results suggest a promising role of ANNs in the development of algorithm for predicting osteoporotic fractures.

Disclosures: Cristina Eller Vainicher, None.


Differences in Skeletal and Non-Skeletal Factors in a Diverse Sample of Men With and Without Type 2 Diabetes Mellitus.Ann Schwartz1, Gretchen Chiu2, Julia Dixon2, Andre Araujo*2. 1University of California, San Francisco, USA, 2New England Research Institutes, USA

Purpose: While fracture risk is higher in patients with type 2 diabetes mellitus (T2DM), patients with T2DM tend to have higher bone mineral density (BMD) than their non-diabetic counterparts. These observations motivate comparisons of both skeletal and non-skeletal factors in subjects with and without T2DM.

Methods: The Boston Area Community Health/Bone (BACH/Bone) Survey is a population-based cross-sectional survey of skeletal health in a random sample of 1,219 Boston men aged 30-79 y. Diabetes status, age and race/ethnicity were obtained via self-report. BMD and body composition were measured by DXA. Physical function was assessed via a composite physical function score derived from walk and chair stand tests. Grip strength was measured with a hydraulic hand dynamometer. Multivariate linear regression was used to examine the association of T2DM status with skeletal and non-skeletal factors.

Results: Of the 1137 men with complete data included in this analysis, the mean age was 48 y. Prevalence of T2DM was 12.5%, with an average duration of disease of 7.4 y. Of the skeletal factors considered, only lumbar spine BMD was significantly higher in men with T2DM. Of the non-skeletal factors considered, both the composite physical function score and maximum grip strength were significantly lower in men with T2DM. As shown in the table, skeletal factors were no longer significantly associated with T2DM status in multiple regression models. Non-skeletal factors including appendicular lean mass, arms lean mass, and maximum grip strength were negatively associated with T2DM after adjustment.

Conclusion: These results suggest that non-skeletal factors such as body composition and muscle strength, which may influence fall risk, could explain higher fracture rates among patients with T2DM.

Multiple regression models showing the association between T2DM and outcomes

Table  .  
  1. Adjusted for age, race/ethnicity, and body mass index

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Disclosures: Andre Araujo, None.


Gender Differences in Factors Associated with Falls in a Population-Based Cohort Study in Japan: The ROAD Study.Shigeyuki Muraki*1, Toru Akune1, Hiroyuki Oka1, Kozo Nakamura2, Hiroshi Kawaguchi3, Noriko Yoshimura1. 1University of Tokyo, Japan, 2The University of Tokyo, Japan, 3University of Tokyo, Faculty of Medicine, Japan

Fall is the most serious factor for fractures in the elderly; therefore, it is important to identify subjects with factors associated with fall. The objective of the present study was to determine factors associated with falls in elderly men and women. From the 3,040 participants in the ROAD study, the present study analyzed 1,180 subjects aged more than 60 years from the mountainous and coastal cohorts (431 men and 749 women; mean age, 71.7 years). At the baseline, physical ability was estimated by measuring grip strength, 6-m walking time, normal step length (NSL), and chair stand time. Further, vertebral fracture (VFx) was assessed by lateral radiographs of the lumbar spine. Lumbar spondylosis and knee osteoarthritis (OA) were defined as a Kellgren/Lawrence grade of ≥3. Falls during the preceding year were evaluated by a self-reported questionnaire. During 1 year, 63 men (14.7%) and 153 women (20.5%) fell once or several times. In men, logistic regression analysis without adjustment revealed that NSL and radiographic VFx at the baseline were significantly associated with falls (odds ratio [OR], 0.94 and 2.21; 95% confidence interval [CI], 0.90-0.99 and 1.19-4.00, respectively), but grip strength, 6-m walking time, chair stand time, knee OA, and lumbar spondylosis were not associated with falls (OR: 0.98, 1.01, 1.03, 0.77, and 1.13; 95% CI: 0.94-1.02, 0.91-1.10, 0.96-1.10, 0.36-1.54, and 0.66-1.94, respectively). Multiple logistic regression analysis after adjustment for age revealed that NSL and radiographic VFx were independently associated with falls (OR, 0.94 and 2.08; 95% CI, 0.89-0.98 and 1.08-3.88, respectively). In women, logistic regression analysis without adjustment revealed that grip strength and knee OA were significantly associated with falls (OR, 0.96 and 1.52; 95% CI, 0.92-0.99 and 1.05-2.20, respectively), but 6-m walking time, NSL, chair stand time, VFx, and lumbar spondylosis were not associated with falls (OR: 1.03, 0.99, 1.02, 1.47, and 1.23; 95% CI: 0.97-1.08, 0.94-1.03, 0.98-1.05, 0.86-2.42, and 0.86-1.75, respectively). Multiple regression analysis after adjustment for age revealed that grip strength and knee OA were independently associated with falls (OR, 0.95 and 1.56; 95% CI, 0.91-0.99 and 1.05-2.30, respectively). In conclusion, this study revealed distinct factors associated with falls in men and women. In men, NSL and VFx were associated with falls, whereas in women, grip strength and knee OA were associated with falls.

Disclosures: Shigeyuki Muraki, None.


See Friday Plenary number FR0355.


See Friday Plenary number FR0356.


Lack of Association Between Osteoporosis and Coronary Artery Disease in Korean Men and Women.Se Hwa Kim1, Soo-Kyung Kim2, Deok-Kyu Cho1, Yun-Hyeong Cho1, Sun-Ok Song*1. 1Kwandong University College of Medicine, Myongji Hospital, South korea, 2Pochon CHA University College of Medicine, South korea

Background: Coronary artery disease (CAD) and osteoporosis are major causes of morbidity and mortality in the elderly. The aim of this study was to investigate whether osteoporosis or osteoporotic fractures was associated with CAD independent of traditional risk factors. Subjects and Methods: Study population consisted of Korean men and women who underwent coronary angiography. The following clinical and demographic parameters were recorded: age, sex, body mass index (BMI), hypertension, diabetes mellitus, coronary artery disease, smoking status. Bone mineral density (BMD) and T-L spine lateral X-ray were taken. Of 485 patients, 250 were taken T-L spine lateral X-ray to assess the vertebral fractures and BMD was assessed using dual energy X-ray absorptiometry (DXA) in 110 patients. Results: Patients were grouped according to the BMD (normal, osteopenia, and osteoporosis). Patients with vertebral fractures were regard as osteoporosis group. Patients with osteoporosis were older and had lower BMI than those with normal or osteopenia. There were not significant differences between three groups in respect to diabetes mellitus, hypertension, hypercholesterolemia, BMD. Prevalence of CAD was not different between the 3 groups. However, prevalence of CAD involving more than 2 coronary arteries in patients with osteoporosis or vertebral fractures was higher than in patients with normal BMD (26% vs. 56% in women, 36% vs. 69% in man). In the logistic regression analysis showed that age and diabetes mellitus were independently associated with CAD. Conclusions: Age and diabetes mellitus were independent predictors of CAD in our study population. However, osteoporosis or vertebral fracture did not show any association with CAD in Korean men and women.

Disclosures: Sun-Ok Song, None.


Modification of the Osteoporosis Patient Assessment Questionnaire Using Item Response Theory Methods.April Naegeli*1, Russel Burge1, Steven Watts1, Timothy Stump2, Deborah Gold3, Stuart Silverman4. 1Eli Lilly & Company, USA, 2Indiana University School of Medicine, USA, 3Duke University Medical Center, USA, 4Cedars-Sinai/UCLA, USA

The purpose of this study was to develop a modified version of the Osteoporosis Patient Assessment Questionnaire (OPAQ 3.0) that reduces respondent burden and specifically assesses the impact of osteoporosis on physical function and mobility for use in clinical trials evaluating drug therapy for the prevention of vertebral fracture.

This study was conducted using the Multiple Outcomes of Raloxifene Evaluation (MORE) trial database. At baseline, 1478 patients were administered the OPAQ 2.0. The OPAQ 2.0 is an 18-domain, 60-item patient reported outcome (PRO) measure used in clinical trials to evaluate health-related quality of life in patients with fractures related to their osteoporosis. Items were evaluated based on the application of a unidimensional, polytomous Item Response Theory (IRT) model and clinical relevance founded on expert judgment. Dimensionality was assessed with a factor analysis considering eigenvalues. A large drop from the first to second eigenvalue with the rest following the scree plot was indicative of a unidimensional scale. Items were retained based on their relatedness to the underlying construct and ability to discriminate through analysis of Item Information and Item Characteristic Curves, respectively. All analyses were conducted using Mplus statistical software.

OPAQ 3.0 contains 6 domains with 21 items retained from OPAQ 2.0: walking and bending (6 items), sitting and standing (3 items), transfers (4 items), backache and pain (2 items), fear of falls (3 items), and independence (3 items). Slight modifcations to item wording and response options were necessary to improve clinical relevance and to capture descriptions as depicted by patients with osteoporosis.

OPAQ 3.0 was developed based on clinical trial responses to OPAQ 2.0 to decrease respondent burden by reducing the number of items from 60 to 21, and to assess the impact of osteoporosis, specifically on physical function and mobility. Next steps for OPAQ 3.0 include confirmation of content validity, and evaluation of psychometric properties (factor structure, reliability, validity, and responsiveness) in targeted clinical trial patient populations for drug therapy aimed to prevent fracture related to osteoporosis.

Disclosures: April Naegeli, Eli Lilly and Company, 3; Eli Lilly and Company, 1

This study received funding from: Eli Lilly and Company


Obesity in Adolescence and Bone Strength in Adulthood.Kirsti Uusi-Rasi*1, Marika Laaksonen2, Olli Raitakari3, Jorma Viikari4, Mika Kähönen5, Harri Sievänen1. 1UKK Institute for Health Promotion Research, Finland, 2University of Helsinki, Finland, 3University oh Turku, Finland, 4University of Turku, Finland, 5University of Tampere, Finland

While obese children have been reported to have lower bone mass for a given weight, obese adults have greater bone mineral density compared with normal weight adults. In this prospective cohort study we evaluated differences in bone cross-sectional size and density in relation to the age of gaining excess body mass.

328 women from the population-based cohort of The Young Finns Study were divided into 4 groups by body mass index (BMI) at the age of 12-15 years (healthy body weight, C-, or overweight/obese, C+) and 24 years later (healthy weigh in adulthood A-, or overweight/obese in adulthood A+). Of 162 overweight women, 49 had been obese since childhood (C+A+) and 113 had gained extra weight in adulthood (C-A+). Of 166 healthy weight women, 24 had been overweight in childhood (C+A-) and 142 had always been healthy weight (C-A-).

Total cross-sectional area (ToA, mm2) and cortical (shaft CoD, mg/cm3) and trabecular (distal TrD, mg/cm3) bone density of the radius and tibia were measured with pQCT at the mean age (SD) of 38 (1.5) years. Between-group differences were evaluated with analysis of covariance.

Mean body height was 166 (6) cm without between-group differences. In healthy weight groups, mean body mass was similar being 62 (6) and 61 (6) kg, but those women who had been obese since childhood weighted more than women who had become obese in adulthood, the mean weights being 89 (18) and (80 (10) kg, respectively. Childhood obesity was associated with larger ToA especially at the tibia, whilst obesity in adulthood predicted higher TrD and lower CoD (Table).

Childhood obesity was associated with larger cross-sections at long bone diaphyses and distal tibia, but not distal radius. Being obese since childhood or gaining excess weight in adulthood may lead to lower cortical and higher trabecular density both at weight-bearing and nonweight-bearing bones. In contrast, it seems that temporary obesity in adolescence, while being associated with increased bone size, may lead to lower trabecular density.

Table  .  
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Disclosures: Kirsti Uusi-Rasi, None.


See Friday Plenary number FR0360.


Serum 25-Hydroxyvitamin D Levels, Mortality and Risk of Non-spine and Hip Fractures in Older White Women: Data from the Study of Osteoporotic Fractures.Marc Hochberg*1, Li-Yung Lui2, Dennis Black3, Jane Cauley4, Peggy Cawthon2, Kristine Ensrud5, Lisa Fredman6, Teresa Hillier7, Steven Cummings8. 1University of Maryland School of Medicine, USA, 2California Pacific Medical Center Research Institute, USA, 3University of California, San Francisco, USA, 4University of Pittsburgh Graduate School of Public Health, USA, 5Minneapolis VA Medical Center/University of Minnesota, USA, 6Boston University School of Public Health, USA, 7Kaiser Center for Health Research, USA, 8San Francisco Coordinating Center, USA

Objective: The associations between low serum vitamin D levels and mortality and fracture risk are inconsistent. The objective of the current analysis was to determine whether low serum levels of 25-hydroxyvitamin D (25[OH]D) were associated with higher mortality and an increased risk of non-spine, including hip, fractures in older white women. Methods: Serum 25(OH)D levels were measured by liquid chromato-graphy-tandem mass spectroscopy in frozen specimens from 5915 white women (mean [SD] age: 76.7 [4.8] years) who completed Visit 4 in the Study of Osteoporotic Fractures between August 1992 and July 1994. Cox proportional hazards models were used to test the association of serum 25(OH)D levels, as either a continuous or categorical variable, with all-cause mortality, and the incidence of non-spine and hip fractures; results are expressed as hazard ratios (HR) with 95% confidence intervals (CI). Results: Mean (SD) serum 25(OH)D level was 23.2 (11.7) ng/ml; quartiles were defined as <=16, 17-22, 23-28 and >=29 ng/ml. 3199 women died during a mean 11.1 (4.5) years of follow-up. There was a significant trend for increasing mortality in lower quartiles of 25(OH)D in age-adjusted but not multivariate (MV)-adjusted models (Table). Women in the lowest quartile of serum 25(OH)D levels had a significantly increased risk of mortality in age-adjusted models compared with women in Q2-4 combined: HR 1.10 (1.02, 1.19). This association was stronger with shorter length of follow-up: HR 1.29 (1.10, 1.51) and 1.15 (1.04, 1.27) after 5 and 10 years of follow-up, respectively. 2174 women sustained a non-spine fracture, including 768 with a hip fracture, over a mean 8.7 (5.0) years of follow-up. Lower serum 25(OH)D levels were significantly associated with a decreased risk of both non-spine and hip fractures in both age-and MV-adjusted models as a continuous but not as a categorical variable (Table). There was a significant trend for decreasing hip fracture risk in lower quartiles of 25(OH)D levels in MV-adjusted models (Table). Similar results were found when data were analyzed using clinically relevant cutpoints (deficient, insufficient and sufficient) for 25(OH)D levels. Conclusion: These data suggest a relationship between low serum 25(OH)D levels and an increased risk for all-cause mortality while, paradoxically, providing evidence for a reduced risk of hip fractures in these older white women with lower 25(OH)D levels.

Table  .  
  1. *P value for trend =0.04

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Adjusted HR (95% CI) for the Association of Serum 25(OH)D Levels with Mortality and Fractures

Disclosures: Marc Hochberg, None.


The Effect of Biological Aging On Bone Mineral Density and Fracture.Kyoung Min Kim*1, Kwang Joon Kim2, Su-Jin Park3, Gadi Jogeswar3, Jami Ajita3, Yumie Rhee4, Sung-Kil Lim5. 1Yonsei University, South korea, 2Severance Hospital, South korea, 3Yonsei university colleage of medicine, South korea, 4IUMS, Yonsei University, College of Medicine, South korea, 5Yonsei University College of Medicine, South korea

Introduction: Bone mineral density is influenced by the dynamics of aging, inflammation and bone remodeling processes. Admittedly, chronological age (CA) being a major determinant of BMD. However, this CA simply measures the amount of time that has gone by since birth. Thus, CA provides only limited information about matters such as declining functional capacity and other properties we associate with aging. Therefore, several previous reports have tried to search the correlations between telomere length, as a marker of biological aging, and BMD. Purpose: We sought to identify the useful candidate biomarkers of aging for evaluation of individual biological capacity, and using these to develop an equation measuring biological age (BA), then to investigate the relationships between individual biological aging with BMD. Results: Among 4763 men and 5275 women aged 39∼70 years-old, we selected 1509 subjects who met the normality criteria of each biochemical variables. Variables showing significant correlations (correlation coefficient >0.15, p<0.05) with CA were selected. 5 variables were selected including systolic blood pressure (SBP), forced expiratory volume in 1 s (FEV1), glycosylated hemoglobin (HbA1c), red blood cell count (RBC) and waist/hip ratio (W/H). Then, Biological aging calculating equation was obtained by multiple regression analysis with using these 5 variables. We calculated each one's BA and the differences between these two ages (BA-CA, Biological Aging Score (BAS)). Individual BAS showed significant correlations with glucose metabolism, pulmonary function, hematological variables, lipid profile and anthropometric parameters after adjusting CA. However, BAS was not associated with BMD, osteoporosis or fracture after adjusting CA unlikely other clinical parameters. Conclusions: No association was observed between BAS and BMD in Korean. This finding can tell us that BMD will be strongly afftected by not BA but CA, the time after birth.

Disclosures: Kyoung Min Kim, None.


See Friday Plenary number FR0363.


Better Skeletal Microstructure Confers Greater Mechanical Advantages in Chinese-American Women than Caucasian Women.Xiaowei Liu*1, Marcella Walker1, Bini Zhou1, Donald McMahon2, Julia Udesky1, George Liu3, John Bilezikian2, X Guo1. 1Columbia University, USA, 2Columbia University College of Physicians & Surgeons, USA, 3New York Downtown Hospital, USA

Despite lower areal BMD (aBMD) by DXA, Chinese-American (CH) women have fewer fractures (Fx) than Caucasian (CA) women. We hypothesized that better skeletal microstructure in CH women could account for this paradox. To address this hypothesis, we applied individual trabeculae segmentation (ITS), a novel image analysis technique, and micro finite element analysis (μFEA), to high-resolution peripheral quantitative computed tomography (HR-pQCT) images of premenopausal CH and CA women.

Age was similar (36 ± 7 vs. 35 ± 4), but height (64 ± 2 vs. 65 ± 3 inches; p=0.02) and weight (125 ± 22 vs. 139 ± 38 lbs; p=0.03) were lower in the CH (n=46) vs. CA (n=49) group. aBMD by DXA did not differ at the spine, hip and 1/3 radius. ITS analysis was applied to trabecular sub-volumes of HR-pQCT (XtremeCT, Scanco Medical; voxel size 82 μm) images at the distal radius (DR) and distal tibia (DT) to quantify trabecular plate-and rod-microarchitecture. Remarkably, CH women had 94% (DR) and 80% (DT) higher plate bone volume fraction (pBV/TV) and 20% (DR) and 18% (DT) higher plate number density (pTb.N) as compared to CA women (p<0.0001). In contrast, rod-like characteristics {i.e. the amount and number of trabecular rod (rBV/TV and rTb.N)} were similar. Thus, the plate/rod ratio was much greater in CH than in CA trabecular bone. Additionally, plate thickness (pTb.Th) was 9% (DR) and 4% (DT) greater in CH women (p< 0.001). Plate-rod and plate-plate junction densities (PR & P-P Junc.D), parameters indicating the trabecular network connections, were 37% and 56% (DR) and 29% and 49% (DT) greater in CH women (p<0.01). These striking differences in trabecular bone translate into 55%-69% (DR, p<0.001) and 29%-54% (DT, p<0.05) higher Young's and shear moduli in the CH vs. CA groups, as estimated by μFEA of HR-pQCT images. Moreover, although CH women have 9% (DR, p=0.03) and 6% (DT, p=0.08) smaller bone size than CA women, thicker cortices (18% and 10% higher in CH vs. CA, p<0.05) and more plate-like trabecular bone lead to 14% (DR) and 8% (DT) greater whole bone stiffness (p<0.05).

Greater microstructural advantages in CH, as shown by these results, may help to account for lower Fx rates in CH women as compared to CA women. 

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Figure Figure 1. (Top) Bigger bone size, thinner cortex, and less dense trabecular bone of (Left) Caucasian than (Right) Chinese-American women illustrated by representative 3D cortical and trabecular bone microarchitecture imaged by HR-pQCT; (Bottom) a cubic trabecular bone volume was extracted and decomposed into individual trabeculae represented by different colors. The same trabecular bone volume was also illustrated by green and red to represent plate-and rod-like trabeculae: Chinese women have significantly greater amount of plate-like trabecular bone than Caucasian women

Download figure to PowerPoint

Disclosures: Xiaowei Liu, None.


See Friday Plenary number FR0365.


Association of Homocysteine, Folate and Vitamin B12 with Bone Mineral Density and Biochemical Bone Turnover in Young Healthy Indians; A Cross Sectional Study.Sushil Gupta1, Nisha Nigil Haroon*2. 1Sanjay Gandhi Post Graduate Institute of Medical Sciences, India, 2University of Toronto, Canada

Purpose: Indians have lower peak bone mass, higher bone turnover and higher prevalence of osteoporosis compared to Caucasians. Hyperhomocysteinemia (HHcy) is linked to fractures. But the association of homocysteine (Hcy), folate and vitamin B12 to bone mineral density (BMD) is controversial. The role of these factors in determining bone health of young adults is not clear. We studied the effect of Hcy, folate and vitamin B12 on BMD and bone turnover in young healthy Indians.

Methods: This cross sectional study was conducted at a tertiary care centre in Lucknow, India and involved 151 young healthy adults. Data regarding demographic, dietary and lifestyle factors were obtained using a questionnaire. Serum chemistry (calcium, phosphorus, albumin, creatinine, total alkaline phosphatase (ALP) and haematocrit), 25(OH)D3, PTH, vitamin B12, folate, Hcy, bone formation (N-MID-Osteocalcin (OC), bone specific alkaline phosphatase (BAP)) and resorption markers (cross laps (CTx)) were measured. BMD was assessed at hip (total hip, femoral neck and trochanter), lumbar spine (L1-L4) and forearm using DXA (Hologic QDR 4500A). Correlations were assessed with Spearman's correlation coefficient. Linear regression was used to find predictors of BMD. Comparisons between males and females were done using Student's t test or Mann-Whitney U test.

Results: The subjects (n=151) had a mean age of 26+5 years. Table 1 depicts baseline data. Only 6% had a recommended B12 intake of 2.4mcg/day. Most subjects (75%) were vegetarians and took less vitamin B12 than non vegetarians (0.6 vs.1.4 mcg, p<0.01). Low BMD was noted in 17% of subjects. The prevalence of biochemical osteomalacia (ALP >150 IU/L), vitamin B12 deficiency (<200 pmol/L), HHcy (>15μmol/L) and hypovitaminosis D (< 20 ng/ml) were 27%, 68%, 71% and 83% respectively. Serum Hcy inversely correlated with vitamin B12 and folate (r=-0.327, -0.224, p<0.05) levels. Vitamin B12, Hcy and folate levels did not correlate with ALP, BAP, OC, CTx and BMD at total hip, femoral neck, trochanter, lumbar spine and forearm. Even in those with Hcy levels in the highest quartile, no correlation was found between Hcy and BMD or bone markers. On multiple linear regression, height, BMI and calcium intake were independent and significant predictors of BMD.

Conclusion: Young North Indian adults have high prevalence of HHcy and vitamin B12 deficiency. Hcy, folate and vitamin B12 are not predictors of bone turnover and BMD in young adults.

Table Table 1. Base line character istics of study subjects.
  1. *p <0.01 (males vs. females). Data are mean ± SD or median (IQ). BAP: Bone specific alkaline phosphatase.

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Disclosures: Nisha Nigil Haroon, None.


Vitamin K Deficiency in Subjects with Severe Motor and Intellectual Disabilities.Akiko Kuwabara1, Akiko Nagae2, Naoko Tsugawa3, Kunihiko Tozawa4, Mari Kitagawa2, Hiroaki Kohno5, Masakazu Miura6, Toshio Okano3, Masao Kumode2, Kiyoshi Tanaka*7. 1Osaka Shoin Women's University, Japan, 2Biwako Gakuen Kusatsu Medical & Welfare Center for Children or Person with Severe Motor & intellectual disabilities, Japan, 3Kobe Pharmaceutical University, Japan, 4Sanko Junyaku Co., Ltd, Japan, 5Orthopaedics, Japan, 6Hokuriku University, Japan, 7Kyoto Women's University, Japan

Purpose: Subjects with severe motor and intellectual disabilities are at high risk of fracture. High incidence of vitamin D deficiency and BMD increase after vitamin D intervention have been reported in these subjects. Regarding vitamin K; another bone-active vitamin, however, only few papers are available. Therefore, we have studied the vitamin K status in these subjects by measuring serum levels of protein induced by vitamin K absence (PIVKA-II) and undercarboxylated osteocalcin (ucOC) as sensitive markers for hepatic and skeletal vitamin K deficiency, respectively.

Methods: Eighty-two subjects with severe motor and intellectual disabilities, mostly due to cerebral palsy, were evaluated for their serum levels of PIVKA-II and ucOC, and their 7-day vitamin K intake.

Results: Serum concentrations of PIVKA-II and ucOC were 60.9 ± 106.5 mAU/mL (median: 29.0) and 5.44 ± 5.70 ng/mL (median: 3.49), respectively. Subjects with oral intake had approximately 3 times higher vitamin K intake (median; 208.5 μg/day) and significantly lower serum levels of PIVKA-II and ucOC than those under enteral feeding. Serum levels of PIVKA-II and ucOC were significantly higher, and vitamin K intake was significantly lower in those with antibiotics use than those without it. Next, subjects were divided into four groups depending on the presence or absence of enteral feeding and antibiotics treatment. In those with both of them, median serum PIVKA-II and ucOC concentrations were 67.0 mAU/mL and 7.02 ng/mL, respectively, which were significantly higher than those in other three groups.

Conclusions: The current subjects had high prevalence of vitamin K deficiency both in the bone and liver, to which enteral feeding and antibiotics use were the contributing factors, and these two risk factors were additive. Thus, subjects with both enteral feeding and antibiotics use were at the highest risk of vitamin K deficiency. Although this is a preliminary cross-sectional study evaluating only the surrogate markers, attention should be paid on the vitamin K deficiency in subjects with severe motor and intellectual disabilities with its possible involvement in the increased fracture risk in these subjects.

Disclosures: Kiyoshi Tanaka, None.


See Friday Plenary number FR0368.


See Friday Plenary number FR0369.


See Friday Plenary number FR0370.


See Friday Plenary number FR0371.


Endogenous Opioid Effects on Bone Reveal a Critical Role of Hypothalamic Neuropeptide Y.Frank Driessler*1, Iris Wong1, Ronaldo Enriquez2, Brigitte Kieffer3, Christoph Schwarzer4, Amanda Sainsbury2, Herbert Herzog2, Jacqueline Center1, John Eisman1, Paul Baldock1. 1Garvan Institute of Medical Research, Australia, 2Garvan Institute, Australia, 3IGBMC, France, 4Institut für Pharmakologie, Austria

Exogenous opioids are known to exert powerful effects on bone mass via endocrine and non-endocrine effects, increasing hip fracture by around 2-fold. We examined a component of endogenous opioid system, the dynorphins, for skeletal effects and the signaling pathway involved. Dynorphins are predominantly expressed within the central nervous system, with actions on pain, addiction and depression. In the hypothalamus, dynorphin and pre-prodynorphin is co-expressed with neuropeptide Y (NPY) and NPY expression is reduced in Dyn-/- mice.

We examined the bone phenotype of Dynorphin knockout mice (Dyn-/-) and the potential involvement of NPY.

Cancellous bone volume was elevated in Dyn-/- mice compared to wild type (wt: 8.8% ± 0.6 vs Dyn-/-: 11.9% ± 1, p<0.02). Osteoclast surface (7.9% ± 0.7 vs 11.9% ± 0.7, p<0.01) and osteoclast number (3.3/mm ± 0.3 vs 4.4/mm ± 0.2, p<0.05) were elevated in Dyn-/- mice. However, these changes were overridden by an increased mineral apposition rate (MAR) in Dyn-/- (1.6μm/d ± 0.1 vs 2.4μm/d ± 0.2, p<0.02).

Dynorphins signal through the kappa opioid receptor (KOR or), however, this receptor does not appear involved in the skeletal changes. There was no skeletal phenotype in KOR-/- mice (wt: 18.3% ± 1.6 vs KOR-/-: 20.2% ± 1.8, ns) or those treated with the KOR antagonist norBNI. Moreover, KOR was expressed in brain but not in bone tissue and KOR agonist treatment induced a response in primary neurons but not primary osteoblasts in vitro, thereby indicating an indirect action.

Loss of dynorphin signalling is known to reduce neuropeptide Y expression in the hypothalamus, a change known to elevate bone formation. Both NPY-/- and Dyn-/- mice have elevated bone volume and bone formation, however, NPY-/-/Dyn-/- double mutant mice showed no further increase compared to single mutant mice (MAR wt: 1.3μm/d ± 0.06, NPY-/-:1.5μm/d ± 0.06, Dyn-/-:1.6μm/d ± 0.08, NPY-/-/Dyn-/-:1.5μm/d ± 0.04). This indicates a critical role for NPY in the transmission of the central dynorphin pathway.

The endogenous opioid system is required for normal bone homeostasis. The dynorphin system, acting via NPY, may represent a pathway by which higher processes including stress, reward/addiction and depression influence skeletal metabolism. Understanding of these interactions may also enable modulation of the adverse effects of exogenous opioid treatment.

Disclosures: Frank Driessler, None.


Reduced Bone Density Concomitant with Metabolic Abnormalities in Type 1 Diabetic (T1D) Patients.Priscille Masse*1, Carole C. Tranchant2, Maisha B. Pacifique2, Karen Ericson3, Sharon M. Donovan4, Edgard Delvin5. 1University of Moncton, Canada, 2Université de Moncton, Canada, 3Indiana University-Purdue University, USA, 4University of Illinois, USA, 5Hopital Sainte-Justine, Canada

Type 1 diabetic (T1D) patients are at risk of osteoporosis and nutritional deficiencies. These risks have never been investigated in parallel. Nutritional deficiencies can aggravate T1D complications. The present study aimed at fully investigating T1D-induced bone disorder by densitometry combined with blood chemistry after the attainment of peak bone mass. Twenty seven (27) insulin-treated T1D female patients, without renal complication, and 32 healthy controls, aged 30-40 yrs, were rigorously recruited. Exclusion criteria were vegetarianism, pregnancy or lactation, BMi>30 and use of drugs susceptible to influence bone metabolism, including estrogen. Bone mineral density (BMD, g/cm2) was measured by Lunar DEXA on trabecular and cortical sites of lumbar vertebrae and femur. Diagnosis of low BMD (osteopenia + osteoporosis cases) was made according to WHO T-scores. The two groups of women were homogenous and comparable. Kidney functions, assessed by urinary albumin/creatinine, did not differ between them. T1D, diagnosed since 21 yrs in average, was fairly well-controlled with HbA1c (7.8%) only slightly above limit. BMD was significantly reduced in the T1D group at 4 lumbar sites (L1, p<0.02;L2, p<0.03;L1-L2, p<0.02;L1-L3, p<0.05) and at the Ward triangle (p<0.04). There was no significant difference for the cortical bone of the diaphyseal femur. The proportion of low BMD (≤ -1.1 SD) cases, when averaging all trabecular sites, was 1.75 greater (p<0.01) in the T1D group. This group had lower protein status for serum transferrin (p<0.02) and IGF-I (p<0.001) but not for serum albumin. Contrarily to other reports, serum Mg and plasma (P) pyridoxal phosphate (PLP), coenzyme in collagen maturation, were not reduced. P-PLP, used as a biomarker of vitamin B6 nutritional status, was near cut-off limit of deficiency in both groups. Serum iron concentration was also reduced in T1D group (p<0.03) although body stores (ferritin) and hemoglobin level were not affected. Whereas serum phosphate was unchanged, serum Ca (adjusted for albumin) was increased (p<0.0003) despite the reduction of 1,25(OH)2D3 (p<0.001). Vitamin D nutritional status, assessed by 25(OH)D3, was adequate in both groups. Significant blood differences were correlated with %HbA1c indicating that the changes evidenced were disease-induced. This study demonstrated metabolic abnormalities associated with T1D that could not jeopardize the quality of bone collagenous matrix, nor explain bone mineral loss.

Disclosures: Priscille Masse, None.


Disuse Osteopenia of the Forearm: What Accounts for Response Variability Following Immobilization?Joseph Spadaro*1, Christina Centore2, Rebecca Hickman3, Jennifer Kelly1. 1State University of New York Upstate Medical University, USA, 2Department of Biomedical & Chemical Engineering, Syracuse University, USA, 3Upstate Medical University, USA

Changes in bone density and geometry, as a result of immobilization, was more closely examined as part of an ongoing study of bone loss after disuse. Although on average a substantial loss of bone density occurs (5-20%) soon after injury/immobilization, a large variability among individuals is observed. In an attempt to better understand and predict individual responses, we examined the “responders” in our cohort and some of the factors that might be involved. Data was available on the extent of loss of bone mineral density (BMD) in the forearm bones during the six months period following forearm immobilization due to Colles' fracture or carpal surgery in 82 subjects, 30 males and 52 females, age 18-80 (the e-Bone Study). No subjects had conditions or medications known to affect bone density. Measurements of BMD (DXA) in the forearm were categorized by the amount of bone loss at 8, 16, and 24 weeks since completion of immobilization (baseline). Here we focused on the distal ulna, which was not fractured, to help eliminate the extra variability inherent in the radius BMD in these individuals.

Those individuals who had subsequent bone BMD loss between >3% at week 8 were considered “responders” to disuse (57% of the subjects) and 26% >10% loss (figure 1). By 24 weeks, this increased to 39% with > 10% losses. On average, the females lost more bone early (8 weeks), while the males had increased losses by 24 weeks. Those whose dominant hand was casted had more delayed ulna bone loss: 7 individuals at week 8 vs. 20 at weel 24. Dietary calcium does not appear to be a strong factor, and increasing age has previously been identified as increasing disuse bone loss.

It appears that using the “responder” approach to study disuse bone loss can be useful in identifying susceptible individuals disposed to larger losses after disuse, but so far the etiological variables appear to be numerous and a combinatorial approach may be beneficial.

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Figure Figure 1. Disuse BMD loss in the distal ulna by gender Disclosures: Joseph Spadaro, None.

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Skeletal Changes in Type 2 Diabetic Goto-KaKizaKi Rats and Their Relationship with Expression of Bone Formation and Resorption Genes.José L. Pérez-Castrillón*1, Jose A. Riancho2, Daniel De Luis3, David Guede-Garcia4, José Ramon Caeiro5, Manuel Gonzalez-Sagrado6, Maria Domingo-Andres7, David Primo-Martin6. 1Hospital Universitario Río Hortega. Instituto Endocrinología. Reticef, Spain, 2Hospital Universitario Marques de Valdecilla.Reticef.Univiversidad de Cantabria, Spain, 3Hospital Rio Hortega. Instituto de Endocrinologia.Reticef, Spain, 4Trabeculae., Spain, 5Trabeculae. Reticef, Spain, 6Hospital Universitario Río Hortega. Reticef, Spain, 7Reticef, Spain

It has been suggested that the risk of fracture is increased in diabetic patients. However, studies on type 2 diabetes have demonstrated decreased, normal or even increased BMD. Overweight and obesity are prevalent in individuals with type 2 diabetes. Obesity per se tends to be associated with increased bone mass and is thus a confunding factor. We used Goto-KaKizaKi rats, a non-obese animal model of spontaneous type 2 diabetes, to characterize the bone properties in type 2 diabetes.

This study was performed to characterize the bone metabolism in five Goto-KaKizaKi (GK) rats, a spontaneous type 2 diabetic model. Bone samples from 5 GK rats and 5 age-matched, non-diabetic Wistar rats were analyzed. All specimens were analyzed by μ-CT at a 9.0 μm nominal resolution with a Skyscan 1172 scanner and associated analysis software. The following parameters were measured in cancellous bone region: bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), structure model index (SMI) and trabecular bone pattern factor (Tb.Pf). Parameters in cortical bone region included cross-sectional thickness (Cs.Th) and 3D cortical thickness (C.Th). Additionally, volumetric bone mineral density (vBMD) was measured in both regions. RNA was isolated from the femoral heads and the expression of several genes involved in bone formation or resorption (BGP, RANKL, OPG, SOST and DKK1) was measured by real-time qPCR. Structural studies showed a significantly decreased trabecular bone volume and interconnectivity of the trabeculae. In addition, the expression of osteocalcin gene was significantly decreased whilst SOST and DKK1 gene expression was significantly increased. In conclusion, the GK non-obese diabetic rat shows a decreased bone mass associated with decreased expression of the osteoblast-derived osteocalcin gene and might be a useful model to unravel the effects of diabetes on bone independent of obesity.

Disclosures: José L. Pérez-Castrillón, None.

This study received funding from: RETICS. RETICEF


See Friday Plenary number FR0376.


See Friday Plenary number FR0377.


See Friday Plenary number FR0378.


Interactions of Nitric Oxide and Insulin-Like Growth Factor I, in Prevention of Bone Loss.Sunil Wimalawansa*. Robert Wood Johnson Medical School, USA

Many therapeutic advances have been made over the past decade in the prevention and treatment of osteoporosis. However, these are expensive and some also have significant adverse effects, and hence simple, cost-effective therapeutic options are warranted. The beneficial effects of estrogen on bone maintenance is at least in part mediated via nitric oxide (NO)/cGMP pathway, and perhaps also via IGF-1. At appropriate doses, nitroglycerin (NG) as a nitric oxide donor was shown to favorably affect both osteoblasts and osteoclasts (i.e., uncoupling these two cell types), and prevention of estrogen as well as glucocorticoid-induced bone losses.

A three-year randomized, doubled-blind, controlled clinical trial was conducted to assess the efficacy NG in preventing bone loss in early postmenopausal women. This study, Nitroglycerin as an Option: Value in Early Bone Loss (NOVEL) was funded by NIAMS. Women were randomized to receive either nitroglycerin ointment or placebo ointment. All women received calcium and vitamin D supplementation. There were no differences in the BMD in the treatment vs. calcium and vitamin D arms. However, taking compliance (∼75%) into consideration, the dose actually used by the study participants was only ∼50% of that was originally intend to use in this study; i.e., a sub-therapeutic dose that would not have even expected to have positive effect on the skeleton.

Nevertheless, a significant increase of serum IGF-1 levels was observed in women who had positive BMD response following NG therapy, but not in the placebo-treated subjects who had gain in BMD. NG-treated subjects with increased BMD had increase of serum IGF-1 levels, 201 ± 25.6 vs. non-responders, 40.2 ± 16.9 ng/mL (p<0.001), and the BMD changes was significantly correlated with the change of serum IGF-1 levels from the baseline (r = 0.5; p<0.01). Whereas, those who were in the placebo group with increased BMD had no change in serum IGF-1 levels (-2.6 ± 24.6 vs. 10.8 ± 13.5 ng/mL, NS; responders vs. non-responders). Previously, we have demonstrated that estrogenic effects on bone can be completely blocked with nitric oxide synthase (NOS) inhibitors such as L-NAME. Current data suggest that NG, in addition to be one of the key final common pathways for positive effect of estrogen in bone, may also involved in enhancing the local production of IGF-1, thereby assisting bone formation that is observed with nitric oxide therapy.

Disclosures: Sunil Wimalawansa, None.


Teriparatide Treatment in Japanese Subjects with Osteoporosis at High Risk of Fracture: Effect on Bone Mineral Density and Bone Turnover Markers during 12-Month, Randomized, Placebo-Controlled, Double-Blind and 12-Month Open-Label Study Periods.Toshio Matsumoto*1, Toshitsugu Sugimoto2, Hideaki Sowa3, Mika Tsujimoto4, Miho Hatano4, Takao Awa4, Noriko Iikuni4, Akimitsu Miyauchi5, Margaret R Warner6, Toshitaka Nakamura7. 1University of Tokushima Graduate School of Medical Sciences, Japan, 2Shimane University School of Medicine, Japan, 3Eli Lilly Japan K.K., Japan, 4Eli Lilly Japan KK, Japan, 5Omura municipal Hospital, Japan, 6Eli Lilly & Company, USA, 7University of Occupational & Environmental Health, Japan

This multicenter study was conducted to assess the safety and efficacy of teriparatide 20 μg/day in Japanese men and women with osteoporosis at high risk of fracture during a 12-month, randomized, double-blind, placebo-controlled treatment period followed by a 12-month, open-label treatment period with all subjects receiving teriparatide. Subjects (93% female; median age 70 years) were randomized 2:1 to teriparatide versus placebo (teriparatide, n=137; placebo-teriparatide, n=70). In the open-label treatment period, 119 subjects continued teriparatide and 59 placebo-treated subjects switched to teriparatide (placebo-teriparatide group). For subjects with measurements at the 12-month timepoint, bone mineral density (BMD) at the lumbar spine (L2-L4), femoral neck, and total hip increased from baseline by 10.04 ± 5.23% (mean percent change ± SD), 2.01 ± 4.63%, and 2.72 ± 4.04% in the teriparatide group and 0.19 ± 4.33%, 0.44 ± 3.97%, and -0.26 ± 3.42% in the placebo-teriparatide group. At the last available measurement through 12 months (LOCF), teriparatide significantly increased BMD compared with placebo at the lumbar spine (p<0.001), at the femoral neck (P=0.015), and at the total hip (p<0.001). For subjects with measurements at the 24-month timepoint, BMD at the lumbar spine, femoral neck, and total hip increased from baseline by 13.42 ± 6.12%, 3.26 ± 4.25%, and 3.67 ± 3.98% in the teriparatide group and 9.11 ± 5.14%, 2.19 ± 4.81%, and 2.46 ± 3.54% in the placebo-teriparatide group. In the teriparatide group, serum procollagen I N-terminal propeptide (PINP) increased significantly from baseline within 1 month and remained elevated at 24 months (median percent change 49.24%, p<0.001); in the placebo-teriparatide group PINP change was -17.23% at 12 months and 76.12% at 24 months (for both times, p<0.001 versus study baseline). The incidence of treatment-emergent adverse events (TEAEs), serious TEAEs, and discontinuations due to TEAEs was comparable in the teriparatide and placebo-teriparatide groups. These lines of evidence suggest that teriparatide 20 μg/day was well tolerated and stimulated bone formation in Japanese subjects with osteoporosis at high risk of fracture during 24 months of treatment.

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Months 0 to 12: Subjects in the Placebo-Teriparatide group received placebo and subjects in the Teriparatide group received teriparatide 20 μg/day. Months 12 to 24: all subjects received teriparatide 20 μg/day.

Forteo 24month figure_ASBMR

Disclosures: Toshio Matsumoto, Ono Pharmaceutical, 2; JAPAN TOBACCO INC, 5; Teijin Limited, 5; Chugai Pharmaceutical Co., Ltd., 5; Eli Lilly Japan KK, 5; Astellas Pharma Inc, 5; Asahi Chemical, 5; Daiichi Sankyo Company, 5


Vitamin D Insufficency as a Possible Explanation for Lack of BMD Increase in Bisphosphonate Pretreated Male Patients with Fractures and PTH 1-84 Therapy.Friederike Kühne1, Judith Haschka1, Miriam Putz1, Astrid Fahrleitner-Pammer2, Heinrich Resch1, Roland Kocijan1, Christian Muschitz*3. 1Medical Department II-The VINFORCE Study Group, Austria, 2Department of Endocrinology-Medical University of Graz, Austria, 3St. Vincent's Hospital, Austria

Purpose: The aim of this prospective open label study is the evaluation of efficacy and tolerability of PTH (1-84) in men at high risk for first or further osteoporotic fracture. Methods: A total of 15 men with a mean age of 60.6 ± 16.2 years was prospectively assigned to PTH (1-84) 100 μg sc daily for 24 months. All patients received 1000mg calcium and 800IE vitamin D supplementation daily. 13 had prior bisphosphonates with a mean duration of 7.6 ± 3.8 years, 9 of them had major osteoportic fractures an, 2 had a bone biopsy (low connectivity). 12 patients had prior vertebral fractures (mean 3 fractures) and 3 patients had prior hip fracture. We investigated DXA (spine & hip), BTMs, fracture status, improvement of daily activities and reduction of pain. Results: For evaluation baseline and 12 months results were compared. Mean levels of PTH decreased from 49.6 ± 16.7 to 36.3 ± 21.0 ng/ml (P=0.030); P1NP increased from 26.38 ± 9.64 to 148.13 ± 35.45 μg/l (P=0.005) and S-CTX increased from 0.29 ± 0.16 to 0.75 ± 0.44 ng(ml (P=0.002). BMD L1-L4 increased 3.6% (P=0.097) while BMD at hip did not change. Subjects were divided into two groups according to the initial vitamin 25OHD3 levels (cut off: 30 ng/ml). 7 out of 15 subjects had vitamin D insufficiency, only 4 of them reached levels above 30 ng/ml during therapy. 2/8 subjects with physiological levels at baseline decreased within 12 months. The mean increase of vitamin D in the group <30 was 4.75 ng/ml while the mean decrease of vitamin D in the group >30 was 12.8 ng/ml (P=0.032). In the group with evidence of vitamin D insufficiency there was no change in lumbar spine BMD while there was a slight decrease of -1.8% in the other group (P=0.044). No hypercalcaemia was observed and no new vertebral fracture occurred in the 15 patients. Patients reported a significant reduction of back pain resulting in an improvement of daily activities (p<0.05). Conclusions: There is a rapid and sustained change in markers of bone turnover and spine BMD indicating an anabolic effect. The majority of the male patients had an initial vitamin D insufficiency and also a large number of patients with initialy sufficient levels decreased during therapy. Our data show that Vitamin D is as important as the osteoinductive effect of PTH for the increase of mineralsation of bone and deficiency leads to a release of the osteoanabolic effect of PTH. An initial boost phase with high dose vitamin D is essential prior to PTH therapy.

Disclosures: Christian Muschitz, None.


A Systematic Review of the Risk of Bisphosphonate-related Osteonecrosis of the Jaw in Osteoporosis.Emileigh Mercer*1, Sook-Bin Woo2, Nathaniel Treister2, Jerry Avorn3, Sebastian Schneeweiss3, Daniel Solomon4. 1Brigham & Women's Hospital, USA, 2Division of Oral Medicine & Dentistry, Brigham & Women's Hospital, USA, 3Division of Pharmacoepidemiology, Department of Medicine, Brigham & Women's Hospital, USA, 4Harvard Medical School, USA

Purpose: We reviewed the epidemiological literature regarding bisphosphonate (BIS) treatment for osteoporosis and its association with osteonecrosis of the jaw (BRONJ) to both understand the methodologies used and describe the results.

Methods: The literature was reviewed for all English language articles using the search terms “osteonecrosis of the jaw,” or one of each “alendronate,” “etidronate,” “ibandronate,” “pamidronate,” “risedronate,” or “zoledronic acid,” and “epidemiology.” We also searched for “bisphosphonate-related osteonecrosis of the jaw” and “epidemiology.” After excluding articles without primary data on the prevalence of BRONJ in patients with osteoporosis, 4 articles were examined for their methodology, including study design, population source, BRONJ definition, treatment type, exclusion criteria, BRONJ confirmation, and treatment duration, as well as their final prevalence estimate of BRONJ.

Results: Of the 4 articles, 2 were from the United States, one from Korea and one from Australia. All 4 studies defined the inclusion for BRONJ cases similarly; either using the AAOMS definition or other broadly accepted criteria. When needed, confirmation of BRONJ was obtained by some combination of medical chart review and follow-up to reaffirm cases. The methods varied widely particularly with respect to the definition of the denominator of BIS-users: 1 study used an insurance database, 1 used hospital medical records, 1 study used an electronic medical record from a dental clinic and 1 used pharmaceutical marketing data. Various methods for collecting cases were used as 2 queried electronic medical records, 1 surveyed dental surgeons and specialists known to treat BRONJ, and 1 surveyed BIS users for dental symptoms. Treatment type also varied across the studies: 2 simply considered alendronate, while 2 others focused on alendronate, risedronate, and ibandronate. The results of each of the studies are shown in the Table. The incidence rate of BRONJ ranged from 0.1 to 1.0 per 1,000 persons based on the 3 strongest studies.

Conclusions: The epidemiologic literature on BRONJ is inconsistent in rigor, possibly explaining the great variation in reported incidence rates. The best studies suggest rates much higher than original estimates. Further work should use standard definitions for BRONJ, utilize appropriately defined denominators, and examine populations not using BIS to determine whether these drugs are truly the primary risk factor.

Table Table 1. Epidemiologic Results of Studies Presenting a Prevalence Estimate for BRONJ in BIS Users
  1. *28 per 100.000 person-years of oral BIS treatment

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Disclosures: Emileigh Mercer, None.


Analgesic Effect of Minodronate, a New Bisphoshonate, on Back and Knee Pain in Elderly Subjects with Osteoporosis and/or Osteoarthritis.Takuo Fujita*1, Mutsumi Ohue1, Yoshio Fujii2, Akimitsu Miyauchi3, Yasuyuki Takagi4. 1Katsuragi Hospital, Japan, 2Calcium Research Institute Kobe Branch, Japan, 3Omura Municipal Hospital, Japan, 4National Hyogo Chuo Hospital, Japan

Purpose In addition to increasing bone mineral density and reducing occurrence of fracture, bisphosphonates were reported to be effective on back and knee pain associated with osteoporosis and osteoarthritis in elderly subjects. The analgesic effect of minodronate, a new bisphosphonate, was tested by electroalgometry (EAM) measuring fall of skin impedance and recording subjective pain by visual rating scale (VRS).

Methods In 22 patients with a mean age of 70 ± 4 years (20 females and 2 males), complaining of back and knee pain both back and knee pain in 21 and back pain alone in 1), 1 mg minodronate was orally administered once daily 30 minutes before breakfast. Osteoporosis by lumbar BMD < 70% of YAM was seen in 27%, possibly underestimated because of osteoarthritic changes (Nathan Score of 2 or higher) was seen in 63% and Kellgren Score for knee osteoarthritis of 2 or higher for spinal osteoarthritis in 77%. Pain was estimated before and after exercise loading; knee bending, walking on a flat surface and up and down stairs and lying down supine on bed and standing up, by measuring the fall of skin impedance using Impedance Meter made by General Devices, Richfield, New Jersey (EAM) and recording subjective pain by VRS scale dividing the distance between unbearable pain and no pain into a 0 to 100.

Results The fall of skin impedance (% of the preload value) employed as an EAM pain index showed a significant positive regression over subjective pain recorded on VRS scale with r=0.484, Y=3.58X + 15.75, p= 0.0221. Paired comparison of pain indices between pre-and post-minodronate treatment revealed a significant analgesic effect 28 day treatment by EAM (p=0.0248) and 56 day treatment by VRS (p=0.0269). Overall paired comparison of pain indices between pre-test and 14∼56 day post-treatment indicated a highly significant decrease of pain by both EAM (p=0.0009) and VRS (p=0.0004).

Conclusion Minodronate significantly alleviated back and knee pain in 22 elderly subjects according to EMS measurement of the fall of skin impedance and VRS recording of subjective pain, the former effect noted after 28 days and the latter after 56 days of treatment

Disclosures: Takuo Fujita, None.


Biphosphonate-associated osteonecrosis of the jaw: An Ontario Survey.Aliya Khan*, Lorena Rios Stange, Nazir Khan. McMaster University, Canada

Purpose: To evaluate the period prevalence of bisphosphonate (BP)-associated osteonecrosis of the jaw (ONJ) seen by oral surgeons in Ontario between 2004 and 2006.

Methods: A survey developed by representatives of the Ontario Society of Oral and Maxillofacial Surgeons was mailed to Ontario oral and maxillofacial surgeons (OMFSs) in December 2006 asking oral surgeons to provide information on cases of ONJ seen in the previous three calendar years (2004 to 2006). OMFSs were subsequently contacted by phone if they had not responded or if they had reported cases of ONJ. The frequency of ONJ in association with BP use was estimated from the number of patients with filled prescriptions in Ontario between 2004 and 2006. The incidence of ONJ was calculated separately for both iv BP use in cancer related bone disease as well as oral or iv BP use for osteoporosis or other metabolic bone diseases

Results: Forty-one cases of ONJ were identified of which 32 cases occurred between 2004 and 2006. Nineteen patients were receiving intravenous BP for cancer related skeletal disease and 13 patients were receiving oral or intravenous BP for osteoporosis or metabolic bone disease. The average annual incidence of BP associated ONJ was 4 per 1000 for cancer patients and 2/100000 for patients with osteoporosis or other metabolic bone disease on oral or intravenous BP. Other risk factors for ONJ were present in all five cases in whom detailed assessment was available. In osteoporosis patients the median duration of BP use was 42 months (36 to 120 months). In patients receiving intravenous BP for cancer related skeletal disease the median duration of exposure to intravenous bisphosphonate therapy was 42 months (11 to 79 months).

Conclusions: The average annual incidence for BP associated ONJ was 4/1000 in cancer patients. In people receiving oral or iv BP for osteoporosis or metabolic bone disease, the average annual incidence was 1/100,000. This study provides an approximate frequency of BP associated ONJ in Canada. These numbers need to be quantified prospectively with accurate assessment of coexisting risk factors.

Disclosures: Aliya Khan, NPS, 2; MERCK, 2; Novartis, 2; Aventis, 2; Amgen, 2

This study received funding from: Novartis, Proctor & Gamble, MERCK


Bisphosphonate Use in Women and Men Who Are at High Risk for New Fractures and Living in Long-Term Care Homes: The Vitamin D Osteoporosis Study (ViDOS).Alexandra Papaioannou*1, Sharon Marr2, George Ioannidis3, Courtney Kennedy2, Lora Giangregorio4, Laura Pickard2, Jenna Johnson1, Glenda Campbell5, Jackie Stroud5, Suzanne Morin6, Robert Josse7, Anna Sawka8, Richard Crilly9, Lehana Thabane2, Lisa Dolovich2, Mary Lou van der Horst2, Norm Flett2, Lynn Nash2, Jonathan Adachi10. 1Hamilton Health Sciences, Canada, 2McMaster University, Canada, 3Sympatico, Canada, 4University of Waterloo, Canada, 5Medical Pharmacies Group Inc, Canada, 6McGill University Health Centre, Canada, 7St. Michael's Hospital, University of Toronto, Canada, 8Toronto General Hospital, Canada, 9University of Western Ontario, Canada, 10St. Joseph's Hospital, Canada

Aim: The ViDOS study is an integrated knowledge translation initiative designed to improve the management of falls and fracture in the long-term care (LTC) setting. One of the study objectives is to increase appropriate prescribing of bisphosphonates in high-risk residents.

Methods: The ViDOS study is a clustered randomized control trial involving a multifaceted integrated disease management process that consists of a small group of physicians, nurses, and pharmacists employed by LTC homes who regularly meet to examine resident care and quality improvement objectives. In intervention homes, these groups attend 3 problem-based learning sessions occurring 5-months apart and led by an Osteoporosis Expert. Control homes receive standard materials given to all LTC homes in the province. The planned recruitment is 40 LTC homes randomized into either intervention (n=20) or control (n=20). Prescribing, falls and fracture data will be collected for both the control and intervention LTC homes in 3 time periods (baseline, follow-up 1 and follow-up 2). Demographic, medications, and disease conditions are collected from the central pharmacy database that has records for all residents living in each home. Medication and co-morbidity data are based on the medication administration records (MAR). This analysis evaluated baseline data and examined the use of bisphosphonate therapy in women and men who are at high risk for new fracture in 16 LTC homes. High risk was defined as residents who had osteoporosis or a prior hip fracture recorded on their MAR sheets. Descriptive statistics are presented for women and men separately.

Results: A total of 1559 medical records for women and 688 for men were analyzed. The mean (SD) age was 83.8 (10.3) years for women and 78.1 (11.8) years for men. Results revealed that 15% (n=236) and 8% (n=119) of women and 4% (n=29) and 2% (n=17) of men had documented osteoporosis or a prior hip fracture, respectively. A total of 50% (118/236) of women and 55% (16/29) of men with documented osteoporosis were taking a bisphosphonate. Furthermore, 40% (47/119) of women and 0% (0/17) of men with a prior hip fracture were taking a bisphosphonate.

Conclusions: A large number of women and men at high risk of fracture in LTC, particularly for those with a prior hip fracture, were not taking bisphosphonate therapy. Ensuring optimal osteoporosis management may reduce the probability of future fractures and the negative consequences associated with fracture.

Disclosures: Alexandra Papaioannou, Amgen, 2; Amgen, 8; Merck Frosst, 8; Novartis, 2; Procter and Gamble, 2; sanofi-aventis, 8; Merck Frosst, 2; Eli Lilly, 8; Servier, 8; Eli Lilly, 2


Bisphosphonate-related Osteonecrosis of the Jaw; Clinical and Radiographical Difference Between the Conventional Chronic Osteomyelitis of the Jaw.Tae-Geon Kwon*1, So-Young Choi2, Chang-Hyeon An3, 1Kyungpook National University, School of Dentistry, South korea, 2Kyungpook National University, School of Dentistry, Oral & Maxillofacial Surgery, South korea, 3Kyungpook National University, School of Dentistry, Oral & Maxillofacial Radiology, South korea

There had been various reports concerning the osteonecrosis of the jaw after receiving the intravenous or oral bisphosphonates. Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is mainly related with various degree of infection of the jaw bone. With the use of antibiotics and improved dental care, conventional osteomyelitis of the jaw is less common these days, but BRONJ is newly emerged in oral and maxillofacial surgery field. PURPOSE: The present study was intended to compare the conventional osteomyelitis and BRONJ on the features of clinical and radiographic finding so that we can establish the optimized treatment for BRONJ. PATIENTS AND METHODS: The CT, Panorama, Bone scan data of 83 BRONJ patients (Male 6, Female 77, and mean age 70.3 years) and 98 conventional chronic osteomyelitis patients (Male 54, Female 44, Mean age 58.0 years) were compared. Destruction of trabeculae or cortex, size of the sequestra, invasion of adjacent structure such as inferior alveolar nerve or sinus, periosteal new bone formation, intramedullary osteosclerosis were investigated. Also correlation of the radiographic or clinical symptoms with biochemical markers CTX (C-terminal telopeptide), Alkaline phosphatase, Osteocalcin, C-reactive protein were evaluated in 32 patients. RESULTS: BRONJ and conventional chronic osteomyelitis (C-OM) showed similar proportion of systemic disease whereas BRONJ occurred more multiple sites than COM. Larger sequestrum (≥15mm), adjacent bone destruction (p<0.05) were more frequent in BRONJ patients. BRONJ patient showed significant lower CTX value (p<0.05). However the biochemical markers used in the study did not correlate with disease severity and or stage of the BRONJ. CONCLUSIONS: According to the previous treatment protocol (AAOMS), conservative treatment was recommended if possible. However, as the BRONJ showed more destructive pattern than C-OM, these results implies the surgery at the proper time might be more effective. Moreover, CTX value can be used as one of the clinical reference but cannot be regarded as a standard for BRONJ progression.

Disclosures: Tae-Geon Kwon, None.

This study received funding from: Brain Korea 21, MRC


See Friday Plenary number FR0387.


See Friday Plenary number FR0388.


See Friday Plenary number FR0389.


See Friday Plenary number FR0390.


Histologic Analysis of the Lateral Femoral Cortex in Two Patients, One with a Biphosphonate Related Femoral Fracture and the Other with a Pending Fracture.Richard Dell1, Eric Eisemon*2, Vincent Vigorita2. 1Kaiser, USA, 2Maimonides Medical Center, USA

Purpose: Several case reports have demonstrated a specific type of femur fracture seen in patients on bisphosphonates. The hallmark of these fractures is a thickened lateral cortex with a lateral projection at the fracture line, which is transverse on the lateral cortex and oblique on the medial cortex. Pre-fracture signs are focal thickening, lateral projections and a radiolucent line on the lateral cortex. The purposed underlying pathology of these fractures is a low turnover state induced by long term bisphosphonate use. We performed a histological analysis of the cortex where the fractures occur.

Methods: Inclusion criteria for our study were atypical appearing femur fractures or pending fracture as described above, low energy mechanism of injury or focal pain, and a documented history of bisphosphonate use. One patient with pre-fracture (Fig 1A) and fracture (Fig 2A) were included in our study. Specimens were obtained during surgery of the lateral cortex at the level of the fracture, along with intramedullary reamings. The specimens were processed in a non-decalcified manner and treated with hematoxylin and eosin, Goldner, and Von Kossa stains.

Results: Specimens consisted of fragments of both cancellous and cortical bone. Samples showed focal osteoblast activity and focal osteoid deposition indicating bone formation activity. In the pre-fracture patient with pre-fracture there was little identifiable osteoclast activity (Fig 1B) and in the patient with fracture osteoclasts were present but appeared detached from the bone surface (Figure 2B). The osteoclasts in both cases appeared rounded, without ruffled borders. There was no evidence of frank osteomalacia in either specimen.

Conclusions: Our findings demonstrate limited osteoclast remodeling but some osteoblast remodeling activity in the cancellous bone but demonstrable abnormal remodeling in the cortical bone. The most interesting finding was the lack of cortical osteoclast activity in one case (Fig 2A) and the abnormal appearance of the cortical osteoclasts in the other case (Fig 2B). Detached from the bone surface and rounded in appearance, the oseoclasts appear dysfunctional. These findings are significant in that they demonstrate abnormal bone remodeling at the fracture and pre-fracture site and are consistent with some known long term effects of biphosphonate therapy which, in part, are felt to cause interference with osteoclast linkage (ligand) to the bone surface.

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Figure Figure 1A.  

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Figure Figure 1B.  

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Figure Figure 2A.  

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Figure Figure 2B.  

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Disclosures: Eric Eisemon, None.


Randomized Placebo-controlled Trial of Risedronate in Patients with Crohn's Disease and Osteopenia.Marieke Pierik1, Daan W Hommes2, Gerard Dijkstra3, Ruud A van Hogezand2, Paul Lips*4, Maurice Russel5, Ad A. van Bodegraven6, C.J. van der Woude7, Lex Van De Langerijt8, Pieter Stokkers9, Geeske M.E.E. Peeters6, Bas Oldenburg10, Coen (J.C.) Netelenbos6. 1Academisch Ziekenhuis Maastricht, Netherlands, 2Leiden University Medical Center, Netherlands, 3Academisch Ziekenhuis Groningen, Netherlands, 4VU University Medical Center, The netherlands, 5Medisch Spectrum Twente, Netherlands, 6VU University Medical Center, Netherlands, 7Erasmus Medical Center, Netherlands, 8Sanofi-aventis, The netherlands, 9Academic Medical Center, Netherlands, 10University Medical Center, Netherlands


Crohn's disease (CD) is associated with increased bone resorption inducing low bone density and increased fracture risk. To assess the efficacy of bisphosphonates to increase bone density in Crohn's disease associated osteopenia, a prospective, randomized, double-blind trial was conducted with risedronate 35 mg weekly versus placebo. The patients originated from clinics of all 8 university hospitals, and one referral hospital in The Netherlands.


Bone mineral density was assessed by dual energy absorptiometry (DXA) in 123 patients with established CD that was in remission. Patients were between 18-60 years of age and had no recent use of glucocorticoids or bisphosphonates, and all were vitamin D sufficient. Crohn's disease Activity Index (CDAI) was calculated, and C-reactive protein was measured to ascertain clinically inactive disease at baseline. DXA was measured at baseline, 12 and 24 months using Hologic densitometers. Daily calcium and vitamin D supplements were provided along with risedronate 35mg or placebo once per week. Activity of CD was evaluated during the 2-years study period. The primary endpoint of study was the change in BMD, expressed as T-score between baseline and 24 months of treatment


Baseline demographics included a patient mean age of 42.7 (SD 13.0) yrs, 46% were male, and at inclusion CRP levels and CDAI were 3 mg/L and 86 in the placebo and 4 mg/l and 76 in the risedronate group, respectively. Patients groups with risedronate and placebo were comparable.

DXA in the lumbar spine at baseline was median 0.93 (IQR 0.88-1.01) g/cm2, T-score -1.28 (SD 0.77) in the placebo group, and 0.93 (IQR 0.89-1.00) g/cm2, T-score-1.30 (SD 0.61) in the risedronate group. The median lumbar BMD change at 24 months was 0.01 (IQR -0.02 -0.03) g/cm2 in the placebo and 0.03 (IQR 0.00 -0.07) g/cm2 in the risedronate group (P= 0.006). The T-score increased at 24 months 0.08 (SD 0.42) in the placebo group and 0.33 (SD 0.46) in the risedronate group (P=0.005). Changes in BMD of the femur were not significant.


Risedronate improves lumbar bone mineral density in CD-patients suffering from CD-associated low bone mass as assessed by DXA.

Disclosures: Paul Lips, Sanofi-Aventis, 2 This study received funding from: Sanofi-Aventis


See Friday Plenary number FR0393.


Validation of Electronic Coding in Women with Diaphyseal Femur Fractures in a Defined Population.Susan Ott*1, Leslie Spangler2, Delia Scholes3. 1University of Washington Medical Center, USA, 2Group Health Cooperative, USA, 3Group Health CooperativeGroup Health Research Institute, USA

Recent reports have questioned whether non-traumatic fractures of the diaphyseal femur are associated with long-term bisphosphonate use. Automated database studies may be useful in evaluating this and estimating incidence. Our study was undertaken to validate electronic coding for femur fractures and was conducted in a non-profit managed care plan that provides comprehensive health care, including medications. We used automated data for 2007 to identify the first occurrence of a relevant ICD9 code in women >45 years (n = 95,765). The ICD9 codes were those for fractures of the subtrochanteric region (820.22, 820.32), the shaft (821.01, 821.11), unspecified part of femur (821, 821.0, 821.00, 821.1, 821.10) or stress fracture, femur (733.97). Medical records were reviewed. Of the 161 women identified by the ICD9 codes, charts had enough data to verify fracture location in 157. This showed that diaphyseal fractures (subtroch or shaft) occurred in 58 cases; 64% of them had a code for either subtroch or shaft, and the remaining 36% had a code for non-specified part of the femur. The code for subtroch was accurate in 11 of 21 cases and shaft in 16 of 32 cases. We also evaluated timing of the fracture. The fracture date was prior to 7/1/06 in 12 of the 161 cases. Altogether, of the cases identified by an electronic search of ICD-9 codes, only 49 (30%) were classed as incident diaphyseal fractures.

In the 49 diaphyseal fractures, we viewed all available images; the following patterns were seen: trauma, periprosthetic, thin “osteoporotic”, thick, and thick with beaking. In 6 cases images were unavailable but radiology reports clearly defined thin or comminuted pattern, and in 4 cases there were no images or detailed descriptions. Of 5 with the thick, beaked pattern, 4 had been taking bisphosphonates (mean 3.8 yrs). In the other 44 cases, 9 had received bisphosphonates (2.4 yrs). Approximately 4500 women had > one oral bisphosphonate prescription filled in 2007. These results from a defined population are consistent with an association between long-term bisphosphonate use and a pattern of transverse femoral fractures with thick cortices and beaking. This relationship was found only if radiographs were viewed.

In this study use of ICD9 codes alone resulted in unsatisfactory confirmation rate of femoral diaphyseal fractures. Our findings support validating ICD9 codes before they are used as a surrogate for the occurrence of a femoral diaphyseal fracture. 

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Disclosures: Susan Ott, None.


Zoledronate Prevents Tibial Bone Loss in Postmenopausal Women with Osteoporosis.Albrecht Popp*, Helene Buffat, Christoph Senn, Isolde Okere, Romain Perrelet, Kurt Lippuner. Osteoporosis Policlinic, University of Bern, Switzerland

Background: Zoledronate (ZOL 5mg i.v., once yearly) reduces the risk of fractures in postmenopausal women with osteoporosis (PMO). In the pivotal HORIZON trial, a gain of bone mineral density (BMD) was demonstrated at the lumbar spine and the hip compared to placebo. Little is known about the effects of zoledronate on BMD at peripheral sites.

Methods: An ancillary investigator-initiated substudy was performed in patients who participated in the PMO-HORIZON-core study at the Osteoporosis Policlinic, University of Bern, Switzerland. Bone density measurements of the distal tibial epiphysis (T-EPI) and diaphysis (T-DIA) were performed at month 0, 6, 12, 24 and 36 according to a previously validated method (Casez JP, J Bone Miner Res 1994; Popp AW, Osteoporos Int 2009) using a Hologic QDR 4500 ATM scanner.

One hundred seventeen women consented to participate in the substudy and were randomised to ZOL or placebo (PLB). Treatment allocation was recently unblinded for fifty-one women (ZOL 23/PLB 28) who did not participate to the extension study (E1) of HORIZON (drop-outs, unsuitable for or unwilling to participate to the extension study). This preliminary analysis includes all patients who received at least one dose of the study medication and had at least one follow-up DXA measurement (ZOL 22/PLB 25). Changes in BMD were compared using ANOVA for repeated measurements. Missing values were imputed by carrying forward the last observation. Values are indicated as means ± SEM.

Results: Baseline characteristics of included patients were comparable with those in the core study and were similar in ZOL and PLB. After 36 months, BMD at lumbar spine increased by 7.5 ± 1.6% (p<0.0001) vs baseline in ZOL and did not change significantly in PLB (+1.9 ± 1.0%). At the total hip, BMD increased in ZOL (+2.6 ± 0.9%, p<0.001) and decreased in PLB (-1.4 ± 0.7%, p< 0.05). Peripheral bone loss at T-EPI and T-DIA was prevented by ZOL (+ 0.9 ± 0.8%; -0.1 ± 0.4%, both n.s. vs baseline), while a significant loss was observed at both tibial sites under PLB (-3.2 ± 1.2%; -1.2 ± 0.5%, both p<0.01 vs baseline).

Conclusion: ZOL prevented peripheral bone loss at the distal tibial sites in postmenopausal women with osteoporosis. This finding may differ from the reported effects with other bisphosphonates.

Disclosures: Albrecht Popp, Received speaker's fees of and/or was member of a advisory board of Amgen, Daiichi Sankyo, Eli Lilly, MSD, Synthes., 9


See Friday Plenary number FR0396.


Compliance and Efficacy of Ibandronate 3 mg iv Quarterly vs. Oral Alendronate-Real World Data with the Non-interventional Study VIVA.Peyman Hadji*. Philipps-University of Marburg, Germany

Purpose: Numerous clinical trials have demonstrated the efficacy of Bisphosphonates in the treatment of postmenopausal osteoporosis. Bisphosphonates have been shown to be an effective treatment with regard to risk reduction of bone loss, osteoporosis related fractures, bone pain and as such are currently the standard of care treatment. However, the translation of clinical study results to real world experience is dependent on treatment compliance which in RCTs is generally high while in clinical practice significantly lower. This is generally true for many chronic diseases where patient adherence to treatment has been shown to be poor, which is particularly the case for oral bisphosphonates. Oral formulations of bisphosphonates are associated with poor gastrointestinal absorption and an increase in gastrointestinal adverse events necessitating complicated dosing regimens. It is well known that suboptimal adherence to bisphosphonate therapy is associated with an increased risk of fracture. In contrast to the poor adherence with oral bisphosphonates, the treatment with Ibandronate iv seems to guarantee an optimal absorption and adherence, because it is applied directly by the physician.

The objective of this non-interventional study is to investigate compliance and persistence of patients with Ibandronate 3 mg iv quarterly vs. alendronate 70 mg weekly in a real world setting. Additionally the management and controllability of intravenous Ibandronate vs. oral alendronate is assessed. Furthermore the impact of compliance and persistence and of intravenous or oral therapy on important factors reflecting real life efficacy like pain intensity, quality of life, mobility and incidence of new osteoporotic fractures will be investigated. Additionally, for the first time relating to PMO, patient questionnaires for the prospective assessment of probable compliance (SSAS and BMQ) are used in this study.

Methods: This non-interventional study has started in Germany end of March 2010. Overall 6.000 patients with postmenopausal osteoporosis will be enrolled by 1.500 to 2.000 office based physicians. Since the interest of this study is focused mainly on Ibandronate, a 3:1 ratio Ibandronate iv: alendronate has been chosen. The observation period per patient is 12 months. Final results are expected in autumn 2012. Together with more details on the study outline first insights on progression of recruitment of this important study will be presented.

Disclosures: Peyman Hadji, None.

This study received funding from: Roche Pharma AG, Germany


Identifying Factors Associated with Patients Making the link Between a Fragility Fracture and Osteoporosis.Rebeka Sujic*1, Monique Gignac2, Rhonda Cockerill3, Dorcas Beaton4. 1St. Michael's Hospital, Canada, 2Toronto Western Research Institute, Canada, 3University of Toronto, Canada, 4Keenan Research Centre, St Michael's Hospital, Canada

Introduction: Patients who associate their fragility fracture with osteoporosis (OP) are more likely to initiate first-line OP treatment. It is not known, however, who is more likely to make this association. The purpose of this study is to examine baseline factors associated with patients making the link between their fragility fracture and OP at follow up.

Methods: Data on a population-based cohort were collected as part of a provincial OP screening initiative targeting low trauma fracture patients over the age of 50. OP screening coordinators collected data at baseline and treatment-naïve, previously undiagnosed patients were followed up at 3 and/or 6 month. Logistic regression was used to identify which modifiable (e.g., patient perception) and non-modifiable (e.g., previous fracture) factors are predictive of patients making an association between their fragility fracture and OP at follow up.

Results: At baseline, 93% (853/916) of patients were unsure or did not believe their fracture was caused by OP. Of these, only 8.8% changed this perception at follow up. Adjusted analyses showed that those who changed were more likely to have a previous fracture OR 2.3 (1.3-4.1), be uncertain about maternal history of fractures OR 2.5 (1.1-5.7), perceive their bones as thin OR 6.9 (3.5-13.4) or be unsure about the quality of their bones OR 2.7 (1.4-5.3) at baseline.

Discussion: Many fragility fracture patients do not associate their fracture with having OP. We identified the baseline characteristics predictive of making the OP-fracture link at follow up. These findings should be incorporated in post-fracture OP interventions to better communicate to patients their risk of future fractures and OP. Future research should support increasing awareness in the patient group likely to make the OP-fracture link and consider using other approaches in those not fitting this profile.

Disclosures: Rebeka Sujic, None.


Influence of a Written or Oral Patient Support Program on Adherence with a Weekly Bisphosphonate in Comparison to Standard Information-Results of the COMBI-Study.Volker Ziller*1, Karina Höntsch2, Ioannis Kyvernitakis2, Berna Seker-Pektas2, Peyman Hadji3. 1Philipps-University-Marburg, Germany, 2Philipps-University Marburg, Germany, 3Philipps-University of Marburg, Germany


In long term medical treatment of postmenopausal osteoporosis an issue of increasing relevance is reduced adherence using oral bisphosphonates. Because reasons for non-adherence are multiple and complex, we designed two interventions intending to inform, motivate and remind patients about osteoporosis, treatment options, side effects problems and solutions and provided counseling contact to health care professionals. The COMBI-Study (Compliance in Osteoporosis treatment in Marburg using Bisphosphonates) was designed as a monocentric, prospective, randomized, partially blinded three armed parallel group study to evaluate the effect of a written versus oral versus standard patient support program on adherence to weekly bisphosphonates.

Material and Methods

180 postmenopausal women with diagnosed osteoporosis according to German guidelines and treatment with a weekly bisphosphonate had been randomized in the three intervention arms. The intervention groups received personalized letters and information leaflets or phone calls by a specially trained study nurse in predefined patterns for one year. Adherence and persistence were measured with a combination of self-report, prescription refill counts and bone marker changes (CTX). For the primary endpoint the three adherence measurements were combined to classify adherent versus non-adherent patients.


Baseline characteristics showed a well balanced randomisation with no significant differences in age, weight, bone mineral density, prevalent fractures, back pain, loss of height and others. In the standard group 53,0%, in the phone group 52,7% and in the letter group 52,6% of patients were classified adherent. The differences were not statistically significant. Furthermore, there were no significant differences in persistence or diverse clinical parameters among the groups.


Improving adherence to medical treatment in osteoporosis treatment is of upmost importance to the individual as well as to health care economic aspects.

The COMBI-Study showed no significant effect on adherence with a complex and multifaceted letter or phone based patient support program in a prospective, randomized setting. More understanding of factors influencing adherence is needed to design and evaluate effective interventions in the future

Disclosures: Volker Ziller, Procter and Gamble Pharmaceuticals, 8; Daichi Sankyo Germany, 8; Lilly Germany, 8; Novartis, 8

This study received funding from: Procter and Gamble Pharmaceuticals


Secondary Fracture Prevention in Home Health Care: Initial Results from a Group Randomized Trial.Meredith Kilgore*1, Ryan Outman2, Kenneth Saag2, Julie Locher2, Jeroan Allison3, Jeffrey Curtis2. 1University of Alabama At Birmingham School of Public Health, USA, 2University of Alabama at Birmingham, USA, 3University of Massachusetts School of Medicine, USA

Purpose: To develop and evaluate a multimodal intervention to increase rates of osteoporosis treatment in high risk patients receiving home health services.

Methods: We developed and tested an intervention to improve preventive care, particularly use of prescription osteoporosis drugs, among patients with a history of fracture receiving home health services. The intervention included an educational component for nurses, a computerized care plan that prompted the nurse to initiate specific actions related to osteoporosis management, easy reference materials for physicians, prepared order sheets to facilitate osteoporosis drug prescription, and patient education materials focused on osteoporosis, fracture risks, fracture prevention, and medication adherence. Field offices (n=23) of a home health agency operating throughout Alabama were randomized to receive the intervention or serve as controls. The primary outcome measure was the rate of osteoporosis medications prescribed among patients with a fracture history. A t-Test of proportions was used to assess difference between groups

Results: We found an absolute difference of 3.9% (NS) between the treatment and control group in post-intervention osteoporosis care. The figure shows the monthly distribution of treatment rates by office before and after the intervention. While all but one of the treatment offices showed an improvement, overall rates were low, and the corresponding rates in the control arm improved as well.

Pre- & Post-Intervention Osteoporosis Medication Prescription Rates in Intervention and Control Groups

A secondary analysis tested whether treatment rates increased when the nursing care plan was activated. In this case the difference was 15.3% (p < 0.0001).

Conclusions: Preliminary results do not demonstrate significant efficacy of the intervention overall, but suggest that improvements in processes of care could have significant effects on appropriate osteoporosis care delivery. The trial is continuing with a reinforcement of the nursing in-service training with audit and feedback to field offices on their relative performance rates, particularly with respect to activating the nursing care plan.

Disclosures: Meredith Kilgore, Amgen, Inc., 2; Eli Lilly, 2; Amgen, Inc., 5

This study received funding from: Amgen, Inc.


What Predicts Inititation of Osteoporosis Treatment after Fracture: Education, Organisation, Socio-economic Status.Nathalie Saidenberg-kermanac'h*. avicenne hospital (AP-HP), France


The reasons of the limited success of initiatives aimed at increasing osteoporosis (OP) care after fragility fractures are still poorly understood. To answer this question we studied a population of patients entering our hospital for fragility fracture that underwent a dedicated intervention program of education on OP and organisation of further OP care. We then analysed patients-and intervention program-related factors predictive of OP investigation and treatment initiation as evaluated 6 months after the fracture event.


349 patients entering Avicenne Hospital (Bobigny, France) for fragility fracture underwent a dedicated program of OP education (structured interview with a trained nurse) and proposition of further OP care: either with their general physician (or private rheumatologist) or at the hospital. In the latter case, the patients were given a date for OP-centred investigation and consultation. Six months after the fracture they were telephonically contacted to know whether they had been investigated and had started a treatment for OP. The patients-and intervention program-related factors predicting the outcome were analysed.


The organisation of further OP care at the hospital yielded the highest probability of being treated, at both univariate (OR 26,74; 95%CI [11,99-59,57) and multivariate analysis (OR 118,09; 95%CI [13,93-1000,92]), while patient's education on OP had a slighter effect (OR 4.74; 95%CI [2,15-10,44]). A low socio-economic status was the patient-related strongest negative predictor of further treatment (OR 0,22 [95%CI 0,09-0,47]).


The organisation of patient's OP care is the strongest determinant of OP investigation and treatment after fracture, and this aspect should be taken care of when attempting to increase OP care in everyday practice. Patients having a low socioeconomic status are less likely to be investigated and treated, and additional effort to properly organise their care are warranted.

Disclosures: Nathalie Saidenberg-kermanac'h, None.

This study received funding from: MSD


See Friday Plenary number FR0402.


See Friday Plenary number FR0403.


See Friday Plenary number FR0404.


See Friday Plenary number FR0405.


A Randomized Controlled Trial of Music-Based Multitask Training on Gait, Balance and Falls Risk.Andrea Trombetti*1, Mélany Hars1, Silvia Del Bianco2, François Herrmann1, Serge Ferrari3, Rene Rizzoli4. 1University Hospital of Geneva, Switzerland, 2Jaques-Dalcroze Institute, Switzerland, 3Geneva University Hospital & Faculty of Medicine, Switzerland, 4University Hospital, Switzerland


Fracture prevention aims at reducing both bone loss and fall risk. Fall prevention includes a large variety of measures with inconsistently demonstrated efficacy. We assessed the effects of a 6-month music-based multitask training program (Jaques-Dalcroze eurhythmics) on gait, balance, and falls risk in community-dwelling elderly at increased risk of falling.


We randomized 134 community-dwellers aged 65 or older (76 ± 7 years), to either a 1-hour weekly eurhythmics (Jaques-Dalcroze) program (n=66) or no intervention (control, n=68) for 6 months. The intervention consisted of multitask exercises performed to the rhythm of improvised piano music. The primary endpoint was the change in gait variability as assessed by quantitative gait analysis recorded on a 10 meter electronic walkway under single and dual task conditions, and balance analysis with a sway measuring portable device, together with a battery of functional tests. Falls were prospectively ascertained using a monthly diary. A carryover effect was measured at 12 months. Analysis was by intention-to-treat.


112 participants (84%) completed the first 6-month period. At that time, the intervention group exhibited significant improvements in gait as compared with controls, with an increase in usual gait speed (adjusted mean difference: 4.7cm/s, 95%CI:0.5-8.8, p=0.03) and a reduction in step and stride length variability (-1.8%, 95%CI:-2.8-0.9, p<0.001; -1.4%, 95%CI:-2.3-0.6, p<0.002, respectively) under dual-task. One-legged stance duration and medio-lateral angular velocity in the intervention group were significantly improved after the exercise program (+0.9s, 95%CI:0.3-1.6, p<0.006; -4.6, 95%CI:-8.6-0.6, respectively), as compared with controls. In the intervention group, there were improvements in the Tinetti score and the Timed Up and Go test. During the intervention period, there were fewer falls in the intervention group (Incidence Rate Ratio: 0.46, p=0.005) and a reduction in the risk of falling (Relative Risk=0.61, p=0.03). Two fractures occurred in the intervention group and 3 in the controls. Benefits of intervention were retained 6 months after the program has ended on gait, balance and fall risk.


This randomized controlled trial in community-dwelling elderly, indicates that a music-based multitask training program improves gait performance under single and dual-task conditions, and balance, as well as reduces both rate of falls and the risk of falling.

Disclosures: Andrea Trombetti, None.


Analysis of Response to Denosumab in Postmenopausal Osteoporosis.Richard Eastell*1, Li-Yung Lui2, Douglas Bauer3, Cesar Libanati4, Steven Boonen5, Ian Reid6, Steven Cummings7. 1University of Sheffield, United Kingdom, 2California Pacific Medical Center Research Institute, USA, 3University of California, San Francisco, USA, 4Amgen Inc, USA, 5Center for Metabolic Bone Disease, Belgium, 6University of Auckland, New zealand, 7San Francisco Coordinating Center, USA

Denosumab is an investigational human monoclonal antibody to RANKL and it reduced the risk of new vertebral, non-vertebral and hip fracture in the FREEDOM Trial. The aim of the present study was 1) to define patterns of response according to total hip BMD change and 2) to identify the characteristics associated with greater or lesser response. FREEDOM was a randomised, placebo-controlled trial of denosumab 60 mg sc or placebo every 6 months in women ages 60 to 90 years with postmenopausal osteoporosis defined as a T-score less than -2.5 at the spine or hip and not less than-4 at either site. All received daily calcium (1000 mg) and vitamin D supplement (400 to 800 IU). We measured 2 bone resorption markers, serum CTX (ELISA, Osteometer) and TRACP5b (immunocapture activity assay, Suomen Bioanalytiikka Oy, Oulu, Finland) in baseline fasting serum samples from 3906 in the placebo and 3902 in the denosumab group. We defined responder thresholds in subjects receiving all six injections (and so were 100% compliant) by calculating the 20th and 80th percentiles respectively for percentage change in total hip BMD in the treated group (80% increased by more than 2.8%) and for the placebo group (80% changed by <1.9%). These numbers were rounded and lesser responders were defined as change less than or equal to 2%, and greater responders more than 2% at 3 years. This threshold was then applied to all patients treated with denosumab in the trial who had hip BMD measurement at 3 years and the table shows that greater responders by BMD were characterised by better compliance and higher baseline bone resorption markers (p<0.01), any associations with age or BMD were small or not significant.

Table Table 1. Baseline characteristics of women with postmenopausal osteoporosis treated with denosumab; change in hip BMD over 3 years in lesser responders ≤2% and in greater responders >2%.
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Disclosures: Richard Eastell, Amgen, Inc., 5

This study received funding from: Amgen, Inc.


See Friday Plenary number FR0408.


Glucocorticoid Use Cancels the Positive Effects of Biologics on Bone Metabolism in Patients with Active Rheumatoid Arthritis.Tadashi Okano*, Masahiro Tada, Yuko Sugioka, Shigeyuki Wakitani, Hiroaki Nakamura, Tatsuya Koike. Osaka City University Medical School, Japan

Rheumatoid arthritis (RA) is associated with systemic bone loss, subchondral bone erosion and cartilage degradation. Biologics therapy has proven efficacious in improving both disease activity and focal bone erosions in patients with RA. We investigated the effects of biologics on bone mineral density (BMD) and biochemical markers in Japanese RA patients. To examine whether treatment with biologics prevents loss of BMD in patients with RA, and to study the changes in markers of bone metabolism during biologics treatment. One hundred and twenty three (103 women, 20 men, 55.8 ± 12.9 years old) patients with active RA, who were treated with biologics (Infliximab:93, Etanercept:27, Tocilizumab:3) during 1 year, were included in this open cohort study. The BMD of the lumbar spine and total hip (DXA: QDR-4500, Hologic) and bone metabolic marker (urinary NTX) were measured at baseline and after at least more than one year. Thirty three patients received bisphosphonates while 84 patients were under oral prednisolone, we also assessed these effect. The BMD of the lumbar spine and hip was unchanged during treatment with biologics. The BMD of the hip in patients who have received predonisolone without bisphosphonates (n=55) showed significant decreases (p<0.01) compared with patients not receiving predonisolone or receiving predonisolone with bisphosphonates. Moreover, the BMD of lumbar spine has increased (p=0.02) in patients who were administered with bisphosphonates (n=32). In whole patients, NTX was significantly decreased from 66.0 ± 67.9 to 51.9 ± 37.7 nmol/mmol CRE (p<0.01). To maintain BMD, dose reduction of prednisolone or the introduction of bisphosphonate should be required, even if biologics are administered.

Disclosures: Tadashi Okano, None.


See Friday Plenary number FR0410.


Postmenopausal Women with PON1 172TT Genotype Respond to Lycopene Intervention With a Decrease in Oxidative Stress Parameters and Bone Resorption Marker NTx.Leticia Rao*1, Erin Mackinnon2, Ahmed El-Sohemy3, Robert Josse4, Venketeshwer Rao3. 1University of Toronto, St. Michael's Hospital, Canada, 2Department of Medicine, St Michael's Hospital & University of Toronto, Canada, 3Department of Nutritional Sciences, University of Toronto, Canada, 4St. Michael's Hospital, University of Toronto, Canada

We have shown recently that lycopene may decrease the risk of osteoporosis through its antioxidative properties, but the mechanism remains unknown. Paraoxonase 1 (PON1) is an HDL-associated, enzyme that contributes to the antioxidant properties of HDL by hydrolyzing the reactive oxygen species hydrogen peroxide that contributes to oxidative stress in the body. Polymorphisms of PON1 have been associated with chronic diseases associated with oxidative stress including osteoporosis. Our objective is to determine whether a polymorphism of PON1 172T[RIGHTWARDS ARROW]A modifies the effect of lycopene intervention on oxidative stress parameters and bone turnover markers in postmenopausal women. Following a one-month washout period, 45 postmenopausal women between 50-60 years of age were supplemented for 4 months with regular tomato juice, lycopene-enriched tomato juice, or lycopene capsules (30, 70 and 30 mg lycopene/day, respectively). Serum samples were analyzed for lycopene, total antioxidant capacity (TAC), the oxidative stress parameters, protein thiols and thiobarbituric acid reactive substances (TBARS), and the bone resorption marker crosslinked N-telopeptides of type I collagen (NTx). Genotyping was performed and carriers of the A allele were compared with those with the TT genotype. Repeated-measures ANOVA was used to determine change over time. Regardless of genotype and type of supplementation, lycopene resulted in significantly increased serum lycopene (both genotypes: p<0.0001), and significantly decreased protein (TT genotype: p<0.005 and A allele carriers: p<0.05) and lipid peroxidation biomarkers (TT genotype: p<0.005 and A allele carriers: p<0.0005). However, in participants with the TT genotype lycopene supplementation resulted in increased TAC (p<0.01) and significantly decreased NTx (p<0.001); an effect not observed among A allele carriers. The decrease in NTx in the TT genotype was inversely correlated with TBARS (p<0.05). This suggests that supplementation with lycopene decreases lipid peroxidation, which may result in decreased NTx in the TT genotype only (p<0.05 for interaction between change in TBARS and PON1 genotype). These findings provide preliminary evidence of how lycopene decreases biomarkers of lipid peroxidation, which may result in decreased bone resorption, and thus may reduce the risk of osteoporosis in postmenopausal women.

Disclosures: Leticia Rao, Research Grants, 2


Relevance of Serum Undercalboxylated Osteocalcin(ucOC) in the Treatment of Women with Osteoporosis.Humiki Hirahara1, Hiromi Yoshikata2, Rituko Kikuchi1, Yoshiyuki Nomura*1, Kentaro Kurasawa1, Osamu Chaki3. 1Yokohama City Univ., Japan, 2Yokohama City Univ., USA, 3Yokohama City University, Japan


Undercarboxylated osteocalcin (ucOC) levels are regarded as a marker of vitamin K status. Recently it was reported that vitamin K status is associated with vertebral fracture of Japanease postmenopausal eldely women, as well as hip fracture. We usually use N-telopeptide of type ?collagen (NTX), Bone alkaline phosphatase (BAP), Deoxypyridinoline (DPD) as bone makers for osteoporosis treatment. Moreover we tried to measure serum ucOC concentration for postmenopausal women with treatment of osteoporosis.


We studied 145 osteoporotic postmenopausal women were treated (mean age, 64.4 ± 9.7 yrs, range, 36-77yrs.) We measured the serum ucOC, urinary NTX, and BMD (Lumber 2∼4) by 6 months. And we were scoring dietary guestionnaire of vitamin K. Then we administered vitamin K2 or guided diet for patients was over 4.5(ng/ml) of serum ucOC.(high level of serum ucOC).

After 3 months from supply by vitamin K, we research serum level of ucOC again.


Serum ucOC level was low by taking drug which is strong suppressive born absorption, bisphosphonate. High serum level of ucOC (over 4.5 ng/ml) was 26.3% of all. Dietary score of vitamin K was correlative to serum level of ucOC. The patients which was low level of urinary NTX and high level of serum ucOC, was 23% of all. This result was discrepant established theory. Therefore we considerd BMD in this group, and it was deceased the percent change of BMD for 3 years. After 3 months from the vitamin K2 intake, serum level of ucOC was decreased and the percent change of BMD was increased.


This study suggests that the vitamin K intake affect to get the bone quantity as for the patients are treating by Bisphosphonate, but serum level of ucOC were high.

Disclosures: Yoshiyuki Nomura, None.


See Friday Plenary number FR0413.


Health-related Quality of Life after Vertebral or Hip Fracture in Women-Short Health Scale Useful for Clinical Practice?Inger Hallberg*, Goran Toss, Anna-Christina Ek, Henrik Hjortswang, Margareta Bachrach-Lindström. Linköping University, Sweden

Introduction: Health-related quality of life (HRQOL) is an important part in clinical practice of osteoporotic fracture assessment. Numerous HRQOL questionnaires are currently at use. However, there is a need for a simple and short questionnaire that still covers the major important health domains. The aim of the study was to validate the Short Health Scale (SHS) in women after vertebral or hip fracture.

Methods: Out of 91 invited women 67 (mean age 75.5, SD 4.6) completed the SHS and 2 other HRQOL questionnaires in a seven-year follow-up after vertebral or hip fracture. SHS was validated and compared with the generic SF-36 and disease-specific Qualeffo-41 questionnaires. The SHS is a four-item self-administered disease-specific health measurement (six-point scale, no problem to very severe problem). Each item represents one of the subjective health dimensions: Distress/Symptoms, Daily function, Worry and General well-being. High scores in SF-36 and low scores in Qualeffo and SHS indicate better HRQOL.

Results: The convergent validity were supported by the correlations (r2) between the SHS and the more extensive SF-36 and Qualeffo-41 corresponding dimensions, such as SHS-Distress/symptoms vs SF-36-Bodily pain (-.71***) and Qualeffo-Backache (.65***); SHS-Daily function vs SF36-Physical function (-.64***), Role physical (-.60***) and Qualeffo-Jobs (.67***) and Mobility (.60***). SHS General well-being correlated well to SF-36-General health (-.72***), Mental health (-.66***) and to Qualeffo-General Health (.79***). The SHS-Worries covers a domain that is less well monitored by the SF-36 or Qualeffo-41. Construct validity using the known-groups approach was supported by the significantly worse score in SHS-distress/symptoms and SHS-daily function in women with vertebral fracture compared to women without vertebral fracture. Similarly women who had suffered new osteoporotic fracture during the last five years had significantly worse score in SHS-Distress/symptoms, SHS-Daily function and SHS-Worry than those without new fracture(s). In conclusion, SHS seems to be valid, is easy and quick to complete and gives a comprehensive overview of the main aspects of the women's subjective health perception after vertebral or hip fracture. It could be a useful tool in clinical practice, but further evaluation in larger study groups is still needed.

Disclosures: Inger Hallberg, None.


See Friday Plenary number FR0415.


Concentrations of the C-3 epimer of 25-hydroxyvitamin D3 in Adult, Child and Neonate Serum Samples.Gary Lensmeyer*1, Michael Poquette2, Donald Wiebe2, Anita Iwanski2, Neil Binkley3., USA, 2University of Wisconsin Hospital & Clinics Clinical Toxicology Laboratory, USA, 3University of Wisconsin, USA

Epimers have identical molecular structure but differ in stereochemical con-gfiguration. Currently, it is accepted that the C-3 epimer of 25-hydroxyvitamin D3 (3 epi-25(OH)D3), is found primarily in neonates. Yet, recently this epimer was detected in a limited number of adult serums but concentrations were not reported. The physiological importance of 3 epi-25(OH)D3 is uncertain, although, it has been reported that the 1,25 dihydroxy metabolite of this epimer is relatively inactive in calcium metabolism but inhibits parathyroid cells in vitro. Importantly, the influence of 3 epi-25(OH)D3 on 25(OH)D test results obtained with various immunochemical and chromatographic assays, and ultimately on the reliability of “25(OH)D” measurement, remains to be defined. We have observed a peak consistent with 3 epi-25(OH)D3 in serum tested with a high-performance liquid chromatography (HPLC) method we developed. Here we report serum 25(OH)D3 and 3 epi-25(OH)D3 concentrations in a cohort (n = 200+; 60% female) ranging in age from neonates to age 80+. To simplify data presentation and analysis, only sera with 25(OH)D3 and no 25(OH)D2 were included. HPLC with ultraviolet detection (HPLC/UV) and tandem mass spectrometry (LC/MS/MS) equipped with cyanopropyl analytical columns were used to baseline-separate and quantitate 25(OH)D3 and its C-3 epimer. Authentic C-3 epimer substance and NIST reference materials were employed to validate the identity of the C-3 epimer chromatographic peak. Between-run precision for the assays was < 7%; the assays were linear up to at least 300 ng/mL. Lower limit of detection for the C-3 epimer on LC/MS/MS was 0.2 ng/mL. The C-3 epimer was detected in >95% of these samples. Concentrations ranged from 3-610 ng/mL for 25(OH)D3 and 0.2-70 ng/mL for 3 epi-25(OH)D3. The relative amounts of the epimer to 25(OH)D3 ranged from 2.5-14%. The relative amount of epimer to 25(OH)D3 increased as 25(OH)D3 increased; the regression analysis relationship was not linear. In sera with the same 25(OH)D3 concentration, the ratio of epimer to 25(OH)D3 varied. In conclusion, 3 epi-25(OH)D3 is present in the majority of serum specimens regardless of age or gender. Importantly, 3 epi-25(OH)D3 is not limited to neonates. Further work must investigate the impact of 3 epi-25(OH)D3 on the various 25(OH)D assays and ultimately what information, if any, the C-3 epimer can afford clinically.

Disclosures: Gary Lensmeyer, None.


Effects of Risedronate with Cholecalciferol in Osteoporosis: Results of a Multicenter, Randomized Controlled Trial.Hoyeon Chung*1, Jung-Min Koh2, Sung-hwan Moon3, Yoon-Sok Chung4, Byung-koo Yoon5, Moo-Il Kang6, Hyun-Koo Yoon7, Hyoung-moo Park8. 1Kyung Hee University, South korea, 2Asan Medical Center, South korea, 3Yonsei University, South korea, 4Ajou University School of Medicine, South korea, 5Sungkyunkwan University, South korea, 6Seoul St. Mary's Hospital, South korea, 7Cheil Hospital & Women's Healthcare Center, South korea, 8Chung-ang University, South korea

Introduction: A randomized, double-blind, prospective, 16-week clinical trial in osteeoporosis was performed to evaluate the efficacy and safety of risedronate with and without cholecalciferol on vitamin D status and bone markers.

Methods: A total of 164 women and men with osteoporosis were randomly assigned to one of two treatment group; risedronate 35mg with cholecalciferol 5600 IU(RIS plus) or risedronate(RIS). Serum 25OH D, PTH, bone markers and muscle function tests were performed at baseline and after 8 and 16 weeks of treatment.

Results: Serum 25OH D increased from 15.9 to 28.2 ng/ml with RIS plus and declined from 16.3 to 14.0 ng/mL with RIS after 16-week treatment. Both treatment groups had significant increase in serum PTH during the study. The RIS group had a significantly greater increase of PTH from baseline at 16-week versus RIS plus group(13.7 ± 17.6 vs 4.7 ± 13.8; p=0.0004). In both groups, serum BSAP and CTX declined rapidly, and to a similar extent and there were no significant differences between the two groups. The result of muscle function test, 8-foot walking test and sit-to-stand test were also no significant differences between the two group after 8 and 16 weeks of treatment. The overall incidence of clinical adverse events was not significantly different between treatment groups.

Conclusion: In osteoporosis patients, this once-weekly risedronate with cholecalciferol provided equivalent anti-resorptive efficacy, improved vitamin D status over 16 week treatment without significant adverse events.

Disclosures: Hoyeon Chung, None.

This study received funding from: Hanlim pharmaceutical Co.Ltd


See Friday Plenary number FR0418.


Weekly Alendronate Plus Vitamin D3 5600 IU vs. Usual Care: Effect on Serum Hydroxyvitamin D in Osteoporotic Postmenopausal Women with Vitamin D Inadequacy-6-month Results of a Randomized Trial.Stuart Ralston*1, Neil Binkley2, Steven Boonen3, Douglas Kiel4, Christian Roux5, Suvajit Samanta6, Elizabeth Rosenberg6, Arthur Santora7. 1University of Edinburgh, United Kingdom, 2University of Wisconsin, USA, 3Center for Metabolic Bone Disease, Belgium, 4Hebrew SeniorLife, USA, 5Hospital Cochin, France, 6Merck & Co., Inc., USA, 7Merck Research Laboratories, USA

Purpose: To compare effects on serum 25-hydroxyvitamin D [25(OH)D] and bone turnover markers in postmenopausal osteoporotic women with low 25(OH)D after treatment with either weekly combination tablets of alendronate 70 mg plus vitamin D3 5600 IU (ALN+D) or referred care (prescribed by patients' personal physicians).

Methods: Postmenopausal women age ≥65 years with osteoporosis (BMD T-score ≤2.5, or ≤1.5 with a prior fragility fracture), 25(OH)D ≤20 ng/mL but ≥8 ng/mL, and increased risk of falls (past history of falls, functional performance, low 25(OH)D) were randomly assigned to receive ALN+D weekly (and 500 mg elemental calcium daily if daily intake was <1000 mg) or referred (provided with a letter stating they had osteoporosis needing treatment) to their personal primary or specialist physicians (who were not investigators in the trial) for osteoporosis drugs and nutritional support with marketed vitamin D and calcium supplements. The primary evaluation reported here is the proportion of patients in each group with 25(OH)D < 20 ng/mL after 26 weeks of treatment. Secondary evaluations compare bone turnover markers. Safety was monitored through adverse experience reporting.

Results: Participants receiving ALN+D (n=257) and referred care (n=258) had similar baseline characteristics (mean age=73 years, 72% Caucasian, mean 25(OH)D=15 ng/mL, and 56% with 25(OH)D≤15 ng/mL). At 6 months, the proportion of women with 25(OH)D<20ng/mL was 7.8% with ALN+D and 31.2% with referred care (p<0.001). Least squares (LS) mean changes from baseline in 25(OH)D at week 26 were 14.1 and 10.7 ng/mL, respectively. Geometric LS mean changes from baseline for serum BSAP were -46% and -39%, and for urine NTX were -58% and -50% with ALN+D and referred care, respectively (P≤0.004 for each difference). Incidence of adverse events and discontinuations due to adverse events were similar among treatments, and consistent with recognized safety profiles.

Conclusions: ALN+D reduced the proportion of postmenopausal, osteoporotic women with 25(OH)D<20 ng/mL more than did referred care (that might have included vitamin D supplementation) prescribed by physicians not directly involved with the investigation. Patients in the ALN+D group had lower BSAP and NTX than did patients in referred care.

Disclosures: Stuart Ralston, Merck, 5

This study received funding from: The study was funded by Merck, the sponsor of the study


See Friday Plenary number FR0420.


See Friday Plenary number FR0421.


Exploring the LRP5 SOST Interacting Surface to Identify Small Molecule Inhibitor of SOST Action.David Halladay*1, Amy Sato2, Yanfei Ma1, Pamela Shetler1, Heather Bullock1, Venkatesh Krishnan1. 1Eli Lilly & Company, USA, 2DePauw University, USA

The Wnt signaling pathway has been shown to be important in bone formation. Specific mutations of the Wnt co-receptor LRP5 results in a high bone mass (HBM) phenotype in humans. Additionally, nonsense mutations within the sclerostin gene (SOST), an inhibitor of the Wnt signaling pathway, resulted in sclerosteosis, characterized by progressive skeletal overgrowth. We have utilized a Fluorescence Resonance Energy Transfer (FRET) based assay to study the interacting surface of LRP5 and SOST. Using a MYC-tagged hLRP5 and HA-tagged hSOST and/or FLAG-tagged hSOST we show specific interactions between these two proteins. We also show specific interactions between Wnt1 and LRP5 using similar methods. Using a stably transfected TCF/LEF reporter cell line transiently transfected with either pSOST-HA or pSOST-FLAG we show a dose dependent inhibition of Wnt3a-CM induced activity and this inhibitory activity was reversed by an anti SOST Ab. To confirm that SOST bound the first propeller domain of LRP5 we tested three HBM mutants located in the first propeller domain of LRP5 (tagged with MYC) and clearly demonstrate decreased ability to bind SOST as compared to wt hLRP5. In order to explore what regions of SOST were important for LRP5 binding we created mutant SOST-FLAG plasmids with various deletions in the second loop. We demonstrate that more pSOST-HA was needed to compete with pSOST-FLAG for MYC-LRP5 binding compared to the pSOST(-ΔLoop2)-FLAG, the largest deletion, indicating that pSOST(ΔLoop2)-FLAG has a lower affinity for MYC-LRP5 than the wild-type pSOST-FLAG. Smaller deletions within the loop 2 of hSOST showed intermediate affinity for hLRP5. Transient transfection of the TCF/LEF reporter stable cell line with wt pSOST-FLAG dose dependently reduced Wnt3a-CM induced luciferase activity, whereas pSOST(ΔLoop2)-FLAG increased Wnt3a-CM induced luciferase activity. The shorter deletions of SOST loop 2 had no inhibitory or stimulatory effect. Here we also show, for the first time, that a small molecule was able to dose dependently disrupt the binding of SOST to LRP5 via this specific interacting surface and as expected this disruptor led to the stimulation of the TCF/LEF reporter activity. Additional characterization of this agent as a potential bone anabolic agent in rodent animal models will be discussed.

Disclosures: David Halladay, None.


Monitoring of Intermittent PTH(1-34) Treatment by Serum PINP in Adult Ovariectomized Osteopenic Rats.Jussi Halleen*1, ZhiQi Peng1, Katja Fagerlund1, Tiina A. Suutari1, Jukka Vääräniemi1, Mari Suominen1, Jukka Rissanen2, Harrie C.M. Boonen3, Trine S.R. Neerup4, Hanne H. Bak4, Jukka Morko5. 1Pharmatest Services Ltd, Finland, 2Pharmatest Services, Limited, Finland, 3University of Copenhagen, Denmark, 4Zealand Pharma A/S, Denmark, 5Pharmatest Services Ltd, Fin

Procollagen I N-terminal propeptide (PINP) is a sensitive marker of bone formation for monitoring the efficacy of treatment with recombinant human parathyroid hormone (1-34) analog [PTH(1-34)] in osteoporotic patients. Recently, a new immunoassay has been developed for serum PINP in rodents, allowing measurement of serum PINP in preclinical rodent osteoporosis models. The purpose of this study was to evaluate the use of serum PINP for monitoring intermittent PTH(1-34) treatment in adult ovariectomized (OVX) osteopenic rats. Study groups included a sham-operated control group and an OVX-operated control group receiving vehicle, and OVX-operated groups receiving daily subcutaneous injections of 1.2, 4.0, 12.0, 40.0 and 120.0 μg/kg human PTH(1-34). Each group contained 12 animals that were 6 months of age at the time of the operations. Dosing was started at 7 weeks after OVX and continued for 6 weeks. Before the start of treatment, the animals were randomized to groups based on their body weights and trabecular bone mineral density values obtained by peripheral quantitative computed tomography (pQCT). PINP values were determined from serum samples collected before the start of treatment, at 2 weeks after the start of treatment and at the end of the study. The PINP values obtained at 2 weeks and at the end of the study were divided by the values obtained in the same animal before the start of treatment to determine the change of PINP in each animal during the treatment. PTH(1-34) treatment showed strong anabolic effects in tibial metaphysis, tibial diaphysis and vertebra based on bone parameters determined by pQCT measurements and static and dynamic histomorphometry. Serum PINP values showed a strong dose-dependent increase by PTH(1-34) treatment both at 2 weeks after start of treatment and at the end of the study. We conclude that serum PINP is a reliable marker for monitoring the effects of anabolic treatment with PTH(1-34) in the rat OVX model, and short-term changes in serum PINP values predict long-term changes in bone parameters obtained by pQCT measurements and histomorphometry.

Disclosures: Jussi Halleen, IDS Ltd, 5

This study received funding from: ZEALAND Pharma A/S


Osteoporosis from type 1 Diabetes is Reversed by Intermittent Parathyroid Hormone Stimulation of Bone Formation.Katherine Motyl*, Laura McCabe. Michigan State University, USA

Type 1 diabetic osteoporosis results from impaired osteoblast activity and osteoblast death. Because diabetes does not affect or reduces resorption, antiresorptive bisphosphonates may not be the most appropriate therapy. Anabolic intermittent parathyroid hormone (PTH) stimulates bone remodeling and increases bone density, although the exact mechanism of its action is unknown and could be altered/defective in type 1 diabetes. Here, we examined the ability of daily 8 μg/kg and 40 μg/kg PTH to counteract bone loss in streptozotocin-induced diabetic BALB/c mice. We found a significant elevation of tibia trabecular bone density parameters with the 40 μg/kg dose in control mice and diabetic mice (compared to their metabolic-state matched vehicle treated controls), and elevation of trabecular bone parameters in diabetic 8 μg/kg treated mice. In particular, bone mineral density (BMD) was increased 17% in 40 μg/kg PTH treated control mice (compared to untreated euglycemic control mice), and 26% in equivalently treated diabetic mice (compared to untreated diabetics). Increased bone density in diabetic PTH-treated mice was due to increased bone formation, mineral apposition, and osteoblast surface, all of which are defective in type 1 diabetes. We also determined that 40 μg/kg PTH treatment reversed preexisting bone loss from diabetes, such that diabetic PTH treated BMD was not significantly different from that of untreated controls. We conclude that intermittent PTH is capable of promoting bone formation under diabetic conditions, even after bone loss has already occurred. Thus, this work warrants further investigation into the use of PTH as an osteoporosis therapy for patients with type 1 diabetes.

Disclosures: Katherine Motyl, None.


See Friday Plenary number FR0425.


Strontium Stabilizes β-catenin by Activating Wnt Signaling.Zhaoyang Li*1, Tao Qiu2, Mei Wan2, Xu Cao3, William Lu4. 1The University of Hong Kong, Peoples republic of china, 2Johns Hopkins University School of Medicine, USA, 3Johns Hopkins University, USA, 4The University of Hong Kong, Hong kong

Strontium (Sr) compounds stimulate bone formation by promoting differentiation of osteogenic bone marrow stromal cells and are used in prevention and treatment of osteoporosis, but the precise mechanisms responsible for their responses in osteoblasts are not known. LRP6-mediated stabilization of ?-catenin has been shown to play an important role in bone cell function. Particularly, PTH induces the association of LRP6 with PTH/PTHrP receptor PTH1R to stabilize β-catenin and its translocation into the nucleus. Here we show that Sr treatment increased the abundance of β-catenin in osteoblastic MC3T3 cells. Calcium sensing receptor (CaSR), a seven-trans-membrane receptor like PTH1R, regulates calcium metabolism. We further examined whether strontium stabilizes β-catenin by activating Wnt signaling. Preosteoblast MC3T3 cells were treated with 6.0 mM SrCl2 and harvested at 0, 5, 15, 30 and 60 minutes after treatment. Sr stimulated the level of β-catenin in a time dependent manner with 10 fold increase at 60 min after treatment, whereas Ca2+ had modest effect on β -catenin stabilization. Phosphorylation of β-catenin by Glycogen synthase kinase (GSK)-3β leads to constantly degradation of β-catenin via the ubiquitin-proteosome pathway and activation of LRP6 results in phosphorylation of GSK3β and stabilizes β-catenin. Western blot analysis revealed that Sr induced phosphorylation of GSK3β, indicating activation of Wnt signaling for β-catenin stabilization. In summary, our data suggests that Sr likely increased the osteoblast differentiation and bone formation through activating Wnt signaling pathway. The stabilized β-catenin protein may accumulate in the nucleus and complex with the TCF/LEF family of DNA-binding transcription factors to enhance gene expression and stimulate bone formation.

Disclosures: Zhaoyang Li, None.


See Friday Plenary number FR0427.


A Novel Bisphosphonate with Potent Anabolic Action.Hisashi Shinoda*1, Keiko Suzuki2, Mirei Chiba1, Shinobu Murakami1, Sadaaki Takeyama3, Yuka Narusawa1, Hiroaki Hirotani1, Shogi Yamada2, Kaoru Igarashi1, Paula Stern4. 1Tohoku University Graduate School of Dentistry, Japan, 2Showa University Graduate School of Dentistry, Japan, 3Hokkaido University Graduate School of Dentistry, Japan, 4Northwestern University Medical School, USA

Bisphosphonates (BPs) are potent inhibitors of osteoclastic bone resorption and have been used for the treatment of various metabolic bone diseases that are accompanied by excessive bone resorption. BPs are chemically characterized by a P[BOND]C[BOND]P bond and this structure allows us to create a great number of variations by changing two lateral side chains on the carbon atom. Many BPs have been synthesized and investigated with respect to their effects on bone. The data show that the pharmacological characteristics of bisphosphonates, such as their potency, mechanism of action, and side effects, vary depending on the structure of the side chains.

[4-(Methylthio)phenylthio] methanebisphosphonate (MPMBP) is a novel synthetic bisphosphonate with an anti-oxidant side chain on the carbon atom of the P[BOND]C[BOND]P bond. During the course of our studies on the structure-activity relationships of various bisphosphonates, we recently found that MPMBP has not only anti-bone-resorbing action, but also potent anabolic action on bone.

Our observations and findings can be summarized as follows: (1) MPMBP dose-dependently increased alkaline-phosphatase activity in a culture of osteoblastic MC3T3-E1 cells. (2) MPMBP increased the synthesis of collagen (type-I) in an organ culture of mouse calvaria. (3) Topical injection of MPMBP to alveolar bone induced prominent increases in both the bone mass and thickness of alveolar bone at the local site of injection in both rats and rabbits. (4) MPMBP increased the mRNA expression of alkaline-phosphatase, type-I collagen, osteocalcin, and bone sialoprotein in MC3T3-E1 cells. (5) In osteoblasts from cultured mouse calvaria, MPMBP inhibited the translocation of NF-κB to the nuclei and enhanced the expression of Fos-related antigen-1(Fra-1), an essential transcription factor involved in bone matrix formation in vivo and in vitro.

Taken together, our findings show that MPMBP can promote bone formation both in vitro and in vivo, and suggest that this compound could be used for the treatment of various bone diseases associated with excessive bone loss, such as periodontitis and osteoporosis. It may also be useful for the promotion of fracture healing, for the maintenance of implants, for the treatment of cleft palate and for various conditions that require the regeneration of bone tissues.

Disclosures: Hisashi Shinoda, None.


Active Site Mutants of Farnesyl Pyrophosphate Synthase Help to Characterise Inhibition by Nitrogen Containing Bisphosphonates.Maria Tsoumpra*1, Bobby L. Barnett2, Richard L. Walter3, Frank H. Ebetino4, Robert G.G. Russell5, Udo Oppermann5, James E. Dunford5. 1Oxford University, United Kingdom, 2Chemistry Department, University of Cincinnati, USA, 3Shamrock Structures, USA, 4Warner Chilcott, USA, 5Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology & Musculoskeletal Sciences, Oxford University, United Kingdom

Farnesyl Pyrophosphate Synthase (FPPS) is a key branch point enzyme in the mevalonate pathway. Selective inhibition of FPPS impairs osteoclast survival and forms the basis of antiresorptive treatment by nitrogen containing bisphosphonates (N-BPs). Crystallographic and kinetic studies suggest that potent N-BPs compete for and finally occupy the allylic substrate binding site. Subsequent binding of isopentenyl pyrophosphate (IPP) results in FPPS isomerization and formation of a tight bound enzyme-inhibitor complex. The present study aimed to investigate the role of FPPS amino acid residues that are strongly implicated in i)stabilization of the carbocation intermediate (lysine 200 or K200) ii)conformational switch upon IPP binding (phenylalanine 239 or F239) and iii)formation of a tight enzyme-inhibitor complex (tyrosine 204 or Y204). Disrupted interactions between the N-BP phosphonate group and the amino group of K200 might be responsible for the increased inhibition constants (Ki) for risedronate (Ris) from 0.34 to 62.9 nM, ibandronate (Ibn) from 3.6 to 18.2 nM, alendronate (Aln) from 56.9 to 82.7 nM and pamidronate (Pam) from 52.2 nM to 460.8 nM in K200 to glycine (K200G) mutant. Replacement of K200 with Leucine (K200L) led to further reduction of N-BP potencies with increased Ki for Ris (75.1 nM), Ibn (140.2 nM), Aln (239.8 nM) and Pam (3717.4 nM). The F239 to alanine mutation (F239A) led to increased inhibition constants for all N-BPs examined (Ris: 65.5 nM, Ibn:15.5 nM, Aln: 75.4 nM, Pam: 945.7 nM), revealing inability to form a tight enzyme: inhibitor complex. Removal of Y204 reduced the Ris potency in phenylalanine mutant (Y204F, Ki= 2.22) as well as alanine mutant (Y204A, Ki=5.18), possibly due to abolishing hydrophobic interactions between the heterocyclic ring of Ris and the phenyl ring of Y204. Furthermore, the decreased Ki for Aln (6 nM) and Pam (19 nM) in Y204F but not in the Y204A mutant suggests formation of novel interactions between the amino group of N-BPs and the phenyl ring of Y204F, previously inhibited due to interference by the hydroxyl group in Y204. Finally, determination of kinetic parameters for all mutants revealed unaltered binding of the FPPS with respect to the allylic substrate geranylpyrophosphate (GPP) but reduced catalytic activity and affinity for IPP. In conclusion, data highlight important aspects of the multiple interactions between N-BPs and side chain FPPS residues that determine the strength of N-BP binding.

Disclosures: Maria Tsoumpra, None.

This study received funding from: Procter & Gamble


See Friday Plenary number FR0430.


See Friday Plenary number FR0431.


See Friday Plenary number FR0432.


A Comparison of the Effects on Bone, Muscle and Fat Tissue between a more Potent Synthetic Androgen 7α-methyl-19-nortestosterone (MENT) and Testosterone: Evidence from a Preclinical Hypogonadal Rat Model.Filip Callewaert1, Maarten Morreels1, Narender Kumar2, Regine Sitruk-Ware2, Frank Claessens1, Steven Boonen3, Dirk Vanderschueren*1. 1Catholic University of Leuven, Belgium, 2Population Council, USA, 3Center for Metabolic Bone Disease, Belgium

Orchidectomy-induced hypogonadism results in muscle frailty, bone loss and gain of fat mass. In contrast with testosterone (T), a synthetic androgen 7α-methyl-19-nortestosterone (MENT) is not amplified in the male reproductive tract since it does not undergo 5α-reduction. In this study, the potential curative properties of MENT as anabolic agent was evaluated and compared with T in a preclinical hypogonadal male rat model. Aged male Wistar rats (11 months) were either orchidectomized (orch) or sham-operated (sham) and left untreated during a period of two months. As expected these orch animals experienced a significant loss of trabecular bone volume (-58% vs. sham) as well as lean mass (-8% vs. sham). On the other hand, cortical bone and fat mass were not significantly altered following 2 months castration. Subsequently, these 13-month-old orch rats were treated with either vehicle, MENT (12 μg/day) or T (72 μg/day) subcutaneously for 4 months via mini-osmotic pumps. Results were compared with age-matched sham or vehicle-treated orch rats (orch). Despite a 6-fold difference in dose, MENT and T equally restored the weight of the ventral prostate to sham level, but MENT increased the weight of the seminal vesicles and the musculus levator ani above sham levels (+20% and +44%, resp.). At the end of the study, trabecular bone volume and lean mass remained significantly lower in orch vs. sham rats (-60% and -16%, resp.). Yet, also cortical bone area was significantly decreased in orch rats at sacrifice (-23% vs. sham), through a lower periosteal but higher endocortical bone formation. MENT and T both fully restored lean mass and trabecular bone volume to sham level. The cortical bone loss was also prevented by MENT and T, however without full recovery to sham level. However, MENT restored periosteal and endocortical bone formation to sham level, while T normalized endocortical bone formation but stimulated periosteal bone formation above sham level (+94%, p = 0.06). MENT and T also normalized the higher fat mass in orch rats, but MENT decreased fat tissue even below sham levels (-24%) and this correlated with lower leptin levels (-43%).

In conclusion, MENT appears a more potent androgen than T in a preclinical model of male hypogonadism, with significantly more of an effect on fat mass. Both MENT and T restore trabecular bone loss and prevent cortical bone loss in aged hypogonadal rats. T showed a superior effect on periosteal bone formation compared with MENT.

Disclosures: Dirk Vanderschueren, None.

This study received funding from: The Population Council, Inc


Equol Decreased Bone Resorption in Equol Non-producing Postmenopausal Japanese Women: a Pilot Randomized Placebo-Controlled Trial.Tomomi Ueno1, Yoshiko Ishimi2, Junko Ezaki3, Shigeto Uchiyama4, Yasuhiro Fujii4, Yuko Tousen*2, Mamoru Nishimuta5. 1Otsuka Pharmaceutical Co., Ltd., Japan, 2National Institute of Health & Nutrition, Japan, 3Food Function & Labeling Program, National Institute of Health & Nutririon, Japan, 4Saga Nutraceutricals Research Institute, Otsuka Pharmaceutical Co., Ltd., Japan, 5Chiba Prefectural University of Health Sciences, Japan

Objective: Equol, a metabolite of isoflavone daidzein, obtained from soy isoflavones may play a critical role in the prevention of bone loss in postmenopausal women. However, clinical trials on the use of equol have not yet been published. This study aimed to investigate the effects of equol on bone metabolism, as well as on serum sex and thyroid hormone levels, in postmenopausal Japanese women.

Methods: We performed a 1-year double-blind, randomized placebo-controlled trial using natural S-equol supplementation in 93 equol non-producing postmenopausal Japanese women at less than 5 years after the onset of menopause. Subjects aged 41-62 years were randomly assigned to the following 4 groups: placebo, 2 mg of S-equol (EQ-2), 6 mg of S-equol (EQ-6), and 10 mg of S-equol (EQ-10); subjects were required to take the supplements once per day.

Results: The mean body weight and height of the subjects were 54.1 ± 8.2 kg and 156.8 ± 4.9 cm, respectively. There were no major differences in body weight and height among the different groups at baseline and after 12 months of equol intervention. Equol intervention increased the concentration of equol in the serum and urine of subjects in a dose-dependent manner. After 12 months of the intervention, urinary deoxypyridinoline (DPD) level significantly decreased to -23.94% in the EQ-10 group compared with that in the placebo group (-2.87%) (p = 0.020). This showed that 10 mg per day of equol supplementation markedly inhibited bone resorption. Additionally, the percent change in the whole body bone mineral density (BMD) in the EQ-10 group was -1.10%, which was significantly different from that in the placebo group (1.88%) (p = 0.027). Thus, a 12-month supplementation of 10 mg per day of equol prevented the decrease in the whole body BMD in postmenopausal women. Serum sex and thyroid hormone concentrations did not differ among the 4 groups after the equol intervention.

Conclusions: This is the first report providing specific evidence of the effect of equol on bone metabolism in humans. The findings of this study suggest that supplementation of 10 mg of equol per day may contribute to the amelioration of bone health in equol non-producing postmenopausal women without causing any adverse effects. Further research is needed to determine the effects of equol on bone metabolism and safety.

Disclosures: Yuko Tousen, None.

This study received funding from: Otsuka Pharmaceuticals Co., Ltd.,


See Friday Plenary number FR0435.


Atorvastatin Attenuates Lrp5/Wnt3a in the eNOS null mouse experimental hypercholesterolemic femurs.Jeffrey Park*1, Malayannan Subramaniam2, John R. Hawse2, Amy Flores1, Thomas C. Spelsberg2, Nalini Rajamannan3. 1Northwestern University, USA, 2Mayo Clinic, USA, 3Northwestern University Medical School, USA

Atorvastatin Attenuates Lrp5/Wnt3a in the eNOS null mouse femurs after experimental hypercholesterolemia Osteoporosis(OP) is the most common cause of morbidity secondary to the increased risk of bone fractures. This study hypothesizes that OP develops secondary to activation of Wnt3a/LRP5 via endochondral bone turnover in experimental hypercholesterolemia in the eNOS null mouse. We tested this hypothesis in the eNO null mouse as a model of oxidative stress. In this study, we utilized eNOS null mice as a model system to determine if a high cholesterol diet leads to osteoporosis and if this process could be revered by the addition of Atorvastatin. The following groups of eNOS null mice were used in this study: control (n=80), cholesterol (n=80), cholesterol + Atorvastatin (n=80). Treatments began at 2 months of age and were continued for a duration of 6 months. At this time, the femurs of all mice were analyzed for the development of osteoporosis using Li-Cor Infrared Imaging Li-Cor imaging uses an infrared technique to determine if a hydroxyapatite bone probe is taken up in active bone turnover. The bone probe is injected 48 hours prior to analysis of the femurs. Additionally, the femurs of 240 mice were isolated and mRNA was extracted for RT-PCR analysis of, Lrp5, Wnt3a, Cbfa1, Osteocalcin, OPN, and Sox 9. RNA isolated from the femurs of cholesterol treated eNOS mice revealed increased expression of Cbfa1, Sox9, OPN, Lrp5 and Wnt3a relative to control mice. Increased expression of these genes was attenuated with atorvastatin therapy. LiCor infrared imaging revealed an increase in bone probe uptake 48 hours after injection into the cholesterol mouse with attenuation with the atorvastatin therapy (p<0.05). (Quantification of results are in Table 1.) Taken together, these data demonstrate that eNOS null mice develop an osteoporotic phenotype following administration of a high cholesterol diet which is reversible by the addition of Atorvastatin. Furthermore, we have shown evidence for an important role for the Wnt3a/Lrp5 pathway in mediating this bone phenotype.

Table Table 1.  
  1. *p<0.05 control versus cholesterol

  2. **p<0.05 cholesterol versus cholesterol + Atorvastatin

inline image

Disclosures: Jeffrey Park, None.


Differential Effects of Odanacatib Compared to Alendronate on Bone Turnover Markers in Adult Ovariectomized Rhesus Monkeys.Le Thi Duong1, Sherri Motzel2, Gregg Wesolowski3, Maureen Pickarski*3. 1Merck Research Laboratories, USA, 2Merck & Co., Inc, USA, 3Merck & Co., Inc., USA

Odanacatib (ODN) is a selective, potent and reversible inhibitor of cathepsin K (CatK) currently in development for the treatment of postmenopausal osteoporosis. ODN (at 2-4μMṁ24hr) was previously demonstrated to fully prevent bone loss and resulted in normal biomechanical properties in ovariectomized (OVX) rhesus monkeys post treatment for 21-mo. Here, the effects of ODN on bone turnover markers were directly compared to that of Alendronate (ALN) in the same preclinical model. OVX-rhesus monkeys (10-22 yr-old) were randomized post-surgery into four groups (N=16/group), and dosed daily by oral gavage with HPMC-AS vehicle (Veh), ODN at 2mg/kg (L-ODN; 11μMṁ24hr), ODN at 8mg/kg (H-ODN; 140μMṁ24hr), or ALN(15μg/kg; 2X/wk, s.c.) in prevention mode for 20 mo. The H-ODN dose was reduced to 4mg/kg (49μMṁ24hr) approximately 7-months post dosing. Treatment with both doses of ODN and ALN resulted in rapid suppression of bone resorption markers, uNTx and sCTx, to 65-75% below baseline levels. ALN consistently reduced serum bone formation markers, BSAP and P1NP, by 60% and 80% of baseline, respectively. ODN displayed significantly less reduction in a dose-reversal manner of BSAP (40% in L-ODN and <10% in H-ODN) and P1NP (60% in L-ODN and 40% in H-ODN) compared to ALN for the first 7-mo. post-dosing. As the H-ODN dose group was switched from 8 to 4mg/kg/d, the degree of reduction in BSAP and P1NP reversed to 30% and 45% reduction from baseline, closer to that of the L-ODN group. The serum marker 1-CTP, a metalloproteinase-generated C-terminal peptide of Col type I and a substrate of CatK, was not responsive to Veh or ALN treatment. Steady state levels of 1-CTP increased by 70% and 120% in the L-ODN and H-ODN groups versus Veh. The selective induction of 1-CTP supported the target engagement of Cat K by ODN in bone. ALN rapidly suppressed Trap-5b levels by 30% within 1.5 mo. post-dosing. However, Trap-5b levels trended to elevate in L-ODN and increased 80% in H-ODN above Veh within 6-mo. post-dosing. Hence, ODN is as effective as ALN in suppressing the bone resorption markers. However, ODN clearly works differently from ALN in displaying significantly less reduction of bone formation markers and increased a marker for osteoclast number. These findings on the effects of ODN on bone turnover markers in OVX-monkeys are consistent with previous observations on the bone formation sparing effects of odanacatib in a PhII study with osteoporotic women.

Disclosures: Maureen Pickarski, Merck & Co., Inc, 3

This study received funding from: Merck & Co., Inc


See Friday Plenary number FR0438.


See Friday Plenary number FR0439.


See Friday Plenary number FR0440.


Bone Quality in Monkeys Treated with ED-71, a Vitamin D Analogue.Marc Grynpas*1, Richard Cheung2, Susan Y. Smith3, Hitoshi Saito4. 1Samuel Lunenfeld Research Institute, Canada, 2Lunenfeld Research Institute, Canada, 3Charles River Laboratories, Canada, 4Chugai Pharmaceutical Company, Limited, Japan

Bone quality was assessed in femurs and vertebrae (verts) by density fractionation, and illustrated by electron backscattering imaging (BSE) on selected tibia and verts. Trabecular connectivity (trab conn) was evaluated in verts by strut analysis of BSE images.

Bones were from 2 groups (10 animals/group) in 2 separate studies. Following OVX, monkeys (China, 9+ years) were gavaged daily with vehicle or ED 71 for 26 weeks at 0.1 or 23 weeks at 0.3μg/kg/day. In vivo results showed ED-71 preserved bone mass due to prevention of OVX-induced increases in the rate of bone remodeling.

Verts/femora were ground and sieved to separate bone particles <20 μm. Bone powder was chemically “fractionated” (<1.6->2.1 g/ml) for verts and (<2.0->2.2 g/ml) femora in a bromoform-toluene mixture by stepwise centrifugation (Grynpas & Hunter 1988). The contribution of each fraction, relative to original unfractionated bone weight, was calculated to determine a mineralization profile.

Tibia cross-sections and verts were fixed, dehydrated, embedded in Spurr resin, polished, carbon coated and examined by BSE. Images from verts were skeletonized and quantified for # of nodes, free-ends and different types of struts to determine trab conn.

To estimate if 2 distributions differ the logit function (LF) of each distribution was calculated. LF: ln= (proportion X) where X is the density cut-off for each group. There was no significant difference between LF of controls vs 0.3 (p=0.144) while a shift in LF between controls and 0.1 was significant (p=0.015) for femurs indicating an increase in mineralization. This was confirmed by BSE of tibiae. The shift in LF for verts was significant between controls and 0.3 (p<0.001) while there was no difference between LF of 0.1 and controls (p=0.294). Results were consistent with increased trab conn as demonstrated by increases in # of multiple points, node-node struts and decreased end points and free-free struts at 0.3. Similar trends were seen at 0.1.

In general, OVX-induced increases in bone turnover results in a shift of the mineralization profile to lower densities due to an increase in newly formed bone. Cortical porosity also increases markedly. Drugs like ED 71 that slow bone turnover tend to shift the mineralization profile to higher density due to an increase in secondary mineralization. In addition, we see decrease trab conn due to loss of trabeculae, a consequence of OVX; this was prevented at the 0.3 dose.

Disclosures: Marc Grynpas, contract, 5

This study received funding from: Chugai Pharmaceutical


See Friday Plenary number FR0442.


Vitamin D Malnutrition is Associated with a Substantially Lower Bone Mass in Northern Chinese Older Postmenopausal Women.Qian Zhang*1, Xiaoqi Hu2, Cuixia Wang2, Ying Liu2, Yifan Duan2, Richard Prince3, Kun Zhu4. 1Institute of Nutrition & Food Safety, Chinese Center for Disease Control & P, Peoples republic of china, 2National Institute for Nutrition & Food Safety, Chinese Centre for Disease Control & Prevention, China, 3Sir Charles Gardner Hospital, Australia, 4University of Western Australia, Sir Charles Gairdner Hospital, Australia

Background: Low vitamin D status may lead to osteoporosis or osteomalacia. There are few data in Northern Chinese older people on the prevalence and predictors of low vitamin D status and its association with bone mass.

Objective: The aims of this study were to examine the prevalence of hypovitaminosis D, to identify predictors of vitamin D status and to evaluate its association with bone mass in Chinese older postmenopausal women.

Design: A total of 446 healthy community-dwelling postmenopausal women aged over 60 years (mean age 68.2 ± 5.6 yrs) living in Beijing, China were recruited from May to July 2008. Serum 25-hydroxyvitamin D [25(OH)D] concentration was determined by RIA in duplicates. Bone mineral density (BMD) at lumber spine, proximal femur, and whole body, and body composition of whole body were measured by a dual-energy X-ray absorptiometry (DXA). Information on lifestyle was elicited by questionnaires.

Results: The average serum 25(OH)D concentration was 15.6 ± 7.0 ng/mL, 19.5% had vitamin D deficiency (25(OH)D<10 ng/mL), 59.0% insufficiency (25(OH)D 10-20 ng/mL) and only 4.9% of subject had 25(OH)D above 30 ng/mL. Predictors of low 25(OH)D in regression analysis (overall R2=0.31) included poor education (β=-4.6, P<0.001), city and apartment living (β=-7.43, p<0.001 and β=-3.8, P<0.001), low dairy intake (β=-1.6, P=0.05), overweight (β=-1.1, P=0.07) and an early summer sample date (β=-2.3, P=0.003), but not out door time, physical activity level, age, height, waist circumstance, body composition indices, smoking and alcohol consumption. Compared to women with vitamin D insufficiency and deficiency, those with 25(OH)D concentrations above 20 ng/mL had significantly higher total hip (5.8-10.1%), femoral neck (6.9-10.4%), total body (4.8-5.9%) and lumber spine (6.37.5%) BMD after accounting for confounding variables including age, sample date, living area, height, weight, physical activity level and calcium intake (all P<0.05).

Conclusions: The prevalence of hypovitaminosis D is alarmingly high in older community-dwelling postmenopausal women living in Beijing. Importantly unlike European population studies low vitamin D status predicted a substantially lower bone mass (5-10%). These findings require urgent investigation to understand the nature of the relationship between vitamin D status and bone structure in Chinese populations.

Disclosures: Qian Zhang, None.


Osteopenia in Gaucher Disease Develops Early in Life: Response to Imiglucerase Enzyme Therapy in Children, Adolescents and Adults.Thomas Hangartner*1, Neal Weinreb2, Paige Kaplan3, J. Alexander Cole4, Andrea Gwosdow4, Pramod Mistry5. 1Wright State University, USA, 2University Research Foundation for Lysosomal Storage Diseases, Inc., USA, 3Children's Hospital of Philadelphia, USA, 4Genzyme Corporation, USA, 5Yale University School of Medicine, USA

Purpose: To analyze the bone mineral density (BMD) of children, adolescents, young and older adults with type 1 Gaucher disease (GD1) at baseline and after treatment with imiglucerase. Methods: The analysis included all patients between the ages of 5 and 50y with GD1 treated with imiglucerase and enrolled in the ICGG Gaucher Registry. Lumbar spine BMD was assessed by dual-energy x-ray absorptiometry (DXA) and expressed as Z-scores. BMD data at baseline and up to 10y on imiglucerase were analyzed in each age group. Correlations were evaluated between BMD and other phenotypic characteristics, i.e., hemoglobin, platelet counts, spleen and liver volumes, bone pain and bone crises. Descriptive statistics were used to calculate mean DXA Z-scores at baseline according to 5-year age categories. Nonlinear mixed effects models were used to analyze DXA Z-scores over time. Results: Baseline hematological and visceral manifestations in the 4 age groups were similar. The most common genotype of patients with bone manifestations was N370S/Other. At baseline, DXA Z-scores were below ≤-1 in 44% of children (ages ≥5 to <12 years; n=43), 76% of adolescents (≥12 to <20 years; n=41), 54% of young adults (≥20 to <30 years; n=56) and 52% of adults (≥30 to <50 years; n=171). Among children with DXA Z-scores below -1 at baseline (n=19), regression models revealed the median DXA Z-scores improved from baseline -1.38 (95% CI -1.73 to -1.03) over 6y of imiglucerase to -0.73 (95% CI -1.25 to -0.21). A similar response was observed in adolescents and both adult groups. Among young adults with DXA Z-scores below -1 at baseline (n=30), regression models revealed that imiglucerase resulted in improvement of bone density from baseline Z-scores of -1.95 (95% CI -2.26 to -1.64) to -0.67 (95% CI -1.09 to -0.26) after 10y of imiglucerase. Conclusions: Low bone density is prevalent in all age groups with GD; it is even more prevalent during adolescence, a developmental period critical to attainment of peak bone mass. Imiglucerase results in amelioration of osteopenia in all age groups but the effect is greater in younger patients. The early onset of osteopenia in young untreated GD patients, with resultant failure to achieve optimal bone mass, and the good response to treatment in these patients suggest that early therapeutic intervention is the best strategy for achieving maximal skeletal outcomes.

Disclosures: Thomas Hangartner, None.

This study received funding from: Genzyme Corporation


See Friday Plenary number FR0445.


See Friday Plenary number FR0446.


Effect of a Single Oral Dose of 600,000 IU of Cholecalciferol on Serum Calciotropic Hormones in Young Subjects With Vitamin D Deficiency: a Prospective Intervention Study.Cristiana Cipriani*1, Elisabetta Romagnoli1, Alfredo Scillitani2, Iacopo Chiodini3, Romano Del Fiacco4, Vincenzo Carnevale5, Maria Lucia Mascia6, Claudia Battista5, Luciano Carlucci6, Cristina Eller Vainicher7, Salvatore Minisola1. 1University of Rome, Italy, 2Casa Sollievo Della Sofferenza Scientific Institute, Italy, 3Department of Medical Sciences, University of Milan, Fondazione Policlinico IRCCS, Italy, 4University “Sapienza”-Rome, Italy, 5Department of Internal Medicine & Endocrinology Casa sollievo della Sofferenza Hospital, Italy, 6Department of Clinical Sciences, University of Rome Sapienza, Italy, 7Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinco Milano, Italy

Purpose: Vitamin D depletion has been reported as a very common condition among adult general population. Neverthless, the effect of vitamin D repletion in young people with low vitamin D status has not been investigated so far. We carried out a prospective intervention study to evaluate short and long-term serum changes of the main parameters of calcium metabolism inducend by a single oral dose of cholecalciferol (600,000 IU) in a sample of young subjects with vitamin D deficiency.

Methods: Forty-eight young subjects (35 females and 13 males; mean age 36.04 ± 8.46 SD yrs, range 25-56 yrs; BMI 24 ± 3.52 kg/cm2) with vitamin D deficiency received a single oral dose of 600,000 IU of cholecalciferol. Serum levels of 25-hydroxyvitamin D [25(OH)D], total calcium [Ca], phosphorus [P], magnesium [Mg] and parathyroid hormone [PTH] were measured at baseline, and at 3, 15 and 30 d. A subgroup of 20 female subjects (mean age 33.15 ± 6.02, range 25-50; BMI 23.8 ± 4.01) were also assessed at 60 and 90 d. In this subgroup serum calcitriol (1,25-dihydroxyvitamin D [1,25(OH)2D]) levels were also measured.

Results: We found a significant change in 25(OH)D levels throughout the entire observation period (p<0.001). 25(OH)D serum levels were significantly increased at 3 d (77.16 ± 30.52 ng/ml; p<0.001) and up to 30d (62.46 ± 26.12; p<0.001) compared to baseline. A significant concomitant decrease in serum PTH concentration was found (p<0.001). This decrease was already significant at 3 d (-14.47 ± 13.89 pg/ml; p<0.001) and up to 30 d (-9.69 ± 16.01; p<0.001). These trends were mantained in the subgroup followed up to 90 d (p<0.001). Mean serum Ca, P and Mg values significantly changed throughout the entire period (p<0.05, p<0.01 and p<0.001 respectively). Ca and P levels were significantly higher at 3 d compared to baseline (9.46 ± 0.32; 4 ± 0.56 mg/dl; p<0.01 for both). On the contrary, mean serum Mg values were significantly reduced at the same time point (1.91 ± 0.18 mg/dl; p<0.01). Serum 1,25(OH)2D concomitantly increased (p<0.001) and was significantly higher than baseline as soon as 3 d of observation (51.03 ± 41.08 pg/ml; p<0.001) and up to 60 d (12.77 ± 24.48; p<0.05).

Conclusions: A single very large oral dose of 600,000 IU of cholecalciferol is useful in rapidly and safely enhancing serum 25(OH)D and in reducing serum PTH in young people with vitamin D deficiency. Serum 25(OH)D seems to play a direct role in modulating PTH secretion, regardless of 1,25(OH)2D and calcium levels.

Disclosures: Cristiana Cipriani, None.


Prevalence and Risk Factors of Low Vitamin D Status among Inuit Adults.Jessy Hayek*, Hope Weiler, Grace Egeland. McGill University, Canada

Background: Low vitamin D status may be a risk factor for many health conditions, including osteoporosis, cardiovascular disease, and cancer. Evidence since the Nutrition Canada Survey (1973) suggests that Aboriginal people have low intakes of vitamin D and are shifting away from the consumption of traditional foods. Further risk factors including age, higher body mass index (BMI), ethnicity, elevated parathyroid hormone (PTH) concentrations and low socio-economic status predispose Aboriginal populations to low vitamin D status.

Objectives: 1) Determine the prevalence of vitamin D deficiency and insufficiency in Inuit adults and 2) Identify risk factors for low vitamin D status (gender, age, vitamin D intake, BMI, household crowding, and socio-economic status.) Methods: 2207 Inuit adults (18-90 y) participated in the 2007-2008 Inuit Health Survey. Households were selected randomly from communities in Nunavut, Nunatsiavut and Inuvialuit Settlement Region. All data were collected in the field and through a mobile research laboratory, the Amundsen Research Ship. Dietary intake was assessed through the administration of a 24 h recall and a Food Frequency Questionnaire (FFQ). Anthropometric measurements and information about household living conditions, supplement use and health status were collected through interviews. Nurses collected blood samples and serum 25(OH)D and PTH were measured by Chemiluminesent technology (Diasorin, Liaison). Statistical analysis, including student t test, ANOVA, chi-square and logistic regression were performed using STATA 10.

Results: At the end of the summer, 72% of Inuit adults had insufficient 25(OH)D concentrations (> 75nmol/L) with a weighted mean of 58.5 ± 33.3 nmol/L. Older adults (> 50 years old) had higher vitamin D concentrations and intake than younger adults (47.6 ± 27.2 vs 83.8 ± 32.4 nmol/L, p<0.05). Men had better vitamin D status and intake than women (60.6 ± 34.2 vs 57.2 ± 32.7 nmol/L, p<0.05). The strongest predictors of vitamin D status among Inuit adults were age and BMI.

Conclusions: This is the first population assessment of vitamin D status in Inuit adults. Based on our results, there is a need for interventions promoting the consumption of traditional foods rich in vitamin D particularly among young adults. Further, assessment of vitamin D status in the winter across wider age ranges is advised.

Disclosures: Jessy Hayek, None.


Propagation of Paget's Disease of Bone after Reaming and Intramedullary Rod Placement for a Fracture.Joseph Shaker*1, David King2, Mercedes Gacad3, Frederick Singer3. 1Medical College of Wisconsin, USA, 2Medical College of Wiconsin, USA, 3John Wayne Cancer Institute, USA

Paget's disease of bone (PDB) has rarely been reported to develop in a new bone after inadvertent use of pagetic bone in a bone graft. We now report a case of PDB developing in the distal tibia after surgery on a pagetic proximal tibia. A 49 year old man developed pain in the right tibia in 1/07. Radiographs suggested mixed lytic/sclerotic PDB involved the proximal tibia. There was no family history of PDB or other bone disease. In 4/07, while playing soccer he turned and fractured the proximal right tibia. He underwent a biopsy and intramedullary rod placement with reaming. Because of postoperative deformity he had a revision with repeat intramedullary rod placement as well as a plate in 5/07. In early 2009 he developed right lower leg pain. In 4/09 his pain became more severe and radiographs revealed new lytic lesions more distally in the right tibia. On examination there was slight warmth over the right tibia with mild tenderness distally. Laboratory data included total serum alkaline phosphatase 144 U/L (40-129), bone specific alkaline phosphatase 37.5 μg/liter (020.1), 25-hydroxy vitamin D 21 ng/ml, creatinine 0.74 mg/dl, and urinary calcium 295 mg per 24 hours. A total body bone scan demonstrated increased uptake in the proximal right tibia as well as in the new lytic lesions more distally located in the right tibia. The initial biopsy (4/07) as well as a biopsy of one of the new lytic areas (8/09) revealed numerous large multi-nucleated osteoclasts, numerous osteoblasts and a fibrovascular marrow characteristic of PDB. Immunohistochemical evaluation of the 4/07 biopsy with an anti-measles virus nucleocapsid protein antibody demonstrated staining of the osteoclasts.

After repletion of vitamin D in 11/09 he was treated with zoledronic acid 5 mg intravenously. Approximately 2 months later, the right tibia symptoms had improved 30-40% and the bone specific alkaline phosphatase had decreased to 12.1 μg/liter (020.1).

This patient presented with PDB in the proximal right tibia and developed lytic PDB in the distal tibia after reaming and intramedullary rod placement for a fracture. The new lytic lesions developed much faster than would be expected from the normal progression of a lytic lesion in PDB. This suggests that the PDB was propagated by the procedure. The explanation for the spread of PDB could be the transposition of bone cells containing measles virus nucleocapsid protein from the proximal to the distal tibia.

Disclosures: Joseph Shaker, None.


Cinacalcet Treatment in Patients with Primary Hyperparathyroidism.Claudio Marcocci*, Luisella Cianferotti, Chiara Banti, Feredica Saponaro, Edda Vignali, Silvia Chiavistelli, Giuseppe Viccica, Filomena Cetani. University of Pisa, Italy

Primary hyperparathyroidism (PHPT) is an endocrinopathy diagnosed by chronically elevated serum calcium and PTH. Parathyroidectomy usually cures the disease, but few treatment alternatives exist for patients who fail surgery, have contraindication or refuse surgery, or do not meet current operative guidelines. Medical therapies capable of reducing serum calcium and PTH and increasing bone mass in PHPT patients are needed. Calcimimetics binding the calcium-sensing receptors (CASR) quickly and directly reduce serum calcium level.

The aim of the present study was to evaluate efficacy, safety, and long-term tolerability of cinacalcet in women with PHTP. We studied 13 patients with PHPT. All subjects had serum calcium levels higher than 11.2 mg/dl. In Europe cinacalcet is approved for reduction of hypercalcemia in patients with PHPT for whom parathyroidectomy is indicated on basis of calcium levels but in whom surgery is clinically inappropriate or is contraindicate. Subjects requiring drugs metabolized by cytochrome P450 were excluded because of cytochrome P450 inhibition by cinacalcet. Women on treatment with bisfosphonate were included in the study. During the initial dose-titration phase, cinacalcet dose increase from 30 mg daily to 60 mg daily. In the maintenance phase, visits were approximately every 4-5 wk and then every 12 -24 wk, during which doses could be adjusted. If a patient developed hypocalcemia (serum calcium <8 mg/dl), cinacalcet treatment was withheld for approximately 1 wk until the next serum calcium measurement was at least 8,4 mg/dl. Blood was drawn after an overnight fast the morning dose of cinacalcet and adverse events recorded. Blood was collected for measurement of serum calcium, ionized calcium, PTH, bone alkaline phosphatase, osteocalcin, urinary and serum cross laps, and 24 h urinary calcium. These parameters were evaluated at baseline and after 6-8 months.

Three out of 13 patients withdrew cinacalcet due to adverse effects (nausea, vomiting and heartburn), and underwent surgery. In the remaining patients, compared with baseline, cinacalcet treatment improved measures of PHPT including reducing serum calcium, ionized calcium and PTH. No changes in bone markers and 24 h urinary calcium were seen.

Treatment of PHPT patients with cinacalcet maintained normocalcemia, reduces plasma PTH, and was well tolerated

Disclosures: Claudio Marcocci, None.


See Friday Plenary number FR0451.


Mutational Analysis of GCMB, a Parathyroid-Specific Transcription Factor, Suggests That it is Not a Frequent Cause of Primary Hyperparathyroidism.Michael Mannstadt*1, Emily Holick2, Wenping Zhao2, Harald Jueppner3. 1Massachusetts General Hospital Harvard Medical School, USA, 2Endocrine Unit, Massachusetts General Hospital, USA, 3Massachusetts General Hospital, USA

Purpose of the study: Primary hyperparathyroidism (PHPT) is a common endocrine disorder characterized by hypercalcemia and elevated PTH levels. In a small subset of parathyroid adenomas, activating mutations of the cyclin D gene have been identified. Many parathyroid adenomas are monoclonal in origin, but the somatic or germ-line mutations that lead to an adenoma have not been identified in the majority of cases. GCMB is a parathyroid specific transcription factor, which causes hypoparathyroidism when inactivated before parathyroid gland development. It continues to be expressed in adult parathyroid glands where its post-natal function remains unknown. However, overexpression of GCMB mRNA has been reported in parathyroid adenomas, and we tested the hypothesis whether acquired activating mutations of GCMB can be found in parathyroid adenomas.

Methods: We studied 30 surgically resected parathyroid adenomas from 30 patients with HPTH. Genomic DNA was extracted and all exons and exon-intron borders of the gene encoding GCMB were sequenced. Nucleotide sequence variations were compared to public databases and novel amino acid changes were tested in vitro by Western blot analysis and Luciferase assay using transfected DF-1 cells.

Results: Several polymorphisms were identified that were previously reported or can be found in public databases. In one of the 30 glands, a novel heterozygous missense mutation, c1144G> A, was identified leading to the substitution of an evolutionary conserved valine at position 382 to methionine (V382M). The presence of this mutation, which was not found in public databases, was confirmed by restriction site analysis. Western blot analysis using mutant GCMB (GCMB-V382M) from lysates from transiently transfected DF-1 fibroblasts demonstrated a protein band that was identical in size and similar in intensity to the wildtype protein indicating that the identified amino acid change does not significantly impair GCMB expression. Transient transfection of DF-1 cells with plasmids encoding wildtype and GCMB-V382M showed a similarly robust, 16-fold increase in luciferase activity. Cotransfection of wildtype and mutant proteins did not change luciferase activity.

Conclusions: In 30 analyzed parathyroid adenomas from patients with primary hyperparathyroidism, functionally important mutations in GCMB were absent. This suggests that mutations in this transcription factor do not seem to play a major role in the pathogenesis of PHPT.

Disclosures: Michael Mannstadt, None.


No Improvement in Carotid Vascular Abnormalities with Parathyroidectomy in Mild Primary Hyperparathyroidism.Tatjana Rundek1, Marcella Walker*2, Chiyuan Zhang2, Donald McMahon2, James Lee2, Jessica Fleischer2, Tamara Taggart2, Julia Udesky2, Ralph Sacco1, Shonni Silverberg2. 1University of Miami, USA, 2Columbia University, USA

We have reported that mild primary hyperparathyroidism (PHPT) is associated with subclinical cardiovascular (CV) abnormalities. This study seeks to determine whether carotid vascular abnormalities are reversible with parathyroidectomy (PTX).

Results: Patients (N=43) with mild PHPT (79% female, age 62 ± 7 yrs, serum calcium 10.6 ± 0.5 mg/dl, PTH 97 ± 47 pg/ml; blood pressure 121 ± 15/74 ± 10 mmHg) had carotid ultrasound prior to and 1-YR post-PTX. BP did not change after PTX. Carotid plaque was present in 42% at baseline and did not regress after PTX (present in 49%, p=0.66). Maximum plaque thickness (MCPT) increased post-operatively (0.86 ± 1.08 to 1.03 ± 1.11: p=0.01). This increase was not associated with baseline calcium or PTH or predicted by postoperative changes in calcium or PTH levels. Mean carotid intima-media thickness [(IMT): 0.96 ± 0.09; normal < 0.90 mm; elevated in 74%] and stiffness (6.3 ± 3.1; normal < 6; elevated in 42%), were increased at baseline. Neither measure, however, was linearly associated with baseline calcium, PTH, 25-hydroxyvitamin D or 1, 25-dihydroxyvitamin D levels, nor did they improve 1 yr after PTX. Other indices of decreased vessel compliance (strain and distensibility) also did not change after PTX. None of the carotid measures improved after PTX in the subsets of patients that were abnormal at baseline.

Conclusions: Patients with mild PHPT have evidence of increased carotid stiffness and IMT, a structural indicator of subclinical CV disease associated with an increased risk of deleterious CV outcomes. These abnormalities did not improve in the first postoperative year after PTX. Plaque thickness increased after PTX, but the increase is not clearly associated with cure of PHPT. Without improvement in these indices, there is no clear suggestion that the presence of carotid vascular abnormalities should be an indication for surgery in PHPT. It will, however, be important to evaluate whether there are beneficial changes in other aspects of the CV system after PTX. 

Table  .  
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Disclosures: Marcella Walker, None.


Parathyroidectomy Does Not Affect Flow-Mediated Vasodilation in Mild Primary Hyperparathyroidism.Angela Carrelli*1, Marcella Walker2, Marco Di Tullio3, Shunichi Homma3, Chiyuan Zhang2, Donald McMahon3, Shonni Silverberg2. 1New York Presbyterian-Columbia University, USA, 2Columbia University, USA, 3Columbia University College of Physicians & Surgeons, USA

Purpose: Severe primary hyperparathyroidism (PHPT) has been linked to impaired endothelial function, which normalizes after parathyroidectomy. Endothelial dysfunction is an important early event in the pathogenesis of atherosclerosis. It is not known if endothelial dysfunction occurs in mild PHPT and if so, whether it is reversed following parathyroidectomy. To address this issue, we measured flow-mediated dilation (FMD) by ultrasound imaging in patients with mild PHPT prior to and 12 months after parathyroidectomy. FMD, a non-invasive technique that estimates endothelial function, quantifies the brachial artery response to reactive hyperemia by measuring the percent change in vessel diameter before and after inflation of a forearm cuff to supra-systolic pressure for 5 minutes.

Results: We studied 43 patients who were typical of those with mild PHPT (79% female, age ± SD 62 ± 7 yrs, serum calcium 10.6 ± 0.5 mg/dl, PTH 97 ± 47 pg/ml). Baseline blood pressure was normal (Table).

At baseline, FMD in these PHPT patients was normal (4.59 ± 3.19%; age matched control population: mean 4.92 ± 3.93%, 25th percentile 2.55, 75th percentile 6.77). Baseline FMD was not associated with serum calcium or PTH levels (calcium: r=0.16, p=0.29; PTH: r=-0.057, p=0.72). Baseline brachial artery diameter prior to cuff inflation was positively associated with PTH levels (r=0.34, p=0.03). Although this finding is consistent with known vasodilatory effects of PTH, this association did not persist after controlling for height and weight.

One yr after parathyroidectomy, FMD remained normal (5.13 ± 4.71%) and was not significantly changed compared to baseline (difference: 0.54%, p=0.49). The change in FMD was not predicted by baseline calcium or PTH levels (calcium: r=-0.28, p=0.06; PTH: r=-0.16, p=0.30), nor was it associated with the change in serum calcium or PTH following parathyroidectomy (calcium: r=0.20, p=0.19; PTH: r=0.21, p=0.17).

Conclusions: FMD is normal in patients with mild PHPT and is unchanged one year after parathyroidectomy. We conclude that mild PHPT does not appear to be associated with the endothelial dysfunction seen in severe PHPT.

Table Table.  
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Disclosures: Angela Carrelli, None.


PTH Reverses the Imbalance between Cortical and Trabecular Bone Compartments in Hypoparathyroidism: A Three-Year Longitudinal HR-pQCT Study.Xiaowei Liu*1, Mishaela Rubin1, Jim Sliney Jr2, Donald McMahon3, David Dempster1, John Bilezikian3. 1Columbia University, USA, 2Columbia University Medical Center, USA, 3Columbia University College of Physicians & Surgeons, USA

Hypoparathyroidism (HypoPT), a disorder of PTH deficiency, is associated with increased bone density and microstructural abnormalities. To address the hypothesis that these skeletal abnormalities of HypoPT can be reversed by PTH administration, we evaluated cortical (Ct) and trabecular (Tb) skeletal microstructure in HypoPT by high resolution peripheral quantitative computed tomography (HR-pQCT; Xtreme CT, Scanco Medical) before and after PTH treatment. This represents the first prospective longitudinal HR-pQCT study of a disorder of defective parathyroid function.

In comparison to age-and sex-matched controls, subjects with untreated hypoPT (n=42, 9 male, 33 female, 46 ± 13 yrs) had significantly (p=0.03) higher Ct area (Ct.Area, +13%) and thickness (Ct.Th, +12%) at the distal radius (DR), but not at the distal tibia (DT). While Ct volumetric BMD (DComp) was similar to controls, Ct perimeter (Ct.Pm) was 14% and 8% higher at DR and DT (p<0.05). Tb area (Tb.Area), Tb vBMD (Dtrab), and Tb microstructure measurements were similar between HypoPT and controls while integral vBMD tended to be higher in HypoPT at DR and DT.

100 μg of hPTH(1-84) was administered every other day for 36 months. HR-pQCT scans were obtained at baseline and after 3, 6, 12, 24, and 36 months. Reductions were found in Ct.Area at DR (24mo, -2%, p=0.08) and DT (36mo, -2%, p=0.03), Dcomp at DR (24&36mo, -1-2%, p<0.02) and DT (24&36mo, -1-2%, p<0.01), Ct.Th at DR (36mo, -3%, p=0.11) and DT (24&36 mo, -2%, p<0.02). In contrast, Dtrab significantly increased at DR (24&36mo, +2-3%, p<0.05), but remained unchanged at DT. As a result, at DR, integral vBMD remained unchanged with Dcomp and Dtrab changing in opposite directions; at DT, vBMD significantly decreased (24mo, 1%, p=0.02). In addition, Tb.Area did not change with PTH. Other Tb microstructure measures, such as Tb number, thickness and spacing, showed no significant change.

In conclusion, PTH deficiency in HypoPT is associated with normal Tb indices but abnormal increases in the Ct bone compartment in the peripheral skeleton as compared to matched controls. PTH treatment is associated with normalization in the area and thickness of the Ct compartment at both DR and DT, along with concomitant decreases in Tb bone density at DR. These data suggest that PTH administration reverses microstructural skeletal abnormalities in HypoPT by restoring the balance between Ct and Tb indices.

Disclosures: Xiaowei Liu, None.


Accumulation of Maillard Reaction Products Involve Bone Fragility in Adynamic Bone Disease.Yoshiko Iwasaki*1, Hideyuki Yamato2, Masafumi Fukagawa3. 1Oita University of Nursing & Health Sciences, Japan, 2Kureha Corporation, Japan, 3Tokai University School of Medicine, Japan

It is well known that adynamic bone disease (ABD) is highly prevalent in dialysis patients. Although patients with ABD have normal bone mineral density (BMD), they have a high incidence of fracture rates. However, it still remains unknown why fracture rates increase in ABD. In this meeting last year, we presented that rat models with ABD have reduced bone strength despite having normal BMD. It was a possibility that this reduction was affected by changing bone chemical composition. On the other hand, several studies determined Maillard reaction products, such as pentosidine, carboxymethyl-lysine (CML), contribute collagen properties. The purpose of this study is to clarify whether Maillard reaction products affect bone strength. As for the ABD model, after male SD rats underwent thyroparathyr-oidectomy (TPTx), they underwent 5/6 nephrectomy (Nx) or sham operations. These rats were continuously infused with rat PTH and injected with L-thyroxin subcutaneously to maintain physiological levels. Therefore TPTx-Nx rats can simulate ABD, because of their low turnover bone associated with the suppression of renal function. Groups were ABD (TPTx-Nx) and control (TPTx-sham). Recently, it has became easier to obtain spectroscopic properties from bone with a laser raman spectroscopy system (Nicolet Almega XR, Thermo Fisher Scientific Co.). We can obtain much information about the matrix in bone and we can also evaluate local change of bone composition by this system. As the results, by means of Raman, pentosidine, which is a non-enzymatic collagen crosslink, and CML revealed higher level in ABD group compared with control group. In comparison to the control group, the accumulation level in ABD of pentosidine and CML was 10% and 15% higher, respectively. And also, enzymatic crosslink was accumulated in the ABD group. In addition, the levels of Pentosidine, CML, and crosslink were correlated with bone viscoelasticity measured by DMA system.

These results suggest that accumulation of Maillard reaction products induced by kidney dysfunction were deterioration mechanical properties of bone. Those matrix changes affect bone fragility and could lead to increased fracture risk without bone loss.

Disclosures: Yoshiko Iwasaki, None.


Anti-Diabetes Drug Class of SGLT1 Inhibitors Increases Bone Mass in Young and Adult Female Sprague-Dawley Rats by Decreasing Bone Turnover.Rana Samadfam*1, Nancy Doyle1, Martin Heinrichs2, Thomas Kissner2, Eckart Krupp2, Susan Y. Smith1. 1Charles River Laboratories, Canada, 2Sanofi-Aventis, Germany

Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia caused by defective insulin secretion, resistance to insulin action, or a combination of both. Due to the important role of bone in energy balance and the cross-talk between nutritional hormones and the skeleton, it is likely that anti diabetic compounds affect bone mass. Treating hyperglycemia with drugs that block intestinal glucose uptake and renal glucose reabsorption via the sodium-glucose transporters (SGLT1, SGLT2) represents a novel approach to diabetes treatment. These studies were designed to investigate the effect of SGLT1 inhibitors on bone and electrolytes in serum and urine. Rats were treated daily with either vehicle control or SAR474832, a selective SGLT1 inhibitor and SAR7226, a mixed SGLT1/2 inhibitor for 28 days. Blood and urine were collected for calciotropic hormone and bone turnover analysis. In addition, bone densitometry (DXA, pQCT) and bone quality (three point bending test and compression test) were assessed. Histology was also performed on excised bones. Treatment with SAR7226 resulted in dose-related, marked glucosuria (the anticipated pharmacological effect), polyuria, and calciuria. Normal serum calcium was maintained while serum phosphorus was slightly increased. PTH and 1,25 vitamin D3 were markedly suppressed as well as markers of bone turnover. These effects were associated with increases in bone mineral density (BMD). Increases in BMD at the spine were positively associated with increases in bone strength. Effects on bone mass were characterized microscopically by increases in trabecular bone. The SGLT1/2 inhibitor SAR7226 markedly influences calcium and phosphorus homeostasis with positive effects on bone mass and strength. Similar effects were observed with the low-absorbable selective SGLT1 inhibitor SAR474832. From these results it is concluded that calciuria observed following treatment with SAR7226 and SAR474832 is not associated with calcium release from bone. These studies highlight the importance of considering evaluations of the skeleton in the safety assessment of compounds affecting calcium homeostasis to provide important safety data. Calciuria and increased trabecular bone was found in toxicity studies in rats with the mixed SGLT1/2 inhibitor SAR7226 and the low-absorbable and selective SGLT1 inhibitor SAR474832.

Disclosures: Rana Samadfam, Contract Research, 3

This study received funding from: Sanofi-Aventis


Assessment of Bone Tissue Composition in Patients Undergoing Dialysis Therapy Using Raman Spectrometry.Shozo Yano*1, Yoshiko Iwasaki2, Akihide Tokumoto3, Hideyuki Yamato4, Toru Yamaguchi1, Toshitsugu Sugimoto5. 1Shimane University Faculty of Medicine, Japan, 2Oita University of Nursing & Health Sciences, Japan, 3Kamifukubara Medical Clinic, Japan, 4Kureha Corporation, Japan, 5Shimane University School of Medicine, Japan

BACKGROUND: It is well-known that adynamic bone disease (ABD) is common in patients undergoing dialysis therapy, and recent findings suggest that low turnover of bone metabolism might decrease the quality of the bone. Although bone biopsy is gold standard for the assessment, the examination is invasive. Thus, FT-IR and Raman spectrometry would be candidates for the substitutive and non-invasive methods, if they can extracorporeally assess the bone characteristics. OBJECTIVE: To analyze bone tissue composition in dialysis patients using Raman spectrometry. SUBJECTS and METHODS: Bone specimen was obtained from 43 patients with maintainance dialysis (HD 37 and PD 6). According to the histomorphometrical analysis, ABD, mild lesion (ML) and osteitis fibrosa (OF) were shown in 14, 16 and 13 patients, respectively. Bone composition was analyzed by Raman spectrometry (Nicolet Almega XR: Thermofisher Scientific Co). RESULTS: Mineral matrix ratio, which was expressed as (PO43-)/Amide I ratio, was the highest in the ABD group among 3 groups. The hydroxyproline/proline ratio implying the maturation of collagen was similar result. Collagen crosslink, a marker of mature crosslink, was shown in ABD>ML>OF whereas the pentosidine/Amide I ratio, a marker of senile crosslink, had a similar tendency. CONCLUSION: Reduced bone mineral content was observed in OF and ML, which was consistent with clinical findings such as decreased bone density associated with secondary hyperparathyroidism. In contrast, in ABD, mature as well as senile crosslink accumulation was found in the highest level among 3 groups. These results were also compatible with clinical findings where the fracture frequency was elevated in patients with lower serum PTH levels, suggesting the association of ABD with skeletal fragility.

Disclosures: Shozo Yano, None.


See Friday Plenary number FR0459.


Volumetric Bone Mineral Density, Geometry and Stiffness Discriminate Vertebral Fracture Status in Patients with Chronic Kidney Disease.Thomas Nickolas*1, Xiaowei Liu2, Valerie Thomas1, Emily Stein3, Adi Cohen1, Ryan Chauncy1, Donald McMahon3, Mary Leonard4, X Guo2, Elizabeth Shane3. 1Columbia University Medical Center, USA, 2Columbia University, USA, 3Columbia University College of Physicians & Surgeons, USA, 4Children's Hospital of Philadelphia, USA

Introduction: In chronic kidney disease (CKD), dual energy X-ray absorptiometry (DXA) measures of lumbar spine (LS) areal BMD (aBMD) provide poor discrimination of vertebral fracture (VFx) status. Discrimination of VFx in CKD patients by high resolution peripheral quantitative computed tomography (HRpQCT, Xtreme CT, voxel size ∼82 microns) has not been evaluated.

Methods: Patients with (n=10) and without (n=38) VFx were enrolled from an ongoing observational study of adults ≥50 years with CKD stages 3-5 not on dialysis. DXA aBMD was measured at LS, total hip (TH), femoral neck (FN) and ultradistal radius (UDR). Total, Cortical (Ct) and Trabecular (Tb) volumetric BMD (D100, Dcort and Dtrab); Ct thickness (CtTh) and periosteal perimeter (CtPm); total, Ct and Tb area (TotArea, CtArea and TbArea); and Tb microarchitecture were measured by HRpQCT at the distal radius (RAD) and tibia (TIB). Bone stiffness and both proximal and distal Ct load share (PCLS and DCLS) were measured by finite element analysis (FEA) of HRpQCT datasets. Results are expressed as Means ± SD, percent differences and areas under the receiver operating characteristic curves (AUC) with 95% confidence intervals (CI).

Results: Age, estimated glomerular filtration rate and BMI were 71 ± 9 years, 34 ± 16 mL/minute and 29 ± 5 kg/m2, respectively. Twenty-one percent were women and 54% were white. Mean T-Scores were above the osteoporotic range (LS: -0.2 ± 1.6; TH: -0.9 ± 1.1; FN: -1.5 ± 1.0; UDR: -1.5 ± 1.4). There were no significant differences in demographics or T-Scores between groups with/without VFx. By HRpQCT of the RAD, patients with VFx had 20% lower D100 (p=0.03), 9% lower Dcort (p=0.01), 27% lower CtTh (p=0.01) and 17% greater TbArea (p=0.006). At the TIB, patients with VFx had 9% lower Dcort (p=0.05), 8% greater CtPm (p=0.0008), 14% greater TotArea (p=0.007), 15% greater TbArea (p=0.006), 31% lower DCLS (p=0.03) and 16% lower PCLS (p=0.01). AUCs for VFx discrimination at the RAD for Dcort (0.74; 0.58-0.91) and CTh (0.76; 0.59-0.92) and at the TIB for Dcort (0.69; 0.49-0.90), CtPm (0.80; 0.67-0.93), TbArea (0.70; 0.55-0.85), PCLS (0.76; 0.60-0.92) and DCLS (0.72; 0.54-0.90) were all significantly better than LS aBMD (0.53; 0.34-0.73).

Conclusions: Volumetric BMD, geometric parameters and whole bone stiffness measured by HRpQCT and FEA provide better discrimination of VFx than LS aBMD in pre-dialysis CKD patients. Longitudinal studies are needed to determine whether HRpQCT can predict VFx in CKD patients

Disclosures: Thomas Nickolas, None.


See Friday Plenary number FR0461.


See Friday Plenary number FR0462.


Gene Analysis To Elucidate the Mechanisms of Tenofovir Mediated Osteopenia.Kim Mansky*, Iwen Grigsby, Lan Pham, Ann Emery, Louis Mansky, Raj Gopalakrishnan. University of Minnesota, USA

Clinical observations have revealed a strong correlation between loss of bone density in HIV-infected individuals, particularly in conjunction with the antiretroviral drug tenofovir, a nucleotide analog that inhibits HIV reverse transcriptase. The most compelling correlations have been observed in clinical studies involving young children and adolescents. The goal of this study was to investigate the in vitro effects of tenofovir exposure on primary osteoblast and osteoclast gene expression in order to gain insights into the potential mechanisms for the loss of bone density as well as to identify potential biomarkers. Primary osteoblasts and osteoclasts were generated from calvaria and bone marrow of wild type mice, respectively. Osteoblast and osteoclast cultures were either untreated or treated with the tenofovir prodrug, tenofovir disoproxil fumarate (TDF) and total RNA were extracted and used for microarray analysis to assess TDF-associated changes in gene expression profiles. Hierarchical clustering and Gene Ontology analysis were performed of the gene expression data from both osteoblasts and osteoclasts. Data from osteoblasts showed broad changes in gene expression profiles, including alterations in genes associated with osteoblast function. Inspection of these results led to the identification of genes that are associated with the Wnt, TGF-beta, Hedgehog, VEGF, B cell receptor, and the Fc epsilon RI signaling pathways. Data from osteoclasts revealed downregulation of Gnas, Got2 and Snord32a in TDF-treated cells. Microarray results were validated by confirming expression of the identified genes by qPCR analysis. To our knowledge these are the first studies, which demonstrate the impact of tenofovir on osteoblast and osteoclast gene expression. Understanding the functions of these genes should help explain the basis for tenofovir-associated loss of bone density in HIV-patients as well as identify potential biomarkers for diagnosis.

Disclosures: Kim Mansky, None.


Hyperkyphosis and Decline in Functional Status in Older Community Dwelling Women: The Study of Osteoporotic Fractures (SOF).Mei-Hua Huang1, Wendy Katzman, DSc2, Steven Cummings3, Deborah Kado*1. 1University of California, Los Angeles, USA, 2University of California San Francisco, USA, 3San Francisco Coordinating Center, USA

Maintaining physical functional ability is an important prerequisite for preserving independence in later life. Hyperkyphosis, or an increased thoracic curvature, is commonly observed in older persons and has been associated with worse self-reported and objectively measured physical function in multiple cross-sectional studies, including the Study of Osteoporotic Fractures. However, it is unknown whether hyperkyphosis may predict worse physical function over time. Therefore, we sought to determine whether hyperkyphosis precedes worse self-reported and objectively measured physical function over an average follow-up of 15 years. The digitized Cobb angle (T4-T12) derived from supine lateral thoracic spine plain films was used to calculate the degree of kyphosis in 1196 older women aged 65 and older who were randomly selected from the original cohort of 9,704 women because they had 15 year clinic visit data available for analyses. Participants were an average of 68.8 years old (SD= 3.3) and had a mean kyphosis angle of 44.6 degrees (SD = 12.1). Multivariable linear regression models were used to evaluate the association between kyphosis measured at baseline and change over 15 years in: 1) self-reported functional status (any difficulty in performing six tasks: walking 2-3 blocks, climbing or descending 10 steps, preparing meals, heavy housework, and shopping); 2) measured walking speed; and 3) grip strength. With each 10 degree increase in kyphosis, women were more likely to decline in functional status15 years later (β estimate = 0.086, p = 0.04), after adjustment for age, clinic, weight, height, physical activity, baseline functional status, baseline vertebral fracture, heel bone mineral density, health status, and arthritis. In similarly adjusted models, with each 10 degree increase in kyphosis, women had a decline in walking speed of 1.4 cm/sec (p = 0.017). There was no significant association between kyphosis and worsening grip strength. Interestingly, in these multivariable models, having a baseline vertebral fracture was not predictive of functional decline (β estimate = -0.023, p = 0.87) nor decline in walking speed (β estimate = -0.006, p = 0.74). Although the effect of hyperkyphosis on future physical functional decline is small in magnitude, in this select group of women survivors followed for 15 years, having hyperkyphosis was a stronger predictor of physical functional decline than was having a baseline prevalent vertebral fracture.

Disclosures: Deborah Kado, None.


Regulation of Energy Metabolism By Osteocytes.Ronald Kwon*, Diana Meays, Natalie Kardos, John Frangos. La Jolla Bioengineering Institute, USA

Bone has been discovered to exert endocrine control of energy metabolism [1], however the cellular mediators of this process have not been fully explored. Recently, Lee et al. implicated osteoblasts in regulating energy metabolism via a mechanism that involves the hormone osteocalcin (OCN) and the enzyme OST-PTP, which alters OCN bioactivity [1]. Given that osteocytes outnumber osteoblasts in vivo and produce high levels of OCN in culture, we hypothesized that osteocytes also exert endocrine control of energy metabolism through this mechanism. In this study, we used mice possessing a diphtheria toxin (DT) receptor transgene driven by the DMP1 promotor (DMP1-DTR) [2] to investigate the effects of DT-induced osteocyte ablation on energy metabolism. 8wk M wildtype (WT) and transgenic (Tg) DMP1-DTR mice were administered a single dose of DT (10ug/kg), and various indices of energy metabolism were quantified 4wk later. By focusing on long-term effects and using a relatively mild DT dose [2], we minimized the potential for confounding effects on energy metabolism by short-term inflammatory processes associated with osteocyte ablation. In the 4wks following DT administration, Tg mice gained significantly less weight per day compared to WT mice (WT: 0.18 ± 0.03g/day, Tg: 0.08 ± 0.03g/day; p=0.03, n=17 per group) despite consuming a similar amount of food (WT: 4.3 ± 0.5g/day, Tg: 5.6 ± 1.5g/day; p=0.47, n=7-9 per group). Fasted levels of blood glucose (WT: 161.3 ± 4.4mg/dl, Tg: 175.1 ± 3.8mg/dl; p=0.03, n=7-9 per group) and serum insulin (WT: 0.18 ± 0.01ng/ml, Tg: 0.24 ± 0.01ng/ml; p=0.02, n=5-7 per group) were significantly elevated in Tg mice. Unexpectedly, serum levels of OCN were not significantly different in Tg mice (WT: 28.8 ± 2.4ng/ml, Tg: 28.5 ± 1.8ng/ml; p=0.93, n=6-7 per group); nor were serum levels of adiponectin (WT: 8.5 ± 0.4ug/ml, Tg: 8.3 ± 0.6ug/ml; p=0.75, n=6-7 per group), which has been shown to be reduced as a result of osteoblast specific-deletion of OCN [1]. This suggests that effects of osteocyte ablation on energy metabolism were not due to reduced levels of circulating OCN. Finally, serum levels of leptin were not significantly different in Tg mice (WT: 3.8 ± 1.0ng/ml, Tg: 4.5 ± 0.9ng/ml; p=0.64, n=7 per group). Together, these data suggest that osteocytes regulate energy metabolism via an unidentified mechanism, either through endocrine signaling, or regulating a cellular intermediary such as osteoblasts.

[1] Lee et al., Cell 2007; [2] Tatsumi et al., Cell Metab 2007

Disclosures: Ronald Kwon, None.


Sex-related Differences in Skeletal Phenotype in a Rat Model of Type 2 Diabetes Mellitus.Maxime Gallant*1, Kathleen Hill1, Jennifer M. Doyle1, Susan Reinwald1, Richard G. Peterson2, David Burr1. 1Indiana University School of Medicine, USA, 2PreClinOmics, Inc., USA

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease that leads to various comorbidities. T2DM is known to increase the risk of fractures with little change in BMD. Men with T2DM are at higher risk for fractures than women with T2DM, and post-menopausal women are at greater risk than pre-menopausal women. We hypothesized that gender differences in bone structure that reduce bone mechanical properties may explain these differences in fracture risk in T2DM.

This study investigates sex differences in bone phenotype in a model of spontaneous T2DM, the ZDSD rat, with non-diabetic CD rats as controls. Animals were fed a high-fat diet to induce T2DM onset. Fourteen weeks after T2DM onset, animals were euthanized and bone phenotype was investigated using dual-energy X-ray absorptiometry, micro-CT imaging and mechanical testing. T-tests were used to test for differences between ZDSD and controls within each sex, and two-way ANOVAs were used to detect main effects for sex, diabetes and their interaction.

Femur and lumbar BMD were lower in male and female ZDSD rats compared to controls. However, when corrected for weight, lumbar BMD was not different between ZDSD and control females. Micro-CT analyses revealed that lumbar spine and femur trabecular bone architecture was compromised in ZDSD rats compared to controls (reduced BV/TV and Tb.Th, increased SMI and Tb.Sp), this deterioration being predominant in males compared to females. Lumbar ventral Ct.Th was lower in ZDSD rats compared to controls, but to a larger extent in males versus females (50 and 15% decreases, respectively). These architectural changes were reflected in vertebrae material properties where ultimate stress and toughness were lower in male ZDSD rats only. Cortical bone analyses showed that only ZDSD male rats had smaller (i.e.: Area/length) bone than their controls and significantly thinner cortices than ZDSD females. The material properties of cortical bone were reduced in ZDSD males but not females, as shown by lower UF, yield force, stiffness, post-yield displacement, toughness and post-yield toughness, indicating a more brittle phenotype in ZDSD males.

These results indicate that, as seen in humans, T2DM exerts a more detrimental effect on bone in males compared to females, suggesting that female sex hormones might play a protective role for bone in T2DM.

Disclosures: Maxime Gallant, None.


The Impact of type 2 Diabetes on Bone Microarchitecture: A Cross-sectional Evaluation in Postmenopausal Women.Janet Pritchard*1, Lora Giangregorio2, Stephanie Atkinson1, Karen Beattie1, Jonathan Adachi3, Dean Inglis4, Alexandra Papaioannou5. 1McMaster University, Canada, 2University of Waterloo, Canada, 3St. Joseph's Hospital, Canada, 4Centre for Appendicular MRI Studies, Canada, 5Hamilton Health Sciences, Canada

PURPOSE: In adults with type 2 diabetes (T2D), fracture risk is elevated despite normal or elevated areal bone mineral density (aBMD). The primary objective of this study was to compare trabecular bone microarchitecture in postmenopausal women with long-standing T2D, to that of postmenopausal women without T2D. The secondary objective was to compare aBMD between groups.

METHODS: Participants with T2D had a self-reported diagnosis of T2D (≥ 5 years), and were recruited from out-patient diabetes clinics. Participants without T2D were recruited through community advertisements. The non-dominant distal radius was imaged using a 1T peripheral magnetic resonance imaging (pMRI) system (OrthOneTM, ONI Inc.). A sagittal localizer scan was performed, followed by a coronal localizer scan to plan the final transaxial slices of the distal radius. The axial MR images (in-plane resolution =195 μm) were segmented at the endosteal surface and parameters of microarchitecture (marrow pore size, BV/TV, Tb.Th, Tb.N, Tb.Sp, free ends) were derived using in-house software. Body composition, lumbar spine aBMD, and proximal femur aBMD were measured using DXA (Hologic, Discovery QDR4500A). The characteristics of participants and all bone variables are reported as mean ± SD for continuous variables, and variables were compared using an unpaired t-test (SPSS v.17.0). A p-value of <0.05 was considered significant for this study.

RESULTS: Participants with T2D (n=29) were similar in age (71.0 ± 4.8 yrs vs. 70.7 ± 4.8 yrs, p=0.87) and number of years since menopause (21.7 ± 6.6 yrs vs. 22.1 ± 7.7 yrs, p=0.84) compared to the participants without T2D (n=26). Participants with T2D had greater body mass index (BMI) (34.6 ± 7.6 kg/m2 vs. 27.9 ± 5.6 kg/m2, p=0.00), but not percent body fat (40.3 ± 6.1% vs. 37.2 ± 6.5%, p=0.06) compared to participants without T2D. The average number of years with a diagnosis of T2D was 16.6 ± 11.1 yrs, and 62.1% (18/29) of participants with T2D were taking exogenous insulin. Table 1 displays bone densitometry and microarchitecture data for the participants.

CONCLUSIONS: Hole size has been shown to be a good predictor of fracture. We have shown for the first time that the average marrow pore size is greater in postmenopausal women with T2D. Therefore, skeletal fragility in postmenopausal women with T2D may be due in part to enlarged perforations between trabecular units in cancellous bone, which constitutes about 50% and 90% of the bone at the hip and spine, respectively.

Table Table 1. A comparison of bone-related parameters in women with T2D and controls
inline image

Disclosures: Janet Pritchard, None.


The Multifunctional Role Of Osteopontin In Diabetic Arteriosclerosis.Jian Su Shao1, Oscar Sierra1, Su-Li Cheng1, Linda Halstead2, Dwight Towler*2. 1Washington University in St. Louis School of Medicine, USA, 2Washington University in St. Louis, USA

Calcification and fibrosis reduce vascular compliance in diabetic arteriosclerosis, increasing lower extremity ischemia. To better understand the role of osteopontin (OPN), a multifunctional protein upregulated in diabetic arteries, we evaluated contributions of OPN in male LDLR-/- mice fed high fat Western diet (HFD), a model of diabetic arteriosclerosis and vascular calcification. OPN deficiency had no impact on HFD-induced hyperglycemia, dyslipidemia, or body composition. However, OPN-/-;LDLR-/- mice exhibited an altered time-course of aortic calcium accrual-reduced during disease initiation but increased with progression-vs. OPN+/+; LDLR-/- controls. Moreover, diabetic OPN-/-;LDLR-/- mice exhibited reduced aortic compliance and diminished hind-limb blood flow. Collagen accumulation, fragmented elastin, and wall thickness were significantly increased in aortas of diabetic OPN-/-;LDLR-/- mice, as were serum levels of P1NP, a biomarker of type I collagen synthesis. VSMC-specific expression of OPN (SM22-OPN transgene) did reduce aortic elastin fragmentation and Col2A1 expression in OPN-/-;LDLR-/- mice, but did not significantly impact aortic calcification, collagen protein accumulation, or arterial stiffness. However, pharmacologic dosing with the pro-inflammatory OPN fragment SVVYGLR upregulated aortic Wnt3a, Wnt7a, and Wnt7b, increased aortic β -catenin protein, induced osteochondrocytic gene expression (Runx2, COL2A1, COL10A1, OSC), and restored early-phase aortic calcification in OPN-/-;LDLR-/- mice on HFD. Thus, OPN has stage-specific roles in diabetic arteriosclerosis. Pro-inflammatory actions promote vascular osteochondrocytic differentiation and calcification with disease initiation-but other actions limit elastin fragmentation, chondroid metaplasia, vascular calcification and fibrosis with disease progression. Complete OPN deficiency yields a net increase in arteriosclerotic disease severity, reducing distal hind limb tissue perfusion in diabetic LDLR-/- mice.

Disclosures: Dwight Towler, None.


The Prevalence of Low Muscle Mass (Sarcopenia) in Individuals with HIV.Bjoern Buehring*, Elisabeth Kirchner, Zhiyuan Sun, Leonard Calabrese. Cleveland Clinic, USA

Purpose: The introduction of highly active antiretroviral therapy to treat HIV infections has turned this disease from a fatal to a chronic process. With the increasing life span changes in bone and fat tissues have became more important. Low bone mineral density (BMD) is common in individuals with HIV and associated with increased fracture risk. Lipodystrophy syndrome leads to metabolic and morphological abnormalities. Less attention has been given to changes in muscle mass in this population. Growing evidence exists that low muscle mass and poor muscle function is associated with increased disability, morbidity, and mortality in older adults. This study aimed to examine the prevalence of low muscle mass as well as low BMD and lipodystrophy in persons with HIV using current DXA definitions. Methods: We invited individuals with HIV on therapy or treatment naïve to undergo DXA whole body composition. We used WHO T-score criteria to assess BMD, a % central fat/% lower extremity ratio to define lipodystrophy and previously proposed sarcopenia criteria (appendicular lean mass/(height)2 and lowest 20% of residuals from regression analysis) to identify persons with body composition abnormalities. Potential risk factors (HIV medication, Age, BMI, length of illness, CD4 count, viral load, testosterone levels, and tobacco use) were assessed through chart review.

Results: 66 males (56 with treatment, 10 treatment naive) volunteered. Treated individuals were older than naive (44 vs 34 years) and had HIV longer (108 vs 14 months). In all 66 males prevalence of sarcopenia was 21.2% and 18.2% depending on the definition used. Low BMD was present in 68.2% (60.6% osteopenia, 7.6% osteoporosis). 53% had lipodystrophy. Persons on treatment had higher prevalence of lipodystrophy than treatment naïve (61.1% vs 20%, p=0.034). Lower BMI was related to presence of sarcopenia. Sarcopenia and lipodystrophy were negatively associated (OR 0.11-0.18, p≤0.012). Risk factors for abnormal BMD were treatment with thymidine analogues and non-nucleoside reverse transcriptase inhibitors. Development of lipodystrophy was associated with multiple HIV drugs. Conclusions: To our knowledge this is the first study to estimate the prevalence of low muscle mass based on DXA definitions in persons with HIV. Sarcopenia is common considering the age of our study population. More research is necessary to reproduce these results and to obtain measures of muscle function as well as outcomes data.

Disclosures: Bjoern Buehring, None.


See Friday Plenary number FR0470.


Low Incidence of Clinical Fractures after Liver Transplantation: An Audit of 531 Consecutive Patients.Melissa Premaor1, Tapas K Das2, Irene Debiram3, Graeme Alexander3, Juliet Compston*4. 1Addenbrooke's Hospital, United Kingdom, 2Liver Unit, Addenbrookes Hospital, Cambridge & Peterborough & Stamford Hospitals NHS Foundation Trust, United Kingdom, 3Department of Medicine-University of Cambridge, United Kingdom, 4University of Cambridge School of Clinical Medicine, United Kingdom

In earlier studies high rates of fracture were reported in patients following liver transplantation. Subsequently, lower doses of glucocorticoids for immunosuppression and the use of bone protective therapy for patients at high risk of fracture have become standard practice. The aim of this study was to document clinical fracture incidence after liver transplantation during the period 1998-2008 in a single transplant centre, following the introduction of a protocol for bone protective therapy in patients at risk. An audit was performed in all patients with a first liver transplant between Jan 1998 and Jan 2008. Records were retrieved from 531 of 592 eligible patients (89.7%), 327 male and 204 female, mean age 51.7 yrs. The median(IQR) duration of prednisolone therapy was 3(3,5) months. Spine X-rays were obtained before transplant in 432 patients and after transplant when clinically indicated. All fractures were recorded and verified radiologically. Bone mineral density was assessed pre-transplant by DXA in 67.4% of patients. The mean follow-up period was 61.4 months (range 0-131). 5.6% of the patients had a history of fracture prior to transplant (76% spine, 8% wrist, 8% lower leg, 8% other). After transplantation, incident clinical fracture was recorded in 15 patients (3.5%) of which 46.7% were vertebral. The median time since transplant to fracture was 26 months (range 2-83). Patients with an incident clinical fracture were significantly older than those without (p=0.002) and significantly more likely to be female (p=0.007). Pre-transplant spine and hip BMD T-scores were lower in patients with fracture [-1.74(2.0) vs -1.16(1.6) and -1.08(1.2) vs -0.65(1.2); p=ns]. A previous fracture was not significantly more common in patients with than without previous fracture [13.3% vs 5.1%]. The median(IQR) duration of prednisolone post-transplant was 4(3,36) months in patients with incident fracture and 3(3,5) months in those without (p=0.016). 60% of patients with incident fracture and 28.3% of those without had received bone protective therapy post-transplantation. Our results demonstrate low rates of fracture in patients undergoing liver transplantation in our centre over the last ten years. Lower doses of prednisolone and administration of bone protective therapy in high risk patients are both likely to have contributed. In addition, the low rate of fractures prior to transplant indicates better bone health prior to transplant.

Disclosures: Juliet Compston, None.


See Friday Plenary number FR0472.


Weekly Alendronate versus Zoledronic Acid For Prevention of Bone Loss During the First Year After Heart or Liver Transplantation.Adi Cohen*1, Emily Stein2, Halley Rogers1, Donald J. McMahon3, Shannon Kokolus3, Polly Chen3, Chiyuan Zhang3, Ronald B. Staron3, Susan Restaino3, Donna M. Mancini3, Robert S. Brown3, Elizabeth Shane2. 1Columbia University Medical Center, USA, 2Columbia University College of Physicians & Surgeons, USA, 3Columbia University, USA

The first year after heart (CTX) or liver transplantation (LTX) is characterized by high rates of bone loss and fractures. Bone mineral density (BMD) declines by 3-10% and vertebral fracture rates vary from 5-30%. Daily alendronate (ALN) reduces rates of bone loss in CTX recipients and quarterly infusions of zoledronic acid (ZA) reduce rates of bone loss in LTX recipients.

We conducted a 1-year, double-masked, active comparator, non-inferiority, randomized clinical trial (RCT) in which we compared rates of bone loss in CTX and LTX recipients assigned to receive ALN (70 mg/week) for the first year or a single infusion of ZA (5 mg). We hypothesized that ZA, administered within 1 month of transplantation (TX) would provide similar protection from bone loss as weekly ALN initiated at the same time. Subjects (n=84) were randomized to ALN or ZA, 25+8 (SD) days after TX. A nonrandomized reference (REF) group of TX recipients (n=25) with BMD T scores >-1.5 was enrolled to provide data on rates of bone loss in TX recipients not treated with bisphosphonates (BP). As vitamin D deficiency was very common (> 70% of participants), all received ergocalciferol 50,000 IU daily for 5 days before the ZA/placebo infusion. All received immunosuppressive therapy (prednisone, calcineurin inhibitors) and daily calcium (945 mg) and vitamin D (1000 IU) during the study. The primary efficacy variable is percent change from baseline in total hip (TH) BMD at 1 year in the ALN and ZA groups. Secondary efficacy variables include percent change from baseline in lumbar spine (LS) and femoral neck (FN) BMD at 1 year, and serum bone resorption markers. Secondary analyses will compare rates of bone loss in the ALN and ZA groups to the REF group.

At baseline, REF and randomized subjects were similar with respect to age (48+15 vs 54+10 respectively, P=0.07); gender (% Male: 77 vs 80; P=NS), race (% Caucasian: 60 vs 68; P=NS); transplant type (% CTX: 63 vs 64; P=NS). Baseline BMD (g/cm2) was significantly higher (all p<0.001) in the REF group at the TH (1.078+0.13 vs 0.923+0.14), LS (1.123+0.15 vs 0.964+0.14), and FN (0.924+0.11 vs 0.793+0.12). The study remains blinded at the writing of this abstract. The last patient will complete the study in July 2010. One-year results comparing rates of bone loss between the ALN and ZA groups, and between the ALN and ZA groups and the REF group will be available and presented at the time of the meeting.

Disclosures: Adi Cohen, None.

This study received funding from: Novartis


N-linked Glycosylation Sites Required for Normal Calcium-sensing Receptor Expression and/or Function.Sarah Brennan*1, Karolina Windloch1, Arthur Christopoulos2, Arthur Conigrave1. 1University of Sydney, Australia, 2Monash University, Australia

The extracellular calcium-sensing receptor (CaR) is a class C G-protein coupled receptor (GPCR) that provides feedback control of calcium homeostasis by inhibiting parathyroid hormone secretion and renal calcium reabsorption. CaRs are activated not only by Ca2+o, but also by L-amino acids and type II calcimimetics, including cinacalcet (Sensipar).

The CaR has eight recognised N-linked glycosylation sites in the extracellular Venus Fly Trap (VFT) domain and loss of 3 or more of these sites has been reported to markedly impair receptor expression [1]. Further investigation of the roles of specific glycosylation sites has led to the identification of two mutants that exhibit notable aberration in calcium-sensing-an inactivating mutation, N468Q and an activating mutation, N130Q.

When transiently transfected in HEK-293 cells, N468Q exhibited a marked reduction in the complex glycosylated 160kDa form of the receptor but apparently normal levels of the immature high mannose 140kDa form, as determined by Western blotting, indicative of retention in the endoplasmic reticulum. In addition, cell surface expression of the mutant was significantly decreased (by ∼70%) compared to wild type in an ELISA-based assay. The proteasome inhibitor, MG132, partially increased surface expression (to ∼50%), implying that proteasomal-dependent degradation may be involved.

The N468Q mutant also showed a marked reduction in potency and maximal agonist effect in response to Ca2+o-mediated Ca2+i mobilization, with a complete loss of oscillatory behaviour. Neither L-Phe (10 mM) nor the type II calcimimetic NPS R-467 (1 μM) were able to rescue this loss of function to Ca2+o.

In contrast, the N130Q mutation increased the Ca2+o potency, with an EC50 for Ca2+o of 2.90 ± 0.2 mM (n = 6). In comparison, the apparent EC50 for Ca2+o in the wild type receptor was 4.0 ± 0.4 mM (n=9) despite similar levels of surface expression. N130 lies in a region, close to the dimer interface between VFT domains, that has been shown to be particularly susceptible to activating mutations (residues 116-131)

In conclusion, individual disruption of the N-linked glycosylation sites at 130 and 468 disturbs the normal Ca2+o-sensing properties of the CaR in HEK-293 cells.

1. Ray K., et al., J Biol Chem, 1998. 273: 34558-67

Disclosures: Sarah Brennan, None.


Glucocorticoids Suppress Bone Formation by Attenuating Osteoblast Differentiation via the Monomeric Glucocorticoid Receptor.Alexander Rauch*1, Ulrike Baschant1, Sebastian Seitz2, Thorsten Schinke3, Michael Amling4, Ulf Lerner5, Jean-Pierre David6, Guenther Schuetz7, Jan P. Tuckermann1. 1FLI Jena, Germany, 2Center for Biomechanics & Skeletal Biology, Germany, 3University Hospital Hamburg, Eppendorf, Germany, 4University Medical Center Hamburg-Eppendorf, Germany, 5University of Umea, Sweden, 6University Erlangen-Nuremberg, Germany, 7DKFZ Heidelberg, Germany

It has been hypothesized that many side effects of glucocorticoid (GC) therapy, such as osteoporosis, depend on dimerization of the glucocorticoid receptor (GR). Using a novel Cre-transgenic mouse line, we now demonstrate that repression of bone formation by GCs is abrogated in the absence of GR expression in osteoblasts since they become refractory to hormone-induced apoptosis and inhibition of proliferation and differentiation. In contrast, mice carrying a dimerization-defective GR mutation (GRdim) display reduced bone formation upon GC treatment, resulting in bone loss in vivo and enhanced apoptosis and suppressed differentiation in vitro. GCs potently suppress osteoclast activity in vivo independent of GR dimerization or GR ablation in the osteoblasts. Therefore GCs act in a cell autonomous manner in the osteoblasts to decrease bone mass and bone formation. The inhibitory effects by GCs on osteoblasts were found to be due to a novel mechanism involving inhibition of cytokines such as interleukin-11 via the monomer GR recruitment to AP-1 responsive elements in the promoter. Moreover supplementation of GC-treated osteoblasts with interleukin-11 in vitro abrogates the suppression of differentiation. Taken together, attenuation of osteoblast differentiation by GR dimerization-independent suppression of osteoblast-derived cytokines contributes, at least in part, for bone loss during GC therapy.

Disclosures: Alexander Rauch, None.


25-Hydroxyvitamin D Reduces Secondary Hyperparathyroidism in Mice with Chronic Kidney Disease Independent of Circulating 1,25-Dihydroxyvitamin D In Vivo.Loan Nguyen-Yamamoto*1, Isabel Bolivar2, Miren Gratton2, Raymonde Gagnon2, Richard Kremer1, Geoffrey Hendy1, David Goltzman3. 1McGill University, Royal Victoria Hospital, Canada, 2McGill University, Canada, 3McGill University Health Centre, Canada

Increases in serum 25-hydroxyvitamin D (25OHD), are associated with reductions in serum parathyroid hormone (PTH) due to the renal and/or local conversion of 25OHD to 1,25 dihydroxyvitamin D [1,25(OH)2D]. However, in vivo evidence of extra-renal conversion of 25OHD to modulate PTH levels is lacking. We examined the influence of vitamin D metabolites on parathyroid and mineral homeostasis in an adenine-treated mouse model of chronic kidney disease (CKD) where renal production of 1,25(OH)2D was severely diminished. We compared this to results in mice expressing the null mutation for the 25OHD-1αhydroxylase enzyme (1αOHase-/-) which exhibit tissue-wide inability to synthesize 1,25(OH)2D. Compared to normal controls, CKD mice had 2.3-fold increases in serum creatinine, 4-fold increases in blood urea nitrogen (BUN), normal serum calcium (Ca)(2.56 ± 0.09mM), elevated serum phosphorus (P)(3.78 ± 0.25mM), and markedly reduced serum 1,25(OH)2D (80.40 ± 12.51pM compared to normal levels of 248 ± 35.21 pM). In 1α OHase-/- mice on a normal diet, serum creatinine and BUN were normal, serum Ca (1.58 ± 0.12mM) and P (2.13 ± 0.07mM) were low and serum 1,25(OH)2D was undetectable (< 25pmol/L). Serum PTH levels were extremely elevated in 1αOHase-/- mice (22-fold), and in CKD mice (11-fold). Administration of 1,25(OH)2D3 (50 pg/g/thrice weekly) for 6 weeks, normalized serum Ca, P and PTH in 1αOHase-/- mice, and increased serum Ca (2.93 ± 0.07) and normalized serum PTH in CKD mice. A very high dose of 25OHD3 (75 ng/g/thrice weekly) also increased serum calcium (2.78 ± 0.09 mM) increased serum P (3.89 ± 0.36 mM) and normalized PTH in 1αOHase-/- mice. This dose of 25OHD3 in CKD mice normalized serum P and PTH with no increase in circulating 1,25(OH)2D. Administration of a lower dose(10ng/g/thrice weekly) of 25OHD3, did not affect the abnormal serum Ca, P and PTH in 1αOHase-/-mice, however in CKD mice, serum P was normalized and serum PTH was reduced by 50% with no increase in circulating 1,25(OH)2D. The in vivo PTH-lowering efficacy of the very high administered dose (75ng/g) of 25OHD3 in mice with tissue-wide absence of the 1αOHase enzyme reflects the capacity of 25OHD3 to cross react with low affinity at the VDR. The in vivo PTH-lowering efficacy of the lower dose (10ng/g) of 25OHD3 in CKD mice with severely impaired renal 1αOHase activity but with intact extra-renal 1αOHase activity indicates that local extra-renal conversion of 25OHD3 is significant in vivo.

Disclosures: Loan Nguyen-Yamamoto, None.


Estrogen Ameliorates Synovial Inflammation and Joint Erosivity in mBSA Induced Monoarthritis.Cecilia Engdahl*, Anna Börjesson, Alexandra Karlström, Catharina Lindholm, Hans Carlsten, Marie Lagerquist. Center for Bone & Arthritis Research, Institution of Medicine, Sahlgrenska Academy at the University of Gothenburg, Sweden

Objective. Estrogen ameliorates the development and progression of collagen II induced polyarthritis, a systemic animal model for postmenopausal rheumatoid arthritis (RA). The aim of this study was to determine whether estrogen ameliorates mBSA induced monoarthritis in the same manner and to elucidate the mechanism behind these protective effects of estrogen.

Methods. Female C57/Bl6 mice were ovariectomized at 3 months of age and mBSA monoarthritis was induced. One knee was injected with mBSA and the control knee with saline. Mice were treated with estradiol (0,9μg/day) or placebo using subcutaneous pellets. At termination, knees were collected for histology, synovial cells were investigated using flow cytometry and serum was analyzed for mBSA specific antibodies.

Results. Treatment with estradiol decreased synovitis (-22%, p<0.05) and dramatically reduced the grade of bone erosion (-60%, p<0.05) in the mBSA-injected knee as compared to placebo treatment. In both estradiol and placebo treated mice, mBSA-injection resulted in an increase in the frequency of lymphocytes (+61% and 83%, respectively, p<0.05) as well as macrophages (+29% and +107%, respectively, p< 0.05) in the synovium compared to saline-injection. Interestingly, a tendency to decrease in the frequency of macrophages was found in the synovium of mBSA-treated knees after estradiol treatment as compared to placebo treatment (-40%, p=0.058). The serum levels of mBSA specific antibodies were not affected by estradiol treatment.

Conclusion. In a model of monoarthritis, estrogen ameliorates the disease by significantly decreasing both the synovitis and the bone erosions. Induction of arthritis by injection of mBSA increases the frequency of inflammatory cells (lymphocytes and macrophages) in the synovium. Interestingly, estrogen reduces the induction of macrophages resulting in decreased frequency in macrophages after estradiol-treatment as compared to placebo treatment. This reduction might be part of the mechanism behind the protective effects of estrogen on synovitis and bone erosions. Increased knowledge about the mechanisms behind the beneficial effects of estrogen is useful in the search for novel treatment against inflammation triggered bone loss.

Disclosures: Cecilia Engdahl, None.


See Friday Plenary number FR0478.


See Friday Plenary number FR0479.


A Transgene Containing the Human Vitamin D Receptor Gene Locus Recapitulates Endogenous Tissue-specific Expression of the Receptor in the Mouse.Seong Min Lee*1, Kathleen Bishop1, Mark Meyer1, Robert Nerenz1, Charles O'Brien2, J. Pike1. 1University of Wisconsin-Madison, USA, 2University of Arkansas for Medical Sciences, USA

The vitamin D receptor (VDR) is a primary determinant of target tissue response to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). In recent studies, we have characterized key regions that regulate both basal and inducible expression of the mouse VDR gene by hormones such as 1,25(OH)2D3, PTH and retinoic acid. These studies led to the development of a mouse transgene capable of recapitulating endogenous VDR gene expression in vivo. While the human VDR gene is highly conserved, several important differences are evident. They include the presence of an additional promoter, synthesis of two gene products, and the presence of novel phosphorylation sites. A number of single nucleotide polymorphisms (SNPs) have also been identified within the locus that are associated with altered bone mineral density and/or increased cancer risk. To explore the human VDR gene, we searched for control regions across the gene locus using ChIP-chip and ChIP-seq analyses and employed antibodies capable of detecting epigenetic histone marks that correlated directly with active regulatory regions. These studies highlighted the two VDR promoters, one near the annotated TSS and the second over 30 kb upstream. They also revealed potential enhancers that were shared in the mouse gene. Additional studies demonstrated that specific regulatory regions bound VDR in bone cells, the estrogen receptor in MCF-7 cells, and c-FOS in activated T cells. To explore determinant of tissue-specific VDR gene expression, we created a BAC clone containing the extended human VDR locus, an HA tag inserted at the VDR translational start site and a luciferase reporter in the last exon, and prepared mouse strains containing germ-line copies of this transgene for subsequent tissue analysis. We contrasted luciferase expression as well as both endogenous and BAC clone-derived VDR expression using immunohistochemical staining procedures. Expression from the VDR BAC clone recapitulated endogenous sites of VDR expression in all tissues examined, including segments of the intestinal tract and sections from the kidney, parathyroid glands, pancreatic islets, hair follicles and the epidermis. Liver and muscle expression of endogenous and recombinant VDR was not detected. These studies support the identification of key regulatory regions within the human VDR gene essential for factor-regulate and tissue-specific expression of the VDR gene both in vitro and in vivo and should permit humanization of the VDR-null mouse.

Disclosures: Seong Min Lee, None.


See Friday Plenary number FR0481.


See Friday Plenary number FR0482.


Phospholipase A2 Activating Protein (PLAA) is Required for 1a,25(OH)2D3-Induced Rapid Membrane Response in Osteoblasts.Maryam Doroudi*, Zvi Schwartz, Barbara Boyan. Georgia Institute of Technology, USA

We have previously reported that phospholipase A2 activating protein (PLAA) is required for 1α,25-dihydroxyvitamin D3 (1,25D3) signaling in growth zone chondrocytes in a rat costochondral growth plate model. After 1,25D3 binds its membrane-associated receptor, Pdia3 (ERp60, ERp57, 1,25-MARRS), PLAA is activated. Cytosolic PLA2 is stimulated, resulting in production of arachidonic acid, which can activate PKC directly. We also shown that caveolae and caveolin-1 are required for rapid 1,25D3-dependent PKC signaling and Pdia3 is co-localized with Cav-1 in the plasma membrane and in lipid rafts. PLAA exhibits homology with the G-protein beta subunit, suggesting that it is also membrane-associated. However, it is not clear whether PLAA is present in caveolae and if it interacts with Cav-1 and Pdia3. MC3T3-E1, a pre-osteoblastic cell line, and MC3T3-E1 cells silenced for PLAA (shPLAA) were treated for 9 or 90 minutes with either vehicle (0.001% ethanol) or 10-8M 1,25D3. Whole cell lysates were collected for immunoprecipitation, caveolae isolation, and analyzed by Western blot. Subconfluent cultures of MC3T3-E1 cells were immunostained against PLAA, Cav-1 and Pdia3, and imaged with confocal microscopy. The effect of 1,25D3 on PLA2 activity was examined by measuring total PLA2 activity in cell lysates using a PLA2 assay kit. Although PLAA, Pdia3, and Cav-1 were detected in the plasma membrane by Western blot, just Pdia3 and Cav-1 were detected in caveolae. Whole cell lysates treated with either vehicle of 1,25D3 for 9 and 90 minutes were immunoprecipitated using PLAA antibody. Cav-1 was detected in all samples analyzed, but Pdia3 was present only after treatment with 1,25D3. Cav-1 was found when immunoprecipitated with anti-Pdia3 antibodies. These observations were confirmed by immunofluorescence staining for Pdia3, Cav-1 and PLAA. shPLAA cells failed to activate PLA2 with 1,25D3 treatment at 9 minutes. Taken together, our results show that PLAA is present in plasma membranes, but not in caveolae, in the absence of 1,25D3. PLAA and Pdia3 interact with Cav-1 in both the control and 1,25D3-treated groups. PLAA interacted with Pdia3 only in the presence of 1,25D3. shPLAA cells did not activate PLA2 with 1,25D3 treatment at 9 minutes. Thus, 1,25D3 initiates conformational changes in membrane proteins, recruiting PLAA to caveolae and initiating the Pdia3 signaling pathway.

Disclosures: Maryam Doroudi, None.