ASBMR 29th Annual Meeting

M372

Factors Influencing Women Persistence with Osteoporosis Treatment After 6 Months of Treatment.

V. Breuil¹, P. Fardellonne*², M. Rossignol*³, B. Cortet*⁴, F. Liard*⁵, F. A. Allaert*⁶, F. E. Cotté*⁷, A. F. Gaudin*⁷. ¹CHRU l'Archet, Nice, France, ²CHU, Amiens, France, ³RRSSS, Montreal, PQ, Canada, ⁴CHRU Roger Salengro, Lille, France, ⁵General Practitionar, Saint-Epain, France, ⁶DIM CHRU, Dijon, France, ⁷GSK, Marly le Roy, France.

A retrospective study carried out by 2029 GPs whose representativeness has been controlled.

The purpose was to describe the persistence of women treated for postmenopausal osteoporosis (PMO) for at least 6 months and factors influencing it. Each of them was requested to describe the first 2 successive postmenopausal osteoporotic women who have been persisting with osteoporosis treatment for at least 6 months during the last year, and to ask them to complete a self administered questionnaire. 3598 women 69.5±8.8 years old were included. Regarding risk factors of osteoporosis, 37.5% had maternal history of osteoporotic fracture, 21.5% an early menopause (<45 y old), 19.6% were smoking more than 20 cig/day, 13.8% had a long duration of steroid use (≥ 6 months), 11.7% had a BMI < 19 kg/m² and 10.0% were immobilized more than 3 months. 71.3% of them had at least one osteoporotic fracture and the average number of fractures was 1.1 ± 1.0. They were located in vertebra (50.0%), wrist (48.9%) and hip (10.1%). 83.5% of women received the same treatment since the diagnostic of osteoporosis and were defined as persistent. Persistence was higher with weekly bisphosphonates (WBP) (89.8%) and SERM (84.6%) compare to daily bisphosphonates (DBP) (41.4%). The main reasons for non-persistence were “Treatment intolerance” for women treated with WBP and SERM (respectively 36.0% and 34.6%) and “Treatment strictness” with DBP (55.5%). Univariate analysis showed that “Consideration of PMO as a serious disease” and “Knowledge of PMO risk factors” were significantly higher in persistent patient comparing to non-persistent one (<0.01). Persistent patients had significantly more medical consultations (p<0.001). Women under 70 years were more persistent than the older (<0.05). With both daily or weekly regimens, logistic regression shows that persistence with bisphosphonates increases with the number of previous fracture (3 vs 0): OR=1.9. IC_95% =]1.1;3.3[ and OR= 1.7, IC_95% = ]1.0;2.8[, respectively.

Strictness of dosing regimen and intolerance on therapy appeared to be the main reasons for discontinuing PMO treatments. Otherwise, improving information of women and acknowledgement of PMO severity and consequences could be the major way for enhancing their persistence on therapy.

Disclosures: V. Breuil, None.

This study received funding from: Laboratory GlaxoSmithKline.

M373

See Sunday Plenary Number S373

M374

Elderly Patients with Non-Hodgkin's Lymphoma Who Receive Chemotherapy at Higher Risk for Osteopenia and Osteoporosis.

M. E. Cabanillas¹, H. Lu*¹, S. Fang*², X. L. Du*². ¹General Internal Medicine, MD Anderson Cancer Center, Houston, TX, USA, ²Division of Epidemiology, The School of Public Health, The University of Texas Health Science Center at Houston, Houston, TX, USA.

In order to determine the risk of osteopenia and osteoporosis associated with chemotherapy among elderly patients with Non-Hodgkin's Lymphoma (NHL), we studied a cohort of 13,570 patients aged ≥65 years with incident NHL in 1992–1999, identified from the SEER-Medicare linked data. We searched for any diagnosis of osteoporosis or osteopenia 1 year prior to and thereafter the diagnosis of NHL, during the 11 years of follow-up. Of 13,570 patients, 60% received chemotherapy. One year prior to the diagnosis of NHL, there were 335 (4.1%) patients who had claims for osteoporosis in the chemotherapy group versus 272 (5%) subjects in the no chemotherapy group (p=0.012). After diagnosis, patients who received chemotherapy had a statistically significant increase in rates of osteoporosis (10.1%) and osteopenia (8.1%) in the chemotherapy group compared to the no chemotherapy group (8.3% and 4.0%, respectively) (p=<0.001). After controlling for ethnicity, age, gender, follow-up time, tumor stage, and geographic location, patients who received chemotherapy had a significantly increased odds ratio of 1.27 (95% CI: 1.12–1.45) for osteoporosis and 1.95 (95% CI: 1.66–2.30) for osteopenia compared to those who did not. In conclusion, this population-based retrospective cohort study demonstrated that chemotherapy was associated with an increased risk of developing osteopenia and osteoporosis in community dwelling elderly patients with NHL. 

Table  .  

Disclosures: M.E. Cabanillas, None.

M375

See Sunday Plenary Number S375

M376

Prevalence and Risk Factors of Osteoporosis and Vertebral Fractures in COPD Males.

E. Casado¹, M. Gallego*², M. Larrosa¹, C. Orellana*¹, R. Valls*³, E. Berlanga*⁴, C. Domingo*², J. Gratacòs*¹. ¹Rheumatology, Hospital Sabadell, Sabadell, Spain, ²Pneumology, Hospital Sabadell, Sabadell, Spain, ³Radiology, UDIAT, Sabadell, Spain, ⁴Laboratory, UDIAT, Sabadell, Spain.

The objective of our study was to determine the prevalence of densitometric osteoporosis (OP) and osteoporotic vertebral fractures in COPD patients. To identify the risk factors for the presence of OP and vertebral fractures in COPD patients.

Inclusion criteria: Male sex, older than 50, COPD defined according to ATS/ERS classification. Patients gave their informed consent to participate in the study. Exclusion criteria: Patients with COPD presenting any other concomitant pulmonary disease. Patients with COPD presenting rheumatologic and/or vertebral disease that may hinder the interpretation of the densitometry. Bone mass measurement was determined by dual-energy x-ray absorptiometry (DXA) at lumbar and femoral sites. OP was defined using the WHO score. X-ray of lumbar and dorsal spine was performed in order to evaluate the presence of vertebral fractures. Analytical parameters that influence the phospho-calcium metabolism were determined: calcemia, phosphoremia, 25-OH-vitamin D, PTH, alkaline phosphatase and testosterone. For each patient, age, weight, height, body mass index (BMI), comorbidities, toxins (cigarettes, alcohol), medications, degree of exposure to sun and daily calcium intake were recorded. Corticosteroids treatments in the previous five years and number of hospitalizations were also recorded.

211 patients were evaluated. Mean age was 66.78 years (50–84). 73 patients had a FEV1 between 50–80% and 138 had a FEV1 <50%. Prevalence of OP and fractures were respectively 41.6% and 33%. Dose of prednisone greater than 675 mg, BMI below 21, low blood levels of 25-OH-vitamin D, and FEV1<30% were associated significantly with OP (p <0.05). When logistic regression was performed only BMI (OR=0.9; 95% CI, 0.84–0.96) and FEV1<30% (OR=3.32; 95% CI, 1.36–8.06) remained in the model. Age, use of corticosteroid and FEV1 <30% were associated significantly with vertebral fractures. When logistic regression analysiswas performed, age (OR=1.06; 95% CI, 1.01–1.1) and FEV1<30% (OR=4.39; 95% CI, 1.66–11.59) remained in the model. In conclusion, the prevalence of OP and vertebral fractures are higher than expected in general population.

COPD severity is an important riskfactor for OP and vertebral fracture.

Disclosures: E. Casado, None.

M337

See Sunday Plenary Number S377

M378

Prevalence and Patterns of Presumed Osteoporosis (OP) Among Older Americans Based on Medicare Data.

H. Cheng, L. C. Gary*, J. R. Curtis, M. L. Kilgore*, K. G. Saag*, H. Yun*, R. Matthews*, S. Swaminathan*, M. A. Morrisey*, E. Delzell*. Univ. of AL at Birmingham, B'ham, AL, USA.

Background OP prevalence is rising nationally, with recent prevalence estimates for older white women, based on femoral neck bone mineral density testing, of 13.6% for ages 60–69, 26.0% for ages 70–79 and 47.9% for ages 80+. Medicare data afford opportunities and challenges for studying the epidemiology of OP at a population level.

Methods We used Medicare claims data to estimate the prevalence of presumed OP in 2004 and to determine the contributions to estimated OP prevalence of claims for OP and related fractures. The Medicare Chronic Condition Data Warehouse provided data for an enhanced 5% national sample of beneficiaries. We studied 1,600,877 beneficiaries who were ≥ 65 years of age in 2004, had 12 months of Medicare Parts A and B coverage and were not enrolled in an HMO. We assessed claims for 1999–2004 to identify cases with three definitions (D) of presumed OP: D1, ≥ 1 physician service claim with a diagnosis code for OP or for fracture sites strongly associated with OP (i.e., hip, spine or wrist) in any year; D2, ≥ 1 claim for OP or for fractures strongly or usually associated with OP (e.g. pelvis, humerus, radius/ulna, femur, tibia/fibula or clavicle); D3, ≥ 1 claim for OP or for fractures strongly, usually or sometimes associated with OP (e.g. ribs, scapula, patella or ankle). Results OP prevalence was 19.1% (D1) to 22.9% (D3) overall and varied by gender, race, age and number of years of data (table). For white women, D1-OP prevalence estimates (by age) were 15.7% (65–69), 26.9% (70–79) and 33.4% (80+). Of all Dl cases, 68% had ≥2 physician service claims, 71.4% had claims with diagnosis codes for OP only, 14.4% had claims for both OP and fracture, and 14.2% had claims for fracture but not OP. Among people with fracture claims, only 50% had a diagnosis code for OP.

Conclusions Estimates of OP prevalence based on Medicare claims were similar to recent prevalence estimates obtained using bone mineral density testing for white women ages 65–79 years and were 20% (D3)-30% (D1) lower for white women ages 80+. Variation in claims-based prevalence by age, gender and race was similar to that found using other methodological approaches. OP-related fractures accounted for a substantial proportion of presumed OP cases. The low proportion of fracture patients with an OP diagnosis code may reflect low rates of OP testing among post-fracture patients.

Prevalence of presumed osteoporosis in 2004 (definition 1)

Table  .  

Disclosures: H. Cheng, Amgen 2.

This study received funding from: Amgen.

M379

See Sunday Plenary Number S379

M380

No Ethnic Differences in Vertebral Fractures Were Found Between Caucasians, Mestizos and Other Ethnic Background in the Latin-American Vertebral Osteoporosis Study (LAVOS).

P. Clark¹, J. Talavera*¹, S. R. Cummings², D. Margarita³, F. Cons-Molina⁴, J. Zanchetta⁵, D. Messina⁶, L. Haddock⁷, S. Ragi⁸, J. Jaller⁹, a. The LAVOS Group¹. ¹Clinical Epidemiology Unit, IMSS, Mexico City, Mexico, ²Coordinating Center, UCSF, San Francisco, CA, USA, ³Clinica de Osteoporosis, Puebla, Mexico, Mexico, ⁴Unidad de Diagnostico de Osteoporosis, Mexicali, Mexico, Mexico, ⁵Instituto de Investigaciones Metabólicas, Buenos Aires, Argentina, ⁶Clinica de Osteoporosis, Buenos Aires, Argentina, ⁷Universidad de Puerto Rico, San José, Puerto Rico, ⁸CEDOES, Brazil, Brazil, ⁹Clinical Epidemiology Unit, Colombia, Barranquilla, Colombia.

Different rates of OP and fractures have been reported between groups with different ethnicity. Our aim was to determine if different ethnic backgrounds were associated with the risk of vertebral fractures in Latin American women.

A population based sampling frames were obtained from 5 countries within the Latin-American Region in women 50 years and older: Argentina, Brazil, Colombia, Mexico and Puerto Rico. A questionnaire to get demographic information, conventional risk factors, ethnic background and some life style characteristics was applied. BMD in two regions and lateral dorsal/lumbar x-rays were obtained in all cases accordingly with international protocols. Digital morphometry was used to determine vertebral deformities by modified Eastell criteria; all readings were concentrated in one center. A total of 1922 women from the five countries were included from which, 969 (50.4%) self-reported to be Mestizo, 748 (38.9%) Caucasians, and (10.7%) others (Back, Amerindians, Asians or native Indians). Prevalence of Vertebral fractures was 15.3% (148) in Mestizos, 15 % (112) in Caucasians and 10.7% (22) in other ethnic backgrounds. Ethnicity did not impact the fracture risk. The observed odds ratio between Mestizo and Caucasian was 0.98 (95% CI 0.74–1.29) and 0.67 (95% CI 0.40–1.10) between Mestizo and others. Conclusions: Ethnicity does not appear to have any impact in the risk of vertebral fractures in community dwelling Latin-American women.

Disclosures: P. Clark, None.

This study received funding from: IOF.

M381

See Sunday Plenary Number S381

M382

Stage-of-Change Model Applied to Osteoporosis Medication Use at the Time of Fragility Fracture.

B. G. Escott*, V. Elliot-Gibson*, E. R. Bogoch, D. E. Beaton. Mobility Program Clinical Research Unit, Department of Surgery, Keenan Research Center, Li Ka Shing Knowledge Institute, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.

The purpose of this study was to explore the readiness of patients who have sustained a fragility fracture to take osteoporosis (OP) medication and to identify differences in patient-related factors associated with the various stages of change.

This was a cross-sectional study of baseline data from a prospective cohort of women >=40 years and men >=50 years presenting to a fracture clinic with a low-trauma fragility fracture. Data collected at baseline via a self-reported survey included demographics, OP risk factors, prior OP investigation, diagnosis, treatment and adherence, OP awareness and health beliefs, and a single-item stage of change question adopted from Mauck (2002). We performed multivariable logistic regression to determine associations between baseline patient-related factors and different stages of change.

629 patients completed survey information and met our criteria. Preliminary analysis of willingness to take OP medication revealed a bimodal distribution at the extremes of Prochaska's stages of change with the majority of patients falling in the pre-contemplation or maintenance stages, the two groups analyzed in this study. 539 of 629 (85.7%) patients were either in the pre-contemplation (n=372 or 69%) or maintenance (n=165 or 31%) stages. Mean age (+/- SD) was 73 years (12) and 81% of subjects were female.

Multivariable analysis (adjusted OR; 95% CI) revealed that age >60 (2.5; 1.1–5.8), female gender (3.5; 1.5–7.5), self-reported history of previous fracture (3.3; 2.0–5.3) and self-described poor bone quality (19.9; 8.4–47.3) were significantly (p<0.05) associated with being in the maintenance stage compared to pre-contemplation stage. Previous BMD and OP diagnosis were very strongly associated with maintenance stage of change so were not included in the model. Fracture site and highest level of education were not significantly associated.

Higher stage of change was strongly associated with OP awareness. Patients already aware and caring for their OP were more likely to be >60, female, have sustained a previous fracture and have greater self-awareness of bone quality. Mauck (2002) found few subjects in the intermediate or lata stages of change in women suffering a hip fracture. In our study, the intermediate stages of change were not populated suggesting a need to target two main groups for intervention: maintainors for reinforcement and pre-contemplators to raise awareness. This health behavior could be explained using a simpler dichotomous model of motivation for health behavior change. Ref: Mauck et al. (2002) Osteoporos Int 13: 560–564.

Disclosures: B.G. Escott, None.

M383

See Sunday Plenary Number S383

M384

Southern California Hispanic Women Osteoporosis Education and Screening Project.

A. E. Focil*. President, California Hispanic Osteoporosis Foundation (CHOF), Oxnard, CA, USA.

This study identified Hispanic women at risk for osteoporosis to determine 1) if providing a fracture risk assessment in addition to osteoporosis education would motivate them to seek medical help and 2) if acculturation of Hispanic women had an impact on health seeking behavior. 318 postmenopausal Hispanic women of low economic/education status were recruited at osteoporosis screening events in Ventura County, CA. Osteoporosis education and written materials were provided in Spanish. After obtaining written informed consent, all participants received 1) a risk factor questionnaire; 2) an acculturation questionnaire; and 3) a heel peripheral bone density report. The latter half of women screened also received an absolute fracture risk report (AFR). A survey 6 months later (via telephone and in-home interviews) assessed osteoporosis awareness, if medical intervention was sought and if osteoporosis therapy was initiated.

At screening, 14% of Hispanic women in this study were taking estrogen (n = 45), 43% calcium (n =137), and 21% vitamin D (n = 67). Overall, 38% of the Hispanic women in the study had low bone mass (LBM)/osteopenia (n = 121), 13% were osteoporotic (n = 43), and 31% of women ≥65 years old were osteoporotic. Follow-up surveys were completed in 201 out of 318 screened women (63%), 143 in the T-score only group (70%) and 58 from the T-score plus an AFR group (50%). There was no difference detected in the response with “seeking medical help” between women classified with LBM/osteopenia (40%) or osteoporosis (41%). The number of risk factors and/or a T-score with AFR did not have an impact on this parameter. Age, risk factors and a T-score plus AFR did not appear to influence physician ordering an axial DXA and/or prescribing an osteoporosis treatment or the likelihood of patients filling their prescriptions. There was a nonsignificant trend in the osteoporosis patients to seek medical help, receive an axial DXA, receive a prescription for osteoporosis threapy (30%), and fill the prescription (19%). The acculturation questionnaire showed that 310 out of 318 (97%) participants did not consider themselves as Americanized (75% viewed themselves as being “very Mexican”), reflecting a fairly homogeneous group of Hispanic women. It was therefore not possible to determine if acculturation of Hispanic women had an impact on health seeking behavior.

Providing an AFR did not appear to influence patient or physician behavior. Future studies need to identify factors that will change Hispanic women's and their physicians' behavior related to osteoporosis.

Disclosures: A.E. Focil, Procter & Gamble Pharmaceutical Unrestricted Research Grant to CHOF 2.

This study received funding from: Procter & Gamble Pharmaceutical Unrestricted Research Grant to CHOF.

M385

Effects of Long-Term Treatment with a Prostaglandin E₂ Receptor Subtype 4 Agonist and PTH at Skeletal Sites with Moderate Osteopenia in Aged Ovariectomized Rats.

J. I. Aguirre, M. K. Altman*, S. M. Vanegas*, M. F. Rivera*, T. J. Wronski. Physiological Sciences, University of Florida, Gainesville, FL, USA.

Prostaglandin E₂ (PGE₂) is a strong stimulator of bone formation with the ability to restore lost cancellous bone mass in osteopenic ovariectomized (OVX) rats. We and others have shown that a PGE₂ receptor subtype 4 agonist (EP4A) stimulates cancellous bone formation at skeletal sites with severe and moderate osteopenia in aged OVX rats. We also observed that, despite the potent anabolic effect, this agonist was unable to restore cancellous bone mass in aged OVX rats at skeletal sites with severe osteopenia even after long-term treatment (11 weeks). The purposes of this study were to determine the effects of long-term treatment with EP4A at skeletal sites with moderate cancellous osteopenia, and to compare its bone-restorative efficacy with that of PTH treatment. Groups of OVX and sham-operated rats were maintained untreated for 1 year posovariectomy (15 months of age) to develop cancellous osteopenia. OVX rats were then treated SC with vehicle, the EP4A CP-734432 (3 mg/kg) daily, or PTH (80 μg/kg) 5 days/week for a period of 11 weeks. Cancellous bone histomorphometry was performed in the lumbar vertebra (LV) and proximal femur (PF). We found that after 11 weeks of vehicle treatment, OVX rats were moderately osteopenic at both the LV and PF with cancellous bone volumes of 23% and 30%, which corresponded to 60% and 65% decreases compared with the sham-operated controls, respectively. As expected, PTH treatment restored cancellous bone mass to near the level of sham-operated control rats at both skeletal sites. In contrast, EP4A treatment failed to increase cancellous bone mass in aged OVX rats at the LV and PF. Interestingly, osteoblast, osteoid, mineralizing, and osteoclast surfaces were significantly increased in rats treated with EP4A at both skeletal sites, but were unchanged or increased to a much lesser extent in OVX rats treated with PTH. Moreover, whereas EP4A induced significant increases in bone formation rate at both the LV and PF (14- and 4.3-fold, respectively), PTH did not induce significant changes in this variable after 11 weeks of treatment. These findings indicate that although EP4A stimulated cancellous bone formation and bone turnover to a greater extent than PTH, the agonist failed to translate this anabolic response into a restorative effect on cancellous bone at moderately osteopenic sites in aged OVX rats. These findings also suggest that the increase in bone resorption induced by EP4A is in balance with the increase in bone formation so that a net increase in cancellous bone mass failed to occur.

Disclosures: J.I. Aguirre, None.

M386

See Sunday Plenary Number S386

M387

KR62980, a Novel Compound, Enhances Bone Formation Through Promoting Osteoblastogenesis and Inhibiting Osteoclastogenesis.

M. Bae, H. Cheon*, J. Lee*, J. Heo*, H. Kim*. Medicinal Science Division, KRICT, Daejeon, Republic of Korea.

The renewal of bone is responsible for bone strength throughout our life as a result of the coordinated actions of two cells, the osteoclast and the osteoblasts. Metabolic bone diseases are considered to be conditions in which this bone resoption-formation balance is lost in favor of increased bone resorption by osteoclasts. Herein, we investigated whether KR62980 exhibits any effects on osteoblast and osteoclast differentiation. KR62980 induced alkaline phosphatase (ALP) activity and increased extracellular matrix calcification in primary osteoblast. Real time RT-PCR analysis revealed that KR62980 also increased the mRNA transcripts of specific genes involved in osteoblastic differentiation, including BSP, OC, ALP and Runx-2. Furthermore, the presence of KR62980 inhibited dose-dependently the osteoclast differentiation by reducing RANKL-induced expression of TRAP and actin ring formation with NFκB inactivation. To reveal sensitivity of KR62980 to changes in ant-osteoporotic activity, 4 week female ddy mice were subjected to ovariectomy (OVX), OVX with daily KR62980 or sham. Mice were imaged on week 8 via Micro-CT. OVX induced a decrease of bone mineral density (BMD) in distal femur over sham (p<0.05), a decrease of BMD which was blocked by daily KR62980 (p<0.01). Taken together, these results suggest that KR62980 deserve attention as potential drugs for treating bone disease.

Disclosures: M. Bae, None.

M388

See Sunday Plenary Number S388

M389

Dual-Action Cathepsin K Inhibitors: Modulation of Human Osteoclast Function.

A. J. Westover*, D. Chagnovich, M. W. Long. Velcura Therapeutics, Inc., Ann Arbor, MI, USA.

Cathepsin K (CTSK), a cysteine protease secreted by osteoclasts (OC), is an attractive target for therapies aimed at osteoporosis and other bone diseases. Moreover, the recent demonstration of CTSK's expression in osteoblast (OB) suggests that other functions exist for this enzyme.

VEL-0230 is a dual-action, orally-available CTSK inhibitor that has an anabolic effect on osteoblasts (stimulation of ex vivo and in vivo bone formation) while also acting to disrupt bone resorptive processes. The effects of VEL-0230 on the differentiation and function of human OC were studied by seeding them onto bovine bone slices in the presence or absence of VEL-0230. Evaluation of VEL-0230's actions on OC differentiation indicated that its continuous presence (10 days exposure) failed to affect M-CSF/Rank Ligand-induced OC differentiation and viability over a range of 10nM to 10uM. The actions of VEL-0230 on OC bone resorption were evaluated via collagen telopeptide CTX1 release into the bone-slice supernatant, coupled with the qualitative assessment of OC motility and pit-formation using Toluidine Blue. Levels of the CTX1 and bone pit depth were strongly inhibited buy VEL-0230 in a concentration-dependent manner. Likewise, human OCs treated with VEL-0230 failed to migrate across bone surface, etching a shallow singular pit rather than forming the distinctive trail partern indicative of OC resorption and migration. VEL-0230's mechanism of action (MOA) was investigated by examining its actions on the intracellular processing of CTSK. Cathepsin K is an autocatalytic enzyme that first cleaves its 38 kDa proenzyme to a 27 kDa active enzyme; a subsequent cleavage of this generates a 25 kDa molecule. Western analysis of VEL-0230 (a cell-permeable compound) treated OC shows a concentration-dependent increase in the relative abundance of the 27kDa and a concomitant reduction in the 25 kDa molecule, with little or no apparent change in the relative abundance of the 38 kDa proenzyme. While the relative abundance of the 27kDa form is high, CTSK enzymatic activity is essentially ablated (70% reduction at 1 uM; 100% at 10 uM). We conclude that VEL-0230 inhibits OC function, reducing pit formation, OC migration and collagen degradation without affecting OC differentiation. Mechanistically, this CTSK inhibitor seems to act after its first autocatalytic cleavage exposes the enzyme's active site, preventing further processing and inactivating the mature enzyme. Coupled with its anabolic actions, VEL-0230 represents a novel, dual-action (anabolic & anti-catabolic) therapeutic candidate for diseases such as osteoporosis. Further, this compound may also have therapeutic indications were cell mobility is a factor.

Disclosures: D. Chagnovich, Velcura Therapeutics 1, 3.

M390

See Sunday Plenary Number S390

M391

Effects of Low Dose Parathyroid Hormone on Body Composition, Bone Mass and Turnover and Ectopic Osteoinduction in a Rat Model for Chronic Alcohol Abuse.

U. T. Iwaniec¹, C. H. Trevisiol*², G. Maddalozzo*¹, R. T. Turner¹. ¹Nutrition and Exercise Sciences, Oregon State University, Corvallis, OR, USA, ²Chemical Engineering, Oregon State University, Corvallis, OR, USA.

Parathyroid hormone (PTH) is used clinically to increase bone mass by enhancing bone formation. PTH is also under investigation as a therapy to accelerate fracture repair. We have recently shown that disuse greatly diminishes the bone anabolic response to low-dose (similar to a human therapeutic dose) PTH, suggesting that “life-style” factors may modulate the skeletal response to bone anabolic therapy. The prevalent use of alcohol may represent one of these factors. Chronic alcohol abuse is associated with osteoporosis and impaired fracture healing. Alcohol consumption also inhibits osteoinduction by demineralized allogeneic bone matrix (DABM); DABM is used clinically to facilitate bone fracture repair. Therefore, the present study investigated the effects of alcohol on the bone anabolic response to low-dose PTH: 1) during normal bone turnover, and 2) in a model of DABM-induced bone formation. Three-month-old male Sprague Dawley rats (4 groups; n=10–11 rats/group) were fed control or Lieber-DeCarli liquid diet with 35% of the calories derived from ethanol. The control rats were pair-fed an alcohol-free isocaloric diet. Following a 1 week adaptation to the diets, the rats were implanted subcutaneously with DABM cylinders prepared from femurs and tibiae of rats fed normal rat chow. The rats were then treated with PTH (1 μg/kg/d, 5 d/wk;+/- alcohol) or vehicle (+/- alcohol) for 6 weeks. Body composition was determined on day of necropsy using DXA. Tibiae were processed for histomorphometry. Bone architecture in DABM implants was evaluated by μCT. PTH treatment increased whole body bone mineral content (BMC) and bone mineral density (BMD). The hormone also increased bone formation and bone area/tissue area in the proximal tibial metaphysis. In contrast, PTH had minimal effects on osteoinduction by DABM. Alcohol consumption decreased whole body BMC, fat mass and % body fat. Alcohol also decreased bone formation and bone area/total area in tibia and impaired DABM-mediated osteoinduction. There was no interaction between PTH treatment and alcohol consumption for any of the endpoints measured. Our results suggest that PTH, at a dose rate that increases bone mass in a normal weight bearing bone, is ineffective in enhancing osteoinduction under non weight bearing conditions. In contrast, alcohol inhibited osteoinduction, and reduced bone formation in the presence or absence of therapeutic PTH. These results provide additional evidence that alcohol impairs bone healing and may reduce the efficacy of drugs intended to reverse bone loss.

Disclosures: U. T. Iwaniec, None.

This study received funding from: NIH, DOD.

M392

See Sunday Plenary Number S392

M393

In Silico Identification of Selective Fibroblast Growth Factor-Receptor Modulators.

M. E. Leal¹, L. S. Holliday², P. A. Ostroy*³, J. I. Aguirre¹, T. J. Wronski¹. ¹Physiological Sciences, University of Florida, Gainesville, FL, USA, ²Orthodontics, University of Florida, Gainesville, FL, USA, ³Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL, USA.

Basic fibroblast growth factor (bFGF) has promise as a therapeutic agent for the treatment of osteoporosis, but induces side effects including anemia in a rat model. We are seeking small molecules that selectively modulate the fibroblast growth factor receptors (FGFRs) that are present in bone cells. The long-term goal is to identify a selective ligand for an FGFR that induces a stimulatory effect on bone formation with minimal adverse side effects in non-skeletal tissues. Our initial approach involves use of an in silico method to identify FGFR-selective small molecule ligands. We then screened these ligands for effects on growth of BaF3 cells that were stably-transfected with FGFR1c, FGFR2b, and FGFR3c, using a metabolic colorimetric assay. Small molecules identified as interacting with specific receptors were then screened for effects on osteoclast formation in mouse marrow cultures. We initially identified 36 small molecules predicted to interact selectively with FGFR1c and FGFR3c. None were agonists, but several inhibited the growth of BaF3 cells by 50–80% in the presence of bFGF and acidic FGF. From these, we identified one small molecule against FGFR1c (LWOH031c) and one against FGFR3c (LWOH193c) that were selective. LWOH031c reduced osteoclast formation by 80% in mouse marrow cultures in the presence of bFGF and other stimulators, but also reduced bFGF-stimulated proliferation of adherent cells. LWOH193c inhibited osteoclast formation by 80–90%, but did not reduce adherent cell proliferation. We are currently testing the effects of these molecules on bFGF-induced proliferation of calvarial osteoblasts and primary chondrocytes. In conclusion, we have identified small molecules that selectively interact with FGFRlc and FGFR3c to block stimulation by bFGF. Interestingly, both reduced osteoclast formation in mouse marrow cultures in response to bFGF and other stimulators of osteoclastogenesis. The inhibitor of FGFR3c had no detectable effect on proliferation of adherent cells. These molecules may be useful pharmacologically as FGFR-selective inhibitors, or may be derivatized to enable them to serve as selective agonists.

Disclosures: M.E. Leal. None.

This study received finding from: University of Florida.

M394

See Sunday Plenary Number S394

M395

Effects of Intermittent Administration of Parathyroid Hormone (1–34 hPTH) on Distraction Osteogenesis in Rabbits.

H. Maruno*¹, S. Ichimura*², T. Oohata*², C. Uchikura*², K. Satomi*². ¹Orthopaedic, Kyorin Univ, Tokyo, Japan, ²Orthopaedic, Kyorin Univ., Tokyo, Japan.

<Objective> Our study examined the effects of intermittent administration of parathyroid hormone (1–34 hPTH) on distraction osteogenesis. <Materials and Methods> An external fixator was applied to 15 immatured white Japanese rabbits and an osteotomy was performed on the right tibia. After a delay of 1-week, the distraction was started at a rate of 0.375 mm twice a day for 2 weeks. Beginning on distraction, hPTH was subcutaneously administered once a day, four days a week for a total of 4 weeks at a dose of 10 μg/kg (group P10) or 30 μg/kg (group P30) to five rabbits per group. Five rabbits received only the vehicle (group C) in the same manner. Seven weeks after osteotomy, the external fixator was removed, and rabbits were sacrificed 1-week later. We analysed the distracted callus by X-ray, DXA, pQCT, and μCT, to evaluated the bone union, BMD, cross section area and the morphology of callus. Three-point bending test was performed to assess biomechanical parameters. <Results> Bone union was found in all rabbits. The average BMD of the distracted callus (mg/mm²) was 321.0, 330.8 and 354.4 for group C, group P10 and group P30, respectively. The total cross section area (mm²) was 63.5, 53.6 and 81.7 and the value of group P30 was significantly larger than those of the other two groups. The cortical cross section area (mm²) of group P30 was largest and was significantly larger than that of group P10. μCT of group C showed the immature trabecular bone in the medullary cavity of the callus and the undefined lamellar formation of cortical bone. On the other hand, in PTH-administered groups, there was less immature trabecular bone in the medullary cavity, and the formation of lamellar structure in cortical bone was accelerated. Three-point bending analysis showed that fracture energy (N•mm) was 168.9, 240.1 and 583.0, respectively, and group P30 demonstrated a significantly larger value than the other two groups. <Discussion> PTH have a remodeling acceleration effect in bone fracture experiments. In relation to distraction osteogenesis, Seebach found an increase in bone mineral content and a stronger callus using rat femur models. The results in this study did not find any significant difference in bone density, but the cross section area and mechanical strength of the distracted callus of group P30 was significantly larger and higher. The morphology of the distracted callus in groups P10 and P30 also suggested a remodeling acceleration effect. We concluded that intermittent administration of PTH could be benefical to shorten the consolidation period of callus formation in distraction osteogenesis.

Disclosures: H. Maruno, Asahi Kasei Pharma Co. 2.

M396

Calcium Absorption from Commonly Consumed Vegetables in Healthy Thai Women.

S. Charoenkiatkul*¹, W. Kriengsinyos*¹, U. Suthutvoravut*², C. M. Weaver³. ¹Institute of Nutrition, Mahidol University, Nakhonpathom, Thailand, ²Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, ³Foods and Nutrition, Purdue University, West lafayette, IN, USA.

The absorbability of calcium from ivygourd, green leafy vegetable (Coccinia grandis Voigt.) and winged bean young pods (Psophocarpus tetragonolobus, (L) DC) were measured in 19 healthy adult women aged 20–45 y, in a three-way, randomized-order, crossover design with an average calcium load of 100 mg and milk as the referent. The test meals were extrinsically labeled with ⁴⁴Ca and given with rice as breakfast after an overnight fast. Absorption of calcium was determined on a blood sample drawn 5 h after ingestion of the test meal. Fractional calcium absorption (X ± SD) was 0.391 ± 0.128, from winged beans, 0.476 ± 0.109 from ivygourd, and 0.552 ± 0.119, from milk. The difference in fractional calcium absorption for these two vegetables was significant (P<0.05) and the fractional calcium absorption from these two vegetables were both significantly lower than from milk. The difference was partly accounted for by the phytate, oxalate and dietary fiber content of the vegetables. However, the bioavailability of these two vegetables, commonly consumed among Thais, was relatively good compared to milk (71–86% of milk) and could be generally recommended to the public as calcium sources other than milk and Brassica vegetables.

Disclosures: S. Charoenkiatkul, None.

This study received funding from: Thailand Research Fund.

M397

See Sunday Plenary Number S397

M398

Natural Plant Extracts, Gs-Ac and -H, Induce Osteoblast Differentiation and Reduce Osteoclast Differentiation.

Y. Chung¹, H. Yoon*¹, S. Yun*¹, S. Yi*¹, Y. Won*², Y. Shin*³, S. Lee*⁴, S. Lee*⁵, N. Song*⁶, J. Ryu*⁶. ¹Endocrinology and Metabolism, Ajou University School of Medicine, Suwon, Republic of Korea, ²Orthopedic Surgery, Ajou University School of Medicine, Suwon, Republic of Korea, ³Endocrinology and Metabolism, Wonju College of Medicine, Wonju, Republic of Korea, ⁴Biochemistry, Eulji University College of Medicine, Daejeon, Republic of Korea, ⁵ISAM Internal Medicine Clinic, Busan, Republic of Korea, ⁶HL Genomics, Yongin, Republic of Korea.

Backgrouond: Bone is a dynamic organ that is continuously remodeled by combinational roles of osteoblasts and osteoclasts. Osteoporosis is a disease of imbalance in bone formation (osteoblast) and resorption (osteoclast), which leads to bone loss. We investigated that effects of natural plant extracts GS-Ac and -H on osteoblast and osteoclast differentiation.

Methods: Osteoblastic differentiation was evaluated with alkaline phosphatase (ALP) and mineralization assays. Osteoclastic differentiation was determined with osteoprotegerin (OPG) and tartrate resistant acid phosphatase (TRAP) staining.

Results: GS-Ac and -H (0.1, 0.05 mg/ml) increased ALP activity as 5–7-fold of control in osteoblast. Dexamethasone decreased ALP activity as 1.5-fold of control and GS-Ac, and — H increased as 2.3–10-fold of Dexamethasone. GS-Ac (0.1, 0.05 mg/ml) and GS-H (0.05 mg/ml) increased mineral nodule formation in osteoblast. OPG concentration was increased as 2.5-fold of control. TRAP-positive cells were decreased in GS-Ac treated osteoclast.

Conclusion: GS-Ac and -H promoted osteoblast differentiation and suppressed osteoclast differentiation.

Disclosures: Y. Chung, HL Genomics 2.

This study received funding from: GRRC Project of Gyeonnggi Provincial Government, Republic of Korea.

M399

See Sunday Plenary Number S399

M400

Efficiency of Dietary Calcium Use for Skeletal Growth and Mineralization in Young Pigs Fed Diets with Various Phosphorus Concentrations.

H. Singh*, D. K. Schneider*, T. D. Crenshaw. Animal Sciences, University of Wisconsin -Madison, Madison, WI, USA.

Efficiency of nutrient use (nutrient retained/consumed) is typically higher in animals fed diets with marginal deficiencies compared with animals allowed adequate intake of the nutrient. However, Ca efficiency was lower in pigs fed diets with a marginal Ca deficiency compared with Ca efficiency of pigs fed diets with excess Ca. The previous study (J Bone Miner Res 20:S193) was designed to assess recovery of skeletal growth in young pigs following a period of deficit growth induced by a Ca deficiency. Dietary P concentrations may have limited the efficiency of Ca use. The current study was designed to assess the impact of dietary P concentrations (either 70, 90, or 120% of requirements) on efficiency of Ca use in young pigs. Diets provided Ca at either 75 or 150% of requirements for each P level. Ca efficiency was estimated from Ca retention predicted with DXA scans (GE Prodigy, using scan modes previously shown accurate for pigs, J Bone Miner Res 20:S332) and dietary Ca consumption over a 27 day trial. In pigs fed diets with 90% of P requirements, Ca efficiency was greater if Ca was limited (75%) than if excess (150%). However, if pigs were fed diets with excess P (120%) no differences in Ca efficiency were detected between Ca levels. Pigs fed diets with only 70% of P requirements failed to gain bone mass over the 27 d trial regardless of the Ca level. Improvements in efficiency of P use were not detected in animals fed diets limited in P regardless of Ca levels. In conclusion, efficiency of Ca use in young pigs is altered by dietary P levels. 

Table  .  

Disclosures: T.D. Crenshaw, None.

M401

Postmenopausal Osteoporosis HRT and Weight Gain.

A. Bazarra-Fernandez*. ObGyn, Juan Canalejo University Hospital Trust, Amparo Lopez Jean 13 4° A, La Coruña, Spain.

Background: Recent studies have found weight gained during menopause increase the risk of high blood pressure, the diabetes, heart disease, and has been strongly linked to increased incidence of breast and other hormone-related postmenopausal malignancies. These healthcare concerns have led to the conception of specific products that target menopausal weight gain.

Aim: Looking over weight gain and osteoporosis treatment in climacteric. Material and methods: 20 women who were 44 to 58 years old have been recruited. BMI was increased to age. Those with an intact uterus have moderate to severe vasomotor symptoms associated with the menopause, moderate to severe symptoms of vulvar and vaginal atrophy and risk of postmenopausal osteoporosis. They were ascribed to equal two 10 women groups. One group was assigned to 2 mg drospirenone /1 mg 17 beta-estradiol hemihydrate. The other group treated with. 40 mg soy bean.

Results: in the women on 2 mg drospirenone /1 mg 17 beta-estradiol hemihydrate medication decreased, moderate to severe symptoms of vulvar and vaginal atrophy vasomotor symptoms associated with the menopause in regard to the other group treated with 40 mg soy bean. They had weight main loss of 3 kg in one year, (P < 0.05).

Conclusions: Human HRT is in relation to decrease osteoporosis. Nobody noted 17 beta-estradiot is in relation to breast cancer. Estradiol is the same oestrogen produced by the ovaries before menopause. Drospirenone has the unique property of reducing water retention often associated with the use of oestrogen and other synthetic progestin hormones. The impact of obesity on hormone replacement therapy is due to many women associate hormones with weight gain. This late medication formula can be beneficial in minimizing uncomfortable symptoms, such as weight gain, hot flashes, night sweats, and mood swings, associated with the natural progression of a woman's life cycle. So, it is due to conduct one great try to make clear and more comprehensible these points.

Disclosures: A. Bazarra-Fernandez. None.

M402

Bone Specific SARMs (Selective Androgen Receptor Modulators) Increased Bone Mineral Density in a Different Manner from Anti-resorptive Agent, Raloxifene with Minimum Effects on the Uterus.

K. Furuva¹, N. Yamamoto*¹, Y. Ohyabu*¹, T. Morikyu*¹, A. Iwata*¹, H. Ishige*², K. Kuzutani*³, Y. Taquahashi*³. ¹Drug Discovery Research Lab., Kaken Pharmaceutical, Co., Ltd., Kyoto, Japan, ²Chemistry Lab., Kaken Pharmaceutical, Co., Ltd., Kyoto, Japan, ³Safty Research Lab., Kaken Pharmaceutical, Co., Ltd., Fujieda, Japan.

Estrogens and androgens are opposite sex steroid hormones and both affect bone metabolism and uterine homeostasis. Raloxifene is a selective estrogen receptor modulator (SERM) which has estrogenic effects on the bone with reduced side effects on the sexual organs. We have been demonstrated a concept of Selective Androgen Receptor Modulators (SARMs) as useful drugs for a treatment of osteoporosis through proving that anabolic effects on the bone can be separated from virilizing effects. This study compared our new SARM Compound R2 with Raloxifene in the effects on the bone and uterus. Female 12-week old Sprague-Dawley rats were ovariectomized (OVX) and were feed with a low calcium diet for 4 weeks to expedite reduction of the bone mineral density (BMD). Oral administration of Compound R2 (1, 10 mg/kg) or Raloxifene (1 mg/kg) were followed once a day for 8 weeks. Compound R2 significantly increased the BMD at the part between the diaphysis and proximal metaphysis of the femurs at 1 mg/kg, whereas Raloxifene slightly increased the BMD at the distal and proximal metaphysis which are cancellous bone rich parts. Concerning to the effects on the uterus, dihydrotestosterone had a major impact on the uterine muscle and also increased the endometrium. Compound R2 did not show any effects on the uterus at 1mg/kg and displayed minimum effects on the uterine muscle at 10mg/kg. On the other hand, Raloxifene displayed slightly thickening of the endometrium. These results demonstrate that Compound R2 is superior to Raloxifene in bone formation and specificity to the bone, suggesting that SARM is promising for the treatment of severe osteoporosis patients without anabolic effects on the uterus.

Disclosures: K. Furuya, None.

M403

See Sunday Plenary Number S403

M404

Effects of Bazedoxifene Acetate on Bone Loss: A 12-Month Study in Ovariectomized Rats.

B. S. Komm¹, P. V. N. Bodine¹, D. R. Minck*². ¹WHMSB, Wyeth Research, Collegeville, PA, USA, ²Drug Safety, Wyeth Research, Chazy, NY, USA.

Bazedoxifene acetate (BZA) is a novel, stringently screened selective estrogen receptor modulator (SERM) currently in development for the prevention and treatment of postmenopausal osteoporosis and in combination with conjugated estrogens for the treatment of menopausal symptoms and prevention of postmenopausal osteoporosis. We examined the effects of BZA on bone mass/composition, biomechanics, and histomorphology in ovariectomized (OVX), 6-month-old female rats. In the active treatment groups, 3 doses of BZA (0.15, 0.3, and 1.5 mg/kg/day) were administered to OVX animals by oral gavage for 12 months. Control groups included an untreated baseline group, untreated sham-operated animals, untreated OVX animals, and an estrogen-treated (17α-ethinylestradiol [EST], 0.03 mg/kg/day) OVX group. Animals assigned to the baseline group received no treatments or procedures and were euthanized within 2 weeks of study initiation for testing. Untreated sham-operated and untreated OVX animals were administered only test vehicle, once daily. Dual energy x-ray absorptiometry (DXA) scans, peripheral quantitative computerized tomography (pQCT), and biomechanical and histomorphometric studies were conducted to assess bone mineral density (BMD), bone mineral content (BMC), and bone architecture and strength. Based on BMD and BMC analyses, BZA protected the skeleton from estrogen-dependent bone loss at several sites, with optimal protection observed with the 0.3 mg/kg/day dose. In vivo DXA data revealed a dose-related response to treatment with BZA for 11 of 12 parameters examined. Treatment with BZA 0.3- and 1.5-mg/kg/day reduced the loss of trabecular BMD at metaphysis (MTraBMDp) observed in OVX controls by 39%-41% and 34%-37%, respectively (P≤0.01), as confirmed by pQCT analysis at the tibia and femur; these doses of BZA resulted in more marked reductions in MTraBMDp loss than EST. A significant difference was observed between the BZA 0.3- and 1.5-mg/kg/day treatment groups and the OVX control group in maximal stress on the lumbar vertebra 4 on biomechanical testing (P≤0.01); BZA also increased maximum load reached in the neck of the femur, but this effect did not reach statistical significance. Histomorphometric analyses showed differential beneficial effects of treatment with BZA in protecting against the deleterious effects of ovariectomy on the skeleton.

We conclude that BZA, a novel SERM, exhibits a promising therapeutic profile based on demonstration of generally dose-related protective effects on several skeletal parameters in this animal model of accelerated bone loss.

Disclosures: B.S. Komm, Wyeth Pharmaceuticals 1, 3.

This study received funding from: Wyeth Pharmaceuticals.

M405

See Sunday Plenary Number S405

M406

Toremifene Preserves Bone Architecture and Strength in Aged Orchiectomized Rat.

S. Raghow¹, J. Jolette*², S. Y. Smith², C. H. Turner³, D. Farrell*², J. T. Dalton*¹, K. A. Veverka¹. ¹Preclinical Research, GTx, Inc, Memphis, TN, USA, ²Preclinical Research, Charles River Laboratories, Montreal, PQ, Canada, ³Preclinical Research, Indiana University, Bloomington, IN, USA.

The ability of toremifene citrate (TOR), a SERM, to prevent bone loss was studied in aging orchiectomized (ORX) Sprague-Dawley rats. Eight-month old males were randomly assigned to each of five groups: baseline, Sham, ORX control and ORX groups treated with TOR at 10 or 30 mg/kg. TOR was given daily by gavage for 12 months starting one day post-ORX. The cancellous bone region of lumbar vertebra (L2) and tibial proximal metaphysis (Cn-Ti) and the cortex of tibio-fibular junction (Cx-Ti) were analysed by histomorphometry in 10 rats/group. Biomechanical tests included femur 3-point bending, femoral neck shear and L4 compression on bones from 10 rats/group. ORX slightly worsened the age-related cancellous bone loss at L2 and Cn-Ti, as measured by BV/TV, and further augmented the bone formation rates (BFR) and bone turnover (MS/BS). In L2 and Cn-Ti, both TOR doses completely prevented the ORX-induced bone loss and increases in MS/BS and BFR. TOR at 10 and 30 mg/kg consistently reduced Oc.S/BS at both tested sites, consistent with its bone antiresorptive effects. There were no adverse histological effects on bone mineralization or collagen arrangements with ≤30 mg/kg TOR doses. ORX slightly decreased bone strength variables at L4. TOR treatment preserved lumbar strength along with maintenance of bone mass. A significant positive linear relationship was established between peak load and total slice BMC (r = 0.60; p<0.01) and BMD (r = 0.69; p<0.001) and total slice BMD vs. Apparent Strength for L4 in compression (r = 0.58; p<0.01) (BMD data, ASBMR 2006). In Cx-Ti, ORX decreased both cortical area and cortical width, primarily due to an expanded medullary cavity. ORX further increased the age-related increase in labeled surface and BFR at the endocortex along with a non-significant reduction of periosteal labeled surface. TOR did not significantly alter cortical geometry in ORX rats but markedly reduced labeled surfaces and BFR at both periosteal and endocortical envelopes. ORX-associated increases in endocortical label-derived variables were entirely nullified by TOR. Treatment with TOR resulted in positive trends on bone strength variables at the femoral shaft compared to ORX controls, attributed to preservation of cortical thickness with bones of slightly smaller diameter. Preservation of bone mass at the femoral neck was associated with preservation of bone strength similar to Sham group. Thus, TOR preserved bone mass and strength at the femur shaft and clinically relevant sites, the femoral neck and lumbar spine, in this rat model of androgen deficiency-induced bone loss.

Disclosures: S. Raghow, GTx, Inc. 1, 3.

This study received funding from: GTx, Inc.

M407

Bone Effects of 17β-Estradiol and Dihydrotestosterone on Bone Turnover in a Mouse Model of Glucocorticoid-induced Osteoporosis.

M. I. Suominen¹, J. P. Rissanen¹, J. Morko¹, L. Ravanti*², P. J. Kallio*², J. M. Halleen¹. ¹Pharmatest Services Ltd, Turku, Finland, ²Orion Corporation, Turku, Finland.

Glucocorticoids induce osteoporosis by decreasing bone formation due to apoptosis of osteoblasts. 17β-Estradiol (E2) protects osteoblasts from glucocorticoid-induced apoptosis in neonatal mice. We investigated whether E2 or dihydrotestosterone (DHT) were able to prevent changes in bone turnover caused by glucocorticoids in young adult male mice. The following experimental groups were included: 1) Control group receiving vehicle; 2) 30 mg/kg/d of prednisolone (p.o.); 3) 30 mg/kg/d of prednisolone and 60 μg/kg/d of E2 (s.c); 4) 30 mg/kg/d of prednisolone and 3 mg/kg/d of DHT (s.c). Each group contained 12 Balb/c male mice that were 12 weeks old at the beginning of the study. The animals were randomized to groups according to bodyweight. Treatment was started at day 1 and continued daily for 4 weeks. For dynamic histomorphometry, tetracycline labelling was performed at day 10 and calcein labelling at day 2 before termination. Parameters of dynamic histomorphometry were determined from trabecular bone and cortical bone at the end of the study. The bone turnover markers serum osteocalcin and serum TRACP 5b were measured at days 0, 14 and 28. Statistical analysis was performed with ANCOVA for follow-up measurements and either ANOVA or Kruskal-Wallis test for end-point measurements after checking the assumptions for normality and homogeneity of variances. Linear contrasts of means or Mann-Whitney test were utilized for pairwise comparisons. Prednisolone decreased parameters of dynamic histomorphometry in both trabecular and cortical bone. E2 was able to completely prevent the decrease in parameters of trabecular bone, whereas DHT decreased the values even further. E2 showed mild effects on parameters of cortical bone, while DHT had no effect. Bone turnover markers were also decreased by prednisolone. E2 increased serum osteocalcin values at day 14, but had no effects at day 28. DHT had no effects on osteocalcin values at day 14, but decreased the values at day 28. TRACP 5b values were increased by E2 and decreased by DHT at days 14 and 28. These results demonstrate that E2 and DHT act with completely different mechanisms on bone turnover in glucocorticoid-induced osteoporosis. E2 can totally prevent glucocorticoid-induced decrease in bone turnover in trabecular bone, whereas DHT decreases bone turnover further. E2 has only mild effects on bone turnover in cortical bone, and DHT has no effect.

Disclosures: M.I. Suominen, None.

M408

Cathepsin K Inhibitors for the Treatment of Osteoporosis.

U. Grabowska*¹, M. Shiroo*¹, T. J. Chambers², B. Samuelsson*¹. ¹Medivir AB, Huddinge, Sweden, ²St George's, University of London, London, United Kingdom.

Osteoporosis results from excessive bone degradation and is an increasingly prevalent disease. It is characterised by low bone mass and deterioration of skeletal tissue architecture which can lead to bone fragility and predisposes an individual to an increased risk of fracture. Cathepsin K is a lysosomal cysteine protease expressed abundantly in osteoclast cells. Numerous lines of evidence support a pivotal role for cathepsin K in bone degradation without negatively impacting bone formation, differentiating this treatment from currently available anti-resorptives such as bisphosphonates. Furthermore this highlights the potential for cathepsin K inhibition as a novel target for the development of osteoporosis therapeutics. Additionally recent scientific literature demonstrates efficacy, in a dose dependent manner, in animal models of osteoporosis upon inhibition of cathepsin K. We have now developed a series of novel, highly potent specific, non-nitrile warhead cathepsin K inhibitors. These compounds were further selected for their high potency for inhibition of bone resorption by human osteoclasts in-vitro and lack of evidence for cellular toxicity. Compound A (K_i: Cath K = 1.7 nM; Cath L, S, B, V & H > 1500 nM; Cath F = 675 nM for the human enzymes) has been taken forward into clinical development.

The pharmacodynamic effect of compound A was evaluated on bone markers of bone resorption in vivo in young male cynomolgus monkeys. Plasma levels of the C-terminal telopeptides of Type I collagen (CTX-I) was used as a collagenous bone resorption marker. Oral administration of compound A (5 mg/kg) to the animals resulted in a rapid reduction in CTX-1 levels within 2 h to a maximum of 50% after 4–8 hours. A multiple dose study with once daily oral administration of compound A demonstrated a reduction of the biomarker where the suppression of CTX-1 was reversible with bone resorption marker returning to pre-treatment levels within 48 h. This quick on-off suppression of bone resorption may give an additional advantage over other osteoporosis treatment.

Disclosures: U. Grabowska, Medivir AB 3.

This study received funding from: Medivir AB.

M409

See Sunday Plenary Number S409

M410

OCT-1547, a Small Molecule Osteoporosis Drug Candidate, Inhibits Osteoclast Differentiation Through Inhibition of PLCγ2 Activation and NFATc1 Induction.

H. Hwang*¹, S. Kim*¹, H. Hwang*¹, S. Ko², D. Kang*³, S. Shim*³, S. Kim¹, J. Kim*¹. ¹Dept. of Pharmacology and Mechanism, Oscotec Inc., Cheonan, Republic of Korea, ²Dept. of Oral Biochemistry, Dankook University, Cheonan, Republic of Korea, ³Dept. of Medicinal Chemistry, Oscotec Inc., Cheonan, Republic of Korea.

We have previously discovered OCT-1547, a small molecule osteoporosis drug candidate, using our proprietary assay system for osteoclast activity, OAAS™. OCT-1547 inhibits osteoclast differentiation and activity at micromolar range of concentration and bone mineral loss in ovariectomized rats. Preclinical safety evaluation for OCT-1547 was almost completed in Aptuit (UK), and phase I clinical trial will start this year. To investigate the action mechanism of OCT-1547, we examined the effect of OCT-1547 on the signal transduction pathway of RANKL-induced osteoclast differentiation. OCT-1547 inhibited induction of NFATc1, which is a critical transcription factor for the osteoclast differentiation. Examination of the upstream signaling components revealed that OCT-1547 markedly inhibited phosphorylation of PLCγ2 and moderately inhibited degradation of IκB and induction of c-fos. However, the interaction of RANKL, RANK, TRAF6, TRAF2, c-SRC, TAB1, TAB2, and TAK1 were not significantly affected. Treatment of PLCγ inhibitor also inhibited osteoclast differentiation and NFATc1 induction. These results support the hypothesis that inhibition of PLCγ2 activation is a critical event in the inhibition of osteoclast differentiation by OCT-1547. Current investigation is focused on understanding the mechanism how OCT-1547 inhibits the activation of PLCγ2 and the effect of PLCγ2 inhibition on the transcriptional regulation of NFATc1 gene expression.

Disclosures: S. Kim, None.

M411

See Sunday Plenary Number S411

M412

Small Organic Molecule Compound Protects Against Inflammatory Bone Loss in a Rat Model of Periodontitis.

R. Jin*¹, Y. Zhang*¹, Y. Liang*¹, X. Li*¹, R. Bhatt*¹, D. Wu*², J. Zheng*³, L. Xu*⁴, L. Golub*⁴, D. Liu¹. ¹Enzo Therapeutics, Inc, Farmingdale, NY, USA, ²Yale University, New Haven, CT, USA, ³St Jude Hospital, Memphis, TN, USA, ⁴School of Dental Medicine, Stony Brook, NY, USA.

The canonical Wnt signaling pathway is initiated by the binding of Wnt proteins to receptor complexes consisting of LDL receptor-related protein (LRP) 5/6 and frizzled protein. An antagonist of the Wnt pathway, Dikkopf protein (Dkk), binds to the third YWTD repeat domain of LRP5 and Kremen, resulting in the removal of this co-receptor with inactivation of the Wnt pathway. Both human and mouse genetic studies provide supporting evidence that LRP5 has an important role in the regulation of bone remodeling. Hypomorphic or null alleles lead to early onset of osteoporosis; whereas, other LRP5 mutation alleles are associated with high bone mass. Our innovative approach combines structural biology, computational screening, and biological assays to identify small molecule compounds that are capable of disrupting LRP5-Dkk interactions and reversing the inhibitory effect of Dkk on Wnt signaling. We have employed a periodontitis model of bone loss to evaluate the effect that our small molecule compound has on protecting against bone resorption. In this model, lipopolysaccaride (LPS) was used to initiate inflammatory-induced alveolar bone loss. Prior to administering LPS between the first and second molar of the maxillae of Sprague Dawley rats, animals were pretreated with a small molecule test compound (SMTC) or vehicle by oral gavage. Animals were then exposed to LPS every other day and treatment with the SMTC was continued daily. Jaws were removed after 10 days, defleshed and stained with Loeffler's methylene blue in order to identify the cemento-enamel junction (CEJ) as a reference point to measure bone height. Histological analysis clearly showed significant bone resorption and root furcation in the LPS-treated animals, and little bone resorption in the LPS plus SMTC-treated animals. Linear measurements from the CEJs to the alveolar bone crest showed a mean bone loss of 0.94 ±0.08 mm in LPS-treated animals; 0.59 ±0.04 mm in LPS plus SMTC-treated animals; and 0.54 ±0.04 in control animals. There were statistically significant differences between the LPS group and the LPS plus SMTC group (p = 0,00006) and between the control group and LPS group (p = 0.00003). As an indicator of protection, there was no significant difference between the control group and the LPS plus SMTC group (p = 0.18). These data clearly show that SMTC protects against bone resorption in an animal model of endotoxin-induced bone loss. This SMTC may represent an attractive potential new class of therapeutic agents for clinical use, and continued investigation is warranted.

** Equal Contribution

*** Corresponding Author

Disclosures: D. Liu, Enzo Therapeutics, Inc 3.

This study received funding from: Enzo Therapeutics, Inc.

M413

Vitamin D Supplementation and 250HD Levels in Long Term Care Residents.

R. Crilly*¹, M. Mason*², M. Kloseck*³. ¹Department of Medicine, University of Western Ontario, London, ON, Canada, ²Department of Health and Rehabilitation Sciences, Faculty of Health Science, University of Western Ontario, London, ON, Canada, ³Faculty of Health Sciences, University of Western Ontario, London, ON, Canada.

Osteoporosis is common in long term care patients who contribute about 30% of all hip fractures. Vitamin D supplementation has been shown to reduce hip fractures in these patients. Recent information in the community dwelling elderly has suggested that the doses of vitamin D used need to be higher to achieve the blood levels required to reduce the PTH levels. In LTC patients, where sunshine is a rarity, we hypothesized that the supplementation dose would need to be even higher. To date, 41 patients of 4 nursing homes have consented to participate. Patients were either on no supplementation, 400 IU per day or 1000 IU per day. Patients were on various doses of calcium supplementation (range 0–1500). No change in dose of D or calcium had been made for at least 3 months. Blood was drawn for 25 OHD and PTH levels. Patients with renal impairment shown by a creatinine over 130 nmol/L were excluded. 14 patients were on no vitamin D supplementation, 13 were on 400 IU and 14 on 1000IU. Patients were aged 84.4 ± 6.7 yrs SD (range 71–98 yrs.), with no age difference between the groups, had been in the institution for 35.2 months ± 30 SD (range 9–151) and, where applicable, on the current vitamin D dose for 11.3 ± 1.77 months SD. Mean levels of 25OHD in the three groups were 48.29 ± 19.07; 83.46 ± 21.75; and 87.4 ± 15.7 nmol/l for the three groups, no supplement; 400IU; and 1000IU respectively. The level for each treatment dose differed from the no-treatment group (p<.001) but not from each other. Parathyroid hormone levels on the other hand were similar in all groups (4.41± 2.0; 6.02 ± 4.16; 4.81± 2.87 for the zero, 400 and 1000 dose groups respectively), and there was no correlation between 250HD levels and PTH levels overall. Mean levels of calcium supplementation was lower for those on zero and 400IU of vitamin D (405 ± 356 vs. 320 ± 473 mg/day) compared to those on 1000IU (769 ± 525mg/day, p=.044 and .031 respectively). The lower PTH levels in those on no, or lower, vitamin D doses could not be explained by calcium supplementation levels, which was lower in those groups. We conclude on the basis of these unexpected preliminary results that the level of 250HD levels achieved in LTC residents is adequate on 400IU of vitamin D, that the PTH levels are lower than expected in those on no vitamin D, and calcium supplementation does not account for this finding.

Disclosures: R. Crilly, Alliance for Better Bone Health 2.

This study received funding from: Alliance for Better Bone Health.

M414

Age Modifies the Impact of Vitamin D3 Supplementation on the 25-OH Vitamin D/ Parathyroid Hormone Ratio.

H. J. Florez*, E. P. Cherniack*, O. Gómez-Marin*, B. A. Roos*, S. Levis, B. R. Troen. Miami VAMC, University of Miami Miller School of Medicine, Miami, FL, USA.

Both 25-hydroxyvitamin D (250H-D) and parathyroid hormone (PTH) are associated with functional outcomes in the elderly. Recent evidence suggests that the 250H-D/PTH ratio predicts functional recovery after hip fracture in older patients [1]. In a pilot study we evaluated supplementation with 2,000 IU/day of cholecalciferol (D3) for 6 months in elderly men at the Miami VA Medical Center. Participants were randomized to either placebo [n= 17, age (mean ± SD: 79±4 years] or D3-treatment [n=17, age: 80±4 years]. No significant baseline differences were observed between participants aged 80 years and older compared to those age 70 to 79 years in 250H-D (28.7±9.1 vs. 29.7 ±7.3 ng/ml, p=0.7), PTH (48.6±22.1 vs. 42.1±19.6 pg/ml, p=0.4), or the 250H-D/PTH ratio (730±409 vs. 893±571, p=0.3). After 6 months of treatment, 250H-D was higher in both D3-treated age subgroups compared to the placebo subgroups (Table), while no significant differences in PTH values were observed between age and treatment groups. D3 treatment increased the 250H-D/PTH ratio compared to placebo participants, and the changes in this ratio were significantly higher only among those aged 80 years and older (619±126 in D3-treated vs. — 64.5±1336 in placebo participants, p<0.001). These results suggest that D3 treatment improves 250H-D levels in all elderly participants. However, the differences in the 250H-D/ PTH ratio suggest that D3 treatment may provide additional functional benefits in those aged 80 and older. Further studies to characterize the response to D3 therapy in this elderly group are warranted.

Table  .  

[1] Di Monaco M et al., 25-Hyproxyvitamin D, parathyroid hormone, and functional recovery alter hip fracture in elderly patients. J Bone Miner Metab 2006; 24:42–47

Disclosures: H.J. Florez, None.

M415

See Sunday Plenary Number S41S

M416

Orally-active Nonsecosteroidal Vitamin D Agonists Increased Bone Mass in Ovariectomized Rats without Causing Hypercalcemia.

S. Harada*¹, S. Takeda*¹, A. Sugita*¹, M. Ishigai*², H. Kashiwagi*³, T. Takahashi*³, T. Tamura*¹, K. Morikawa*³, E. Ogata*⁴, F. Ichikawa*¹. ¹Pharmaceutical Research Dept. 1, Fuji Gotemba Research Labs. Chugai Pharmaceutical Co., Ltd., Gotemba, Japan, ²Pre-clinical Research Dept, Fuji Gotemba Research Labs. Chugai Pharmaceutical Co., Ltd., Gotemba, Japan, ³Chemistry Research Dept. 1, Fuji Gotemba Research Labs. Chugai Pharmaceutical Co., Ltd., Gotemba, Japan, ⁴Cancer Institute Hospital, Tokyo, Japan.

Secosteroidal vitamin D receptor (VDR) agonists have been used for treatment of psoriasis, secondary hyperparathyroidism and osteoporosis. However, their efficacy is limited due to the development of hypercalcemia. Recently, several nonsecosteroidal compounds were demonstrated to be active as VDR agonists. To identify VDR agonists with less calcemic effects than those of secosteroidal structures, we designed and synthesized about seven hundred nonsecosteroidal VDR agonists using co-crystal structure of VDR and its agonists. In VDR agonists, there were good correlations between epithelial calcium channel 2 (ECaC2) induction in colon cancer cell line (Caco-2) in vitro and urinary calcium excretion in vivo, and also between osteocalcin (OC) secretion in MG63 osteoblast cell line in vitro and response of bone mineral density (BMD) in vivo. Therefore, nonsecosteroid VDR analogs were first tested for their abilities to induce ECaC2 mRNA in Caco-2 and to induce the secretion of osteocalcin from MG63. Forty compounds which showed strong OC induction with less ECaC2 induction were selected for in vivo evaluations. Six compounds (e.g.CH5013072) significantly increased BMD of spine and femur without inducing hypercalcemia in ovariectomized rats after daily oral administration. Moreover, they decreased bone resorption parameters but had little effect on bone formation parameters. Thus, these nonsecosteroidal VDR agonists will be promising candidates as orally-active and less calcemic drugs for osteoporosis. 

Table  .  

Disclosures: S. Harada, None.

M417

Differential Effects of Alfacalcidol in the Distal Tibial Metaphyses of Orchidectomized and Aged Male Rats.

X. Y. Tian¹, X. O. Liu¹, H. Y. Chen¹, R. B. Setterberg*¹, M. Li², W. S. S. Jee¹. ¹Division of Radiobiology, University of Utah School of Medicine, Salt Lake City, UT, USA, ²Department of Cardiovascular and Metabolic Disease, Pfizer Global Research and Development, Groton, CT, USA.

The purpose of the study was to compare the effects of alfacalcidol (ALF), a vitamin D analog, on the cancellous bone of distal tibial metaphysis in aged male and orchidectomized (ORX) rats. The distal tibia fuses its epiphyses at 3 months and is primarily a fatty marrow site with a cancellous bone histomorphometric profile similar to that in human iliac crest sites. Seventy male Sprague-Dawley rats were sham or ORX at 18 months of age and then orally treated with vehicle or ALF at 0.1 or 0.2μg/kg/d, 5 days per week for 12 weeks. Double fluorescent-labeled undecalcified distal tibiae were processed for bone histomorphometry. An age-related increase in bone turnover without bone loss was seen in aged rats. Despite further increased bone turnover, ORX did not cause bone loss in aged rats. Treatment of aged rats with ALF at 0.1 and 0.2μg/kg/d decreased %eroded perimeter (Er.Pm), mineralizing surface (MS/BS), surface referent bone formation rate (BFR/BS), and bone volume referent bone formation rate (BFR/BV), resulting in non-significant increases in ratio of mineralizing surface/eroded surface (MS/Er.Pm) and trabecular area (Tb.Ar). These data indicate that 12 weeks of treatment with ALF had a positive but moderate effect on the balance between bone formation and resorption in the aged male rats. Treatment of ORX rats with ALF at 0.1 μg/kg/d caused similar changes in the Er.Pm, MS/BS, BFR/BS, and BFR/BV but significantly increased MS/Er.Pm and Tb.Ar. Although treatment of ORX rats with ALF at 0.2μg/kg/d decreased Er.Pm (-67%), it did not alter MS/BS, BFR/BS, and BFR/BV, resulting in significant increases in MS/Er.Pm (+220%) and Tb.Ar (+47%) compared with aged rats treated with vehicle. When compared with vehicle-treated ORX rats, ALF significantly decreased Er.Pm (-73%), MS/BS (-46%), and BFR/BV (-58%) whereas increased MS/Er.Pm (+173%) and Tb.Ar (+51%). These data revealed that ALF treatment suppressed bone resorption below normal level while maintaining bone formation at normal level, leading to a positive balance between bone formation and resorption and consequently increased bone mass in ORX rats. Thus, the positive effect of ALF is more pronounced in the distal tibia of ORX rats with a higher bone turnover than sham controls. The differential effects of ALF on bone in sham and ORX rats suggest that vitamin D analog such as alfacalcidol is more efficacious at a predominantly fatty marrow skeletal site with high bone turnover.

Disclosures: X. Y. Tian, None.

M418

The Effect of Oral Ibandronate Following Weekly PTH: PTH Once Weekly Research Follow-up (POWR II).

D. M. Black¹, L. Palermo¹, T. F. Hue¹, S. Majumdar¹, M. L. Bouxsein², C. J. Rosen³. ¹University of California, San Francisco, San Francisco, CA, USA, ²Beth Israel Deaconess Medical Center, Boston, MA, USA, ³MECORE, St. Joseph Hospital, Bangor, ME, USA.

Use of alendronate for one year following one year of daily PTH in the PaTH study led to large increases in BMD during the second year, in postmenopausal women. We recently reported the results from the PTH Once Weekly Research (POWR) study, which demonstrated that PTH 1–84 used once weekly (100 μg) led to an increase of about 2% in DXA spine BMD versus placebo. POWR II is a follow-up study, in which all POWR participants were offered open-label monthly ibandronate (150mg) for 12 months. In the POWR study, 50 women were randomized (1:1) to either one year of PTH 1–84 injections (100 μg weekly, after a 1 month daily loading period) or to placebo. At the end of the year, 48 adherent women were offered 1 year of ibandronate (150 mg/monthly). 43 women chose to take the ibandronate and are included in this analysis. The main endpoint was changed in DXA spine BMD; secondary endpoints (not reported here) included hip BMD, QCT (hip and spine) and MRI (wrist) assessed trabecular BMD. We hypothesized that those who had been on weekly PTH would experience larger rises in BMD than those on placebo.

During the one year on ibandronate, the group formerly taking weekly PTH had a 1.1% increase in spine BMD compared to 3.1% increase in those who were on placebo injections (p=0.07). Cumulatively over two years (months 0–24), the group given weekly PTH followed by ibandronate increased 2.8% compared to 3.2% for those who had been given placebo then ibandronate (see Figure). Results for other endpoints were similar; showing no significant difference in the change in BMD during the second year regardless of earlier treatment with weekly PTH.

Over 24 months, mean BMD change in women on weekly PTH followed by ibandronate was the same as those on placebo followed by ibandronate. The weak anabolic effect of once weekly PTH achieved after 12 months, was insufficient to yield the subsequent BMD gain at 24 months, as seen with bisphosphonate use after daily PTH therapy in PaTH.

Disclosures: D.M. Black. Roche/GSK 2, 5: NPS Pharmaceuticals 5; Novartis Pharmaceuticals 2; Merck & Co., Inc. 8.

This study received funding from: NIAMS-NIH (POWR), Roche (POWR II).

M419

See Sunday Plenary Number S419

M420

Characteristics of Postmenopausal Women Treated with PTH(1–84) in the TOP Study: Relationship of Baseline Serum and Urine Ca Values to Hypercalcemia and Hypercalciuria.

H. A. Bone¹, S. Greenspan², S. Morris³, J. Bilezikian⁴. ¹Michigan Bone and Mineral Clinic, Detroit, MI, USA, ²Osteoporosis Prevention and Treatment Center, Pittsburgh, PA, USA, ³NPS Pharmaceuticals, Parsipanny, NJ, USA, ⁴Columbia University College of Physicians and Surgeons, New York, NY, USA.

The TOP study, an 18-month, multinational, randomized, double blind, placebo-controlled trial, assessed the ability of recombinant human PTH(1–84) to reduce vertebral fracture incidence in postmenopausal osteoporotic women. 2679 subjects received daily Ca (700 mg) and vitamin D (400 IU) supplements in addition to either PTH(1–84) 100 μg SC daily or placebo. Inclusion criteria included a serum Ca ≤10.7 mg/dL and a urinary Ca to creatinine ratio of <1.0 mmol/mmol at screening, values that generally exceed the upper limits of normal for adults. These limits contrast with criteria for other studies of PTH-like peptides, e.g., teriparatide fracture study [PTH(1–34)] and the PaTH study [PTH(1–84)] in which subjects were excluded if baseline serum and urine Ca values were above normal to any degree (e.g. subjects must have serum calcium <10.2 mg/dL and urinary calcium <300 mg/24 hr). Of the 1286 subjects treated with PTH, 16% had serum Ca >10.2mg/dL and 10% had urine Ca >300 mg/24 hr at baseline, values that would have excluded them from participation in the other trials alluded to. Moreover, 3.1% of subjects had both hypercalcemia and hypercalciuria. Subjects treated with PTH(1–84) with a baseline urine Ca >300 mg/24 hr had an incidence of hypercalciuria at least 2-fold higher than in those with baseline urinary calcium <300 mg (>70%). Similarly, individuals treated with PTH(1–84) with baseline serum Ca >10.2 mg/dL had ∼2-fold higher likelihood of hypercalcemia (47%). These results help to explain the substantially higher risk for either hypercalcemia and or hypercalciuria in TOP as compared to the other trials. Although subjects who experienced hyperacalcemia or hypercalciuria benefited from PTH by increases in BMD at multiple skeletal sites and a reduced incidence of vertebral fractures, these data emphasize the critical role that inclusion and exclusion criteria play in the interpretation of PTH safety profiles.

Disclosures: H.A. Bone, NPS Pharmaceuticals 5.

This study received funding from: NPS Pharmaceuticals.

M421

See Sunday Plenary Number S421

M422

Early Adjuvant Therapy with Teriparatide after Major Orthopaedic Surgery of Complicated Fractures of Long Bones in Postmenopausal Women: Preliminary Clinical Results.

C. F. Corradini*¹, F. M. Ulivieri*², C. A. Verdoia*¹. ¹Orthopaedic and Traumatologic Clinic, Studies University of Milan, Milan, Italy, ²Nuclear Medicine, Policlinico Mangiagalli IRCSS Foundation, Milan, Italy.

INTRODUCTION: In the last years the postmenopausal population is increasingly afflicted by complicated fractures of long bones. These are represented by two main clinical situations: the sixties woman without any apparent bone metabolic problem that after a comminuted fracture of long bones suffers a series of complication as late consolidation or non union; the second is an eighties with a new on precedent fracture or periprosthetic fracture. In both the untreated metabolic impairment of bones creates always a trouble to the orthopaedic surgeon because of altered mechanical resistance. So the exigency to improve the bone quality has suggested the use of an anabolic agent with a direct action on osteoblasts.

The aim of the study was to evaluate if the adjuvant therapy with teriparatide may improve the clinical outcome of complex fractures in postmenopausal osteoporotic women.

CASES REPORT: we studied a consecutive series of 7 female patients between 63 and 94 years-old presenting a complex fracture of long bones. Biochemical determinations of bone turnover and calcium homeostasis were obtained on admission and 4, 12 and 24 weeks later. Rx of affected segment was repeated at least 2, 4, 6 months. BMD was measured by DXA during hospitalization and at 6 month.

They received daily subcutaneous teriparatide (25 microg) per day, 1000 mg calcium and 400–1200 IU of vitamin D daily as oral supplementation for 6 months from 15 days by operation.

FINDINGS: Four periprosthetic of the femur, one late consolidation of neck treated with intramedullary nail, one non union of the radio-ulna treated with plaques and screws, one re-fracture of the tibia. At 6 month all patients were cured in a range of 12–20 weeks in average 14. The vitamin D was at lower levels but the supplementation was sufficient to normalize. The other biochemical variables of bone formation and resorption peaked within the consolidation process and at 6 months were indistinguishable from baseline. Subjects increased bone mass by 11.4+/-2.4% in the spine, 3.4+/-2.0% in the total hip.

CONCLUSIONS: These data show that an early adjuvant therapy with teriparatide of complicated fractures in postmenopausal women permits an bone consolidation and functional recovery. Nevertheless further studies in humans will be required to define optimal efficacy, it is intriguing the possibility to obtain the fracture healing with a bone anabolic agent.

Disclosures: C.F. Corradini, None.

M423

See Sunday Plenary Number S423

M424

Changes in 25-hydroxy- and 1,25-dihydroxyvitamin D During Treatment with Teriparatide.

F. Cosman¹, B. Dawson-Hughes², P. Chen³, J. H. Krege³. ¹Clinical Research Center, Helen Hayes Hospital, West Haverstraw, NY, USA, ²Jean Mayer US Department of Agriculture, Human Nutrition Research Center on Aging, Tufts University, Boston, MA, USA, ³Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA.

Teriparatide (TPTD) treatment improves bone density, architecture, and strength. The objective of this study was to evaluate changes in 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)₂D) in placebo-controlled TPTD trials in postmenopausal women (1)(n=1637) and men (2)(n=437) with osteoporosis. Subjects were supplemented with 400–1200 IU of vitamin D daily at least 1 month before baseline samples were obtained and then randomized to daily teriparatide 20 (TPTD20) or 40 (TPTD40) mcg or placebo. 250HD was measured at baseline and 12 months and 1,25(OH)₂D was measured at baseline, 1, 3, 6, and 12 months in a subset of subjects. Lumbar spine BMD was measured at baseline and 18 months. At 12 months, 250HD concentrations significantly declined (p<0.05 vs placebo) with TPTD20 treatment in women and men (Table). Concentrations of 1,25(OH)₂D increased significantly in both women and men (Table). Peak 1,25(OH)₂D increments occurred after 1 month with smaller increments persisting throughout 12 months of TPTD20 treatment. Results for subjects treated with TPTD40 were similar to those with TPTD20 (data not shown). Serum phosphorous did not change significantly in any group. The decline in 25OHD at 12 months and the increment in 1,25(OH)₂D at 1 month explained only 4% and 7%, respectively, of the variance in BMD response to TPTD. The mechanism for the changes in vitamin D levels may relate to TPTD-induced stimulation of 1-hydroxylase resulting in conversion of 25OHD to 1,25(OH)₂D. We conclude that treatment with TPTD decreases 25OHD and increases 1,25(OH)₂D.

Table Table.. Changes from baseline in levels of 25OHD (ng/mL) and 1,25(OH)₂D (pmol/L) in women and men treated with teriparatide

* p<0.05 compared to placebo. Results are median (IQR). 1. Neer et al. 2001 N Engl J Med 344:1434–1441. 2. Orwoll et al. 2003 J Bone Miner Res 18:9–17.

Disclosures: F. Cosman, Eli Lilly and Company 2, 5, 8.

This study received funding from: Eli Lilly and Company.

M425

See Sunday Plenary Number S425

M426

Serum Calcium Values After 4 and 24 Weeks of Treatment with Full-Length Parathyroid Hormone PTH(1–84) of Postmenopausal Women with Primary Osteoporosis: The First 125 Patients.

M. Diaz-Curiel*. Servicio de Medicina Interna. Fundacion Jimenez Diaz Madrid. Cátedra de Enfermedades Metabólicas, Unidad de Enfermedades Metabolicas Óseas, Madrid, Spain.

Background: The human recombinant parathyroid hormones (PTHs) represent a class of potent anabolic agents for the treatment of primary osteoporosis in postmenopausal women. All forms of PTH administration in this treatment increase serum calcium. However, prior clinical trials with PTH(1–84) resulted in markedly different incidences of hypercalcemia, with the incidence much higher in TOP then in PaTH. The PEAK (Preotact after a brEAK) trial employed inclusion/exclusion criteria aligned with those of the PaTH trial, therefore permitting an assessment of their impact on the incidence of hypercalcemia.

Method:The PEAK study is an open label, international, multi centre, parallel group, phase III b, randomised trial, investigating lumbar spine BMD changes in postmenopausal women with primary osteoporosis the first year of which all patients are treated with PTH(1–84). The trial will enrol 390 postmenopausal women aged more than 50 years with primary osteoporosis with a lumbar spine T score ≤ −3.0 SD. Consistent with PaTH (but not TOP), woman with documented baseline hypercalcaemia or hypercalciuria, hyperparathyroidism or severe vitamin D deficiency will be excluded from participation in the study.

Results: 125 patients had reached 4 weeks of treatment with PTH(1–84). The majority (81%) of patients had a serum calcium below the upper limit of normal. 14% had mild elevations in serum calcium. 5% was above 2.67 mmol/l and only one of these had marked elevations of serum calcium (>=3.00 mmol/l).

Fourteen patients had reached 24 weeks of treatment with PTH(1–84). The majority of those patients had a serum calcium below the upper limit of normal. 

Table  .  

Conclusion: The majority of postmenopusal women with primary osteoporosis treated with PTH(1–84) for 4 weeks had a normal total serum calcium.

Disclosures: M. Diaz-Curiel, Servier, Roche, MSD and Lilly 8; Nycomed 5.

This study received funding from: Nycomed.

M427

See Sunday Plenary Number S427

M428

A Meta-Analysis of Individual Patient Data: Significant Reduction in Non-Vertebral Fractures With High-Versus Low-Dose Ibandronate.

J. D. Adachi¹, G. Wells*², S. E. Papapoulos³, A. Cranney, for the SCIENCE Meta-analysis Group*². ¹McMaster University, Hamilton, ON, Canada, ²Ottawa Health Research Institute, Ottawa, ON, Canada, ³Leiden University Medical Center, Leiden, The Netherlands.

Ibandronate (IBN) 2.5mg daily, providing an annual cumulative exposure (ACE) of 5.5mg (0.6% oral bioavailability), significantly reduces vertebral fracture risk by 62% (p=0.0001) in postmenopausal osteoporosis and non-vertebral fracture risk by 69% (p=0.013) in high-risk patients (baseline femoral neck BMD T-score <−3).¹ Non-vertebral fracture efficacy was not shown in the overall study population who were relatively low risk.¹ Analyses of biochemical marker and BMD data obtained with high IBN ACE (≥10.8mg) predict that the licensed doses (monthly oral 150mg, quarterly i.v. 3mg [100% bioavailability]) would have significant antifracture efficacy.² To test this, individual patient data meta-analyses were used to assess the effect of different doses of IBN on non-vertebral fractures.

The analyses included all randomized, controlled trials of IBN; variable doses of IBN based on ACE were explored: 12mg, ≥10.8mg, ≤7.2mg and 5.5mg (ACE for daily oral 2.5mg). Here we present a time-to-event analysis conducted using Kaplan-Meier methodology comparing high ACEs (≥10.8mg) with lower ACEs with data taken from MOBILE and DIVA; hazard ratios (HRs) were derived from a Cox model with adjustments for clinical fracture, age, bone mineral density and study, first with the full model and then stepwise.

A significantly reduced rate of non-vertebral fractures was seen when combined high doses (ACE 12mg and ≥10.8mg) were compared with ACE 5.5mg (table). In addition, there was a dose-response effect with increasing ACE (7.2–12mg) compared with ACE 5.5mg. Similar results were seen when ACE ≥10.8mg was compared with ACE ≤7.2mg (HR: 0.634; 95% CI: 0.427, 0.943; p=0.0243). Adjustment for covariates had minimal effect.

Overall, treatment effects on non-vertebral fractures were dose dependent. A significant effect on non-vertebral fracture risk reduction was seen when IBN doses providing ACE ≥10.8mg were compared with ACEs 5.5mg and ≤7.2mg. These data indicate improved fracture efficacy for the licensed oral and i.v. doses versus oral daily dosing.

• 1.
  Chesnut CH, et al. J Bone Miner Res 2004;19:1241–9.
• 2.
  Papapoulos S, Schimmer R. Ann Rheum Dis 2007; ePub ahead of print.

Table  .  

Disclosures: J.D. Adachi, F. Hoffmann-La Roche Ltd/GlaxoSmithKline 5.

This study received funding from: F. Hoffmann-La Roche Ltd/GlaxoSmithKline.

M429

Fragility Fracture Increases Antidepressant Use among Women 65 Years of Age and Older.

J. D. Adachi*¹, N. N. Borisoy*², C. R. Purple², D. T. Gold*³. ¹McMaster University, Hamilton, ON, Canada, ²Procter & Gamble Pharmaceuticals, Mason, OH, USA, ³Duke University Medical Ctr., Durham, NC, USA.

A fragility fracture is often the initiating event in a spiral of life-style altering comorbidities; these may include depression. The objective of this study was to assess antidepressant use among women ages 65 and over before and after fragility fracture in 2 integrated administrative medical claims databases (Ingenix Lab/Rx™ and Medstat MarketScan®).

A retrospective cohort study was conducted among women with a new non-traumatic closed fracture verified with a diagnostic code between July 1, 2000 and December 31, 2003. The study included fractures at 9 anatomical sites: hip, femur, tibia/fibula, humerus, clavicle, pelvis, forearm, wrist, and spine. The cohort was observed 6 months prior and 6 months after the fracture to identify women who had antidepressant prescriptions.

A total of 19,554 women with a new fragility fracture were identified in the databases during the study period. The mean age was 79 years (SD=8). Nonvertebral fractures represented 77% of all index fragility fractures in this population. Overall, 3.7% more women were using antidepressant drugs 6 months after fragility fracture compared to 6 months before the fracture (p<0.0001). Women with a fragility fracture used 108 (SD=60) more prescriptions for antidepressants per 1,000 women after their fracture than before (p<0.0001). Specifically, women with fractures of the hip, tibia, pelvis, or spine had the highest significant increases in antidepressant use than women with other fractures (figure).

In this study, fragility fractures had a significant effect on antidepressant use. Women with a fragility fracture were more likely to use antidepressants after their fracture than before with significantly more prescriptions. The increased use of antidepressants might indicate a decreased quality of life associated with a fracture. A treatment for osteoporosis that reduces fracture incidence should reduce the cost associated with prescriptions for antidepressants as well. Therefore, this cost reduction should be considered in the cost-effectiveness analysis of treatments for osteoporosis.

Figure. Antidepressant prescriptions difference (per 1,000 fractured women): 6 months after fragility fracture compared to 6 months before the fracture

Disclosures: J.D. Adachi, Procter & Gamble Pharmaceutical 5.

M430

See Sunday Plenary Number S430

M431

Comparison of Estrogen, Raloxifene and Bisphosphonate Administration for Prevention of Bone Loss in Climacteric Women Complicated with Osteopenia.K. Aisaka, K. Nagasaka*, S. Arita*, K. Itabashi*, V. Takane*, Y. Ikezuki*, R. Matsuoka*, K. Kohda*. Obstetrics & Gynecology, Odaira Memorial Tokyo Hitachi Hospital, Tokyo, Japan.

Objective: Bisphosphonate (BIS) and SERM (Selective Estrogen Receptor Modulator, raloxifene) have been widely used for the prevention of bone loss in Japan. On the other hand, the hormone replacement therapy (HRT, using estrogens and progestogens) also performed to improve the QOL of the climacteric women even after the WHI report. The present study was performed to compare these medications for the treatment of bone loss and climacteric symptoms.

Subjects & Methods: The ethics committee of our hospital consulted on the protocol and was approved before beginning of the study. 198 patients of the climacteric women complicated with osteopenia were subjected (53.6 ± 3.9 years old). Then, the subjected patients were divided three groups at random with the enough informed consents. Group A: Administrated BIS (risedronate 2.5mg/day), 76 cases, group B: Administrated SERM (raloxifene 60mg/day), 64 cases, and group C: HRT (conjugated equine estrogen 0.625mg + medroxyprogesterone acetate 2.5mg/day), 58 cases. The changes of BMD values (DXA method), urinary NTx and plasma BAP levels were compared among them.

Results: The percent changes of BMI in the group A, B, C were 1.19 ± 0.64, 1.06 ± 0.36, 1.02 ± 0.54% (6 months after), and 1.33 ± 0.53, 1.17 ± 0.46, 1.15 ± 0.60% (12 months after), respectively, and there was a significant increase in the group A (p<0.025). The urinary levels of NTx in the group A decreased significantly by the treatment compared to another groups (0, 1, 6 months after treatment): A: 48.6 ± 15.8→33.1 ± 18.7→24.3 ± 13.7, B:50.9 ± 21.4→42.9 ± 29.6→34.5 ± 18.5, C: 47.3 ± 20.2→44.5 ± 23.8→35.7 ± 20.1 nmolBCE/nmol. cre., p<0.025–0.001). No significant changes were observed in plasma BAP levels among these three groups. The symptom of hot flush was suppressed in the group C, while increased in the group B. Conclusion: It was concluded that the inhibition effect toward bone resorption was the strongest in BIS administrated group. The improvement of the QOL in climacteric women was much better in the HRT group. From these results, BIS should be used for the patients of severe osteopenia, and the HRT should be considerable to precede the QOL of the climacteric women if they had no risks of the breast cancer.

Disclosures: K. Aisaka, None.

M432

See Sunday Plenary Number S432

M433

Risedronate Treatment in Type 2 Diabetic Men with Primary Osteoporosis: A Three-Year Longitudinal Study.

L. J. L. Ascanio*. Endocrinology, Endocrinologycal Foundations, Caracas, Venezuela.

Bisphosphonates have been widely used in the treatment of osteoporosis in women, whereas until now there have been few data on their use in men. The aim of this study was to evaluate the effect of a 3-year risedronate treatment on bone mineral density (BMD) and quantitative ultrasound (QUS) in type 2 diabetic men with primary osteoporosis. We studied 77 osteoporotic men (aged 57.1 ± 10.8 yrs) who completed a 3-year treatment with risedronate (35 mg/ once a week) plus calcium (1000 mg/day) (n = 39), or calcium alone (n = 38). At baseline and at a 12-month interval, we measured BMD at the lumbar spine and femur (femoral neck and total hip) by DXA (Hologic) and speed of sound (SOS), broadband ultrasound attenuation (BUA) and Stiffness (S) at the os

calcis by Achilles plus (Lunar). Risedronate treatment had significantly increased lumbar spine BMD by 4.2% at year 1, by 6.3% at year 2, and 8.8% at year 3. BMD at the femoral neck and total hip had increased by 2.1% and 1.6% at year 1, by 3.2% and 2.9% at year 2, and by 4.2% and 3.9% at year 3, respectively. BUA and Stiffness showed a significant increase in the risedronate-treated group at year 2 (3.2% and 4.9%, respectively) and at year 3 (3.8% and 6%, respectively). BMD at the lumbar

spine showed the best longitudinal sensitivity whereas longitudinal sensitivity of both QUS at the heel and femur BMD were similar. In conclusion, this study confirms that Risedronate represents an important therapeutic advance in the management of male osteoporosis. BMD at the lumbar spine appears to be the best method for monitoring the effect of risedronate on bone mass in osteoporotic men with type 2 diabetic.

Key words: Male osteoporosis Risedronate Bone mineral density Quantitative ultrasound

Disclosures: L.J.L. Ascanio, None.

M434

See Sunday Plenary Number S434

M435

Mechanistic Bases of Bone Mineral Density Increase During Alendronate Therapy.

D. Vashishth¹, P. Chavassieux², G. Boivin², P. D. Delmas². ¹INSERM Unite 831 Universite de Lyon France & Rensselaer Polytechnic Institute, Troy, NY, USA, ²INSERM Unite 831 Universite de Lyon, Lyon, France.

An increase in the mean degree of tissue mineralization (DMB) occurring through secondary mineralization has been proposed to increase bone mineral density (BMD) during bisphosphonate (BP) therapy [1]. In this study we conducted additional analyses on human iliac crest biopsies obtained as part of alendronate (ALN) clinical trials [2] to identify the mechanistic bases of BMD increase.

Out of a group of 16 patients on a three-year ALN-therapy [1], we identified two groups of 5 patients each showing lower (8.5%) and higher (13.3%) bounds of BMD increase but no difference in baseline BMD. For all 10 patients, previously prepared microradiographs were reanalyzed to measure the mean degree of tissue mineralization (DMB) at the osteonal and interstitial compartments in both cortical and cancellous bone tissues. Based on a moving average analysis, six fields each of cortical and cancellous bone tissues were randomly selected for measurement from each biopsy. The average values for patients in each group within osteonal and interstitial compartments were compared between and across cortical and cancellous bone tissues. All DMB measurements were also tested for correlation with standard bistomorphometric measures of bone structure (BV/TV, Tb.Th, Tb. Separation. Tb.N), osteoclast activity (EV/BV, E-Depth, Oc#/BS), osteoid (OS/BS, OV/BV, OTh), and bone formation (MAR, BFR/BS, FP) reported previously [2].

The low-BMD-gain group demonstrated no difference between the osteonal and interstitial bone compartments within cortical or cancellous bone tissues but demonstrated a higher DMB in cancellous than in cortical tissue (p<0.05). In contrast, the high-BMD-gain group showed higher DMB in interstitial than in osteonal compartment for both cortical and cancellous tissues as well as a higher DMB in cancellous than in cortical tissue (p<0.05). Out of all the DMB measures in cortical and cancellous tissues, only cortical bone interstitial level DMB correlated to bone formation rate (BFR/ BS) (r = −0.86; p = 0.006) and formation period (FP) (r = −0.75; p = 0.04).

In conclusion, this study demonstrates that the effects of ALN-therapy are more evident in cancellous than in cortical bone. Moreover since the interstitial level DMB is at least partially dependent on the duration of secondary mineralization, and negatively correlated to formation parameters, slow bone formation rate and longer bone formation period produce conditions conducive to complete secondary mineralization and consequently larger BMD gain.

References: [1] Boivin et al. Bone. 2000 5 : 687-94. [2] Chavassieux et al. JCI 1997 100: 1475-80.

Disclosures: D. Vashishth, Merck 5.

This study received funding from: INSERM, France & NIH Grants AR49635, AG20718.

M436

Bisphosphonate Use Increases in Patients with Osteoporosis Following an Integrated Osteoporosis Educational Intervention: Canadian Quality Circle (CQC) National Project.

B. Kvern¹, G. Ioannidis², A. Papaioannou², L. Thabane*², A. Gafni², A. Hodsman³, D. Johnstone⁴, L. Salach*⁵, F. Jiwa*⁶, J. D. Adachi². ¹University of Manitoba, Winnipeg, MB, Canada, ²McMaster University, Hamilton, ON, Canada, ³University of Western Ontario, London, ON, Canada, ⁴P&G Pharmaceuticals, Toronto, ON, Canada, ⁵Ontario College of Family Physicians, Toronto, ON, Canada, ⁶Osteoporosis Canada, Toronto, ON, Canada.

Quality Circles (QCs) methodology was used to improve primary care physicians' (PCPs) management of osteoporosis in accordance with the Osteoporosis Canada 2002 Guidelines. The QCs project involves a small group of people from comparable work environments who identify and analyze work related problems and recommend solutions. The study consists of five phases: wave I data collection, 1st educational intervention, wave II data collection, 2nd educational intervention, and wave III data collection. During the educational intervention, QC's met to discuss how they managed osteoporosis and to participate in an osteoporosis workshop. This interim analysis (wave I & II) evaluated the change in treatment in patients with osteoporosis (defined as a BMD t-score <−2.5). According to the guidelines, therapy should be initiated if a patient has a BMD t-score in the osteoporotic range. A total of 340 (wave I) and 301 (wave II) PCPs formed 34 QCs. For each wave, PCPs gathered data from different patients via chart reviews and a standardized collection form. A total of 8376 (wave I) and 7354 (wave II) patient records were selected at random and analyzed. All patients were women 55 years and older. The generalized estimating equations (GEE) approach was used to evaluate differences in bisphosphonate use in patients with osteoporosis pre and post educational intervention. The cluster variable for the GEE model was physician and the analysis was adjusted for patient's age, fracture status, family history of fracture, early menopausal status and other major and minor risk factors for fracture. An exchangeable correlation matrix was used for the analysis. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. A total of 1774 and 1272 patients had osteoporosis during wave I and wave II, respectively. Of these, 1545 (87.1%) during wave I and 1148 (90.3%) during wave II were treated with a bisphosphonate. The adjusted likelihood of bisphosphonate use increased following the educational intervention for patients with osteoporosis (OR 1.30; 95% CI: 1.06, 1.61). In conclusion, a majority of patients with osteoporosis are being treated with bisphosphoante therapy. However, bisphosphonate use increased in patients with osteoporosis following the educational workshop. The use of bisphosphonates may reduce the future fracture risk of these high risk patients.

Disclosures: B. Kvern, Alliance for Better Bone Health 5, 8.

This study received funding from: Alliance for Better Bone Health.

M437

See Sunday Plenary Number S437

M438

Early and Sustained Non-Vertebral Fracture Efficacy with Risedronate.

R. Lindsay¹, C. Roux*², J. D. Adachi*³, X. Zhou*⁴, A. Grauer⁴, N. B. Watts⁵. ¹Helen Hayes Hospital, West Haverstraw, NY, USA, ²Cochin Hospital, Rene Descartes University, Paris, France, ³McMaster University, Hamilton, ON, Canada, ⁴Procter & Gamble Pharmaceuticals, Mason, OH, USA, ⁵University of Cincinnati, Cincinnati, OH, USA.

Previous analyses have shown non-vertebral fractures account for 77% of the osteoporosis-related fractures and over 90% of costs. Predicting an osteoporosis-related fracture is not possible. Therefore, treating with an agent that provides both early and sustained fracture protection will provide maximum benefit to patients. The objective of this analysis is to examine whether the non-vertebral fracture efficacy of risedronate meets these criteria.

Analysis population included 1169 postmenopausal women from VERT and ROE & RON trials with low BMD (LS T-score <−2.5 SD), who were treated with at least one dose of either placebo or risedronate 5mg daily. The mean age of the population was 67 years and 58% of the patients had at least one prevalent vertebral fracture at baseline. The fracture endpoint was radiographically confirmed osteoporosis related non-vertebral fracture. Risk difference of osteoporosis-related non-vertebral fracture between the placebo and risedronate 5mg groups was estimated using the difference of the Kaplan Meier (KM) estimators from month 3 through 36. The standard error of the risk difference was approximated by the square root of the sum of the variance of the KM estimators of the placebo and risedronate 5mg groups. The upper and lower bound of the 95% confidence intervals were calculated as the mean plus and minus 1.96 times the standard error of the estimated risk difference.

The findings are consistent with the rapid onset of fracture risk reduction with risedronate treatment. Relative to placebo, risedronate significantly reduced the risk for non-vertebral fracture starting from Month 6 with an absolute risk reduction of 1.8% (95% CI: 0.4%, 3.3%). The magnitude of the difference in the risk for non-vertebral fracture between the placebo and risedronate 5mg groups increased during the first 2 years and remained constant during the 3^rd year of the trial with an absolute risk reduction of approximately 4%. In summary, the results confirmed the risedronate rapid protection benefit against non-vertebral fracture as early as Month 6. Further, the risedronate anti-fracture efficacy was sustained through out the remaining study period.

Disclosures: R. Lindsay, Procter & Gamble Pharmaceuticals 5.

M439

So How Was It? Patient Opinions on Osteoporosis Interventions in the Fracture Clinic Setting.

D. E. Beaton, E. R. Bogoch, R. Sujic*, V. Elliot-Gibson*. Mobility Program, St. Michael's Hospital, Toronto, ON, Canada.

Our study aims to understand factors that influence fragility fracture patient's adherence with St. Michael's Hospital Osteoporosis Exemplary Care Program's recommendations for further osteoporosis testing and treatment. Our previous research indicates that coordinator-based interventions are an effective way of preventing future fractures. The results of this study will help identify key variables to consider when evaluating the Ontario Osteoporosis Strategy's Fracture Clinic Screening Program which is based on the coordinator model.

This is a qualitative study using focus-group methodology. Out of 45 patients who were eligible based on the study criteria, 24 patients participated in five focus groups. Transcripts of the five focus groups were transferred to N-Vivo for storing and sorting the data. Transcripts were coded for content and links between descriptive labels were made. By using qualitative method, we are hoping to explore the impact of the Osteoporosis Exemplary Care Program in a much more contextualized fashion.

Emerging results support Anderson's behavioural model of health care utilization. Perceived need and susceptibility were associated with osteoporosis prevention efforts and treatment adherence as reported by the patients involved in the focus groups. The most frequent barriers that patients identified in diagnosis and treatment of osteoporosis included the perceived lack of clear and reliable information regarding the nature of BMD testing and proper treatment as well as the lack of general practitioner's recommendations about osteoporosis care and prevention. Most often cited facilitators of osteoporosis testing and treatment included thorough follow up by an osteoporosis coordinator, accessibility and ease of BMD testing and general practitioners' recommendations for treatment and testing.

General practitioners were named as key influence over the initiation of prevention and treatment of osteoporosis.

Disclosures: D.E. Beaton, None.

M440

See Sunday Plenary Number S440

M441

Improvement of the Persistence with Teriparatide in Postmenopausal Osteoporosis: The French Experience of an Education Program.

K. Briot¹, P. Ravaud*², S. Liu-Léage*³, C. Roux¹. ¹Rheumatology, Cochin Hospital, Paris, France, ²Biostatistics and Epidemiology, Bichat Hospital, Paris, France, ³Lilly France, Suresnes, France.

Several anti-osteoporotic treatments have been proved effective in decreasing the risk of fractures but available data suggest low adherence and persistence rates in patients taking medications for osteoporosis. This can result in failure in treatment efficacy. Several strategies have been developed to improve adherence and persistence. Teriparatide, prescribed 20 μg/day injected subcutaneously, is an anabolic treatment licensed for established postmenopausal osteoporosis. Education program has been developed after its launch, to help elderly women to deal with the pen take and consequently better follow the prescribed treatment. The objective is to assess the efficacy of an education program to improve the persistence with teriparatide in postmenopausal osteoporotic women. This education program has been created by the promotor of teriparatide since its launch of teriparatide in France in September 2004 and it is proposed to each woman who begins the teriparatide. The program includes injection technique learning, osteoporosis education, observance and persistence assessment. Women are interviewed by regular calling of a nurse, each month the first year and every 3 months the 6 following months. Data about persistence and side-effects are available for the period September 2004 to December 2006. Persistence is defined as the percentage of patients still on treatment at the end of the 18-month course. Since the launch of teriparatide in France in September 2004, 4518 postmenopausal women (mean age 73.6 ± 11.4 years) with osteoporosis (lumbar spine and/or femoral T score ≤-2.5) and vertebral fractures (4 fractures on average) have participated to the program. At the end of the year 2006, 1951 women have been followed at least 18 months. Of these 1951 women, persistence at 18 months was 82.6%. Main reasons for discontinuation, as declared by the patients, were side-effects (43.8%), wish of the patient (23.8%), physicians' decisions (17%) and deaths (5.3%). Persistence has been compared to the data of the French universal health insurance system; and it has been estimated that persistence at 18 months was closed to 0% for women who have been prescribed teriparatide without any education program. This study shows that an education program can highly improve the persistence with teriparatide at 18 months. Persistence is greater than that of existing oral therapies for osteoporosis, and this high persistence should improve the effectiveness of this therapy.

Disclosures: K. Briot, None.

M442

The Impact of GI Medication Usage with Regard to Discontinuation and Restart Behavior with Oral Bisphosphonates in Three Large US Physician Groups.

M. B. Nichol*¹, W. W. Chan*², T. Dow*¹, K. H. Kahler², S. Bamford*³, L. D. Marks*⁴, C. Parise*⁴, G. Darah*⁵. ¹JMRG Consulting, Encino, CA, USA, ²Novartis Pharmaceuticals Corp., East Hanover, NJ, USA, ³Physician Associates, Pasadena, CA, USA, ⁴Sutter Medical Group, Sacramento, CA, USA, ⁵ProMedica, Toledo, OH, USA.

Purpose: Clinical studies have demonstrated that women at risk of osteoporotic fracture reduce the risk of new osteoporotic fracture with bisphosphonate treatment. However, observational studies have also shown high discontinuation rates with these oral medications. This study investigated the impact of GI medication usage with regard to discontinuation and treatment re-initiation.

Methods: The study was based on a retrospective claims review of data from three large US physician groups. The groups represent a diverse patient base in terms of geographic and organizational considerations. Specifically, the groups are based in Northern and Southern California and Ohio; one is a network of independent physicians in private practice, and two are not-for-profit integrated health care networks. We evaluated the integrated administrative dataset for health plan eligibility as well as drug utilization information.

Results: 5,737 patients were selected for analysis. 4,074 patients (71.0%) initiated therapy on alendronate, and 1,663 patients (29.0%) initiated therapy on risedronate. One-third of women were aged 45 to less than 60, 40% were aged 60 to less than 75, with the remainder 75 and older, with no statistically significant differences by index medication. Approximately 20% of women used GI medications during the one year study period. Duration of GI therapy averaged 176 days (SD 126), with statistically significant differences seen by drug when the medical groups were combined (p = 0.017). Specifically, risedronate initiators averaged 194 days of GI therapy (SD 125) and alendronate initiators averaged 174 days (SD 127). Women typically added GI treatment after approximately three months (mean 93, SD 100) for both medications. Logistic regression analysis showed concurrent GI use was associated with a 74% greater likelihood of discontinuation with bisphosphonate treatment (95% CI: 1.36, 2.22) and a decreased likelihood of re-initiation of bisphosphonate treatment (Odds Ratio 0.76, 95% CI: 0.51, 1.14).

Conclusion: These data revealed that approximately 20% of bisphosphonate users were on GI therapy during the study period. However, rather than acting as a mitigating agent that was associated with better compliance, it appears that GI usage reflected side effect problems that jeopardize persistence. This area deserves further study to determine the direction of cause and effect.

Disclosures: W.W. Chan, Novartis Pharmaceuticals Corp. 1, 3.

This study received finding from: Novartis Pharmaceuticals Corp.

M443

See Sunday Plenary Number S443

M444

Longitudinal Patterns of Adherence with Bisphosphonates.

J. R. Curtis¹, A. Westfall*¹, H. Cheng¹, E. Delzell*¹, K. Lyles², K. G. Saag¹. ¹University of Alabama at Birmingham, Birmingham, AL, USA, ²Duke University, Durham, NC, USA.

Background: oor adherence to bisphosphonates (BPs) appears associated with a greater rate of fracture (Fx). Misclassification of adherence and the impact of acute Fx on adherence may compromise interpretation of findings. We examined longitudinal patterns of BP adherence using less restrictive definitions than in some past studies that included an arbitrary definition of discontinuation. We also determined the impact of adherence associated with hip Fx.

Methods: Using claims data of a large consolidated health plan database, we identified persons initiating BPs and calculated adherence as a Medication Possession Ratio (MPR), allowing for BP switching. We compared adherence among individuals initiating weekly vs. daily BPs. MPR was examined at 1,2, and 3 yrs and was compared among those with no fracture or with hip Fx. We also examined MPR in the 3 and 6 months immediately prior to a hip Fx compared to the 3 and 6 months following this Fx to determine how measured adherence changed following a Fx.

Results: We identified 87,099 new BP users with ≥ 1 year of follow-up. Mean ± SD age was 60.1 ± 7.8 years. The proportion with MPR ≥ 80% was 44% (Figure), 39%, and 36% at 1, 2, and 3 yrs, respectively. Among persons with MPR ≥ 80% at yr 1, 80% of these had MPR ≥ 80% at yr 2. Those who were initially prescribed weekly BPs had higher 1 yr MPR than those initially prescribed daily BPs (mean = 45% vs. 38%, p < 0.001).

Among those with a hip Fx, MPR at years 1, 2, and 3 was 9%, 12%, and 10% lower compared to those without Fx (p < 0.001 for each), and MPR in the 3 and 6 months following the hip Fx was −9% and −6% compared to before the hip Fx (p < 0.0001 and < 0.0001).

Conclusions: Using a definition of adherence that allowed for switching to different dosages or formulations of BPs, adherence was somewhat better than in past studies but remained suboptimal. Adherence at year 1 was a strong predictor of adherence in subsequent years. Adherence in the 3 and 6 months following a hip Fx was significant lower than immediately prior to the fracture, which may be real or an artifact of a lack of pharmacy data capture during periods that patients are hospitalized or in rehabilitation settings. Analyses that fail to consider adherence in a time-dependent fashion, before Fx occur, may magnify the apparent differences in Fx risk between adherent and non-adherent persons.

Disclosures: J.R. Curtis, Merck 2, 5, 8; Proctor & Gamble 5, 8; Novartis 2: Amgen 2.

This study received funding from: Novartis.

M445

A Year-long Study to Demonstrate the Bone Reversal Efficacy of Dried Plums in Postmenopausal Women.

B. H. Arjmandi¹, S. Hooshmand¹, S. C. Chai¹, R. L. Saadat*¹, L. Devareddy*¹, K. Brummel-Smith*². ¹Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL, USA, ²Department of Geriatric, Florida State University, Tallahassee, FL, USA.

Our findings in rat models of osteoporosis as well as a short-term human study suggest that dried plums are highly effective in preventing and reversing ovarian hormone-deficiency-associated bone loss. Dried plums are rich in polyphenolic compounds and other nutrients that contain high antioxidant properties. Antioxidative properties of dried plum, in part, may be responsible for prevention of bone loss that is in part due to the rise in oxygen-derived free radical formation. It is necessary to confirm the findings of animal studies as well as positive effects of dried plum on biomarkers of bone formation in a relatively long-term clinical study. For this purpose, we are conducting a completely randomized comparative-controlled study to test the hypothesis that daily consumption of dried plum increases bone mineral density (BMD) of osteopenic women 1 to 10 years postmenopausal. So far, we have recruited 85 (n=144) subjects who are not on hormone replacement therapy and have been randomly assigned to one of two treatment groups: dried plum (100 g/d) or dried apple (comparative control). All study participants are receiving 500 mg elemental calcium plus 250 IU vitamin D daily. BMD and bone mineral content (BMC) of lumbar spine, forearm, hip and whole body are being assessed at baseline and at the end of the study using dual-energy x-ray absorptiometry. Blood and 24-hr urine samples (baseline, 3-, 6-, and 12-month) are also assessed. Physical activity and dietary confounders will be examined as potential covariates. This study will provide critical data about an acceptable and efficacious food alternative to reverse bone loss in menopausal women. Based on our preliminary data, we anticipate subjects that are consuming dried plum will have an average gain of 3% BMD at all sites after one year. The urinary and blood interim analyses will be presented at the time of poster presentation.

Disclosures: B.H. Arjmandi, None.

M446

Analgesic Efficacy of Calcitonin for Vertebral Fracture Pain.

A. Bazarra-Fernandez*. ObGyn, Juan Canalejo University Hospital Trust, Amparo Lopez Jean 134° A La Coruña, Spain.

Background: Fractures, especially vertebral fractures, are a common complication of osteoporosis, leading to significant pain.

Aim: To compare the pain release induced by osteoporotic vertebral fracture, through teriparatide and teriparatide plus calcitonine.

Methods: We performed a study to compare the analgesic effect of 20 mcg teriparatide versus 20 mcg teriparatide plus metered dose intranasal spray 200IU/activation calcitonin in two groups between postmenopausal women undergoing osteoporosis with vertebral fracture. A 10-point visual analog pain scale (1 = least to 10 = most painful) and a four-point pain grade (grade 1 = least to grade 4 = most painful) were used to measure the pain perception.

Results: The mean pain scores for the teriparatide and teriparatide plus calcitonin were 2.3 ± 1.1 and 8.5 ± 1.1, respectively (P<0.05), while the pain grades for teriparatide and teriparatide plus calcitonin were 1.5 ± 0.3 and 3.5 • 0.4, respectively (P<0.05). In teriparatide group, analgesics were requested, but in teriparatide plus calcitonin group no analgesics were requested (P<0.001).

Conclusion: Using teriparatide is more expensive than other osteoporosis treatment. Studies show that taking a bisphosphonate with hormone replacement therapy (HRT), results in increased bone mass when compared to taking either a bisphosphonate or estrogen alone. Besides, calcitonin is better for osteoporotic vertebral fracture pain release than HRT. However, a larger investigation will be needed to achieve more significant case number.

Disclosures: A. Bazarra-Fernandez, None.

M447

See Sunday Plenary Number S447

M448

The Correction of BMD Measurements for Bone Strontium Content.

G. M. Blake. Nuclear Medicine Department, King's College London School of Medicine, London, United Kingdom.

Strontium ranelate is a new oral treatment for osteoporosis associated with large increases in bone mineral density (BMD) compared with alternative therapies such as bisphosphonates. Much of the BMD increase during strontium ranelate treatment is a physical effect caused by the increased attenuation of X-rays due to the accumulation of strontium in bone tissue. The aim of this study was to assess the contribution made by bone strontium content (BSC) to the overall BMD increase by evaluating the percentage F of the BMD change explained by the physical presence of strontium in bone. A value of F less than 100% would provide evidence of the anabolic effect of strontium ranelate as an additional factor contributing to the overall BMD increase. Studies of mixtures of strontium hydroxyapatite (SrHA) and calcium hydroxyapatite (CaHA) scanned on a variety of dual-energy X-ray absorptiometry (DXA) systems show that a 1% molar ratio of SrHA/(CaHA+SrHA) causes a 10% overestimation of BMD. The correction of spine BMD measurements for the physical effects of strontium depends on knowledge of two further factors: (1) bone biopsy measurements of iliac crest BSC; (2) the ratio R of BSC at the DXA site to BSC at the iliac crest measured in animal studies. We used clinical trial data and values of R_spine measured in studies of monkeys and beagle dogs to determine values of F_spine for 1,2 and 3 years treatment with strontium ranelate. Based on the average value of R_spine = 0.7 for male and female monkeys we found values for F_spine = 75%-80% for 1, 2 and 3 years of treatment. Using the value of R_spine = 1.0 from the beagle study gave values of F_spine = 100%. Although values of F_spine as low as 40% are possible, we conclude that the most likely figure is 75% or greater. However, it is apparent that there are large uncertainties in the correction of BMD results for the effect of bone strontium and that the most important of these is the inference of BSC values at DXA sites from measurements of bone biopsy specimens.

Disclosures: G. M. Blake, None.

M449

Calcium Supplementation Improves Lipid Profile But Does Not Decrease the Incidence of Cardiovascular Events in Postmenopausal Women.

M. J. Bolland*, B. Mason*, A. Horne*, R. Ames*, A. Grey, G. Gamble*, R. Doughty*, A. Barber*, I. R. Reid. Department of Medicine, University of Auckland, Auckland, New Zealand.

Previously we have shown that calcium supplementation produces beneficial changes in lipids that might impact upon the incidence of cardiovascular disease. We set out to determine the effect of calcium supplementation on myocardial infarction (MI), stroke, sudden death, and lipids in post-menopausal women.

We carried out a 5 year randomized, placebo-controlled trial of 1g daily calcium citrate supplementation in 1471 post-menopausal women (mean age 74y). MI, stroke, and sudden death events were recorded during the study. Hospital admissions for cardiovascular events were also obtained from a national database. All events were adjudicated by a physician and a cardiologist or neurologist. Serum lipids were measured annually in a substudy of 223 women.

There were increases in the number of women in the calcium group who during the study reported MI (32 vs 14, P = 0.007), stroke (42 vs 38, P = 0.19), and the composite endpoint of MI, stroke or sudden death (70 vs 45, P = 0.015). After adjudication of events and identification of additional unreported events from the national database, the increases in number of women in the calcium group with MI (31 vs 21, P = 0.16), stroke (34 vs 25, P = 0.23), and the composite of MI, stroke or sudden death (60 vs 50, P = 0.32) persisted but were not statistically significant. The relative risk of MI was 1.2 (1.0–1.5), stroke 1.2(0.9–1.6), and the composite of Ml, stroke or sudden death 1.1 (0.9–1.3). There were trends toward an increased rate of MI in the calcium group (11.1 vs 6.6 /1000 patient-years, P = 0.06), stroke (11.4 vs 7.8 /1000, P = 0.18), and the composite of MI, stroke, or sudden death (23.2 vs 16.3 /1000, P = 0.05).

There were trends toward a greater decrease in LDL from baseline in the calcium group over 5 years (9.5% vs 6.2%. P = 0.05) and a greater increase in HDL (10.4% vs 9.8%, P = 0.11). There was a greater increase in the HDL:LDL ratio over 5 years in the calcium group (P = 0.015).

In summary, 5 years of calcium supplementation, lg daily, in postmenopausal women improved lipid profile but did not reduce the number of cardiovascular events. In fact, there were trends towards an increase in the rate of cardiovascular events in women receiving calcium.

Disclosures: M.J. Bolland. None.

This study received funding from: Health Research Council of New Zealand.

M450

The Effects of UV-B Light on Serum Vitamin D Levels in Humans.

L. A. G. Annas¹, R. P. Heaney¹, M. J. Barger-Lux*¹, C. Huerter*², R. Lund*³. ¹Osteoporosis Research Center, Creighton University, Omaha, NE, USA, ²Dermatology Department, Creighton University, Omaha, NE, USA, ³Nephrology Department, Creighton University, Omaha, NE, USA.

We report results of vitamin D levels after 4 weeks of exposure to graded doses of UV-B light delivered by a light booth. The subjects (n = 69, age 19–49 yr, females = 41, males = 28) were healthy indoor workers with limited non-solar sources of Vitamin D.

Data were gathered from September through June for 2 consecutive years (71% completed the study from January through May). We determined BMI, 25(OH)D, vitamin D, Ca²⁺ and PTH at baseline. The subjects had 90% of their body exposed to broad-band UV-B light from a UV light booth 3 times a week for 4 weeks (12 treatments) in doses ranging from 20mJ to 80mJ per treatment. Serum Vitamin D was drawn at baseline, after 4 weeks of UV-B treatment, and again 4 weeks after completion of UV-B treatment.

At baseline, 80% percent of the subjects had undetectable vitamin D levels. The subjects' median serum vitamin D level was 0 nmol/L (0, 0 interquartile range). At 4 weeks, after 12 treatments with UV-B light, the median vitamin D level was 31.0 nmol/L (19.0, 42.5 interquartile range). Four subjects' vitamin D levels remained at 0 after UV-B treatment despite a median rise in their 25(OH)D levels of 26.6 nmol/L.

At week 8, the median vitamin D level decreased to 0 nmol/L (0, 5.9 interquartile range) with 57% of subjects' levels reaching 0 nmol/L.

The vitamin D response to UV-B light correlated positively with dose of UV-B light and the 25(OH)D level at week 4 and inversely correlated with age, body surface area, and body mass index.

In conclusion, serum vitamin D levels increase in response to UV-B light, but the body needs constant vitamin D input to maintain vitamin D levels. The serum vitamin D levels are quickly depleted by either storage by the body or conversion by the liver to 25(OH)D.

Disclosures: L.A.G. Armas. None.

This study received funding from: Dialysis Clinics, Inc., Endocrine Fellows Foundation.

M451

Daily Versus Monthly Oral Vitamin D2 and D3: Effect on Serum 2SOHD Concentration.

N. Binkley, D. Gemar, R. Ramamurthy, D. Krueger, M. K. Drezner. University of Wisconsin Osteoporosis Research Program, University of Wisconsin-Madison, Madison, WI, USA.

It is widely assumed that ergocalciferol (D2) is less effective than cholecalciferol (D3) in maintaining normal serum 25 hydroxyvitamin D [25(OH)D] levels. However, data supporting this belief remains limited. The common suboptimal adherence to daily supplements confounds this problem further since clinicians may elect to utilize high-dose vitamin D supplementation monthly. As only ergocalciferol is available as a high-dose prescription preparation in the US, evaluation of intermittent high-dose D2 supplementation on 25(OH)D status is required. Moreover, the logical hypothesis that appropriate timing of 25(OH)D measurement is required, such that a trough 25(OH)D value is obtained when dosing monthly, has not been tested. With these related considerations in mind, the purpose of this year long randomized, double-blind, placebo-controlled prospective clinical trial is to evaluate the effect of daily dosing of 1,600 IU D2 or D3 versus monthly dosing of 50,000 IU D2 and D3 on serum 25(OH) D concentration and concurrently to investigate the potential importance of obtaining trough 25(OH)D levels in adults over age 65 years. Study inclusion criteria include community dwelling men and women age > 65 years with a serum 25(OH)D concentration between 10–60 ng/ml who were willing to use sunscreen when exposed to the sun. Those with hypercalcemia, hypercalcuria, renal failure, use of drugs interfering with vitamin D metabolism or a history of nephrolithiasis were excluded. Serum 25(OH)D was measured by reverse phase HPLC. At this time, 20 participants, age 74.3 ± 1.8 years (mean/SEM) have been randomized. At baseline, their serum 25(OH)D concentration was 31.1 ± 2.2 ng/ml. After one week of study, serum 25(OH)D concentration increased (p < 0.001) by 8.6 ± 0.6 ng/ml and 6.7 ± 1.4 ng/ml at three and seven days following receipt of 50,000 D3, but was not different from baseline in those receiving daily therapy with D2 or D3 or monthly D2. Our preliminary observations support the possibility that therapy with D3 may be more effective. However, as this study is ongoing, additional data, including the effect of intermittent monthly dosing, and the impact of specimen acquisition time relative to monthly dosing of D2 and D3, on serum 25(OH)D concentration will be reported.

Disclosures: N. Binkley, None.

M452

See Sunday Plenary Number S452

M453

Effect of Alfacalcidol on Volumetric Bone Mineral Density Measured by pQCT in Alendronate-Treated Postmenopausal Women with Osteopenia or Osteoporosis: 1 Year Interim Analysis of the ALFA Study.

O. Bock¹, H. Boerst*¹, M. Runge*², G. Beller*¹, F. Touby*¹, J. Tuerk*², P. Martus*³, E. Schacht*⁴, J. Hashimoto*⁵, D. Felsenberg¹. ¹Centre for Muscle and Bone Research, Charité — Campus Benjamin Franklin, Berlin, Germany, ²Centre for Muscle and Bone Research, Aerpah Kliniken Esslingen-Kennenburg, Esslingen, Germany, ³Institute of Biostatistics and Clinical Epidemiology, Charité — Campus Benjamin Franklin, Berlin, Germany, ⁴ZORG — Zurich Osteoporosis Research Group, Zurich, Switzerland, ⁵Bone Disease Area Department, Chugai Pharmaceutical Co. Ltd., Tokyo, Japan.

The purpose of the ALFA (Alfacalcidol & Falls) study (three-year prospective, randomized, double-blind, placebo-controlled trial) is to evaluate the effect of alfacalcidol 1 μg daily on the number of falls in postmenopausal, alendronate-treated, osteopenic or osteoporotic women. A pre-planned one year interim analysis was performed to examine the effect of alfacalcidol on bone mineral density (BMD) by DXA and pQCT.

A total of 278 postmenopausal women (mean age 73.7 years, SD 4.8) received either alfacalcidol 1 μg or placebo daily, in addition to alendronate 70 mg weekly and calcium 500 mg daily. Volumetric BMD (vBMD) at standardized sites of radius and tibia were measured by pQCT (Stratec XCT-2000) at baseline and after 3, 6, 9 and 12 months of treatment.

Baseline characteristics of patients, including age, BMI, bone markers, BMD at lumbar spine (mean T-Score −2.33 vs. −2.40 SD) and hip (mean T-Score −1.40 vs. −1.45 SD) were not significantly different between the two groups. 

Table  .  

A slight, but significant increase of cortical vBMD at the mid shaft radius and the 14% and 38% measurement sites of the tibia was detectable after one year of additional alfacalcidol medication versus placebo in alendronate treated patients. Similar results were found for the trabecular vBMD at the tibia. Results were not significantly different for trabecular vBMD at the radius and cortical vBMD at the 66% measurement site of the tibia.

For the first time, our data showed an additional, potentially beneficial, effect of alfacalcidol 1 μg daily in alendronate-treated postmenopausal women not only on lumbar spine BMD measured by DXA (as reported earlier), but also on several volumetric BMD parameters at the appendicular skeleton. Their long-term change and possible impact on bone strength of the peripheral skeleton will be monitored in the actually ongoing study for three years.

Disclosures: O. Bock, None.

This study received funding from: Chugai Pharmaceutical Co., Ltd (Japan), GRY-Pharma GmbH (Germany) and Teva Pharmaceutical Industries (Israel).

M454

Bone Mineral Density (BMD) and Serum Vitamin D Levels in Patients with Chronic Obstructive Pulmonary Disease (COPD) Without Glucocorticoid Use.

C. Franco*¹, C. A. M. Kulak¹, P. Gomes*¹, S. C. Radominski², V. Z. C. Borba¹, C. L. Boguzewski*¹. ¹Endocrinology, UFPR, Curitiba, Brazil, ²Reumathology, UFPR, Curitiba, Brazil.

The evaluation of BMD in patients with COPD is restricted to those in glucocorticoid use or with risk factors. It is not known if COPD per se affects bone mass and increases the risk of fracture. The purpose of this study was to evaluate the BMD, vitamin D levels and spirometric parameters in a group of COPD patients without systemic glucorticoid (GC) use. We examined 28 women and 21 men (65,4 ± 9,2 years old) with COPD without chronic systemic GC use. Age below 25 years old and patients with any other cause for osteoporosis were excluded. Patients answered to a questionnaire and were submitted to DXA (Hologic QDRW1000) of lumbar spine and femur. The diagnosis of COPD was done by spirometry and blood was collected for laboratory exams. Past history of fractures were present in 22, 4%. The lumbar spine BMD (g/cm²) was 0,794 ± 0,169, femoral neck 0,704 ± 0,143, total femur 0, 78 ± 0, 15. Low bone mass was presented in 79,6% and 51% presented osteoporosis. There was a correlation between spirometry and BMD: lumbar (R = 0, 38 p <0, 01), femoral neck (R = 0, 40 p = 0, 01), total femur (R = 0, 36 p <0, 01). The mean levels of 25 OH vitamin D (25-OHD) was 20,8 ± 1,0 ng/dl, (28, 6% were 25-OHD insufficient; 6% were 25-OHD deficient and 67% had secondary hyperparathyroidism). Patients with oxygen saturation <88% presented lower 25-OHD levels, and who stopped smoking > 49 months had better BMD. In regression analysis, weight, current smoking, sex and time without smoking were related to the worsening of BMD (R² = 0,7) These data suggest that COPD patients present low bone mass, vitamin D insufficiency and increased fracture risk, independent of the GC use.

Disclosures: V.Z.C. Borba, None.

M455

Remodelling of Bone Microarchitecture in Aging Mice Is Associated with Changes in Ephrins Gene Expression.

E. N. Bianchi*, R. Rizzoli, S. L. Ferrari.. Div. of Bone Diseases, Geneva University Hospital, Geneva, Switzerland.

Continuous bone remodelling leads to prominent losses of trabecular microarchitecture in aging C57BL/6J mice, which mimics the deterioration of bone structure observed in post-menopausal women. The molecular mechanisms involved in this process however remain poorly understood. Binding of ephrin (Efn) B2 to the ephrin receptor (Eph) B4 was recently reported to induce stimulation of osteoblast differentiation and inhibition of osteoclastogenesis. We hypothesized that a decline in ephrins expression could be implicated in the changes of bone microarchitecture observed in aging mice. For this purpose, female C57BL/6J mice were sacrificed at 4 and 12 months of age, and trabecular (Tb) and cortical (Ct) bone microarchitecture analyzed by microCT (Scanco40) at distal and midshaft femur, respectively. RNA was extracted from the same regions of interest, i.e. the diaphysis (Dia) after flushing of the bone marrow and the metaphysis-epiphysis (Met) of femur. Expression levels of Efn B1 and B2 and Eph A3, A4, B2, B3 and B4 genes were determined by quantitative RT-PCR (TaqMan MGB probes).

Tb BV/TV decreased by 60% between 4 and 12 months of age (p = 0.0001) associated with a decline in Tb number and connectivity, and an increase in Tb separation (all p<0.001). Meanwhile, Ct volume increased by 20–30%. Overall, Efn and Eph expression levels were higher in Met than Dia and in young compared to older mice. More precisely, expression of EfnB2 decreased by 65% (p<0.0001) in Met and 40% (p<0.02) in Dia with aging, whereas its receptors Eph B2, B3 and A4 decreased by 40% to 70% (p<0.05) in both compartments. In contrast, significant down-regulation (-35% to −50%, p<0.02) of EfnB1 and EphB4 occurred specifically in Met, but not in Dia. We then investigated expression of additional genes associated with osteoclast (OPG, RANKL and TRAP) and osteoblast/osteocyte activity (IGF-1 and SOST). OPG RANKL and TRAP were more prominently expressed in Met than Dia (p<0.005), and both the RANKL/OPG ratio and TRAP gene expression were significantly down-regulated with age. In contrast, IGF-1 and SOST mRNA levels were twice as high in Dia than Met, and decreased only marginally with age in Dia.

In summary, reciprocal changes in bone geometry and trabecular content and distribution occur in the femur of aging mice. Neither the changes of OPG RANKL and TRAP, not those of IGF-1 and SOST can explain the age-related decline of trabecular microarchitecture. However, the decline of ephrins and ephrin receptors gene expression, particularly of EfnB1 and EphB4 in the Tb compartment, might favor uncoupling of bone formation from resorption and remodelling of skeletal microarchitecture.

Disclosures: E.N. Bianchi, None.

M456

Low Vitamin D Level and High Bone Turnover in Aged Type 2 Diabetic Patient.

P. Chung., J. Chung*, D. Myung*, D. Cho*, M. Chung*. Internal Medicine, Chonnam National University Medical School, Gwangju City, Republic of Korea.

Bone mineral density in type 2 diabetic patient is known to be variable; such as increased, decreased, or not different from normal subject. Levels of bone turnover markers are also conflicting while several histomorphometric data shows low bone turnover status in diabetic bone. However, recent epidemiologic data showed that osteoporotic fracture rate is increased in type 2 diabetes even in patients with normal or high BMD possibly due to poor bone quality. Bone metabolism in diabetes is thought to be affected by many factors including glycemic control status, presence of late chronic complications, disorder of calcium and vitamin D metabolism. In this study, we investigated the vitamin D status and bone turnover rate in 53 type 2 diabetic postmenopausal women (69.9 ± 8.l years, Hbalc was 8.2 ± 2.3%) and 34 non-diabetic postmenopausal women (66.8 ± 8.4 years).250HD, serum CTX and other laboratory data were aquired after overnight fasting. BMD was measured at the lumbar spine and femur using dual energy X-ray absorptiometry (Lunar Prodigy Advance). Body mass index and years since menopause were not different between two groups. The 250HD level was significantly lower (p = 0.001) in diabetic women (23.l ± 11.4 ng/ml) compared to normal subject (32.6 ± 12.3 ng/ml). The serum CTX was significantly higher (p = 0.013) in diabetic women (0.61 ± 0.42 ng/ml) compared to non-diabetic women (0.39 ± 0.29 ng/ml). However, the BMD at the spine and femur were not significantly different between two groups. These results suggest low serum vitamin D level and high bone turnover in aged type 2 diabetic patient may contribute to increased probability of osteoporotic fracture.

Disclosures: D. Chung, None.

M457

Multifactorial Etiology of Weak Bones in Type 1 Diabetes (T1DM).

K. K. Danielson*¹, M. E. Elliott², M. Palta*³. ¹Section of Pediatric Endocrinology, University of Chicago, Chicago, IL, USA, ²Pharmacy Practice Division, University of Wisconsin, Madison, WI, USA, ³Population Health Sciences and Biostatistics & Medical Informatics, University of Wisconsin, Madison, WI, USA.

Our research has demonstrated bone mineral density (BMD) and bone formation to be lower in women with T1DM than in matched controls. We also found that the earliest etiologic effects of T1DM on bone appear at the level of bone turnover. We therefore attempted to delineate the potential pathophysiologic mechanisms altering bone turnover in T1DM. The study included premenopausal women with T1DM enrolled in the Wisconsin Diabetes Registry (n = 89). Each participant completed questionnaires, a physical exam, and blood draw to measure HbA1c, parathyroid hormone (PTH), insulinlike growth factor 1 (IGF-1), vitamin D, estradiol, osteocalcin and NTx. An Uncoupling Index (UI) was calculated as the difference between osteocalcin and NTx z-scores. Urinary albumin excretion (UAE; n = 61) was collected as a measure of kidney function during the three years prior to the study (mean = 1.7 years). The study was approved by the University of Wisconsin institutional review board. Mean age was 28 years (18–45 years) and 98% were Caucasian. Mean disease duration was 16 years and current mean HbA1c was 8.1%. Only 9.8% demonstrated early kidney impairment with elevated UAE (≥20 μg/min). A negative UI was exhibited by 66.3% of the sample, indicating bone resorption was outpacing formation. The final multivariable regression model for bone formation demonstrated several potential etiologic factors to be independently associated with osteocalcin (ng/ml): HbA1c (%; β = −0.3), PTH (10 pg/ml; β = 0.4), IGF-1 (10 ng/ml; β = 0.1), and logUAE (μg/min; β = 0.3). The HbA1c coefficient did not attenuate when PTH, IGF-1, or UAE was added to the final model indicating they did not mediate the association between glycemic control and bone formation. The final model for bone resorption (NTx [nmol BCE/L]) also demonstrated a positive association with logUAE (β = 1.6). Lastly, the UI (z-score) was independently associated with PTH (β = 0.2) and IGF-1 (β = 0.1). In conclusion, uncoupled bone turnover in T1DM, encompassing low formation and net resorption, appears to be related to the poor glycemic control and low IGF-1 and PTH levels associated with T1DM. The progression of kidney disease, even the early stages as detected in our sample by slightly elevated UAE, may then accelerate this uncoupled bone turnover. These factors may eventually culminate in the low BMD and increased fracture risk seen in T1DM. Fortunately, this multifactorial process provides several potential targets for intervention.

Disclosures: K.K. Danielson, None.

This study received funding from: American Diabetes Association.

M458

Relationship of Bone Turnover Markers, Ovarian Hormones and Cytokines During Menopause.

M. P. Desai*¹, V. R. Taskar*², M. Khatkhatay*¹. ¹Molecular Immunodiagnostics, National Institute for Research in Reproductive Health, Mumbai, India, ²Clinical, Streehit Karini, Mumbai, India.

Estrogen influences bone remodeling, by modulating the secretion of cytokines. Its deficiency at menopause causes accelerated bone loss and subsequently osteoporosis. The aim of the study was therefore to characterize the changes in cytokines and ovarian hormonal profiles and correlate with bone turnover markers and bone density. This would aid in identification of women at risk for osteoporosis and preventive therapy could be initiated. We studied a cross-sectional age stratified population sample of 124 premenopausal women (age: 21–40yrs), 68 perimenopausal women (age: 36–44 yrs) and 188 postmenopausal women (age: 41–75 yrs) not using any oral contraceptives or on hormone replacement therapy. Serum calcium, phosphorus and PTH were estimated to assess the bone homeostasis while bone mineral density (BMD) of lumbar spine and femoral neck was measured by DEXA. The women in the groups were classified into normal, osteopenic and osteoporotic based on BMD measurements and their ovarian hormonal status. Urinary estrone glucuronide (E₁G) and follicle stimulating hormone (FSH) were estimated by in-house assays, whereas serum interleukins (IL1, IL6 and TNFα) by optimized EIA assays. The bone markers such as Osteocalcin (OC), bone specific alkaline phosphatase (BSAP), urinary type 1 collagen telopeptide (CTx) and pyridinium crosslink de-oxypyridinoline (Dpd) by ELISA kits. The perimenopausal women showed 30% higher CTx excretion (528 ± 126 μg /mol Cr, p<0.01), 28% higher Dpd levels (5.02 ± 1.20 nMol / mol Cr, p<0.01), increased levels of urinary FSH (38.6 ± 3.84 m IU / mg Cr, p<0.0001) and 1L6 (11.68 ± 2.61 ng/ml, p<0.05) with osteopenic changes on BMD whereas E₁G, bone formation markers and BMD though low did not show any significant change. In the osteopenic menopausal women all the bone markers were significantly elevated indicating increased rate of bone remodeling. Of the three cytokines, IL6 increased significantly (15.85 ± 3.5 ng/ml, p<0.0001) correlating well with increased bone turnover (CTx 1126 ± 362 μg /mol Cr and Dpd 8.49 ± 2.65 nM/ mol Cr) while there was marginal increase in TNFα levels (6.5 pg/ml; p < 0.0056) only in the osteoporotic women. A significant drop in E₁G (8.99 ng/mgCr; p<0.0001) and BMD measurements with rise in IL 6 was also observed in these women. The data suggests that elevated cytokines (IL 6 and TNFα) and bone resorption markers (CTx and Dpd) are associated with rapid of fall of estrogens in the first decade of menopause leading to postmenopausal osteoporosis. Combination of hormonal profiles, bone resorption markers and cytokines will thus aid in identifying women at risk for osteoporosis.

Disclosures: M.P. Desai. None.

This study received funding from: Department of Family Welfare, Government of India.

M459

See Sunday Plenary Number S459

M460

Relationships Between Leptin, PTH, Vitamin D and Bone Metabolism Markers in Patients with Hip Fracture.

A. A. Fisher*¹, E. K. Southcott*¹, S. L. Goh*², W. Srikusalanukul*¹, M. W. Davis*¹, P. N. Smith*². ¹Department of Geriatric Medicine, The Canberra Hospital, Australian National University, The Canberra Hospital, Woden, ACT, Australia, ²Department of Orthopaedic Surgery, The Canberra Hospital, Australian National University, The Canberra Hospital, Woden, ACT, Australia.

The relationship between leptin and bone metabolism is controversial. The study aim was to investigate leptin-skeletal interactions in older patients with hip fracture (HF), a population not previously studied.

In 207 consecutive patients (mean age 82.1 ± 7.9 years; 75.8% women) with low-energy trauma HF (119/88 cervical/trochanteric) serum concentrations of leptin, 25 hydroxy-vitamin D [25(OH)D], parathyroid hormone (PTH), calcium, phosphorus, magnesium, osteocalcin (OC), bone-specific alkaline phosphatase (BAP) and urine excretion (normalised for urinary creatinine) of free deoxypyridinoline (DPD) and N-terminal cross-linked telepeptide of type 1 collagen (NTx) were measured and clinical data collected prospectively.

Elevated PTH (> 6.5pmol/L) was present in 53%, 25(OH)D insufficiency ( < 50nmol/L) in 81.6%, excessive bone resorption (increased DPD and/or NTx excretion) in 93.7% and low bone formation (low OC and/or BAP) in 59.2%. Leptin (log-transformed) was significantly and positively correlated with OC (r = 0.18; p = 0.006) and negatively with NTx (r = −0.17: p = 0.015) and DPD (r = — 0.14: p = 0.037). In both cervical and trochanteric HF groups, leptin was positively associated with OC, but the association with DPD was significant only in trochanteric HF (r = −0.30; p = 0.009). Trochanteric compared to cervical HF patients have higher levels of PTH (7.9 ± 6.0 vs 5.9 ± 3.8 pmol/L; p = 0.005), but the concentrations of leptin, 25(OH)D and bone turnover markers were similar. Only in cervical HF patients were leptin and PTH positively correlated (r = 0.26; p = 0.005) and 25(OH)D levels were negatively and significantly correlated with NTx (r = −0.25; p = 0.016), DPD (r = −0.31; p = 0.002) and BAP (r = −0.24; p = 0.012). Other bone metabolism parameters were not associated with leptin in neither group.

These findings suggest that in HF patients there exists a complex relationship between leptin and calciotropic factors, that serum leptin may independently contribute to bone remodelling and is likely to exert different effect on cortical and trabecular bone compartments.

Disclosures: A.A. Fisher, None.

M461

Do Periosteal and Intracortical Responses to Ulnar Fatigue Loading Differ Between Old and Young Rats?

J. B. Jones*, R. T. Kitterman*, S. D. Mendenhall*, J. G. Skedros. Dept. of Orthop. Surgery, Univ. of Utah, SLC, UT, USA.

Age-related skeletal deterioration results not only in decreased bone mass but also bone quality. Inadequate modeling responses and/or incomplete infilling of resorption cavities following bone fatigue contribute to this decline in bone mechanical properties. Using the rat ulna fatigue model, we hypothesized that older adult rats would show less periosteal woven bone apposition and resorption space infilling when compared with younger adult rats following mechanical loading. With IACUC approval, 28 male F344 rats (14, 5 month-old, 14, 15 month-old) were obtained from the National Institute on Aging (Bethesda, MD). Eight rats of each age formed the experimental groups and the remaining were used for load/strain calibration. Right forearms were cyclically loaded [Uthgenannt & Silva, J Biomech, 2006] and the contralateral ulnae served as controls. Tetracycline was injected 13 and 3 days prior to sacrifice at 18 days post-loading. Ten transverse sections were then cut from mid-third ulnar diaphyses and mounted on slides. Resorption space areas (Rs.Ar/Ct.Ar), infilling areas (Rs.Inf.Ar/Ct.Ar), and periosteal woven bone area (PWb.Ar/Ct.Ar) were quantified. Kruskal-Wallis ANOVA was employed with significance set at p<0.05. Contrary to our hypothesis that older rats would exhibit less periosteal bone apposition, a larger amount was observed when compared to younger rats (see Table).

Additionally, total resorption area was twice as large in older rats. However, when resorption space infilling area was considered, the older rats showed significantly less infilling than younger rats (33% vs. 55%, see Table). These data suggest that older rats show an imbalance between intracortical resorption and bone formation which leads to net bone loss–a similar phenomenon experienced by aging humans. The finding that older rats exhibit more periosteal woven bone than younger rats implies the possibility of a compensatory mechanism that counteracts the increased cortical porosity. Although a similar interaction between remodeling and modeling has been suggested in humans, the modeling response is relatively subtle [Ural & Vashishth: J Orthop Res 2006]. This demonstrates a limitation when attempting to extrapolate the capacity of bone modeling in 15-month old rats to that in aging humans. 

Table Table:. Periosteal modeling, intracortical resorption, and intracortical infilling by age in the rat

Disclosures: J.B. Jones, None.

This study received funding from: OREF Grant 01–024.

M462

Increased Active Osteoclasts Are Associated with Acute Cancellous Bone Loss in Adult Mice Exposed to Ionizing Radiation and Musculoskeletal Disuse.

H. Kondo¹, R. Mojarrab*¹, A. Wang*¹, J. Phillips*¹, E. A. C. Almeida*¹, D. J. Loftus*¹, E. Morey-Holton*¹, R. K. Globus*¹, W. Vercoutere*¹, C. Limoli*², N. D. Searby*¹. ¹Bone and Signaling Lab, NASA Ames Research Center, Moffett Field, CA, USA, ²Radiation oncology, University of California Irvine, Irvine, CA, USA.

Spaceflight challenges the skeletal health of astronauts by exposure to radiation in microgravity. Hamilton et al. (JAP (2006) 101:789) showed that irradiation alone decreases bone mass in growing mice 4 mo after exposure to 2 Gy vs. controls, as does musculoskeletal disuse. We hypothesize that radiation and disuse share cellular and molecular mechanisms to cause rapid bone loss in the adult. To define dose (1–2 Gy) and time dependence (3 or 10 d) of skeletal responses to radiation, 4 mo old C57/B16 mice were Cs¹³⁷ gamma-irradiated (IR), then marrow cells analyzed for viability by FACS and bones for structure by microCT. In a second study, mice were hindlimb unloaded (HU) or normally loaded (NL) for 7 d; half the mice from each group were IR with 2 Gy 4 d into the unloading period. Bones were analyzed for structure (microCT), osteoclast surface (histomorphometry) and oxidative damage to lipids (peroxidation by malondialdehyde assay). As expected, HU for 7 d decreased cancellous bone compared to NL. Surprisingly, only 3 d after exposure, IR (2 Gy) caused a loss in cancellous BV/TV(∼20%), trabecular number, and connectivity similar to 7 d HU. By 10 d after IR, BV/TV was 25% lower than controls at 1 Gy and 35% lower at 2 Gy, showing both dose and time-dependent effects of IR. Cancellous bone loss was similar in mice exposed to IR during HU, compared to mice treated with either IR or HU alone. IR transiently reduced (by 65% at 3 d) numbers of viable marrow cells due to increased apoptosis. IR elevated (by 36%) lipid peroxidation in bone. Osteoclast surface per bone surface increased 113% due to IR, 100% due to HU, and 153% due to both IR and HU compared to NL, indicating stimulated resorption. Hydrogen peroxide treatment of cultured bone marrow cells caused a dose-dependent increase in TRAP+ cells, inhibited by the anti-oxidant, alpha-lipoic acid. Together, these results showed that IR and HU caused similar detrimental effects on cancellous bone structure of adult mice, and shared the cellular mechanism of increasing active osteoclasts. In the short-term, continuous HU did not alter the structural changes caused by radiation, possibly due to depletion of osteoclast precursors by unloading or irradiation alone. Finally, radiation caused oxidative stress in bone tissue that may contribute to increased resorption leading to rapid bone loss. These results have potential clinical relevance to astronauts as well as to cancer patients treated therapeutically with radiation.

Disclosures: H. Kondo, None.

This study received funding from: NASA.

M463

The Periosteal and Endosteal Modeling Process Is Affected by Estrogen Differently in Response to Fatigue Loading Than During Development.

S. D. Mendenhall*, J. B. Jones*, R. T. Kitterman*, J. G. Skedros. Dept. of Orthop. Surgery, Univ. of Utah, SLC, UT, USA.

Females are “set up” during skeletal development to have fragile bones later in life. This results primarily from the inhibitory effect of estrogen on periosteal modeling during bone mass acquisition, causing women's bones to be less robust than men's. Delayed pubertal development in female rats increases periosteal bone growth, but results in a minor decline in endosteal modeling [Yingling: Tran Orthop Res Soc 2007]. However, little is known about the effects of estrogen on periosteal/endosteal modeling responses to fatigue loading in rats. Using the rat ovariectomy (Ovx) model, we tested the hypothesis that estrogen-deficient rats would exhibit less endosteal modeling and more periosteal modeling following fatigue loading than Ovx rats that were estrogen-repleted (Ovx-Est). With IACUC approval, 36 five-month-old female F344 rats were obtained-6 for load/strain calibration and 15 for each of the experimental groups. Following Ovx, right forearms were cyclically loaded [Uthgenannt & Silva, J Biomech, 2006] and the contralateral ulnae served as controls. The Ovx Est group received daily β-Estradiol (0.05 mg/kg) injections. All animals received tetracycline injections 13 and 3 days prior to sacrifice at 18 days post-loading. Ten transverse sections were cut from mid-third ulnar diaphyses and mounted on slides. Endosteal woven bone area (End.Wb.Ar), periosteal woven bone area (Per.Wb.Ar), and total woven bone area (T.Wb.Ar) were then quantified. Kruskal-Wallis ANOVA was used with significance at p<0.05. As expected, the Ovx rats had less endosteal bone growth when compared to Ovx-Est rats. Surprisingly, the Ovx-Est rats produced nearly twice as much periosteal woven bone than did Ovx rats (see Figure  ). These results show an interesting interaction between hormone status and fatigue-induced modeling responses in the rat. Although estrogen inhibits periosteal bone growth during development, heavy mechanical stimuli seem to reverse this role, thereby maximizing the amount of new bone growth on periosteal and endosteal surfaces. Similar anabolic effects of estrogen have been reported in women on estrogen replacement therapy [Vedi et al.: Osteoporos Int 1999]. Further work is needed to validate the rat Ovx model for evaluating novel therapeutic agents that target estrogen's actions on bone.

Figure Figure:. Woven Bone Response by Estrogen Status

Disclosures: S.D. Mendenhall, None.

This study received funding from: OREF Grant 01–024.

M464

An Acute 7 Gray Dose of Gamma-Radiation Induces a Profound and Rapid Loss of Trabecular Bone In Mice.

J. S. Willey*¹, D. S. Gridley*², M.J. Pecaut*², R. W. Norrdin*³, T. A. Bateman¹. ¹Bioengineering Department, Clemson University, Clemson, SC, USA, ²Department of Radiation Medicine, Loma Linda University, Loma Linda, CA, USA, ³College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.

Fracture rates of irradiated bones are elevated in patients receiving radiation therapy for a primary tumor as well as palliative care treatment. The mechanism for the elevated risk of fracture is unclear. Osteoblast death and damaged vascularity have been documented in patients as well as animal models, and contribute to atrophy after irradiation. But the effects of radiation on osteoclast number and function are not well defined. We investigated the effects of acute, high-dose radiation exposure on trabecular and cortical bone to document bone quantity and structural properties at an early time-point post-irradiation. Nine-week old C57BL/6 mice (n = 6) received 7 Gray (Gy) gamma-radiation, with nonirradiated controls (n = 6). Fourteen days after exposure the animals were euthanized, hind limbs removed, and tibiae analyzed via microCT and histomorphometry. Statistical analyses were performed using t-tests to compare the groups. Profound loss of trabecular bone within the metaphysis was observed. Significant reduction in BV/TV (-54%), Conn.Dens (-69%), and Tb.N (-26%) were observed in irradiated animals compared with control. No differences were observed in cortical parameters. Decreased growth plate cellularity, disorganized chondrocyte arrangement, and a terminal bar of bone along the distal physeal border indicated slow growth among all animals. Oc.S/BS and ES/BS were similar between both groups, with TRAP staining confirming presence of multinucleated osteoclasts. The large difference in the amount of bone between groups and evidence together with evidence of slow growth suggests bone loss as opposed to reduced bone growth. Evidence of stabilized resorption at sacrifice indicates that large declines in bone quantity occurred via increased resorption very early after exposure. Radiation-induced reduction in bone quantity and microarchitecture via elevated resorption could reduce bone quality and may provide insight into the nature of increased fracture rates among radiation therapy recipients.

Disclosures: J.S. Willey, None.

This study received funding from: NSBRI.

M465

Reduced Levels of Serum Insulin Like Growth Factor 1 in Male with Idiopathic Osteoporosis.

J. Dewailly*¹, I. Legroux-Cerot*¹, M. D'Herbomez*², X. Marchandise*², D. Dewailly*³, B. Duquesnoy*¹, B. Cortet¹. ¹Rheumatology, CHRU Hopital Salengro, Lille, France, ²Service de Médecine nucléaire, CHRU Hopital Salengro, Lille, France, ³Endocrinology, CHRU, Lille, France.

Objective: The pathophysiology of idiopathic male osteoporosis remains unknown. The histomorphometric studies suggest reduction of bone formation. A disturbed secretion of IGF-1 could be involved in male idiopathic osteoporosis since IGF-1 has a stimulative effect on the osteoblastic proliferation and on the bone tissue synthesis. Few studies have evaluated the relationship between the circulating concentrations of IGF-1 and bone mineral density, particularly in osteoporotic men, and the results are controversial. The aim of our study was to evaluate the involvement of IGF-1 in idiopathic male osteoporosis and the relationships between bone density and the serum levels of IGF-1 and sex hormones.

Materials and methods: Serum levels of IGF-1, estradiol, testosterone, SHBG and bone mineral density at lumbar spine, femoral neck and total hip were compared between 79 men with idiopathic osteoporosis and 26 healthy subjects. Inclusion criteria were a densitometric osteoporosis defined by a T-score ≤ −2.5 SD and /or an osteoporotic fracture. The osteoporotic patients were included after exhaustive work-up excluding the principal causes of secondary osteoporosis.

Results: A significant reduction in the mean serum IGF-1 levels was found in osteoporosis patients (p = 0.02) that persisted after adjustement for BMI. However, no correlation was found between bone density and the serum IGF-1 levels. The mean SHBG level was higher in osteoprotic patients (p = 0.001) thus yielding a reduction in the mean Free Testosterone Index (FTI, total testosterone / SHBG) (p = 0.002). These differences remained significant after adjustement for BMI. After adjustment for the FTI, the mean IGF-1 levels between patients and controls were not significantly different. However, after adjustment for the FTI and the BMI in patients, the IGF-1 level was significantly related to the presence of an osteoporotic fracture, thus indicating an independent effect of the IGF-1 level on fracture risk.

Conclusion: Our study confirms the presence of reduced serum IGF-1 levels in male idiopathic osteoporosis. This could be the cause or the consequence of a disturbance in sex hormones with increased SHBG serum level. This confirms the important role of SHBG in the pathophysiology of male osteoporosis.

Disclosures: J. Dewailly, None.

M466

See Sunday Plenary Number S466

M467

Birth Weight Predicts Lean Body Mass and Thus Bmc in Healthy Men at Peak Bone Mass — Results from the Odense Androgen Study.

L. Frederiksen*, T. L. Nielsen*, K. Wraae*, H. Claus*, M. Andersen*, K. Brixen*. Endocrinology, Odense University Hospital, Odense, Denmark.

Birth weight has been associated with low bone mass in later life. Previous studies, however, have relied on self-reported data on birth weight, included select populations, or been of a limited size. Moreover, it is unclear if the association between birth weight and bone mass is be mediated by body weight, lean body mass, or fat mass.

We hypothesize that birth weight is associated with peak bone mass in men independent of current lean body mass and body weight.

The Odense Androgen Study is a population-based, prospective, observational study on the inter-relationship between endocrine status, body composition, muscle function, and bone metabolism in young men. In brief, 3000 males aged 20–30 years were randomly selected from the civil registration database in Funen County, Denmark, and invited by mail to participate in the study. A total of 2042 men returned the questionnaire, 783 gave written informed consent to participate in the study and the data are presented here. Bone mass measurements (spine, hip, and whole body) were performed using a hologic-4500a densitometer. Data on birth weight, length at birth, and gestational age was retrieved in a national database covering all birth clinics in Denmark in the current period. The relationship between Birth weight, BMC, Lean body mass and fat mass as tested using multiple regression analysis is shown in the table as partial correlation coefficients Data on birth weight and birth length were available on 754 participants while gestational age was available on 263 participants (those 20–24 years old). In bivariate analyses, BMC at whole body, spine, and hip were significantly associated with current body weigth (R = 0.50 to 0.30, p<0.001) and birth weight (R = 0.17 to 0.09, p<0.01). Birth weight was also significantly associated with current body weight (R = 0.09, p<0.01), current lean body mass (R = 0.15, p<0.001) but not current fat mass (R = −0.04, NS). No significant difference was found in BMC at any skeletal site in participants born prematurely (n = 26) and those born at term (n = 237). 

Table  .  

*p<0.05, **p<0.0l, ****p<0.001

Birth weight predicts peak bone mass, but this seems to be due to correlation between birth weight and body weight (and lean body mass) in young adult life.

Disclosures: L. Frederiksen, None.

M468

See Sunday Plenary Number S468

M469

Aberrations in Wnt/β-Catenin Signaling Driving HAART-induced Bone Loss In Vitro.

J. S. Butler¹, P. P. Doran*¹, R. T. Moon*², J. M. O'Byme*³, W. G. Powderly*¹. ¹Mater Misericordiae University Hospital, UCD School of Medicine & Medical Science, Dublin, Ireland, ²Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, WA, USA, ³Dept of Trauma & Orthopaedic Surgery, Royal College of Surgeons in Ireland, Dublin, Ireland.

The advent of highly active anti-retroviral therapy (HAART) had dramatically decreased the rate of HIV-related mortality and significantly extended the life span of patients with AIDS. A variety of metabolic side effects are associated with these therapies, one of which is metabolic bone disease. A higher prevalence of osteopenia and osteoporosis is reported in HIV-infected patients receiving anti-retroviral therapy relative to patients not on therapy. Thus, we decided to investigate the role played by Wnt/β-catenin signaling in Protease Inhibitor (PI) induced bone loss in vitro.

Our objectives were to assess the gene expression profile of primary human osteoblasts (HOBs) exposed to Pls with a view to identifying key genes driving HAART-induced bone loss and to assess the effects of silencing the Wnt antagonist Dickkopf-1 (Dkkl) on the bone profile of primary human osteoblasts (HOBs) exposed to 5μM of IDV and RTV in vitro.

Primary human osteoblasts (HOBs) were cultured in vitro and exposed to 5μM of IDV and RTV over a time course of 4hr, 12hr, 24hr and 48hr. Dkkl expression was silenced using small interfering RNA (siRNA). Quantitative RT-PCR was performed to confirm gene knockdown. Control and Pi-treated HOBs were compared with respect to bone turnover. Markers of bone turnover analyzed included alkaline phosphatase activity, calcium deposition and osteocalcin expression, along with cell proliferation and cellular apoptosis. Global changes in HOB gene expression were elicited by IDV and RTV. Development associated gene pathways were co-ordinately dysregulated with the expression profile of key genes of the Wnt Pathway significantly altered. Dkkl expression in HOBs was increased in response to IDV and RTV exposure with an associated reduction in alkaline phosphatase activity and calcium deposition. Silencing of Dkkl expression, as confirmed by quantitative RT-PCR, was associated with an increase in alkaline phosphatase activity and calcium deposition, along with increased cell proliferation and reduced cellular apoptosis.

Dkkl is an antagonist of Wnt/β-catenin signaling and plays a key role in regulating bone development and remodeling. Silencing the expression of Dkkl in primary human osteoblasts has been shown to rescue the effects of Pi-induced bone loss in vitro. The pharmacological targeting of the Wnt/β-catenin signaling pathway offers an exciting opportunity for the development of novel anabolic bone agents to treat HAART associated osteopenia/osteoporosis.

Disclosures: J.S. Butler, None.

M470

Bone Mineral Density and Prevalence of Osteoporosis and Fractures in Psoriatic Arthritis.

M. Romera, N. Busquets*, J. Rodríguez-Moreno*, C. Gómez-Vaquero*, X. Juanola*, J. M. Nolla*. Rheumatology, Hospital Universitari de Bellvitge, L'Hospitalet, Spain.

Systemic diseases, mainly those involving joints, are known to produce secondary osteoporosis. However, there is little information available about bone mineral density (BMD) and the prevalence of osteoporosis and fractures in patients with psoriatic arthritis (PsA).

The purpose of the study is to evaluate BMD and to quantify the prevalence of osteoporosis and clinical fractures in PsA.

BMD was determined by DXA (Hologic QDR 4.500) in all the patients with PsA regularly evaluated in a tertiary university hospital who were willing to participate in the study; patients with axial involvement were excluded. All patients underwent a physical examination and a general analytics. We gathered demographic and clinical variables related with BMD and risk of fractures. We also recorded the antecedent of clinical low impact fracture. The activity of PsA was assessed by means of the Modified Health Assessment Questionnaire (HAQm) and the Disease Activity Score 28 (DAS28). The population of reference to calculate T-score and Z-score proceeds from a Spanish database. The diagnosis of osteoporosis was made according to the recommendations of the International Society for Clinical Densitometry.

One hundred and fifty-five patients were included (64 postmenopausal women, 26 premenopausal women and 65 men) with a mean age of 62 ± 8, 39 ± 7 and 55 ± 14 years, respectively. The clinical forms of PsA were: 1% monoarticular, 44% oligoarticular and 55% poliarticular.

BMD in males under 50 years old was lower than BMD of healthy males of the same age (p < 0.05); there were no differences in the other groups of patients. BMD related in a statistically significant way to age, body mass index (BMI), time since the beginning of PsA, number of tender joints, HAQm and DAS28.

The prevalence of osteoporosis in postmenopausal women was 28%; in males above 50 years old, 10%. Fifteen percent of the postmenopausal women and 20% of the males under 50 years old had a BMD below the expected range for their age.

Thirteen percent of the patients had had a clinical fracture. The most frequent localization of the fracture was forearm (7%), followed by vertebra (1%) and hip (1%). Clinical and demographic data were similar in patients with and without fracture. Postmenopausal women had a significantly higher prevalence of fractures.

As a conclusion, patients with PsA, mainly postmenopausal women, have a high prevalence of osteoporosis. BMD correlates with age, BMI and activity of the disease. The frequency of clinical osteoporotic fractures is not negligible. The factors associated to fractures in PsA are similar to those in general population.

Disclosures: C Gómez-Vaquero, None.

M471

See Sunday Plenary Number S471

M472

Associations of Osteoporotic Spinal Deformity with Back Strength Among Elderly Women in Japan and the United States.

M. Hongo¹, M. Sinaki², N. Miyakoshi¹, Y. Shimada*¹, E. Itoi*³. ¹Department of Orthopedic Surgery, Akita University, Akita, Japan, ²Department of Physical Medicine and Rehabilitation, Mayo Clinic, Rochester, MN, USA, ³Department of Orthopedic Surgery, Töhoku University, Sendai, Japan.

Kyphosis is associated with diminished daily physical function, increased risk of falls, and increased mortality risk. Back extensor strength (BES) is an important factor in maintaining the sagittal alignment of spine and preventing vertebral fractures. When back exercises are prescribed, it is important to know the associations of the sagittal alignment with BES. However, there is a controversy in the literature regarding the influence of the thoracic and lumbar spinal curvatures on BES or quality of life. We hypothesized that the regional or ethnic factor may influence the relationship between thoracic and lumbar sagittal curve, BES and other factors. The purpose of this cross-sectional study was to assess the associations of osteoporotic spinal deformity with back strength in elderly women in Japan and the United States. Using the same inclusion criteria, 104 Asian women residing in Akita, Japan and 102 Caucasian women residing in Minnesota with postmenopausal osteoporosis were selected for this study. Kyphosis angle of the thoracic and lumbar spine were measured with lateral radiographs. Isometric BES was evaluated using a custom-made dynamometer. Correlations between the kyphosis angle, isometric BES, bone mineral density, physical activity score or quality of life score, the number of vertebral fractures, and grip strengths were analyzed. No significant difference was found in thoracic kyphosis between Akita and Minnesota (43.1° and 44.1°, respectively), whereas lumbar kyphosis angle in Akita was significantly larger than in the Minnesota (-15.4° and −54.0° p<0.0001). In Akita, BES showed significant negative correlation with lumbar kyphosis angle (r = −0.492, pO.0001), but no correlation with thoracic kyphosis angle (r = −0.004, p = 0.97). In Minnesota, BES showed significant negative correlation with thoracic kyphosis angle (r = −0.372, p<0.0001), but no correlation was found with lumbar kyphosis angle (r = −0.087, p = 0.362). In addition, quality of life score in Akita demonstrated significant correlation with lumbar kyphosis, but not with thoracic kyphosis. Physical activity score in Minnesota showed significant negative correlation with thoracic kyphosis, but not with lumbar kyphosis. In conclusion, there was a significant difference in lumbar kyphosis angle between the two countries. Lumbar kyphosis angle has more significant influences in maintaining BES and quality of life among women in Akita, Japan compared with those in Minnesota.

Disclosures: M. Hongo, None.

M473

Secretion of Adipokines and Hypertrophic Changes in Bone Marrow Adipocytes.

A. Hozumi*¹, M. Osaki*², H. Shindo*². ¹Orthopaedic surgery, Ngasaki Memorial hospital, Nagasaki, Japan, ²Orthopaedic surgery, Ngasaki University, Nagasaki, Japan.

Recent analysis of gene expression in subcutaneous and visceral adipose tissue has shown that 20 to 30% of proteins expressed in adipose tissue are associated with genes coding for secretory proteins, and that adipocytes secrete a series of physiologically active substances such as adiponectin, leptin, tumor necrosis factor alpha (TNFα), and plasminogen activator inhibitor type 1 (PAI-1), regulating vital functions [1-7]. Thus, subcutaneous and visceral adipose tissue is regarded as an endocrine organ, and its importance is emphasized. No study has investigated the physiological function of adipocytes present in the bone marrow.

In this study, we established a simple method for the primary culture of bone marrow adipocytes with a suspension culture line, and quantified adipokine gene expression and secretory proteins released in suspension.

(Subjects) The subjects were 8 patients (8 joints) with coxarthrosis or femur neck fracture, with a mean age of 72 years. Subjects comprised 1 male and 7 females. Bone marrow adipocytes were isolated from bone marrow fluid collected on prosthesis insertion.

(Methods) After bone marrow fluid was treated with an enzyme, adipocytes were isolated by centrifugation, and used as an experimental system employing the floating culture method. These adipose cells were histopathologically examined by oil red O staining and Mayer's hematoxylin staining. In addition, we investigated morphological changes in the adipocytes 7 days after dexamethazone (DEX) administration. We examined PPARγ, adiponectin, leptin, PAI-1, and TNFα mRNA expressions by RT-PCR 24 hours after culture. Concerning adiponectin, PAI-1, and TNFα release in culture medium, we quantified protein levels by ELISA.

(Results) We confirmed that the isolated adipocytes were histologically mature, and that they comprised a uniform cell population. At the gene expression level, various adipokines were detected in all patients, as demonstrated for subcutaneous and omental adipocytes. We confirmed the secretion of adiponectin, PAI-1, and TNFα in culture medium. Seven days after DEX administration, hypertrophic change of adipocytes was observed in comparison with the control group.

(Discussion) In this study, we initially established a method for the primary floating culture of bone marrow adipocytes. In addition, various cytokines were detected in bone marrow adipocytes, as demonstrated for subcutaneous and omental adipocytes.

This procedure may be useful for investigating mature bone marrow adipocytes.

Disclosures: A. Hozumi. None.

M474

RANKL Signaling in Human Osteoclasts: Effect of P62 Mutations.

E. Chamoux¹, J. N. Couture*¹, B. G. Lemenchick*¹, J. P. Brown*², S. Roux¹. ¹Rheumatology, Faculty of Medicine, Sherbrooke, PQ, Canada, ²GRMO, CHUL Research Center, Quebec, PQ, Canada.

The discovery of mutations in Paget's disease of bone (PDB), characterized by an increased number of overactive osteoclasts (OC), has now targeted p62 as an important player in RANKL signaling. To date, all the mutations identified in p62 prevent its Ubiquitin Binding Associated (UBA) domain from being functional, thus potentially affecting its degradation and/or signaling capacities. Studies on rodent models concluded that p62 forms complexes with the atypical ζPKC and other partners after RANKL stimulation. However, the precise role of p62 in osteoclast physiology is not clearly understood yet. For our study, we build p-EGFP-C2 plasmids containing p62 wild-type (p62^wt), p62 modified with the most common mutation observed in PDB patients (p62^P392L) and p62 with a deleted UBA domain (p62^dUBA). Human OCs differentiated from cord blood monocytes with RANKL were typically able to resorb around 15% of a bone slice surface. This area was more than 3 times larger for osteoclasts transfected with p62^wt or p62^P321. Interestingly, Ocs transfected with p62^dlUBA did not resorb bone more than non-transfected Ocs or Ocs transfected with an empty vector. DAPI staining showed that apoptosis occurred in 44.8 ± 6.1% of non-transfected Ocs 24h after RANKL withdrawal. Similar results were seen in Ocs transfected with p62^wt or an empty vector. In contrast, apoptosis was decreased in cells transfected with p62^P3921 or p62^dUBA (14.l ± 5.7% and 8.3 ± 4.5% respectively), which suggests that p62 mutations increase cell survival. To better understand early events occurring after RANKL stimulation, we used an antibody recognizing ζPKC when phosphorylated on the Ser410 (hallmark of its activation) in immunofluorescence studies. RANKL stimulation led to the relocalization of P-ζPKC from the membrane to the perinuclear area (15 min), then within nuclei (1h). If no difference was observed in cells transfected with p62^wt, OCs expressing p62^P3921 in contrast exhibited P-ζPKC in their nuclei prior to any stimulation by RANKL, and no change was noticed after RANKL addition. We finally determined that RANKL stimulation induced an important intracellular Ca²⁺ mobilization with an amplitude of 356,5 ± 34,4 pM Ca²⁺, which was not observed in OCs transfected with p62^P3921. Our results underline strong relationships between p62 mutations and modified osteoclast behavior resembling to PDB. We also provide new insights in early events occurring after RANKL stimulation of human osteoclasts, noticeably a possible constitutive activation of ζPKC, which may help to explain the increased survival and resorption observed in mutated OCs.

Disclosures: E. Chamoux, None.

This study received funding from: CHIR.

M475

Functional Consequences of P62 Mutations in PDB Osteoclasts.

J. N. Couture*¹, E. Chamoux¹, J. Morissette*², J. P. Brown*², S. Roux¹. ¹Rheumatology, Faculty of Medicine, Sherbrooke, PQ, Canada, ²GRMO, CHUL Research Center, Quebec, PQ, Canada.

Mutations in the gene encoding SQSTM1/p62 have been linked to Paget's disease of the bone (PDB), a chronic focal skeletal disorder characterized by an increased number of overactive osteoclasts (Ocs), but the functional consequences of these mutations still need to be understood. All the PDB-associated p62 mutations were mapped to the ubiquitine-associated (UBA) domain of the protein. Studies in rodents have shown that p62 forms complexes with signalling partners upon RANKL stimulation but its exact role in the Ocs physiology remains to be elucidated. For this study, we conducted experiments on osteoclasts-like cells differentiated from peripheral blood mononuclear cells isolated from PDB patients previously identified with the p62^P3921 mutation. We compared our results to normal human osteoclasts differentiated from cord blood monocytes (CBMs), a well established model. PDB Ocs were able to resorb cortical bone when differentiated with 25, 50 or 100 ng/mL RANKL during 21 days. The resorbed surface was at least 3 times greater than that for CBMs with the same doses of RANKL added throughout the differentiation. We hypothesised that increased cell survival may in part explain this hyper-resorption so, using DAPI staining, we evaluated PDB Ocs response to some of the major apoptotic signals for human Ocs. PDB Ocs and CBMs were deprived of growth factors for 72h, then a Fas-activating antibody (400 ng/ml), TGFβ1 (2 ng/ml), and M-CSF (25 ng/ml) were added where appropriate for 24h. 58 ± 2.3% of CBMs vs 12.5 ± 4.5% in PDB Ocs were apoptotic in controls. This percentage was 75.2 ± 3.6% for CBMs in the presence of anti-Fas (400 ng/mL) vs 16.3 ± 4.5% for PDB Ocs. In the presence of TGFβl (2ng/mL), apoptotic CBMs were 78.4 ± 3.6% vs 28.9 ± 5.6% for PDB Ocs. Finally, 25.1 ± 5.6% of CBMs were apoptotic with M-CSF (25 ng/ml) vs 17.2 ± 3.7% for PDB Ocs. To determine if cellular signalisation upon RANKL activation was affected, we conducted immunofluorescence studies on p62, PKCζ and the activated form of PDK1. In non-treated cells, p62 has a cytoplasmic localisation but reaches the nuclei within 60 minutes after RANKL addition. We also observed that PKCζ, pPDK1 were present in the nucleus of PDB Ocs prior to any stimulation. In normal human Ocs, these proteins reach the perinuclear region upon stimulation but are cytoplasmic otherwise. Together, these results strongly suggest that PDB Ocs carrying p62^P3921 are more resistant to apoptosis and display an altered RANKL signaling compared to normal human osteoclasts This could explain, at least in part, their functional over-activity.

Disclosures: J.N. Couture, None.

This study received funding from: CHIR.

M476

A Randomised, Active-controlled, 6-month Study of Once Weekly 280 mg Oral Buffered Alendronate Solution for Treatment of Paget's Disease of Bone.

M. J. Hooper¹, A. Faustino*², I. R. Reed³, D. Hosking*⁴, N. Gilcrest*⁵, P. Selby*⁶, M. Wu*⁷, G. Salzmann*⁷, J. West*⁷, A. Leung*⁷. ¹University of Sydney, Sydney, Australia, ²Servimed, Lda, Lisbon, Portugal, ³Auckland Hospital, Auckland, New Zealand, ⁴Nottingham City Hospital, David Evans Medical Research Centre, Nottingham, United Kingdom, ⁵Princess Margaret Hospital, Canterbury Geriatric Medical Research Trust, Christchurch, New Zealand, ⁶Manchester Royal Infirmary, Vitamin D Research Group, Manchester, United Kingdom, ⁷Merck & Co, Merck Research Laboratories, Rahway, NJ, USA.

Although daily doses of oral bisphosphonates are a safe and effective treatment for Paget's disease of bone (PDB), some patients may experience upper gastrointestinal adverse effects (UG1 AEs) or find the dosing requirements onerous and become noncompliant. A once weekly (OW) oral dose of bisphosphonate in buffered solution (OBS) may be as effective, better tolerated and more convenient than daily dosing.

During a 6-month, randomized, double-blind, active-controlled trial, we compared an alendronate (ALN) 280-mg OW OBS with an ALN 40-mg/day tablet for treating PDB. Patients were randomized (2:1) to ALN 280-mg OW OBS or ALN 40-mg/day. The primary endpoint was the mean percent decrease in total serum alkaline phosphatase (SAP) from baseline at 6-months.

At Month 6, the mean change from baseline in SAP was −71.76% with a 95% CI of (–76.77, −65.67) in the ALN 280-mg OW OBS group and −73.18% with a 95% CI of (–79.52, −64.89) in the ALN 40-mg/day tablet group. There was no significant difference between groups during the 6-month period. There was a higher incidence of clinical AEs in the ALN 280-mg OW OBS (78.6%) vs. the ALN 40-mg/day tablet group (66.7%), including drug related AEs (47.6% and 9.5%, respectively), that led to study discontinuation (19.0% and 9.5%, respectively). There was a higher incidence of laboratory AEs in the ALN 280-mg OW OBS group (16.7%) vs. the ALN 40-mg/day tablet group (4.8%), including drug related AEs.

Although ALN 280-mg OW OBS was as safe and effective as ALN 40-mg/day in reducing SAP in patients with PDB, ALN 40-mg/day tablet appears to be better tolerated than ALN 280-mg OW OBS.

Disclosures: M.J. Hooper, Merck & Co. 2.

This study received funding from: Merck & Co.

M477

See Sunday Plenary Number S477

M478

Long-term Effects of Single Zoledronate or Neridronate Infusion in Paget's Disease of Bone.

D. Merlotti, L. Gennari, F. Valleggi, V. De Paola, G. Martini, A. Avanzati*, R. Nuti. Internal Medicine, Endocrine-Metabolic Sciences and Biochemistry, University of Siena, Siena, Italy.

Aminobisphosphonates actually represent the most common treatment for Paget's disease of bone (PDB), with the potential for sustained remission. Intravenous regimens with different compounds demonstrated improved efficacy and compliance with respect to oral regimens. However, there have been few head to head randomized trials comparing intravenous bisphosphonates, and it is not demonstrated if these drugs differ in therapeutic efficacy. We recently performed a randomized study comparing intravenous pamidronate to zoledronate or neridronate in 90 subjects with active PDB. We report the results from the trial and the post-trial follow up of patients. At baseline patients were randomly assigned to receive either a 4 mg infusion of zoledronic acid (n = 30) or a 30 mg infusion of pamidronate for 2 consecutive days every 3 months (n = 60). After 6 months non-responders to pamidronate were crossed over to zoledronate (n = 18) or neridronate (n = 15, infusion of 100 mg for 2 consecutive days) treatment. Blood samples were collected at baseline and after 1, 3, 6, 12, 15, 18, and 24 months. No bisphosphonate was given after the cross-over, during the extension study. At 6 months, normal ALP levels were achieved in 93% of patients in zoledronate group and in 35% of patients in pamidronate group. Normalization of ALP levels was maintained in 79% and 65% of patients in the zoledronic acid group, after 12 and 24 months follow-up, respectively, while loss of therapeutic response was observed in 2/30 (6%) at 24 months. Among non-responders patients to pamidronate, 14/15 (93%) in the neridronate group and 17/18 (94%) in the zoledronate group achieved a therapeutic response after 6 months from the cross-over. Similar normalization rates were also observed between neridronate (80%) and zoledronate (83%) treated subjects at 6 months. Moreover, at 18 months from cross-over treatment (corresponding to 24 months from the baseline visit) ALP normalization was maintained in 60% and 77% of patients in neridronate or zoledronate group, respectively. In conclusion, single neridronate and zoledronate infusion produced a rapid and sustained control of bone turnover in up to 90% of PDB patients non-responders to pamidronate. This effect was largely independent of pre-treatment disease activity and prior bisphosphonate therapy.

Disclosures: D. Merlotti, None.

M479

Measles Virus Nucleoprotein (MVNP) Enhances NFATc1 Activation During Osteoclastogenesis in Paget's Disease.

A. Sarmasik*¹, Y. Hiruma¹, S. Okumura*¹, J. P. Brown², J. J. Windle³, G. D. Roodman⁴, N, Kurihara¹, D. L. Galson¹. ¹Ctr for Bone Biol, Dept of Med, Univ of Pittsburgh, Pittsburgh, PA, USA, ²Laval Univ, CHUL Res Center, Quebec City, PQ, Canada, ³Human Genetics, Virginia Commonwealth Univ, Richmond, VA, USA, ⁴Univ of Pittsburgh & VA Pittsburgh Healthcare System, Pittsburgh, PA, USA.

The mechanisms responsible for the dramatically increased numbers of hypermultinucleated osteoclasts (OCL) in Paget's Disease (PD) are just beginning to be understood. To explore the role of the critical osteoclast transcription factor NFATcl in PD, we determined if NFATc1 expression and activity were affected by measles virus nucleocapsid protein (MVNP), which is implicated in PD. Both OCL precursors from PD patients and normal human OCL precursors transduced with MVNP (MVNP-CFU-GM) are hyper-responsive to RANKL and form pagetic-like OCL in vitro. Furthermore, we have found that in the absence of added RANKL, both OCL precursors from PD patients and MVNP-CFU-GM have an increased amount (2-3 fold) of NFATcl protein. Transient co-transfections of an NFAT-responsive CTR P3 promoter reporter and MVNP- and NFATc1-expression vectors into RAW264.7 cells reveal that MVNP (2 fold alone) and NFATcl (50 fold alone) cooperate to activate the P3 reporter 250 fold. Additionally, RANKL treatment of RAW264.7 cells co-transfected with the P3 reporter and MVNP demonstrated that MVNP also cooperates with RANKL (2.5 fold alone) to activate this reporter 15 fold. However, MVNP expression could not help NFATc1 overcome lack of the key NFAT-binding site in the P3 reporter, suggesting that NFAT binding is required for MVNP to cooperate with NFAT to activate the P3-reporter. Comparative EMSA analysis of NFAT activation in osteoclastogenesis cultures using bone marrow monocytes (BMM) from wild-type and TRAP promoter-driven MVNP transgenic mice (MVNP-Tg) reveal that both TNF-α and RANKL induction of NFATcl activation was increased in MVNP-expressing cells. However, CsA blocked MVNP + RANKL from generating multinucleated TRAP+ OCL, implying that MVNP effects require an intact NFAT activation pathway. A constitutively-active NFATc1 (caNFATc1) retrovirus can induce wild-type mouse BMM to differentiate into OCL in the absence of RANKL at a low level with increased differentiation in the presence of suboptimal levels of RANKL. caNFATcl transduction into BMM from MVNP-Tg mice revealed cooperative enhancement of OCL differentiation by caNFATc1 and MVNP at no and suboptimal RANKL with more and larger OCLs formed than in the presence of either alone. These data demonstrate that NFATc1 is increased in PD and that MVNP can synergize with NFATcl to upregulate OCL differentiation in PD. Hence, inhibition of NFATc1 activation may be a possible therapeutic target for PD patients.

Disclosures: A. Sarmasik, None.

This study received finding from: NIH grant P01-AR049363.

M480

Enhanced Levels of FGF-2 and SOCS Signaling Modulates RANK Ligand Expression in Patients with Paget's Disease.

K. Sundaram¹, J. Senn¹, D. S. Rao², S. V. Reddy¹. ¹Children's Research Institute, Medical University of South Carolina, Charleston, SC, USA, ²Henry Ford Hospital, Detroit, MI, USA.

Paget's disease of bone (PD) is a chronic focal skeletal disorder characterized by excessive bone resorption followed by disorganized new bone formation. We previously reported measles virus nucleocapsid (MVNP) gene expression in patients with PD. MVNP expression in osteoclast lineage results in Pagetic phenotype in osteoclasts and suggested a pathophysiologic role for MVNP in PD. Enhanced levels of IL-6, M-CSF and endothelin-1 have been associated with PD, and are implicated in pathogenesis. Also, RANK ligand (RANKL), a critical osteoclastogenic factor expressed on marrow stromal/osteoblast cells is unregulated in PD. We have recently demonstrated that heat shock factor-2 (HSF-2) is a downstream target of fibroblast growth factor-2 (FGF-2) to induce RANKL expression in stromal preosteoblast cells. In this study, we hypothesized that FGF-2 modulates suppressors of cytokine signaling (SOCS) levels to induce RANKL gene expression in PD. We found a significant increase (2.5-fold) in serum FGF-2 levels in patients (n = 8) with PD compared with normal subjects (n = 10). These results are consistent with previous reports that FGF-2 mRNA expression is elevated in pagetic osteoclasts and bone marrow cells. Interestingly, conditioned media obtained from MVNP transduced normal human bone marrow cells showed a significant increase (3.2-fold) in the level of FGF-2 compared to empty vector transduced cells. Confocal microscopic analysis further demonstrated that MVNP conditioned media (20%) stimulates HSF-2 nuclear translocation in normal human bone marrow stromal/preosteoblast cells, which was blocked by a neutralizing antibody against FGF-2. We further show that FGF-2 stimulation increased SOCS-1 mRNA (5.2-fold) expression in these cells. We next examined if SOCS play a role in FGF-2 signaling to modulate RANKL gene expression in PD. Real-time PCR analysis demonstrated a significant increase in the levels of SOCS-1 (3.4-fold) and SOCS-3 (3.5-fold) mRNA expression in pagetic bone marrow mononuclear cells compared to normal subjects. In addition, co-expression of SOCS-1 with hRANKL gene promoter-luciferase reporter plasmid in marrow stromal cells demonstrated a significant increase (3.0-fold) of RANKL gene promoter activity without FGF-2 stimulation. These data suggest that enhanced levels of SOCS expression, FGF-2 signaling and cross-talk mechanisms may play an important role in modulating RANKL gene expression in PD. The identification of SOCS and FGF-2 involvement in RANKL gene expression may lead to novel therapeutic targets to control excessive bone turnover in PD.

Disclosures: K. Sundaram, None.

This study received finding from: NIH.

M481

Cardiovascular Manifestations of Primary Hyperparathyroidism.

J. Fleischer, M. D. Walker, S. Homma*, T. Rundek*, W. Inabnet*, D. J. McMahon, A. Tineo*, R. Liu*, R. Sacco*, S. J. Silverberg. Columbia University, New York, NY, USA.

Severe primary hyperparathyroidism (PHPT) is associated with cardiovascular (CV) calcification and increased CV mortality. We initiated this prospective observational study of patients who meet NIH surgical criteria and undergo surgery (PTX) to determine whether the mild, frequently asymptomatic PHPT seen today still poses a risk to the CV system, and whether identified abnormalities are reversible.

Patients are evaluated pre-PTX and for 2-yrs post-PTX. Baseline data on the first 29 patients (72% female; age ± SD 61 ± 7 yrs, range: 48-75) and 1 yr post-PTX data (n = 5) are available. Baseline serum calcium was 10.5 ± 0.6 mg/dl and PTH 121 • 50 pg/ml. Blood pressure was 121 ± 15/75 ± 11 mm Hg. Structural changes typical of classical PHPT were uncommon: 5 had valvular calcifications and 4 had increased left ventricular (LV) mass. LV function and wall motion were normal in all. Carotid ultrasound showed plaque in 11 (38%). Carotid intima-medial thickness (IMT), a subclinical marker of atherosclerosis was 0.98 ± .10 mm in the 75^th ( ± 12) percentile of an age/sex/race matched control population. While structural abnormalities were uncommon, functional tests of the heart (diastolic function by mitral pulse wave velocity, deceleration time, tissue doppler E velocity), peripheral vasculature (endothelial function by brachial artery flow-mediated dilation, FMD) and carotid (distensibility [Table]) were revealing. Carotid stiffness and FMD were abnormal. In this small group, FMD improved (4.8 ± 4.9 to 8.5 ± 11.7, p = 0.08) after PTX. Although mean values for other measures of CV function were normal, 27/29 (93%) had at least one test and 16/29 (55%) had 2 or more tests outside the normal range. 

Table  .  

Higher PTH levels were associated with decreased vascular compliance (carotid stiffness: R = 0.37, p = .07; and strain R = −0.61, p = .0009), while serum calcium levels did not predict any CV measures.

In summary, these patients with mild PHPT had abnormal endothelial function and increased carotid stiffness, both of which are risk factors for CV events. Vascular compliance was lowest in those with highest PTH levels. Although the structural CV hallmarks of severe PHPT were not commonly seen, 93% of patients with mild disease had one or more functional CV abnormalities, some of which may improve with PTX. Confirmation and extension of these findings could alter the guidelines for management of PHPT.

Disclosures: J. Fleischer, None.

M482

See Sunday Plenary Number S482

M483

Vitamin D Status Is Inversely Associated with Blood Pressure: Results from the Third National Health and Nutrition Examination Survey.

S. E. Judd¹, H. M. Blanck*², M. S. Nanes³, T. R. Ziegler*³, P. W. F. Wilson*³, V. Tangpricha³. ¹Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA, USA, ²Division of Nutrition and Physical Activity, Centers for Disease Control and Prevention, Atlanta, GA, USA, ³School of Medicine, Emory University, Atlanta, GA, USA.

The prevalence of both hypertension and vitamin D insufficiency are high in the United States. Recent clinical trials and animal studies have suggested that vitamin D insufficiency may be associated with elevated blood pressure. Using cross-sectional data, we sought to determine whether 25(OH)D levels were related to systolic blood pressure in the National Health and Examination Survey (NHANES) III (1988-1994). We excluded individuals currently taking anti-hypertensive medications to minimize the confounding of blood pressure treatment. This study was conducted in adult blacks and whites with available serum 25(OH)D determinations (N = 7699). Blood pressure was classified using the 5 categories from the Joint National Committee 7 with a sixth category added to distinguish normotensives (<110 mmHg) from those with high-normal blood pressure (110-119 mmHg). We used predicted marginals to estimate the conditional means of serum 25 hydroxy vitamin D [25(OH)D] and to test for trend across blood pressure categories. Lower 25(OH)D concentrations were significantly associated with a higher blood pressure category in whites (p < 0.001). This trend held upon stratification by age, gender, and BMI except in persons <50 years old. Such an association was not seen in black individuals; however, 92% were vitamin D insufficient, thus limiting the range of values with which to detect an association. Vitamin D insufficiency is associated with higher blood pressure in U.S. whites. Such an association was not seen in U.S. blacks given a higher prevalence of vitamin D insufficiency in this population. These data suggest the need for greater testing, detection and treatment of vitamin D insufficiency in U.S. adults for potential cardiovascular benefits. Further, these data suggest a strong interaction between vitamin D and hypertension suggesting that vitamin D repletion may be beneficial in lowering blood pressure.

Disclosures: S.E. Judd, VA Foundation Grant 2.

This study received funding from: VA Foundation Grant.

M484

See Sunday Plenary Number S484

M485

Molecular Analysis of Histologically Atypical Parathyroid Adenomas: Incipient Malignancies or Fundamentally Benign?

K. B. Lauter¹, L. J. Krebs*¹, M. R. Rubin², G. G. Fernandez-Ranvier*³, O. H. Clark*³, S. J. Silverberg², A. Arnold¹. ¹University of Connecticut School of Medicine, Farmington, CT, USA, ²Columbia University College of P&S, New York, NY, USA, ³UCSF/Mt. Zion Medical Center, San Francisco, CA, USA.

Whether atypical parathyroid adenomas represent an early stage of parathyroid carcinoma or are fundamentally benign neoplasms is unknown. Atypical parathyroid adenomas possess histological features commonly found in parathyroid cancer (e.g. fibrous bands, mitoses), but lack the definitive diagnostic criteria of distant metastases or invasion of surrounding structures. Mutations of the HRPT2 tumor suppressor gene, encoding parafibromin, are common in clear-cut parathyroid carcinomas and are rare in typical benign parathyroid adenomas. Thus, analysis of non-familial atypical adenomas for HRPT2 mutations could provide insight into their nature and malignant potential.

We examined 13 sporadic atypical adenomas for HRPT2 mutations by direct sequencing of the full 17-exon coding region and splice sites, and for parafibromin expression by immunohistochemistry using antibody 2H1 (Santa Cruz). We found no HRPT2 mutations in any of the 10 fully sequenced atypical adenomas, in striking contrast to the >75% prevalence of inactivating HRPT2 mutations previously detected in definite parathyroid malignancies using identical methodology. Moreover, parafibromin staining was performed in 12/13 atypical adenomas and was strongly or largely positive as quantified with Image J software in all 12. This pattern was similar to the positive staining in 21 typical parathyroid adenoma controls and markedly contrasted with the reduced parafibromin staining we observed in three metastatic/invasive parathyroid carcinoma controls.

The absence of detectable HRPT2 inactivating mutations, supported by parafibromin expression patterns, strongly suggests that most non-familial atypical adenomas are fundamentally different from parathyroid carcinomas and, despite their suspicious histologic features, may not share the latter's malignant potential. Our results imply a biological validity for using stringent criteria in defining parathyroid cancer, and suggest caution when considering post-surgical adjuvant therapies for patients with atypical tumors. Our findings also raise the possibility that HRPT2 inactivation might be crucial for conferring the capacity to invade surrounding tissues or to metastasize, whereas histologic features like fibrous bands and mitoses may occur in the absence of HRPT2 inactivation. In summary, disparate patterns of HRPT2/parafibromin status suggest a fundamental difference between atypical sporadic parathyroid adenomas and carcinomas.

Disclosures: K.B. Lauter, None.

M486

Intravenous Administration of Pamidronate Decreases Serum Levels of FGF23 Rapidly in Patients with Osteogenesis Imperfecta.

T. Kitaoka¹, N. Namba¹, K. Miura*¹, T. Kubota¹, H. Hirai*¹, S. Nakajima¹, T. Yamamoto², K. Ozono¹. ¹Department of Pediatrics, Osaka University Graduate School of Medicine, Suita, Osaka, Japan, ²Department of Pediatrics, Minoh City Hospital, Minoh, Osaka, Japan.

Background: Fibroblast growth factor 23 (FGF23) is a recently discovered phosphate (P)-regulating factor responsible for several disorders associated with hypophosphatemia. FGF23 decreases renal P reabsorption by down-regulating sodium P cotransporter type IIa and 25-hydroxyvitamin D-1-alpha-hydroxylase. However, the details of FGF23 regulation and action remain unclear. In this study, we examined the change of FGF23, P, calcium (Ca) and Ca-regulating hormones during the treatment of pamidronate for patients with osteogenesis imperfecta (OI). Subjects & Methods: Six patients with OI (1.4 to 12.1 year old) were included in the study. All patients were treated cyclically with pamidronate for 3 consecutive days, according to the protocol reported by Rauch et al (J Clin Invest 2002). No other medications were taken during intravenous administration. Blood samples were collected at pre- and post-drip infusion of pamidronate at the first and second cycle of treatment. We evaluated the time-course of serum Ca, P, intact PTH (iPTH), 1,25-dihydroxyvitamin D (1,25(OH)₂D) and intact FGF23 (iFGF23) (ELISA, Kainos Laboratories, inc.) values on each cycle. Written informed consent was obtained from the parents. Results: During the first cycle of treatment, serum Ca levels decreased significantly from day 2 post-infusion (-0.84 mg/dl, p < 0.05), and serum P levels also decreased from day 1 post-infusion (-0.77 mg/dl, p < 0.05). Both iPTH and 1,25(OH)₂D levels increased significantly from day 3 pre-inrusion (+140.2 pg/ml and +31.0pg/ml, respectively). In contrast, serum iFGF23 levels decreased from day 2 pre-infusion (-6.75 pg/ml, p < 0.05). During the second cycle, iFGF23 declined from the day 1 post-infusion, while serum Ca and P decreased significantly from day 3 pre- and day 1 post-infusion, respectively. Discussion: The reduction in serum Ca and P levels following pamidronate infusion was associated with the inhibition of bone resorption. Interestingly, FGF23 decreased rapidly after pamidronate infusion. It is feasible that the hypophosphatemia attenuated the levels of FGF23. However, it is also possible that pamidronate may directly suppress FGF23 secretion as well as bone resorption, since the decrease in FGF23 at times preceded hypophosphatemia.

Disclosures: T. Kitaoka, None.

M487

See Sunday Plenary Number S487

M488

A Severe Hypertension: A Possible Complication of McCune-Albright Syndrome.

Y. Ohata*¹, T. Yamamoto¹, T. Michigami², K. Satomura*³, S. Ida*³, K. Ozono⁴. ¹Pediatrics, Minoh City Hospital, Minoh, Japan, ²Bone and Mineral Research, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan, ³Pediatrics, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan, ⁴Pediatrics, Osaka University Graduate School of Medicine, Suita, Japan.

We describe a female case of McCune-Albright syndrome (MAS) complicated by severe hypertension. She had a previous history of Cushing syndrome and was undertaken surgical removal of bilateral adrenal glands when she was 10 months old. An activating mutation of the Gsα protein (Arg 201 to His) was proved in the stored frozen adrenal tissues. We first observed severe hypertension with the systolic blood pressure of 240 mmHg when she was 9 years old. Laboratory investigation revealed that the levels of serum calcium and phosphate were low (8.5 mg/dl, and 2.9 mg/dl, respectively), and serum alkaline phosphatase was extremely high (8720 U/l). Then, urinary excretion levels of norepinephrine and dopamine were extremely increased by 10 and 4 folds compared to the average levels of the age matched controls respectively. Meta-iodobezylguanigine (MIBG) scintigraphy proved neither pheochromocytoma nor the residual adrenal glands. Iatrogenic hypertension by the supplemental mineral corticoid was implausible because of the normal plasma rennin activity. Hypertension was ameliorated gradually by the combination therapy of several antihypertensive medications including an angiotensin receptor blocker, a calcium blocker, an alfa blocker and diuretics. Since the Gsα protein regulates the norepinephrine synthesis in the sympathetic nodes, it is plausible that an activating mutation of the Gsα protein in MAS complicated by a Cushing syndrome is associated with the cause of hypertension due to the overproduction of norepinephrine, because both of the sympathetic nodes and the adrenal glands in medulla have the similar origins in the process of embryological development. Also this rare case suggested the possible role of Gsα genes in the primary hypertension.

Disclosures: Y. Ohata, None.

M489

See Sunday Plenary Number S489

M490

Acro-osteolysis and Calcinosis — Possible Role of the Sympathetic Nervous System (SNS) in Pathogenesis and Treatment with Beta-blockers.

M. Patel¹, C. Senger*², P. Malleson*², S. Ichikawa³, M. J. Econs³, K. Mulpuri*². ¹Medical Genetics, University of British Columbia, Vancouver, BC, Canada, ²University of British Columbia, Vancouver, BC, Canada, ³Indiana University, Indianapolis, IN, USA.

A 9 year old healthy girl presented with a 1.5 year history of idiopathic, progressive distal acro-osteolysis of all toes. Dystrophic calcification of adjacent soft tissues caused swelling and pain with periodic rupture of a whitish chalky substance through the dorsal skin of the toes. These findings were associated with slightly tight distal phalangeal skin and distal erythema of the fingers and toes. No systemic abnormalities of bone or mineral ion homeostasis were present, no serologic or histologic findings consistent with rheumatologic disease were present and sequencing of the GALNT3 and FGF23 genes did not real mutations in the coding regions or at the intron-exon boundaries. Surgical decompression of one toe demonstrated homogeneous calcification surrounded by reactive macrophages. Analysis of a remnant distal phalanx demonstrated abundant periosteal osteoclasts and absence of osteoblasts. As the cellular findings in bone were consistent with hyperactivation of the SNS, the patient was started on 1.5 mg/kg/day propranolol. This was increased every 2 weeks until beta-blockade occurred, confirmed by an absence of exercise-induced tachycardia, at a final dose of 9 mg/kg/day. Treatment for one year was associated with regression of nascent calcification, arrest of osteolysis and partial regrowth of actively resorbing distal phalanges. The biopsied phalanx re-grew and showed no recurrence of acro-osteolysis on treatment.

Disclosures: M. Patel, None.

M491

Low Total and Bioavailable Testosterone Does Not Play a Role in Renal Bone Disease in Men on Hemodialysis.

U. Gruntmanis¹, C. V. Odvina², K. Sakhaee*², J. E. Zerwekh². ¹Department of Medicine, Division of Endocrinology, UT Southwestern Medical Center and Dallas Vetreans Affairs Medical Center, Dallas, TX, USA, ²Center for Mineral Metabolism and Clinical Research, UT Southwestern Medical Center, Dallas, TX, USA.

In previous studies as many as 60% of men on hemodialysis (HD) are found to be hypogonadal. Yet role of low testosterone in complex pathogenesis of renal bone disease has not been defined. In this cross-sectional study our primary goal was to determine if men on HD with low total testosterone (testosterone below 300 ng/dl) have lower BMD than eugonadal men. Second goal was to find out if there is a difference between serum bone formation and resorption markers (bone specific alkaline phosphatase (BSAP), osteocalcin (OC), type 5b tartrate-resistant acid phosphatase (TRAP5b)) between hypogonadal and eugonadal men on HD. Only BSAP and TRAP5b are not dialyzable and therefore are good markers of bone turnover for this population. We analyzed data on 83 men, 52 African Americans, 20 Hispanic, 9 Caucasians and one Indian American. 50 and 33 men were eugonadal and hypogonadal, respectively. Mean age 52.8 ± 8.2, weight 187.6 ± 40.8 lbs, HD length 30.8 ± 26.1 month and was not different between hypogonadal and eugonadal groups.

Results: BMD at lumbar spine (LS), femoral neck (FN), total hip (TH), distal forearm (DF) and BSAP, TRAP5b, OC levels were not different between hypogonadal and eugonadal groups. 43.8% and 82.1% of men were found to be vitamin D deficient (25-D <20 ng/ml) or insufficient (25-D<30ng/ml), yet low 25 hydroxy vitamin D (25-D) level did not correlate with intact PTH, BMD at LS, FN, TH, DF and BSAP, TRAP5b, OC levels in our study.

Length of HD did not correlate with weight, total testosterone, bioavailable testosterone, estradiol, 25-D, BMD or bone turnover markers. Weight had very strong positive correlation for BMD at LS (p = 0.01), FN (p < 0.0001), TH (p < 0.0001) and DF (p = 0.068) but negative correlation for BSAP (p < 0.009), TRAP5b (p < 0.03), OC (p = 0.4). Intact PTH (i-PTH), had positive correlation with BSAP (p = 0.001), OC (p < 0.0001), TRAP5b (p = 0.08). BSAP as expected, had negative correlation with BMD at TN (p = 0.02), DF (p = 0.05), but no correlation at FN (p = 0.08), LS (p = 0.3). TRAP5b also had negative correlation with BMD at FN and TN (p = 0.04) but no correlation at DF (p = 0.49) and LS (p = 0.07).

Conclusions: Total testosterone level does not appear to play significant role in renal bone disease in our study population. We postulate that men on HD who are heavier have lower bone formation and resorption and as a result have higher BMD. This observation did not change after results were adjusted for total testosterone, bioavailable testosterone, estradiol, i-PTH, 1,25-D and 25-D levels.

Disclosures: V. Gruntmanis, None.

This study received funding from: National Osteoporosis Foundation and GCRC USPHS grant M01-RR00633.

M492

Administration of Pravastatin Inhibits Suppressed Osteoblast Dysfunction and Bone Turnover in Uremic Rats.

Y. Iwasaki*¹, H. Yamato², M. Fukagawa³. ¹Health Sciences, Oita University of Nursing and Health Sciences, Oita, Japan, ²Development, Kureha Special Laboratory Co, Ltd., Fukushima, Japan, ³Nephrology & Dialysis center, Kobe University School of Medicine, Kobe, Japan.

HMG-CoA reductase inhibitors (statins) are widely used for treatment of hypercholesterolemia. Recent studies reveal that statins stimulate bone formation and suppress bone resorption. To evaluate the effect of pravastatin, which is a major statin, on bone formation in low-turnover bone disease with renal failure, we demonstrated bone histomorphometry on our model rats which have low turnover bone. Male SD rats underwent thyroparathyroidectomy (TPTx). The TPTx rats 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. TPTx-Nx rats simulate adynamic bone disease which is a major type of renal osteodystrophy in chronic dialysis patients. Six weeks after the second Nx, TPTx-Nx rats were assigned to receive pravastatin (Daiichi-Sankyo, Co, Ltd., Japan) at a dosage of 3 or 30 mg/kg body weight per day or vehicle in the diet. By bone histomorphometry, bone formation rate in the TPTx-Nx with vehicle group was reduced to 8% of the TPTx-Sham group. The reduction of bone turnover was ameliorated by pravastatin administration in a dose dependent manner. In the high dose group, the level of osteoblast surface recovered was up to 40% of the TPTx-Sham group. The Serum biochemical parameters, including lipid metabolic parameters, did not change among all the groups. Gene expression of oraganic anion transporter (OAT)-3, which mediates cellular uptake of pravastatin, was expressed in rat tibiae. In vitro study, we also determined the effect of co-culturing with pravastatin and indoxyl sulfate (IS), which is one of the uremic toxins, on cultured primary osteoblasts. It is known that IS is taken up via OAT-3 and increases free radical production and suppresses cell viability. Free radical production induced by the addition of IS was decreased in the presence of pravastatin in primary cultured osteoblasts. Suppressed osteoblasts cell viability ameliorated in a dose dependent manner. OAT-3 mRNA was expressed in cultured osteoblast. These results suggest that pravastatin ameliorates bone turnover, induced at least in part by suppressed intracellular free radical production and improve osteoblast function.

Disclosures: Y. Iwasaki, None.

M493

See Sunday Plenary Number S493

M494

Tartrate-Resistant Acid Phosphatase Isoforms in End-Stage Renal Disease.

A. Janckila¹, B. Price*², E. Lederer², L, Yam*¹. ¹Medicine, VA Medical Center, Louisville, KY, USA, ²Medicine, University of Louisville, Louisville, KY, USA.

End-stage renal disease (ESRD) is often complicated by chronic inflammation and osteodystrophy. Tartrate-resistant acid phosphatase (TRACP) isoforms 5a and 5b may be suitable serum markers to gauge inflammation and bone resorption respectively.

Methods: Serum was collected from 73 patients (38 male, 35 female) with ESRD just prior to hemodialysis. The mean age was 56 ± 16 years and the mean time on dialysis was 6 ± 5 years. A control group consisted of 36 healthy adults (8 male, 28 female) aged 43 ± 11 years. TRACP 5a and 5b isoforms were estimated by in-house immunoassays. C-reactive protein was estimated by an in-house, high-sensitivity assay using commercially available antibodies and standards. Commercial kits were used to estimate other inflammation and bone markers including fetuin-A, bone-specific alkaline phosphatase (bALP) and cross-linked N-telopeptides of type-1 collagen (NTx). Intact parathyroid hormone (iPTH) levels were determined by radioimmunoassay. Bone mineral density (DEXA of calcanus) was determined at the time of study.

Results: Mean levels of all serum markers were significantly elevated in ESRD except fetuin-A, which was significantly lower than control. Absolute BMD (g/cm2) was no different than control, but the mean T-score was significantly lower in ESRD. TRACP 5a protein correlated with CRP but not fetuin-A or any other bone marker. CRP and fetuin-A were inversely correlated. TRACP 5b correlated positively with all bone markers and inversely with BMD, but not with any inflammation marker. Neither iPTH nor NTx were significantly associated with BMD.

Conclusion: These preliminary data suggest that TRACP isoforms 5a and 5b may be useful markers to estimate the degree of inflammation and bone resorption respectively in ESRD patients on chronic hemodialysis.

Disclosures: A. Janckila, None.

M495

Albuminuria Predicts Bone Loss in Older Adults: The Rancho Bernardo Study.

S. K. Jassal*¹, D. von Muhlen², E. Barrett-Connor². ¹Medicine, UCSD & VASDHS, San Diego, CA, USA, ²Family and Preventive Medicine, UCSD, San Diego, CA, USA.

Purpose: There is interest in the association between kidney function and bone health, but prior studies have focused on estimates of creatinine clearance or glomerular filtration rate as predictors. We investigate the cross-sectional and longitudinal association between albuminuria and bone mineral density (BMD), bone loss, and osteoporotic fracture in older community-dwelling adults.

Methods: A cross-sectional and prospective study from the Rancho Bernardo Study. Between 1992 and 1995, 1713 participants (average age 71.3 +/- 11.1 years) completed standardized questionnaires, physical examinations, laboratory testing and bone densitometry; 1023 participants returned for a follow-up visit in 1997-1999, an average of 4.1 (+/-0.9) years later.

Results: Using previously established sex-specific cut-offs, albuminuria (urine albumin/creatinine ratio (ACR) ≥ 17mg/g in men and ≥ 25mg/g in women) was present in 148 men (22%) and 143 women (13.8%) at baseline. In cross-sectional analyses, there was a significant linear association between log transformed ACR (log ACR) and hip BMD in men (Beta (SE) = −0.021 (0.010), p = 0.034) and women (Beta (SE) = −0.053 (0.008), p < 0.001). These associations were no longer significant after adjusting for age and body mass index (BMI). In prospective analyses, there was an average annual bone loss at the hip of 0.6%. There was a significant association between baseline log ACR and four-year bone loss in men (Beta (SE) = −0.237 (0.089), p = 0.008) and women (Beta (SE) = −0.332 (0.0110), p = 0.003). These associations persisted after adjusting for age and BMI in both men (Beta (SE) = −0.212 (0.010), p = 0.018) and women (Beta (SE) = −0.254 (0.114) p = 0.026). At baseline, 180 of 1713 participants (11%) reported at least one clinical fracture of the hip, femur, forearm or wrist; 79 (8%) reported at least one new clinical fractures during follow-up. Log ACR was associated with prevalent clinical fractures in women OR (95% CI) = 1.67 (1.19-2.35), but this did not persist after adjusting for age and BMI. Log ACR was not significantly associated with incident clinical fractures.

Conclusions: ACR was not independently associated with hip BMD in cross-sectional analyses. Baseline ACR, however, predicted four-year bone loss at the hip in both sexes independent of age and BMI. Baseline ACR was associated with prevalent but not with incident clinical fracture, perhaps reflecting the multifactorial etiology of fractures beyond BMD.

Disclosures: S.K. Jassal, None.

This study received funding from: Grant AG07181 from the National Institute on Aging and Grant DK31801 from the National Institute of Diabetes and Digestive and Kidney Diseases. Procter & Gamble Pharmaceuticals Unrestricted Educational Grant.

M496

Osteopenia and Lipodistrophy in HIV-infected Patients.

S. Azriel*¹, R. Rubio*², H. Requeio*¹, S. Guadalix*¹, E. Jódar¹, G. Martinez¹, F. Hawkins¹. ¹Endocrinology Service, University Hospital 12 de Octubre, Madrid, Spain, ²HIV Unit, University Hospital 12 de Octubre, Madrid, Spain.

Reduced bone mineral density (BMD) and abnormalities in fat redistribution, glucose homeostasis and lipid metabolism are prevalent among HIV-infected patients on highly active antiretroviral therapy (HAART). The relationship between BMD and changes in regional and whole body composition is unclear.

Patients and methods: This was a prospective cohort study. One hundred HIV-infected Caucasian outpatients were recruited during a period of 24 months and were studied over 2 years of follow-up. Exclusion criteria were any condition that could cause loss of BMD and use of medications that interfere with mineral metabolism in the past 6 months. 68% of the patients were males, mean age of the cohort was 40 years (SD: 9), mean BMI was 24 kg/m³ (SD: 3.6), 95% had undetectable virus load .77%t were taking a HAART regimen (65% included a protease inhibitor), 18% bitherapy (2 nucleoside reverse-transcriptase inhibitors) and 5% were ART-naïve. BMD (lumbar spine, femoral neck, total hip and radium) and body composition (total body fat mass, percent total fat, trunk fat mass, percent trunk fat, lean mass) were assessed by DXA Hologic QDR 4500. Osteopenia/osteoporosis (op/OP) was defined according to WHO criteria. Lipodystrophy was classified according to clinical criteria (patient's self report and physician's evaluation).

Results: The prevalence of op (at any site) in the cohort was 63.6% and 13.1% of OP. Alterations in body fat distribution were described in 38% of the patients: 35% lipoatrophy, 16% lipohypertrophy and 13% mixed anomalies. In the univariate statistical analysis, patients with low lumbar spine BMD (<–1 SD T-score) showed lower total (kg) and % body fat mass (p = 0.06 and p = 0.049, respectively), and trunk fat mass (kg, %; p = 0.039 and p = 0.047). Patients with low femoral BMD (at the femoral neck and total hip) also had lower total body fat mass (p = 0.013 and p = 0.003, respectively). There was no independent association between op/OP and type or duration antiretroviral therapy and lipodystrophy at any site. In the multivariate logistic regression analysis, the total body fat mass was a protective factor for bone loss at lumbar spine [OR: 0.89, 95% CI: 0.82-0.96, p = 0.003, for each additional kg]. No significant changes in BMD and in body composition were found after 2 years of follow-up.

Summary: Prevalence of op in HIV-infected patients is high, irrespective of the antiretroviral therapy. Lumbar op is associated with low total body fat mass. Op does not progress after 24 months.

Disclosures: S. Azriel. None.

This study received funding from: Fundación Mutua Madrileña (project n^o 2005-072).

M497

Anti-TNF Treatment in Rheumatoid Arthritis Patients Does Not Alter the 1-Year Change in Spine and Hip Bone Mineral Density Compared to Control Patients.

P. Boyesen*¹, E. Haavardsholm*¹, G. Haugeberg*², T. Kvien¹. ¹Dept of Rheumatology, Diakonhjemmet sykehus, Oslo, Norway, ²Dept of Rheumatology, Sørlandet hospital, Kristiandsand, Norway.

An increased bone loss is observed in rheumatoid arthritis (RA) patients. Observational uncontrolled studies have suggested that anti-TNF treatment counteract this loss. The aim of this study was to compare one-year change in hip and spine bone mineral density (BMD) between RA patients receiving anti-TNF treatment and historical RA controls.

Twenty-nine RA patients with clinically active disease starting with anti-TNF treatment were included in the intervention group and 253 RA patients were identified as controls from the Oslo RA register. The controls were matched on a group level for disease duration and age. The BMD of hip and spine was measured by dual energy x-ray absorptiometry (DXA) on the same Lunar Expert machine. The anti-TNF group was examined at baseline and 12 months, the controls were assessed in 1996 and 1998 as part of an epidemiologic study.

Mean (SD) age was 52 (6.2) and 55 (6.2) years in the anti-TNF group and control group respectively, disease duration 10 (6.2) and 10 (6.2) years, 82% and 83% were females, 90% and 49% were rheumatoid factor (RF) positive, mean (SD) disease activity score (DAS-28) was 5.3 (1.3) and 4.7 (1.1), Modified Health Assessment Questionnaire (MHAQ) score 1.7 (0.5) and 1.6 (0.4), ESR 31.8 (19.6) and 21.4 (15.8) mm/hr and 28-swollen joint count 9.8 (5.6) and 8.4 (6.0). The only group differences were RF (p < 0.001), ESR (p = 0.02) and DAS-28 (p = 0.01) (Chi square, t-test). 45% and 42% used disease modifying antirheumatic drugs, 14% and 22% estrogens and or bisphosphonates, 52% and 45% were currently using corticosteroids in the anti-TNF group and control group respectively (Chi square test).

At inclusion 55% and 39% were osteopenic, 3% and 13% osteoporotic in the anti-TNF group and the control group respectively. Annual BMD change was computed. BMD of spine and hip were similar between the groups at baseline (t-test). The one-year change was not statistically significant different between the groups. The mean (SD) change in total hip BMD was −0.9% (3.3) and −0.2% (2.6) (p = 0.17) and the spine BMD (L1-L4) −1.0% (5.1) and −0.2% (2.8) (p = 0.17) for the anti-TNF group and control group respectively. The hip BMD results remained unchanged when correcting for sex, RF, corticosteroids, bisphosphonates, DAS-28 and MHAQ (regression analysis). The adjusted change in BMD at the spine showed an association between increased loss in BMD and anti-TNF treatment (p = 0.047).

In conclusion the one-year bone loss of the hip and spine was similar in RA patients treated with anti-TNF and historical RA controls. This finding may suggest that the mechanisms of bone loss in established RA are not halted by TNF blockage.

Disclosures: P. Boyesen. None.

M498

See Sunday Plenary Number S498

M499

Small Molecule Inducer of Activated CD4+ T Cell Death Inhibits Disease Progression in Collagen-induced Arthritis Models.

J. Han*¹, H. Sung*¹, J. Park*¹, S. Lim*², S. Lee*², S. Cho*³, H. Kim*³, S. Shim*², S. Kim¹, S. Ko⁴, S. Chang*¹, J. Kim*¹. ¹Pharmacology, OSCOTEC Inc., Cheonan, Republic of Korea, ²Medicinal Chemistry, OSCOTEC Inc., Cheonan, Republic of Korea, ³Pharmacokinetics, OSCOTEC Inc., Cheonan, Republic of Korea, ⁴Oral Biochemistry, Dankook University, Cheonan, Republic of Korea.

Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory autoimmune disorder that affects 1% of the adult population worldwide. RA is characterized by the inflammation of synovial joints infiltrated by CD4+ T cells, macrophages, and plasma cells that play major roles in the pathogenesis of the disease. CD4+ T cells responding to an unknown antigen activate macrophages to produce pro-inflammatory cytokines. Therefore, selective elimination of activated CD4+ T cells, but not naive cells, may slow down disease progression with minimum toxicity. To identify small molecules that selectively induce activated CD4+ T cell death, high-throughput screening (HTS) was conducted on 5,680 compounds. From the Hit compound identified from HTS, we generated lead compound (TCK-O-1). TCK-O-1 was found to induce activated T cell death in a dose-dependent manner. The cytotoxic effects of TCK-O-1 to naïve splenocytes were observed only at high concentrations (>10 uM), while selective killing effects on activated CD4+ T cells (up to 60 percent) were observed at low concentrations (<3 uM). In addition, TCK-O-1 did not show cytotoxic effect on other immune effector cell lines. To examine the effect of TCK-O-1 on disease progression in RA, time course studies were conducted in collagen induced arthritic (CIA) mice. TCK-O-1 was orally (40 mg/kg) or subcutaneously (10 mg/kg) administered to CIA mice, once a day for 21 days. Administration of TCK-O-1 significantly suppressed the arthritic index and inhibited the activated CD4+ T cell ratio in peripheral blood mononuclear cells (PBMC), but not other immune cells. Therefore, administration of TCK lead compound effectively inhibited disease progression with minimum toxicity. These results suggest that small molecule inducer of activated CD4+ T cell death may be used for the treatment of rheumatoid arthritis. TCK-O-1 is under development as candidate molecules in search for new anti-rheumatoid arthritis drugs.

Disclosures: S. Chang, None.

M500

Can We Predict Structural Damage Progression at 2 Years in Very Early Arthritis? Value of Bone and Cartilage Markers in the Conservatively Treated Community-based Inceptive VErA Cohort.

A. Daragon¹, M. Brazier*², C. Fèvre*¹, R. Daveau*³, O. Mejjad*¹, P. Boumier*⁴, A. Gayet*⁵, F. Tron*³, C. Zarnitsky*⁶, O. Vittecoq*⁷, X. Le Loët*⁷. ¹Rheumatology, CHU — Hǒpitaux de Rouen, Rouen, France, ²Rheumatology, CHU, Amiens, France, ³Immunology Laboratory, Inserm U 519, Rouen, France, ⁴College of Rheumatology Picardie, Amiens, France, ⁵College of Rheumatology Haute Normandie, Rouen, France, ⁶Rheumatology, Groupe Hospitalier, le Havre, France, ⁷Rheumatology, CHU — Hǒpitaux de Rouen, Inserm U 519, Rouen, France.

Objective: identification of bone and cartilage markers to predict structural damage progression at 2 years in patients with very early arthritis.

Methods: community-based-recrutment (media campaigns) of 310 adults (mean age: 51.8 years; 68% females) with swelling of 2 ore more joints persisting more than 4 weeks, evolving for less than six months (mean: 4.2 months), DMARD and steroids naïve. Then, they were conservatively treated for 2 years. Assessment was made at entry and comprised clinical data (global pain, Ritchie index and swollen joints/44), CRP, ESR, auto antibodies rheumatoid factors (RF) by agglutination tests and IgM, IgA, IgG, isotypes by Elisa, anti-CCP II, serum pyridinoline (PYD) deoxypyridinoline (DPD), CTX1, Cartilage Oligométric Matrix Protéin (COMP), osteoprotegerin (OPG) and RANKL. All patients with ACR RA criteria or undifferentiated arthritis were followed except those classified as having defined rheumatism (DR). Criterion of judgment was structural damage progression of erosion with an increase of at least 1 unit at 2 years (measurement was done according to vdH modified erosion Sharp score by two readers.

Results: At 2 years, 213 patients were assessed (29 refused, 1 lost to follow-up; 1 death, 61 classified as DR, 5 with × ray failure): 42 (19,7%) had erosion progression. With univaried analysis, there was a relationship between erosion progression and COMP (p = 0.017), VS (p = 0.003), IgG and IgA (p = 0.007 and 0.005), and anti-CCP II (p < 10⁻⁴). But OPG, CTX1, PYD, DPD, and CRP were not associated with erosion progression. With principal components analysis 75% patients are correctly classified as progressive or not progressive with these five markers. COMP give a better sensibility (61.5%) than other markers (52%). Results of RANKL will be presented.

CONCLUSION: In patients with very early arthritis, conservatively treated, COMP, anti-CPP II, IgA RF, IgG RF, and ESR, are useful to identify patients who will have structural damage progression at 2 years. This population could be a good target for early aggressive treatment.

Disclosures: A. Daragon, None.

This study received funding from: Fondation pour la Recherche Médicale.

M501

Material Properties of Osteoporotic Ewes.

E. F. Calton*¹, J. MacLeay*², A. Boskey¹. ¹Hospital for Special Surgery, New York, NY, USA, ²Colorado State University, Fort Collins, CO, USA.

The purpose of this study was to establish baseline material properties for osteoporotic cancellous bone from ovine iliac crest biopsies. Skeletally mature (4-7 yo; 169 ± 29 lb) Rambouillet-Columbia cross-ewes (n = 18) were administered a diet to induce metabolic acidosis (MA) and create bone loss similar to that seen in human osteoporosis [MacLeay et al. 2004 J Bone Miner Metab 22:561-8]. Bone densitometry (DXA) measurement of the 4 most caudal vertebrae lumbar spine was performed using a Hologic Delphi QDR dual-energy x-ray absorptiometer (Hologic). Transiliac crest biopsies (8mm dia.) were removed and placed in ethanol. Micro-CT data were collected on the cortical and cancellous regions separately for volumetric bone mineral density measurements. Biopsies were bisected axially with a low-speed saw; half was saved for embedding and histological sectioning and half was prepared for infrared (IR), x-ray diffraction (XRD), and chemical analysis of Ca and PO₄. On the latter half, the cortical ends were removed with the low speed saw. Cancellous bone was defatted using a 1:1 mixture of Chloroform and Methanol and lyophilized 16 hours. The dried and defatted cancellous bone was pulverized using a Spex Mill CertiPrep Freezer/Mill. The XRD line width of the c-axis 002 peak was measured using Cu Kα radiation (Bruker AX-8 powder diffractometer, Bruker). FTIR absorbance spectra were collected at 4 cm⁻¹ on pulverized bone formed in KBr pellets (1 wt%). Spectra were baseline corrected and integrated areas were taken for three spectral regions: matrix amide I (1595-1720 cm⁻¹), mineral phosphate v1, v3 (900-1200 cm⁻¹), and carbonate v2 (855-890 cm⁻¹), and area ratios (mineral:matrix and carbonate:phosphate) were calculated. Peak height ratios 1030:1020 cm⁻¹ and 1660:1690 cm⁻¹ were calculated as a measure of crystallinity and collagen cross-linking (maturity), respectively [Paschalis et al. 2001 JBMR 16:1821-8]. Acud hydrolyzed samples were used for phosphate concentration determined by colorimetric assay and for Ca concentration measured by atomic absorption (Perkin Elmer AAnalyst 100). The values obtained from these measurement methods provide baseline values for cancellous bone from sheep iliac crest biopsies. These values are being used to assess the efficacy of therapeutic interventions on sheep with reduced bone mass and may be useful in determining similar effects in humans with osteoporosis. 

Table  .  

Disclosures: E.F. Calton, None.

This study received funding from: NIH Grant AR043215.

M502

Finite Element Models More Accurately Predict Structural Behavior of Human Cancellous Bone When Using Specimen-Specific Tissue Properties.

J. H. Cole¹, E. R. Myers*², M. C. H. van der Meulen¹. ¹Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY, USA, ²Biomedical Mechanics and Materials, Hospital for Special Surgery, New York, NY, USA.

Osteoporosis affects cancellous bone tissue properties through altered mineralization and changes in chemical composition, which may compromise its structural behavior. Experimental assessments of tissue properties are limited primarily to localized in vitro measurements that likely do not fully capture the in vivo spatial variations. Finite element (FE) models that simulate tissue heterogeneity over a larger region of bone can be used to investigate changes in tissue properties non-invasively. This study examined the effect of changes in tissue material properties on the structural behavior of cancellous bone using architecture- and material-based FE models. Cylindrical cores were drilled from the center of the T12 and L2 vertebrae of 13 female and 11 male human cadavers, ages 56-92, and scanned using micro-computed tomography (microCT) at an 11.6-μm resolution. Cores were fixed in brass endcaps and tested to failure at 0.5% strain/s, and apparent stiffness was computed. Micro CT bone voxels were coarsened to 68 μm and directly converted to 8-noded linear brick elements for the gage length region of each cancellous core. A variable tissue modulus (E) was assigned for three sets of models: universal homogeneous (Homo) with E = 20 GPa, specimen-specific homogeneous (SS Homo) with E = f(mean micro CT mineral content), and specimen-specific heterogeneous (SS Hetero) with E = f(micro CT mineral content at each voxel). A uniform compressive strain of 0.25% was applied to the models, and the resulting apparent stiffness and element tissue strains were computed. FE-predicted stiffness was compared to experimentally-measured stiffness using simple linear regression for each set of models. A significance level of 0.05 was used. Repeated measures ANOVA was used to assess the effect of the assigned modulus on the predicted stiffness and tissue strains Overall, the heterogeneous tissue modulus ranged 5.2-36.6 GPa and averaged 9.9-13.0 GPa over all models. Model-predicted apparent stiffness was 0.13-1.50 GPa for Homo, 0.10-0.79 GPa for SS Homo, and 0.09-0.79 GPa for SS Hetero models. Compared to Homo models, SS Homo and Hetero models predicted cancellous bone stiffness more accurately (slopes = 0.36, 0.78, 0.78) and explained more variability (r² = 0.29, 0.42, 0.42). A virtual biopsy modeling method allows tissue property variations to be examined non-invasively and may help predict bone fragility in the aging population.

Disclosures: J.H. Cole, None.

M503

Regional Variations in Three-Dimensional Microstructural Properties of Proximal Femoral Trabeculae with Aging.

Y. Won¹, B. Min*², Y. Chung³, W. Cui*¹, M. Baek*¹, H. Kim*⁴. ¹Orthopaedics, Ajou University School of Medicine, Suwon, Republic of Korea, ²Orthopaedics, Keimyung University School of Medicine, Daegu, Republic of Korea, ³Endocrinology and Metabolism, Ajou University School of Medicine, Suwon, Republic of Korea, ⁴Orthopaedics, Yonsei University School of Medicine, Seoul, Republic of Korea.

Purpose: The purpose of this study was therefore to explore region-dependent changes in the 3D microstructure of trabecular bone in human proximal femur, with respect to aging.

Materials and Methods: A total of 162 trabecular bone cores were obtained from six regions (femoral head, superior and inferior regions of the neck and superior/middle and inferior regions of the trochanter) of twenty-seven normal femora of Korean male cadaver donors, aged 40-90 years. These specimens were scanned using high-resolution micro-computed tomography (micro-CT). The following 3D microstructural parameters were calculated: bone volume fraction (BV/TV), trabecular number (Tb.N), thickness (Tb.Th) and separation (Tb.Sp), structure model index (SMI), and degree of anisotropy (DOA).

Results: The results showed that the trabecular morphology changed significantly with age, as well as varied from different regions of the proximal femur. There was a significant decrease in bone volume fraction and an almost identical decrease in trabecular thickness associated with aging at any region. Regional analysis demonstrated a significant difference in BV/TV, Tb.Th, DOA, Tb.Sp, Tb.N between superior and inferior neck, as well as a significant difference in BV/TV, Tb.Sp, DOA, SMI, Tb.N between superior and inferior trochanter.

Conclusions: Age-related changes in bone loss and trabecular microstructure within the male proximal femur are not uniform in this Korean cadaveric population. As a result of mechanical and age-related adaptation, significant regional variations in microstructural properties of trabecular bone are likely to important factors affecting the mechanical properties of the proximal femur.

Disclosures: W. Cui. None.

M504

See Sunday Plenary Number S504

M505

Calcium Density of Bone Correlates with Trabecular Bone Architecture.

N. L. Fazzalari¹, B. Ma*¹, P. Sutton-Smith*¹, J. S. Kuliwaba¹, R. Phipps², I. H. Parkinson*¹, A. Badiei*¹. ¹Bone and Joint Research Laboratory, Institute of Medical and Veterinary Science and Hanson Institute, Adelaide, Australia, ²Procter & Gamble Pharmaceuticals, Mason, OH, USA.

Many of the skeletal risk factors for fragility fracture have been defined (bone mass, bone geometry, bone architecture, bone mineralisation and bone remodelling). The amount of bone and the degree of mineralisation have been shown to influence the ultimate failure strength of bone independently. In this study we tested if there was any relationship between calcium density of bone (D_Ca = BV/TV x %calcium) and trabecular bone architecture. Intertrochanteric cancellous bone cores were obtained from patients undergoing hemi-arthroplasty surgery for a subcapital fragility femoral fracture (N=40; 26f, 14m, mean age ± SD of 81 ± 9 years), and from controls at autopsy (N=10; 2f, 8m, 76 ☆ 14 years). All samples were imaged by micro CT (Skyscan, Belgium) to determine 3D measures of trabecular bone volume (BV/TV), thickness (Tb.Th), separation (Tb.Sp) and number (Tb.N). An age matched subset of 18 fracture cases (13f. 5m, 82 ± 8 years) and 4 control cases (If, 3m, 79 ± 9 years) were resin-embedded for quantitative backscattered electron imaging (qBSEI) of the degree of mineralisation.

The trabecular bone architecture of the fracture and nonfracture control group was not significantly different. For the pooled group data, Tb.Sp increased exponentially as BV/TV decreased (y = 3.2 x^−0.5, P < 0.001). Between each group the rate at which Tb.Sp increased was more than three times greater for the fracture group in contrast to the control group. In the pooled group data of the qBSEI analysis, BV/TV did not correlate with %Ca indicating that the measures of bone volume and tissue level mineralisation were independent variables in the bone structural hierarchy. D_Ca did positively correlate with Tb.Th and according to the magnitude of r² accounted for greater than 25% of the variability in Tb.Th. In contrast, BV/TV accounted for about 10% and %Ca did not account for any variability in Tb.Th. Collectively, these data suggest that Tb.Th may be reactive to changes in D_Ca through an interaction between BV/TV and %Ca. It is understood that increased fragility fracture risk is associated with decreased BV/TV and reduced mineralisation of the bone tissue but possible secondary consequent changes in bone architecture have not been investigated to date. D_Ca, a composite bone material property provides a novel perspective of bone quality that might help provide a better understanding of fracture risk and treatment efficacy.

Disclosures: N.L. Fazzalari, Procter & Gamble 2, 5.

This study received funding from: Alliance for Better Bone Health.

M506

Role of Microarchitecture on Whole-Vertebral Body Biomechanical Behavior.

A. J. Fields*, S. K. Eswaran*, T. M. Keaveny. Mechanical Engineering, University of California, Berkeley, Berkeley, CA, USA.

Trabecular microarchitecture can explain variations in biomechanical properties of isolated trabecular bone specimens, yet the role of microarchitecture on whole-bone biomechanical behavior remains unclear. For the spine, the influence of microarchitecture might be obscured by such factors as cortical shell thickness and overall bone size. The purpose of this study was to assess the role of trabecular microarchitecture with and without such factors on whole-vertebral mechanical behavior.

Human T9 whole vertebral bodies (posterior elements removed) from male (n=5) and female (n=13) cadavers (87.4±5.0 years) were micro-CT scanned at 30-micron resolution. Internal regions (approximately 15×20×10 mm³) of trabecular bone were analyzed for the standard model-independent microarchitectural parameters (Table). An average cortical shell thickness (Ct.Th) was also measured. Linear, isotropic finite element models (60-micron element size), containing 30–80 million elements each and with assumed uniform bone tissue material properties, were created for each whole vertebra from the micro-CT scans and subjected to uniform compression loading to compute whole-vertebral stiffness (K_FE).

Results indicated that total bone volume (BV) was the best independent predictor of whole-vertebral stiffness, followed by cortical thickness (Ct.Th) and then trabecular spacing (Tb.Sp) and SMI (Table). BV/TV was not an independent predictor Using stepwise multiple regression with all parameters, the model included BV, Tb.Sp, and Conn.D (R²=0.77->0.86->0.92); when BV was excluded, the model included Ct.Th and SMI (R²=0.45->0.62); and when Ct.Th was also excluded, the model included SMI and Tb.Sp (R²=0.35->0.55).

While these finite element models did not contain any tissue mineralization effects and this elderly cohort was limited in size, this study is unique since it included measures of microarchitecture and whole-vertebral mechanical behavior for the same specimens. We conclude that trabecular microarchitectural parameters have the potential to improve predictions of whole-vertebral biomechanical behavior, although their influence interacts with other vertebral features. Ongoing efforts are extending these studies to a larger and younger cohort. 

Table Table:. Pearson correlation coefficients (n=18)

Disclosures: A.J. Fields, None.

This study received funding from: NIH AR49828.

M507

Withdrawn.

M508

See Sunday Plenary Number S508

M509

Assumptions and Limitations in Image-Based Strength Assessment of Bone.

T. N. Hangartner. BioMedical Imaging Laboratory, Wright State University, Dayton, OH, USA.

The goal of many bone measurements is the assessment of the likelihood of future fracture. The most commonly used surrogate for strength is density, which can be measured through the attenuation of x-rays. Mechanical testing of machined bone specimens as well as whole bones usually shows an initial linear behavior between load and deformation, leading to a region of reduced resistance to load and eventually reaching the breaking point. Although the initial slope of these curves is tightly related to the material properties of the bone as well as to the architecture and can be estimated with finite-element models, the breaking point is influenced by non-linear features and differs by age and disease state, usually not taken into account.

Computed tomography, through its three-dimensional representation of the bone, has the highest potential to provide the architectural information needed for accurate mechanical simulation. Although average density of the various compartments like trabecular and cortical bone has proven clinically relevant, strength calculations based on the distribution of the bone material are expected to better represent the strength of an individual bone. It is critical, here, to appropriately convert the CT-measured density values to local elastic moduli, a relationship that almost follows a square law. One specific parameter used by some investigators, the strength-strain index, assumes a linear relationship and may, thus, not adequately represent the strength of the bone.

Dual-energy absorptiometry is a two-dimensional imaging method that reflects the third, collapsed dimension in the density value measured at each point in the image. For purely axial loading, as could be assumed in a simplified model of a vertebra, the resistance to loading would be related to the bone mineral content. In the femur, however, the loads are excentrically applied to the femoral shaft axis, and, thus, major bending stresses occur in the femoral neck. The resistance to a load applied vertically to the center of the femoral head is dictated by the distribution of the bone material in a vertical plane going through the point of force application and the femoral shaft. The projection image of a properly rotated femur provides just that information, and strength calculations based on dual-energy absorptiometry scans have shown tighter relationships with fractures in vitro than simple density measurements. However, current methods assume a linear relationship of density and elastic modulus. Results might be improved if an appropriate power-based relationship is assumed.

Disclosures: T.N. Hangartner, None.

M510

A New Bone Diagnostic Instrument: the Osteoprobe II.

P. K. Hansma, P. J. Turner*, B. Drake*, J. Adams*, J. LeLujian*, A. Proctor*, F. Garza de Leon*. Physics, University of California, Santa Barbara, CA, USA.

The Osteoprobe II can measure the material properties of bone even if the bone is covered in tissue because of its novel probe assembly. The probe assembly consists of a reference probe, a 22 gauge hypodermic syringe, and a test probe, a small diameter sharpened rod, which slides through the inside of the syringe. The probe assembly is inserted through the skin to rest on the bone. The distance that the test probe is indented into the bone can be measured relative to the position of the reference probe, which rests on the surface of the bone. Complete force vs. distance curves for bone indentation cycles are automatically generated and analyzed to obtain the material properties of the bone. Cyclic force vs. distance curves reveal differences for bones that differ in their resistance to fracture. The resistance of bone to continuing indentation with cycling is less for bone that has less resistance to fracture, for example, bone that has been baked or irradiated. The resistance of human bone to continuing indentation with cycling is less for bone from an elderly donor than for bone from a young donor. This is consistent with data showing that the fracture toughness is lower for bone from elderly donors compared to bone from young donors². The long range goal is to develop a diagnostic tool to help physicians assess fracture risk in their patients: specifically the contribution to fracture risk due to deterioration of materials properties such as fracture toughness. The availability of this tool could also help in the development of new therapies to mitigate or reverse deterioration.

• 1.
  Hansma, P.K., Turner, P., & Fantner, GE. Bone Diagnostic Instrument. Review of Scientific Instruments 77, 075105 (2006)
• 2.
  Nalla, R.K., Kruzic, J.J., Kinney, J.H. & Ritchie, R.O. Effect of aging on the toughness of human cortical bone: evaluation by R-curves. Bone 35, 1240 (2004).

This research was supported, in part, by a grant from the NIH: ROI GM 065354–05

Disclosures: R.K. Hansma, None.

M511

Lrp5 Gene Expression and Bone Turnover with Disuse.

G. K. Alvarez, B. T. Hackfort*, B. McGuckin*, M. P. Akhter, D. M. Cullen. Osteoporosis Research Center, Creighton University, Omaha, NE, USA.

The Lrp5 receptor (low density lipoprotein like receptor protein) is associated with canonical Wnt signaling and bone formation. The G171V mutation in LRP5 is associated with high bone mass and greater sensitivity to increase mechanical loading. Conversely, disuse results in bone loss due to increased osteoclast activity and decreased osteoblast activity. The hypothesis for this study was that variation in Lrp5 expression and specifically the GI7IV mutation are associated with differences in bone response to disuse. We examined the response in adult virgin female (4.5 ± 0.4mo) mice to four weeks of hindlimb suspension in three mice genotypes: WT (wild type C57B16, Lrp5+/+), KO (single knock out Lrp5+/-), and HBM (high bone mass due to GI7IV mutation). Mice within each genotype were randomly assigned to suspension (N=46) or control (N= 43). All mice were allowed free access to food and water. The protocol was approved by the University IACUC. Calcein was injected 10 and 3 days before tissue collection and histomorphometry performed on undecalcified femur cortical bone. Measurements included Area (total, cortical), mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS). Differences among genotypes and due to disuse were analyzed by GLM in SPSS with P<0.05. There were no differences among groups in initial body weight (25.6 ±2.6 g). The controls maintained their weight while the hindlimb mice lost an average of 3.6 g over four weeks. Results are presented in the table below. HBM mice had greater total and cortical bone area in the control femur and this difference was maintained after disuse. Only the KO group showed a significant decrease in cortical bone area. Cortical femur after hindlimb suspension had formation two to four fold lower in all genotypes. Mice with the HBM mutation and Lrp5+/+ maintained cortical bone despite a decrease in bone formation with disuse while the Lrp5+/- mice lost bone with the same relative decrease in formation. These results show that Lrp5 gene may protect against bone loss with removal of force. Lrp5 in regulation of osteoclast activity may be a secondary role in addition to the primary role of bone cell proliferation and differentiation. The disuse-related effects of the GI7IV mutation were not discernable in this study since bone areal changes in control mice were small. 

Table  .  

Mean (S.D.), a different from HBM P<0.03, b different within genotype due to hindlimb, p<0.05

Disclosures: G.K. Alvarez, None.

This study received funding from: Nebraska Cigarette Taxes.

M512

See Sunday Plenary Number S512

M513

Effect of Hindlimb Unloading on Bone in Two Strains of Mature Mice.

B.M. Boudignon*, P. Kurimoto*, B. Orwoll*, B. Halloran. Endocrine Unit, Veteran Affairs Medical Center San Francisco, San Francisco, CA, USA.

Hindlimb unloading is a good model of to study the effects of loss of weight bearing on bone. Numerous studies have shown that hindlimb unloading induces osteopenia, decreases bone formation and increases bone resorption. These studies, however, focused mostly on rapidly growing animals and only a few mouse strains have been compared in adult animal. To examine the effects of hindlimb unloading and mouse strain on bone in mature mice, male C57BL6 and DBA/2 animals, 6 months of age, were permitted normal ambulation or were unload for 1, 2 or 4 weeks using the tail suspension method. The femoral distal metaphysis was analyzed using microCT analysis and histomorphometry. Bone marrow cells were collected to examine calcium nodule and osteoclast formation. Basal BV/TV was 50% lower in DBA/2 than C57B1/6J mice and didn't decrease even after 4 weeks of suspension. C57B1/6J mice lost a significant amount of bone after 2 weeks of suspension and this was maintained through week 4. Calcified nodule number decreased dramatically one week after unloading and remained low through week 4. Osteoclast number increased markedly in both strains of mice and normalized after 4 weeks of suspension in the DBA/2 mice but not in the C57B1/6J. Osteoclasts with more than 50 nuclei, believed to be the most active bone resorbing cells, were most increased (nearly 4 times) the first week after hindlimb unloading. Our results show that hindlimb unloading decreases bone mass in mature C57B1/6J mice but not in DBA/2 mice. That basal bone mass was low in the DBA/2 mice might explain why BV/TV was unaffected by unloading. The cell cultures however demonstrated a dramatic effect of unloading on osteoblasts formation and function and osteoclasts formation in both strains. Our results demonstrate that the loss of bone induced by skeletal unloading is strain dependant and in mature mice is associated with changes in both osteoblast and osteoclast function.

Disclosures: B.M. Boudignon, None.

M514

See Sunday Plenary Number S514

M515

Seasonal Changes in the Bone Mineral Density of a Non-hibernating Arctic Rodent Species: The Northern Red-backed Vole, Clethrionomys rutilus.

K. T. Stevenson¹, J. D. Mayfield*¹, B. M. Barnes*¹, I. G van Tets*². ¹Biology & Wildlife, University of Alaska Fairbanks, Institute of Arctic Biology, Fairbanks, AK, USA, ²Biological Sciences, University of Alaska Anchorage, Anchorage, AK, USA.

Arctic rodents use a variety of over-wintering strategies to survive the seasonal arctic environment. Some rodents hibernate. These animals decrease their heart rate, metabolic rate, and body temperature, and in some species may not move, eat, drink, urinate, or defecate for five to seven months. Other rodents, “non-hibernators”, maintain a constant body-temperature during winter, but reduce their activity and change their diet, social behavior, and physiology in the months leading up to and throughout winter. Both types of rodents could potentially provide models for studying disuse osteoporosis in humans. However, the coupling of reduced activity with maintained metabolic rate and body temperature in non-hibernators resembles the situation of the human patients more closely. Our aim was to determine whether and how bone mineral density (BMD) changed with season in a non-hibernating arctic rodent, the northern redbacked vole, Clethrionomys rutilus. We used dual-energy X-ray absorptiometry (DXA) to measure seasonal changes in the BMD of adult C. rutilus carcasses collected in Alaska at different times of the year. There was no significant effect of season on the BMD of female voles. The BMD of males, however, was highest in spring and fall and lowest in summer and winter, suggesting a decrease in bone strength in male voles as the result of reduced winter activity and an increase in bone strength associated with the onset of the breeding season. Recruitment of younger adults having lower BMDs and the loss of older adults with higher BMDs in summer is likely to have been responsible for the lower BMD values in summer. This evidence of reduced BMD during times of naturally low activity for male C. rutilus suggests that these non-hibemating arctic rodents could serve as models for studies of disuse osteoporosis in humans. To further test this, we will dissect the bones out of the vole carcasses and repeat our analyses on these isolated bones. We will also use DXA to measure the effect of hibernation on long bone remodeling in captive arctic ground squirrels, Spermophilus parryii, to test the suitability of hibernating rodents for similar research. Continuing research on the effects of season and activity on mineral metabolism and deposition in arctic rodents is likely to improve our understanding of disuse osteoporosis.

Disclosures: K. T. Stevenson, None.

This study received funding from: NOAA CIFAR International Polar Year, National Science Foundation, HSF Alaska EPSCoR, The American Society of Mammalogists.

M516

A Novel Partial Weight Suspension System Simulating Mars Gravity Leads to Reduced Bone Mass and Strength in Mice.

E. B. Wagner*¹, W. Tan*², K. C. Gosselin*², M. L. Bouxsein³. ¹Division of Health Sciences and Technology, Harvard-MIT, Cambridge, MA, USA, ²Massachusetts Institute of Technology, Cambridge, MA, USA, ³Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA.

The musculoskeletal effects of partial weightbearing, such as expected on the Moon (16%) and Mars (38%), are yet to be quantified. Our novel model of Partial Weight Suspension (PWS) provides the first mechanism for investigating chronic, titrated quadrupedal loading in mice. Suspension is provided by combination of a custom forelimb vest and tail wrap, and can be adjusted to support studies within +/- 5% of a given static loading level. In this study, we examined musculoskeletal adaptation to Mars-analog (38% weightbearing) levels. Adult female BALB/cByJ mice (n=32, 10 weeks old) were randomly assigned to the BASELINE sacrifice group (n=8), MARS suspension group (n=9), JACKET full-weightbearing control group (n=5), or group-housed AGE matched control group (n=10). JACKET mice were singly housed in identical cages and wore the same forelimb vest to mimic any stress induced by the PWS system. JACKET mice were pair-fed based on consumption of MARS, and AGE animals were group housed and fed ad libitum. After 21 days, animals were sacrificed, the gastrocnemius muscle was weighed, and the bone architecture in the distal femur, femoral midshaft, and proximal tibia were assessed by microCT. Femora were tested in 3-point bending to determine biomechanical properties. We found that: 1) Body weight gain did not differ between MARS and AGE- matched controls after an initial adaptation period, suggesting low levels of systemic stress; 2) Gastrocnemius weight and bone architecture did not differ between AGE and JACKET controls; 3) Gastrocnemius weight was significantly decreased in MARS vs AGE-controls (-21.8%, p<0.05); 4) Femoral trabecular BV/TV and mid-shaft cortical thickness were significantly reduced in MARS animals vs controls (BV/TV −20.6%, p<0.01; Cort. Th −8.9%, p<0.001); 5) Bending rigidity and ultimate moment were significantly reduced in MARS animals relative to controls (BR: −13.8%, p<0.005; UltMom: −19.9%, p<0.01). In summary, MARS loading leads to significant muscle and bone loss. Interestingly, trabecular bone loss under MARS weightbearing appears to be attenuated relative to previous reports of bone loss associated with hindlimb suspension.

Disclosures: E.B. Wagner, None.

This study received funding from: NASA.

M517

Fluid Shear Stress and Mechanical Strain Induce a Similar Osteogenic Gene Response via Divergent Signaling Pathways.

N. Case¹, Z. Xie*¹, B. Sen*¹, M. Ma*¹, H. Jo*², J. Rubin¹. ¹Medicine, UNC, Chapel Hill, NC, USA, ²BME, GATech/Emory, Atlanta, GA, USA.

Mechanical loading influences on bone remodeling are well-established with physiologic loading causing net bone gain. The specific forces generated during loading have been postulated to cause equally specific responses: indeed, the few direct comparisons between strain and shear stress have suggested that these forces are not interchangeable. Our previous work has shown that mechanical strain promotes bone formation through repression of RANKL, a necessary factor for osteoclastogeneis, as well as stimulation of Runx2 and osterix, genes that induce osteoblast differentiation and activity. We here ask whether this coordinated regulation of multiple genes associated with bone remodeling is specific to strain: does shear activate the same pattern of osteogenic gene response? Using a modified cone-and-plate shear device, we evaluated the effects of steady fluid flow on regulation of bone remodeling effector genes in the pre-osteoblast CIMC-4 cell line. RANKL mRNA expression was decreased to 0.11 ± 0.03 fold, Runx2 mRNA expression was increased 1.56 ± 0.13 fold and osterix mRNA expression was increased 3.37 ± 0.41 fold in cultures subjected to flow overnight (3 experiments, n=11) as compared to no-flow cultures (p<0.01 all genes). The magnitude of shear-induced changes was > strain effects: RANKL repression was 2–3 times lower, Runx2 expression was unchanged and osterix induction was 2 times higher in flow compared to changes observed in strained cultures. We next investigated potential signaling pathways involved in flow-mediated gene regulation. Signaling pathway inhibitors evaluated included MEK inhibitors, PD98059 and U0126, the PI3-kinase inhibitor LY294002, the nitric oxide synthase inhibitor L-NAME and thapsigargin, which empties intracellular calcium stores. While we have previously shown that PD98059 inhibits strain effects on RANKL, Runx2 and osterix, this MEK inhibitor did not affect shear-induced gene changes. As well, shear regulation of RANKL and Runx2 was not altered by treatment with any other inhibitor. Interestingly, shear upregulation of osterix was inhibited by U0126, LY294002 and thapsigargin, but not by L-NAME, indicating that osterix regulation by flow involves at least PI3-kinase and calcium signals. In sum, these data suggest that fluid shear stress and mechanical strain can elicit similar, if not equivalent, cellular responses and that the same biological endpoint can be reached through different means. In conclusion, this work suggests that there is no one dominant biophysical force guiding the response to skeletal loading; rather, a variety of inputs/signaling pathways converge to elicit a summed response that is anti-catabolic and pro-anabolic in bone cells.

Disclosures: N. Case. None.

This study received funding from: NIAMS, NIH.

M518

See Sunday Plenary Number S518

M519

A Novel Role for Nuclear Factor of Activated T Cell (NFATcl) in Bone Mechanotransduction.

A. B. Celil Aydemir*¹, S. Lee*¹, T. R. Gardner*¹, J. M. Ahn*², F. Y. Lee¹. ¹Orthopaedic Surgery, Columbia University, New York, NY, USA, ²Hallym University, Chuncheon of Gangwon, Republic of Korea.

Bone adapts to the changes in its microenvironment. It has been shown that osteoblasts alter cytokine expression in response to mechanical stimuli. However, signal transduction pathways that mediate bone cell response to mechanotransduction have not been elucidated at the transcriptional level. One of the early events of mechanotransduction is the elevation of intracellular calcium, which is upstream of calcium/calcineurin and NFAT signaling. Calcium/calcineurin signaling activates NFATcI, which is a transcription factor. Our bioinformatics analysis shows the presence of putative NFATcI binding motifs in the promoter region of Cox2, a well-known mechanotransduction mediator. We hypothesize that calcineurin/NFATcI signaling mediates mechanotransduction in osteoblasts. In this study, we show that fluid shear stress (FSS) induces Cox2 and TNFa expression in bone marrow derived human mesenchymal stem cells (hMSC) and mouse preosteoblasts. Osteoblasts and osteocytes are known to be subject to fluid shear stress in vivo. Cells were subjected to a sinusoidal fluid shear stress of ±10 dynes/cm² at 1Hz for 15minutes. Concurrently, a clinically relevant hydrostatic pressure of 0.138MPa (1380 dynes/cm²), comparable to pressures found in the hip joint space during gait, was applied to the cells. Proinflammatory gene expression and TNFa release in hMSC were both found to increase in this loading environment. Further, NFATcI translocates to the nucleus in response to fluid shear stress in hMSC. A peptide inhibitor of NFATcI, Vivit, inhibits the FSS-induced proinflammatory gene expression and NFATcI nuclear translocation. Our results implicate the involvement of the calcineurin/NFATcI axis as a novel mediator of bone cell response due to mechanical stimulation.

Disclosures: A.B. Celil Aydemir, None.

This study received funding from: NIH, OREF.

M520

Fluid Flow Shear Stress Promotes Intracellular Assembly and Formation of Connexin 43-forming Channels in Osteocytes.

S. Burra¹, A. J. Siller-Jackson¹, L. F. Bonewald², E. Sprague*³, J.X.Jiang¹. ¹Biochemistry, University of Texas Health Science Center, San Antonio, TX, USA, ²Oral Biology, School of Dentistry, University of Missouri, Kansas City, MO, USA, ³Radiology, University of Texas Health Science Center, San Antonio, TX, USA.

Connexin 43 (Cx43) is the major gap junction and hemichannel forming protein in primary osteocytes and osteocyte-like cell line MLO-Y4. Osteocytes are the mechanosensory cells; mechanical signals travel from one osteocyte to other through gap junctions formed at the tips of the dendritic processes of osteocytes. Recent studies by our laboratory and others show that in addition to form gap junction channels, Cx43 forms functional hemichannels, an apposed of halves of gap junctions. Fluid flow shear stress induces the opening of hemichannels associated with the release of prostaglandins. Cx43 protein is abundantly expressed intracellularly as well as along the cell surface. We have previously shown that FFSS appears to enhance the migration of intracellular Cx43 protein towards plasma membrane, resulting in an increase in Cx43 protein on the cell surface. To determine the molecular mechanism of assembly and formation of gap junctions and hemichannels in response to FFSS, sucrose gradient sedimentation analysis was conducted. FFSS induced the assembly of Cx43 into hexamers, the oligomeric forms of Cx43 in forming gap junctions and hemichannels. The localization of Cx43 and its response to FFSS was determined by colocalization analysis with intracellular organelle marker proteins including calnexin (endoplasmic reticulum (ER) marker) and 58K Golgi protein (Golgi marker). Cx43 co-localized with both ER and Golgi markers at static conditions. Cx43 protein plaques, indicative of assembled channels, predominantly localized in the Golgi. Interestingly, upon mechanical stimulation by FFSS, the colocalization of Cx43 plaques in Golgi diminished; instead Cx43 appeared to migrate towards the cell surface. The levels of Cx43 protein on cell surface was quantified by biotinylation approach. When MLO-Y4 cells were subjected to FFSS and the then incubated for various periods of time, surface expression of Cx43 gradually increased over the time and reached maximum after 2 h of incubation. Together, these results combined with functional studies show that FFSS stimulates the assembly and formation of Cx43-forming gap junctions and hemichannels, which will accommodate the response to mechanical stimulation in osteocytes.

Disclosures: S. Burra, None.

This study received funding from: National Institutes of Health.

M521

In Vivo Tibial Compression Stimulates Bone Formation.

D. M. Cullen, Q. K. Alvarez, B. T. Hackfort*, B. McGuckin*, M. P. Akhter. Osteoporosis Research Center, Creighton University, Omaha, NE, USA.

Increased exercise stimulates bone formation, but is difficult to model in vivo. Popular noninvasive models include tibial four-point or cantilever bending and ulna compression, but they are limited by the unique load distributions, sample regions, and potential injury. Two papers in Bone 2005, by Fritton and Desousa introduced tibial compression. They presented strain gage data, area, and BMC changes, but did not provide sufficient information to determine appropriate load ranges and sample sites for bone histology. The purpose of this study was to document the bone formation response along the tibia and to demonstrate the load response curve to increasing forces. The load apparatus was modeled after DeSousa with the knee and ankle positioned for force application in the vertical plane with an Enduratec (ELF3200) device. Twenty female C57B16 adult mice (5.5±0.4 mo, 30.0±3.2g) were randomized to loads 8, 9, 10, or 11N and then 100cycles at 2 Hz was applied Mon-Wed-Fri for 3 weeks. Midtibial strains ranged from 1800 to 2500μϵ. Mice received calcein injections 10 and 3 days before euthanasia to label bone formation for dynamic histomorphometry. Animals loaded from 8–11 N showed no signs of injury. In a test of 12 N loads, 20% of the animals had a stress fracture by day 3 and the group was stopped. Bone sections were selected relative to the tibia fibular junction (TFJ, Fig) with one distal (3 mm) and three proximal (1, 3, 5 mm) sections measured. Differences were seen within animals for loaded and non loaded legs and section location, and among load groups at the periosteal and endocortical surfaces for mineralizing surface (MS), mineral apposition rate (MAR), bone formation rate, and woven bone formation. For all endpoints the responses tended to increase with increased load and the differences in load response were greatest at 3 mm proximal to the TFJ. Difference (Loaded — nonloaded) in periosteal bone formation rate (MS x MAR) is shown in the Figure. Formation increased as applied force increased with the greatest differences seen 3 mm proximal to the TFJ where multiple intergroup comparisons were significant (P<0.01). This study demonstrates that the most load sensitive region for cortical bone histomorphometry is ∼ 1 mm proximal to the TFJ, the same region sampled for tibial four-point bending. This area is easily located, reproducible and has a fairly constant shape.

Disclosures: D.M. Cullen, None.

This study received funding from: Grant #AR051365 from NIAMS.

M522

Is Golf a Good Sport for Bone Health in the Elderly? A DPX and High Resolution pQCT Evaluation at both Radius and Tibia Bone Sites.

T. Thomas¹, G Ntougou Assoumou Hourfil*², M. Zouch*², L. Vico². ¹Rheumatology Department — University Hospital, LBTO INSERM U890, Saint-Etienne, France, ²LBTO INSERM U890, Saint-Etienne, France.

A regular practice of golf may be beneficial for preserving a good bone health in the elderly population, both in the lower limbs because of ground impacts during long walks and in the upper limbs because of impacts transmitted by the club during the swing. Therefore, we conducted a prospective cross-sectional study in men (age range: 60–75) comparing a group of 15 golfers, playing their sport 8 to 12 hours a week for the last 2 years, to a group of 15 walkers, with a similar weekly practice and a group of sedentary men, all apparied for age. Evaluation of bone status was performed using DPX (Hologic Delphi 4500 QDR) and HR-pQCT (Scanco Xtreme CT) for microarchitecture analysis as well as biochemical parameters on fasting blood samples collected early morning. Daily calcium intake and physical activities were assessed by validated questionnaires. The 3 groups were not different in terms of height, weight, BMI and daily calcium intake.

Using DPX, we observed that bone mineral content of mid diaphysis radius was significantly higher in the golfer group than in the other groups. There was no statistically significant difference in bone mineral content both at the distal radius and dominant lower limb. The HR-pQCT analysis showed significant differences between groups in central trabecular density (Dinn), total trabecular density (Dtrab) and trabecular bone volume (BV/TV) both at the dominant radius and tibia. Surprisingly, these parameters were higher in the walker group than in the other groups. On the other hand, cortical density at both bone sites was higher in golfers with a large inter-individual variability.

Several factors may partly explain the higher values in trabecular parameters in the walker group. Indeed, total and sport-related annual physical activities were significantly higher in this group compared to the others. Those of golfers were not significantly higher than sedentary group levels. Past physical activity of walker group also was very significantly higher than golfers one while this parameter was significantly lower in sedentary group compared to the others. In addition, testosterone level also was significantly higher in walkers than in other groups and testosterone was significantly and positively related to Dinn (r = 0.41, p<0.05).

In summary, our results suggest that golf practice has beneficial effect on the cortical envelope both at the radius and tibia bone sites. The role of past and actual global physical activity seems preponderant on trabecular envelope. Further studies are necessary to confirm a definitive benefice of golf practice on bone health in the elderly.

Disclosures: T. Thomas, None.

This study received funding from: University Hospital of St-Etienne.

M523

See Sunday Plenary Number S523

M524

Eight Months of Twice Weekly Ten Minute Jumping Activity for PE Warm Up Improves Bone in Adolescent Boys and Girls: The POWER PE Study.

B. K. Weeks, C. M. Young*, B. R. Beck.. School of Physiotherapy and Exercise Science, Griffith University, Gold Coast, Australia.

High intensity skeletal loading during growth may be an effective strategy to maximise bone accrual and reduce fracture risk in old age. Preventing Osteoporosis With Exercise Routines in Physical Education (POWER PE) was an 8-month randomised controlled school-based exercise intervention designed to apply known principles of effective bone loading to practical opportunities to improve life long musculoskeletal outcomes. We replaced regular warm up activities with jumping in the twice-weekly PE classes of early high school students to observe the effect on bone accrual in adolescent boys and girls. A total of 99 adolescents (46M:53F, 13.8 ± 0.4 years) volunteered to participate in the yearlong study. Most were classified as Tanner IV (53%). Intervention group subjects performed ten minutes of supervised jumping activity in place of regular PE warm up activities. Control subjects performed regular PE warm-up activities directed by their teacher. Anthropometrics, Tanner staging, peak height velocity, muscle strength and power, flexibility, and bone mass (DXA and QUS) were measured at baseline and follow-up. Geometric properties (such as FN cross-sectional moment of inertia and LS index of bone strength) were estimated from DXA measures. Physical activity and dietary calcium were determined by questionnaire. There were no differences in any measured variable between control and intervention groups at baseline. No group differences were detected for 8- month change in anthropometric, maturity, strength, or flexibility variables for boys or girls. At eight months, boys in the intervention group had experienced significant improvements in calcaneal BUA (+5.0%, p = 0.012), and fat mass (-10.5%, p = 0.023), while controls did not (+1.4% and −0.8% respectively). Girls in the intervention group, however, experienced significant improvements in FN BMC (+13.9%, p = 0.05) and LS BMAD (+5.2%, p = 0.04), which were not observed in controls (+4.9% and +1.5% respectively). Other bone strength parameters improved significantly for both groups, such that between group comparisons of percent change revealed significant intervention effects only for WB BMC (+10.6% vs +6.3%, p = 0.029) for boys. Short duration, regular jumping activity during adolescence appears to improve bone accrual in a sex-specific manner. Boys improved whole body bone mass and BUA, and reduced fat mass, while girls improved bone mass at the hip and spine.

Disclosures: B.K. Weeks, None.

M525

See Sunday Plenary Number S525

M526

Knee Loading Stimulates Wound Healing in Mouse Femora.

P. Zhang, Q. Sun*, C. H. Turner, H. Yokota. Biomedical Engineering, Indiana University Purdue University Indianapolis, Indianapolis, IN, USA.

Knee loading is a recently developed loading modality capable of inducing anabolic responses in murine femora and tibiae. The object of this study was to examine whether this loading modality would be useful in stimulating bone healing in mouse femora.

Thirty-seven C57/BL/6 female mice (∼ 14 weeks of age; and a body weight of ∼ 20 g) were used for the study. The procedures performed in this study were in accordance with the Institutional Animal Care and Use Committee guidelines. A surgical hole of 0.5 mm in diameter was generated in the left and right femora. The hole penetrated through the anterior and posterior surfaces at 50% (∼ 8 mm) distant from the distal femoral end. From the fourth postoperative day, knee loading was conducted with a custom-made piezoelectric loader, and 0.5-N loads were laterally applied to the left knee at 15 Hz for 3 min/day for 3 consecutive days. The contralateral femur was used as sham-loaded control. Animals were sacrificed 1, 2, or 3 weeks after the surgery. The healing process was evaluated using μCT (n = 20), pQCT (n = 7), and histomorphometry with calcein labeling (n=10).

Compared to sham control, the loaded samples clearly showed load-driven enhancement of wound healing. First, knee loading significantly reduced the size of surgical wounds by 14% (p < 0.05), 24% (p < 0.01), and 32% (p < 0.001) in 1, 2, and 3 weeks after the surgery, respectively. Second, pQCT data showed that the total bone density (BMD) was increased from 571 ± 19 mg/cm² (control) to 686 ± 19 mg/cm² (loading) (p < 0.01), and the total bone content (BMC) was elevated from 3.05 ±0.12 rag/mm (control) to 3.42 ±0.11 mg/mm (loading) (p < 0.05). Similarly, the cortical bone density was increased from 1057 ± 19 mg/cm² (control) to 1136 ± 10 mg/cm² (loading) (p < 0.01), and the cortical bone content was elevated from 2.25 ±0.11 mg/mm (control) to 2.73 ±0.11 mg/mm (loading) (p < 0.01). Lastly, bone formation near the wound was stimulated by loading.

In summary, the current study demonstrates that knee loading is capable of enhancing a healing process in the mouse femur. The described knee loading modality might provide a novel strategy to develop mechanical therapies to accelerate bone healing.

Disclosures: P. Zhang, None.

This study received funding from: NIH.

M527

Sex-Specific Bone Surface Changes During Adolescent Growth: pQCT Analysis of the Mid-Tibia.

Y. Ahamed*, P. M. L. Cooper, H. A. McKay. Orthopaedics, University of British Columbia, Vancouver, BC, Canada.

The classic studies of Garn and colleagues reported that although growing boys and girls can both exhibit endosteal and periosteal apposition within the diaphysis of the second metacarpal; girls undergo greater endosteal apposition while boys undergo greater periosteal apposition. This suggests a strength advantage for boys as adding bone to the endosteal surface is less mechanically advantageous. Further, this biological “preference” has been viewed as contributing to women's increased bone fragility. Garn's classic studies were conducted using planar measurement techniques and thus were unable to directly assess bone cross-sectional geometry. In a previous study by our group, changes in bone surfaces at the mid-tibia over 20 months (2001–2003) in boys and girls were directly assessed by peripheral quantitative computed tomography (pQCT). This study did not detect any evidence of endosteal formation in either sex in this young sample (mean age = 13.5 (±0.55) years in 2003) regardless of maturity level. The aim of our current study was to extend this previous work by examining sex-specific changes in bone surfaces across 56 months. We tested the hypothesis that girls add more bone on the endosteal surface compared with boys during adolescence. Subjects were a part of The University of British Columbia Healthy Bones Study. There was no difference between control and intervention groups for pQCT outcomes at the tibia four months after cessation of the exercise intervention. Therefore, both groups were collapsed for the current study which included 69 participants (38 girls, 31 boys) for whom 6 consecutive mid-diaphyseal pQCT (Stratec XCT 2000) scans were available (2001–2006). Mean ages were 12.0 years (±0.55) at baseline and 16.5 (±0.56) years as of June 2006. Total bone area (ToA) and medullary canal (MedA) were measured using Stratec XCT software version 5.50. Repeated measures univariate analysis of variance with sex as a factor showed significant increases in ToA and MedA (p<0.001) in both sexes. Over 56-months ToA increased 42% in boys and 16% in girls. In boys, MedA increased a total of (32%) while the change in MedA for girls was less pronounced (7% increase). Our current findings differ from Garn and colleagues' earlier studies of the second metacarpal but are consistent with our early findings of this cohort when they were less mature that found no evidence of endosteal apposition at the midshaft of either sexes' tibiae. This indicates that endosteal formation of bone does not occur uniformly throughout the skeleton during adolescence and, therefore, may not underlie differences in fragility between the sexes.

Disclosures: Y. Ahamed, None.

This study received funding from: Michael Smith Foundation for Health Research.

M528

See Sunday Plenary Number S528

M529

Distribution of Bone Measures in Children at 11 Years of Age.

J. C. Tomer*¹, M. C. Willing², T. L. Burns*³, E. Letuchy*¹, K. F. Janz*⁴, T. Marshall*⁵, J. M. E. Gilmore*⁵, J. J. Warren*⁵, S. M. Lew⁵. ¹Epidemiology, University of Iowa College of Public Health, Iowa City, IA, USA, ²Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA, USA, ³Epidemiology, University of Iowa Carver College of Medicine, Iowa City, IA, USA, ⁴Health and Sports Studies, University of Iowa College of Liberal Arts, Iowa City, IA, USA, ⁵Preventive and Community Dentistry, University of Iowa College of Dentistry, Iowa City, IA, USA.

The purpose of this study is to describe and characterize factors of bone development. A cohort of 481 children (251 girls and 230 boys) was evaluated for bone measures of area, bone mineral content (BMC) and density (BMD) using an Hologic 4500a. The children ranged in age from 10.5 to 12.4 years with a mean of 11.2 years. Even within this restricted age range BMD had a wide distribution. 

Table  .  

The coefficient of variation was 12.7 for hip BMD and 14.3 for spine BMD. Values for BMC were 21.5 (spine), 22.9 (hip), 17.7 (whole body) and 20.9 (whole body without head). Correlations of BMC between hip and other sites were higher (r=0.801) than correlations for BMD (r=0.659). Hip was more strongly correlated with total body BMC than spine was. Regression models for bone measures with gender, height, weight, and dichotomized Tanner stage as predictors showed that weight and height are the most important predictors of BMC levels. For hip and spine BMC and BMD, gender is also highly significant; Tanner stage is statistically significant (p<0.05) for spine BMC and BMD, whole body without head BMC and hip BMD, and marginally statistically significant (0.05 < p-value < 0.10) for hip BMC and whole body BMC. Overall, gender, body size and Tanner stage explain 56% of variation in spine BMC, 61% of variation in hip BMC, and 71 and 77% of variation in whole body BMC with and without head; they explain between 36 and 41% of variation in BMD measures. Cross-classification of quartiles adjusted by age, height, weight, gender and Tanner Stage showed that hip BMC and whole body BMC without head had the highest agreement for boys and for girls it was spine and whole body BMC. Ouartile agreement for boys for BMD was 41.7% and for girls it was 43.4%. For the lowest quartile the lowest agreement was between hip and spine. These results show disparity in bone measures by site and suggest differential factors may be related to development.

Disclosures: J.M.E. Gilmore, None.

M530

See Sunday Plenary Number S530

M531

The Independent Contribution of Physical Activity to Bone Mass During Growth.

M. Burrows¹, A. Baxter-Jones², H. M. Macdonald*¹, R. Mirwald², H. McKay¹. ¹Orthopaedics, University of British Columbia, Vancouver, BC, Canada, ²College of Kinesiology, University of Saskatchewan, Saskatoon, SK, Canada.

A few long-term prospective trials have demonstrated the relationship between physical activity and absolute values for bone mass in children. However, the unique contribution of physical activity to the bone gain trajectory (change in bone mass) is not well understood. We assessed bone mineral content velocity (Δ BMC (g/yr), Hologic QDR4500W) at the lumbar spine (LS), proximal femur (PF), femoral neck (FN) and total body (TB) in 361 boys and girls over 7 years (Healthy Bones Study). We obtained parental consent to assess these children aged 9–12 years at baseline. Lean mass (g) and fat mass (g) were obtained from TB scans. Physical activity score (PA score) was determined from a previously validated self-report questionnaire (PAQ-C). To control for the well-documented maturational differences between adolescent boys or girls of the same chronological age, we determined biological age (chronological age at peak height velocity (APHV determined from cubic spline fits) — chronological age at the time of measurement). If APHV had not occurred it was predicted using an anthropometric based regression equation (Mirwald, 2002). We used multi-level modeling to assess the independent contribution of physical activity to change (Δ) in BMC. Table 1 provides significant predictors (with coefficients) for PF and FN BMC Δ Most notably, in girls each increment increase in PA score was associated with a 21.6g increase in BMC Δ at the FN (p<.05). In boys, each increment increase in PA score predicted a 0.55g increase in BMC Δ at the PF (p<.05). PA was a significant predictor of BMC Δ at the TB for girls only (p<05) [Δ TB BMC = 324(51) + 2370 + 104(6.5)yrstart_ij + −13.8(2.I)yrstart2_ij + −5.9(1.0)yrstart3_ij + 7.9(2.3)Δheight_ij + 0.02(0.003)Δlean mass_ij + 21.6(8.9)physical activity score_ij + e_oij]. Physical activity was an important predictor of bone mineral accrual across 7 years especially at weight bearing sites in boys and girls.

Table Table 1.. Multi-level modeling significant predictors of BMC change in boys and girls

Disclosures: M. Burrows. None.

This study received funding from: Canadian Institute of Health Research.

M532

Determination of Bone Mass and Size in Term, Near-Term, and Preterm Boys.

H. Abou Samra*¹, D. Stevens*², T. Binkley¹, B. L. Specker¹. ¹EA Martin Program in Human Nutrition, South Dakota State University, Brookings, SD, USA, ²Pediatrics, Sanford Hospital Medical Center, Sioux Falls, SD, USA.

The purpose of the study was to determine whether there are differences in bone mass and size among prepubertal boys who were bom term (> 37 weeks), near-term (NT: 34 to 37 weeks), or at preterm (FT, < 34 weeks gestation) and whether differences in strength or activity explained bone differences. Total body (TB), spine (LS) and hip DXA and pQCT measures of the distal tibia were obtained on 24 prepubertal boys aged 5.7 to 8.3 years. In multiple regression analysis, after adjusting for current weight, height, age, percentage body fat (%BF), and jump power, term boys had greater cortical thickness (p=0.03) and area (p=0.01), higher trabecular volumetric bone mineral density (aBMD) (p=0.05), TB BMC (p=0.01), and total hip BMD (p=0.01) than PT boys and higher TB BMC (p = 0.01), TB bone area (p=0.04), total hip BMC (p =0.02) and BMD (p=0.01), and femoral neck BMC and BMD (both, p=0.02) than NT boys. There were no differences in activity measures among gestation groups and no group-by-activity interactions. In conclusion, term boys have greater bone size and mass than PT boys and higher bone mass than NT boys at several bone sites. Activity measures did not differ among gestation groups and did not explain bone differences. Our findings, if confirmed in larger populations, may have significant implications for bone health in near- and preterm children, a group that is thought to represent approximately 11.3% of all births.

Disclosures: H. Abou Samra, None.

This study received funding from: EA Martin Endowed Program.

M533

See Sunday Plenary Number S533

M534

Timing of Peak Bone Mass and Bone Mineral Density Are Influenced by High-Level Physical Activity in Young Adults of Both Sexes.

S. Breban*, C. Chappard, C. Jaffre*, C. Benhamou. CHR Orleans, INSERM Unit U658, Orleans, France.

Optimization of Peak Bone Mass may help to prevent osteoporosis and sport is known to be beneficial for bone tissue by increasing Bone Mineral Density (BMD). No study has reported effects of physical activity on Peak bone Mass. Thus, the aim of this cross-sectional study was to analyse the influence of physical activity on timing and level of Peak Bone Mass.

70 girls and 90 boys aged 17–28 years participated in this study. The sample included 40 athlete-girls and 60 athlete-boys participating in weightlifting, ball collective sports or judo for more than 6 hours per week (9.6±3.5 hours per week for women and 10.4±3.7 for men). They were age and sex matched with control girls (n=30) and boys (n=30). Bone Mineral Content (BMC, g) and BMD (g/cm²) were measured by DXA (Delphi, Hologic ®, Waltham, MA), at lumbar spine (LS) and non dominant femur (total and femoral neck (FN)). We also evaluated whole body (WB) BMC and BMD, with derived analysis of trunk, lower and upper limbs. Height, weight and body composition were derived from these DXA measurements. The timing of Peak Bone Mass was analysed by the inflexions of the age/BMD curves at all bone sites. Our variables were best fitted by second order polynomial regression equations. Thus, the model was expressed as y=ax²+bx+c. The age of maximal bone mass was determined as the maximum of the curve, i.e. when x = -b/2a. Athletes had higher BMD measurements than controls in both sexes at all bone sites (p<0.05). Consequently all the Peak Bone Mass levels were significantly higher in athletes than controls. BMD peaks were achieved around 21.5 (FN), 22 years (LS), and 24–25 years (WB, upper and lower limbs) in athlete girls; BMD peaks were reached before 17 years for all bone sites except for LS (21.8 years) in control girls. In athlete boys, BMD peaks were reached before 17 years for upper and lower limbs, and ranged from 21.0 years (FN) to 25.5 and 26.0 years respectively for WB and LS. Finally, in control boys, BMD peaks were reached before 17 years at all bone sites. Timing of Peak Bone Mass was different according to bone sites, sex and athletic or non athletic status. Thus, those results confirm that weight-bearing physical activity lead to a better bone status compared to non athletes, in both sexes and even after puberty. Moreover, our data suggest that the Peak Bone Mass was not reached at all bone sites before 17 years: thus, it is yet possible to optimize BMD and Peak Bone Mass in young adults between 17 and 28 years. High level weight-bearing physical activity seemed to induce a delayed and higher Peak Bone Mass compared to controls.

Disclosures: S. Breban, None.

M535

See Sunday Plenary Number S535

M536

Fat Mass Is Inversely Related to Subsequent Change in Bone Size and Mass in Young Prepubertal Children.

K. S. Wosje¹, P. R. Khoury*¹, S. R. Daniels*². ¹Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA, ²The Children's Hospital, Denver, CO, USA.

Recent literature suggests that whole body fat mass (FM) may be positively related to periosteal bone formation in children. FM was reported to be positively associated with subsequent 2-year changes in whole body bone area (BA) and bone mass (BMC) in 9 year-olds, but this has not been examined in younger children. We hypothesized that there would be a positive relationship between FM at age 3.5 y and changes in BA and BMC from age 3.5 to 7 y, adjusting for race, sex, socioeconomic status [household income (HHI); N = 104 <50,000, N = 117 >50,000], exact age at baseline, change in whole body lean mass (LM), and change in height. 221 children [176 white (45% girls), 45 black (60% girls)] with data available from whole body DXA (Hologic 4500A) scans done within 3 months of both 3.5 and 7 years of age (interval 3.5 ± 0.2 y) as part of a longitudinal study of adiposity development were included. All 442 scans were confirmed by visual inspection of printed output to have no major limb or trunk movement. For the DXA measures of BA and BMC, the skull was excluded because the skull-inclusive whole body data may be affected by differences in relative head size over time within children. LM, height, BA and BMC were expressed as percentage change (Δ) from age 3.5 to 7 y. Data are mean ± SD. In bivariate analysis, FM at age 3.5 y was inversely associated with ΔBA (p<0.001) and ΔBMC (p<0.01). Blacks had greater ABA (32 ± 8 vs. 28 ± 6%, p<0.001) and ΔBMC (91 ± 15 vs. 80 ± 13%, p<0.001) than whites; neither ΔBA nor ΔBMC differed by sex or HHI (all p>0.2). In all children, ΔLM was not associated with ΔBA (p=0.1) but was positively associated with ΔBMC (p<0.001). As expected, exact age at baseline was inversely related to ΔBA and ΔBMC (both p<0.001); and Δheight was positively associated with ΔBA and ΔBMC (both p<0.001). In the final multiple regression model, FM at age 3.5 y remained inversely related to ABA (p<0.001) and ABMC (p<0.001) after adjusting for race, exact age at baseline, ΔLM and Δheight. There was no FM-by-race or FM-by-sex interaction in relation to ΔBA (both p>0.07) or ΔBMC (both p>0.4). Similar findings were obtained when LM, height, BA and BMC were expressed as absolute rather than percentage change from age 3.5 to 7 y, and baseline BA or BMC was included in the model. Our findings were opposite of our hypothesis and conflict with a prior report of a positive relation between FM and subsequent change in BA and BMC among older prepubertal children. Although our study provides indirect evidence that FM does not appear to be positively related to periosteal bone formation during the younger prepubertal years, studies with measures of cortical bone dimensions using pQCT would provide clarification.

Disclosures: K.S. Wosje, None.

This study received funding from: NIH.

T001

Prostaglandin D₂ in Human Bone Formation and Remodelling In Vivo.

M. A. Gallant*, C. Wollsén*, E. Chamoux, J. Parent*, S. Roux, A. J. de Brum-Fernandes.. Division of Rheumatology, Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, PQ, Canada.

Prostaglandin D₂ (PGD₂) is a lipid mediator implicated in several physiological and pathological events. We have previously showed that human osteoblasts produce PGD₂ and that this mediator decreases osteoprotegerin production through the activation of the DP receptor while it decreases RANKL secretion and increases osteoblast migration through the activation of the CRTH2 receptor, both present on osteoblasts (Gallant et al, JBMR 2005, 20:672–81). These results led to the hypothesis that PGD₂ could be an autacoid implicated in bone remodelling and exerting a positive feedback on osteoblasts. Our objective was to determine, in humans, if the PGD₂ stable metabolite 11β-PGF_2α was increased in conditions of high bone remodelling and formation in vivo.

We determined the levels of urinary 11β-PGF_2α and serum bone-specific alkaline phosphatase (BAP) and C-terminal collagen I telopeptide (CTx) in 17 patients with traumatic fracture 6 weeks post-fracture as well as in age- and sex-matched controls. Three patients with active untreated Paget's disease of bone and matched controls were also studied.

The age in patients with fracture varied from 18 to 46 (average 29.4 ± 11.8 years old). Levels of 11β-PGF_2α were significantly increased in patients with of bone fractures compared to age- and sex-matched controls (59.86 ± 6.37 vs 41.26 ± 5.54 ng/mmol creatinine). No significant increases in BAP or CTx were observed in the whole fracture cohort. Men and women fracture subgroups both showed significantly elevated 11β-PGF_2α levels compared to their respective controls (62.66 ± 13.51 vs 35.75 ± 9,03 and 57.90 ± 6.02 vs 33.16 ± 3.74 ng/mmol creatinine for men and women, respectively). The women fracture subgroup presented a significant increase in CTx excretion compared to control (0.499 ± 0.095 vs 0.239 ± 0.021 ng/ml) but that was not observed in the men subgroup. Correlation analyses showed no significant correlation between 11β-PGF_2α levels and BAP or CTx in the whole cohort. In patients suffering from Paget's disease of bone, urinary levels of 11β-PGF_2α were also significantly higher than the controls, however the sample size prevented us from performing statistical analysis. As expected in PDB patients, the level of BAP was also higher than the controls, indicating a high level of bone formation in these patients. These results support our working hypothesis that PGD₂ could be implicated in the control of bone anabolism and remodelling in vivo in humans. They also suggest that the urinary levels of 11β-PGF_2α could be a sensitive marker of osteoblast activity.

Disclosures: M.A. Gallant, None.

This study received funding from: CUGR. URSC.

T002

Bone Turnover in Late Gestation and Six Months Postpartum.

J. P. Greeves¹, J. Achten*², A. Jeukendrup*³, W. D. Fraser⁴. ¹QinetiQ Ltd, Farnborough, United Kingdom, ²Clinical Sciences Research Institute, Coventry, United Kingdom, ³University of Birmingham, Birmingham, United Kingdom, ⁴University of Liverpool, Liverpool, United Kingdom.

Pregnancy and the postpartum period are associated with a loss of bone mass. The aim of the present study was to monitor changes in bone turnover from late gestation to six months postpartum. The study was approved by the North Birmingham Local Ethics Committee.

Early morning blood samples were obtained after an overnight fast in 15 Caucasian pregnant subjects (Preg) at 31 wk gestation (Gest) and at 8, 17 and 26 wk postpartum (PP). Samples were obtained at the same time points in seven non-pregnant controls (Con) during the follicular phase of the menstrual cycle. At 8 wk PP, ten Preg subjects were breast feeding, four were bottle feeding and one was mixed feeding. At 26 wk PP, four Preg subjects were breast feeding and three were mixed feeding. All subjects were non-smokers and followed a standardised diet for 24-h prior to blood sampling. Pregnant and control subjects were matched for age (mean (SD): 30.5 (3.3) vs 29.8 (3.2) y), pre-pregnancy body mass index (22.2 (0.5) vs 24.1 (2.1) kg m²)and leisure time physical activity (437 (198) vs 409 (196) kcal·d⁻¹, respectively). Blood samples were analysed for markers of bone resorption (β-CTX), bone formation (P1NP and Bone ALP), and OPG.

Mean (1SD) data for Preg and Con are shown in the Table. There was a significant increase in β-CTX and P1NP from Gest to the PP period in Preg (p<0.05) but no change in Con. In Preg, β-CTX increased from Gest to 8 wk PP (p<0.001) and 17 wk PP (p<0.05), and P1NP remained elevated at all PP time points (p<0.05). Bone ALP increased significantly in Preg from Gest to 8 wk PP (p<0.05). OPG decreased during PP compared with Gest (p<0.01), and was unchanged in Con.

In conclusion, bone turnover was significantly elevated during the postpartum period, compared to 31 wk gestation. Elevated bone turnover helps to provide sufficient calcium for breastfeeding. OPG is the decoy receptor for RANKL and the decrease to control levels postpartum be required to allow RANKL to stimulate osteoclast-mediated bone resorption 

Table Table:. Markers of bone turnover (β-CTX, P1NP am) bone ALP), and OPG at 31 wk gestation Gest) and during postpartum (PP)(mean (1SD))

significant difference from Gest (p<0.05)< ^<(p<0 01); ^<(p<0 001). significant difference from Preg (p<0.05)< ^<(p<0 01);^<(p<0.001).

Disclosures: J. P. Greeves. None.

This study received funding from: The Human Capability Domain of the UK Ministry of Defence Scientific Research Programme.

T003

The use of Statins to Enhance Bone Mineralisation and Formation in 2D and 3D Tissue Engineered Constructs.

S. Griffiths, S. Cartmell. Institute of Science and Technology in Medicine, University of Keele, Stoke on Trent, United Kingdom.

The culture of human bone cells for tissue engineering has great potential as a therapy for patients suffering from bone loss due to disease or trauma. This study has looked at using simvastatin, for the novel use of enhancing human bone cell tissue engineered constructs in vitro. Statins usually prescribed to lower cholesterol, can also enhance bone differentiation, maturation and matrix formation, with increased expression of BMP2 mRNA locally. Our study used simvastatin as a media supplement for in-vitro 2D and 3D primary human osteoblast seeded constructs, utilising gene expression analysis via Real-time RT-PCR. picogreen DNA assays, MicroCT and calcium analysis. Results of 2D cultures show low statin concentrations (0.001 microM) have a significantly increased proliferative effect in 3 (p=4.5E-05) and 7 day cultures (p=2.5751E-05), with a dosage dependent increase in BMP2 mRNA expression. This effect becomes significant after 1 microM, though smaller doses also appear to have an enhancing effect (although this may be a result of increased proliferation). Higher optimised simvastatin concentrations (5microM) significantly reduce osteoblast proliferation (p=0.007), whilst gene expression showed significant increases in BMP2 and osteopontin mRNA suggest an increase in osteoblast differentiation and maturation at 7 days. This may be a result of the statin mechanism interacting with cell cycle regulators, pushing the osteoblasts into the maturation stage. 3D scaffold cultures showed increasing volumes of mineralised matrix production per cell in primary human cell cultures, using 5microM simvastatin as a media supplement. Optimised concentration/timing was then used in 2D cell culture, to maximise proliferation and mineralisation of bone constructs. Mineralisation and calcification were analysed using calcium assay/Von-Kossa staining, whilst picogreen DNA analysis was used for quantitative proliferative data. A further extension of this optimised experiment on a 3D hydroxyapatite scaffold is ongoing, with further analysis via MicroCT and microarray. Results suggest that various statin concentrations could be used with non-diseased bone tissue to enhance proliferation, differentiation and maturation of bone cells, when introduced at various culture periods to enhance mineralisation of extracellular matrix in bone tissue engineered constructs

Disclosures: S. Griffiths, None.

T004

Tartrate-Resistant Acid Phosphatase Is a Negative Regulator of Osteoblast Differentiation.

H. C. Roberts*¹, R. Crossland*¹, T. M. Cox*², M. J. Evans*³, A. R. Havman¹. ¹School of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU, United Kingdom, ²Addenbrooke's Hospital, University of Cambridge, Cambridge CB2 2QQ, United Kingdom, ³School of Biosciences, University of Cardiff, Cardiff CF10 3US, United Kingdom.

Tartrate-resistant acid phosphatase (TRAP) is a molecule important in both the skeleton and the immune system. It is an iron containing protein expressed by osteoclasts, macrophages, dendritic cells and other cell types including osteoblasts. TRAP is secreted by osteoclasts during bone resorption and its serum activity correlates with the level of resorptive activity in certain bone diseases. We have shown using mice lacking TRAP (-/-) that TRAP is important for the normal development of the skeleton since the gross morphology of the bones in TRAP-/- mice is abnormal. Femoral bones lacking TRAP were stronger and more mineralized and the rate of collagen turnover was increased. Femurs from knockout mice have increased tensile strength, increased MMP-2 activity and contain more total collagen cross-links. Osteoclasts from TRAP-/- animals demonstrate reduced bone resorption in vitro. In this study we aim to determine the role of TRAP in the osteoblast.

We have isolated bone marrow from both TRAP-/- and WT mice and used them in comparative studies. Alkaline phosphatase (ALP) activity was measured enzymatically using p-nitrophenyl phosphate as the substrate, and ALP protein by flow cytometry. Bone marrow cultures were grown to confluency (D-MEM/FCS/antibiotics). The cells were reseeded and the medium supplemented with 50μg/ml ascorbic acid and 50mM β-glycerophosphate. At selected times cells were analysed for (i) cell viability using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT)-dye reduction assay. (ii) ALP activity, (iii) mineral deposition (iv) cbfa-1 expression.

Single-colour flow cytometry using a monoclonal antibody to ALP showed that the TRAP-/- cultures had increased numbers of ALP positive cells compared to wild-type (2.4 fold, p<0.05). TRAP-/- bone marrow cells showed an increase in ALP activity compared with WT (1.8 fold, p<0.05). The rate of cell growth in the TRAP-/- cultures was significantly reduced (p<0.05) and cellular morphology was altered. In cultures of TRAP-/- osteoblasts ALP levels were higher and mineralisation occurred earlier after 3–4 days compared with 7–10 days for WT osteoblasts. Conventional PCR demonstrated an increase in the expression of cbfal and osteocalcin in TRAP-/- osteoblasts.

In conclusion, TRAP-/- mice have abnormal osteoblast function leading to an increased rate of differentiation and enhanced mineralisation. This work suggests that TRAP is a negative regulator of osteoblast activity.

Disclosures: A.R. Hayman, None.

This study received funding from: The Wellcome Trust.

T005

Independent of Their Resorptive Activity, Osteoclasts Secrete an Activity Promoting Bone Formation and Canonical Wnt Signalling in Osteoblastic Cells.

K. Henriksen¹, A. V. Neutzskv-Wulff*¹, K. D. Hausler*², M. Ciccomancini*², C. Christiansen³, M. Gillespie², T. J. Martin², M. A. Karsdal¹. ¹Nordic Bioscience A/S, Herlev, Denmark, ²St. Vincent's Institute of Medical Research, Melbourne, Australia, ³Center for Clinical and Basic Research, CCBR, Ballerup, Denmark.

Some osteopetrotic mutations lead to low bone resorption, increased numbers of osteoclasts and increased bone formation, whereas other osteopetrotic mutations lead to low resorption, low numbers of osteoclasts and decreased bone formation. These findings indicate that the osteoclasts independent of their resorptive activity could be sources of anabolic signals for the osteoblasts.

The aim of the current study was to investigate whether osteoclasts secrete bone anabolic signals, and to elucidate the signal transduction involved. Conditioned media from mature human osteoclasts cultured on either bone slices or plastic were collected. Measuring TRACP and CTX-I validated osteoclast maturity and resorption. Conditioned media were applied to cultures of MC3T3-E1 preosteoblasts, followed by bone formation assessment by Alizarin red and Von Kossa staining after 20 days' culture. We assessed key osteoblast regulatory pathways by using UMR 106.01 cells transiently transfected with several reporter constructs. These were the TOPFlash vector, which contains 8 TCF/LEF response elements, the osteocalcin promoter with x6 tandem OSE repeats (6 x OSE), NFAT, AP-1 and NFkB. In each case appropriate positive control stimuli were used.

Conditioned media from osteoclasts cultured on either bone or plastic stimulated nodule formation by the MC3T3-E1 cells to levels comparable to stimulation with 10ng/mL BMP-2. Conditioned media from osteoclasts cultured on both bone and plastic specifically induced activation of the TCF/LEF response system at a level comparable to induction by 20ng/mL of Wnt3A. The Wnt 3a and conditioned medium signals were equally inhibited by addition of either 100ng/mL of DKK1 or sclerostin, consistent with activation of the canonical Wnt signaling pathway. No activation of the OSE, NFAT, NFkB, or AP-1 reporters was detected, suggesting specific wnt activity.

We present evidence that osteoclasts, independent of their resorptive activity, secrete an activity that stimulates osteoblastic bone formation. The same media contains an activity that signals through the wnt activation cascade, indicating a wnt as a possible anabolic product, i.e a potential coupling factor, produced by the osteoclasts.

Disclosures: K. Henriksen, None.

T006

Alpha1 and Beta2 Adrenergic Agents Modify RANKL/OPG Expression and Cell Proliferation in Human Osteoblasts and Osteoblastic Cells.

H. H. Huang, T. C. Brennan*, M. M. Muir*, R. S. Mason. Department of Physiology and Bosch Institute, University of Sydney, Sydney, Australia.

Previous studies have shown that central control of bone mass is mediated via beta2 receptors of the sympathetic nervous system ⁽¹⁾. Whether alpha adrenergic receptors are expressed in human bone cells is controversial ^(1,2). Takeuchi et al ⁽³⁾ showed that epinephrine increased both RANKL and OPG mRNA expression in murine osteoblast-like cells via beta and alpha adrenergic receptors. The aim of this study is to further investigate alpha and beta adrenergic receptors in human bone cells. Human osteoblastic MG63 cells and primary human fetal bone cells (FBC) were cultured in DMEM with 10% FBS then adapted to serum-free medium for 24 h before treatments were added. Alpha1b and beta2 adrenergic receptors were expressed in MG63 cells and FBC as determined by RT-PCR (alpha1b, beta2) and sequence analysis (alpha1b). In FBC, 10–⁵M cirazoline (specific alphal agonist) increased OPG and RANKL mRNA expression by 1.43 (p<0.01) and 1.36 (p<0.001) times respectively, while only high-dose cirazoline (10–³M) up-regulated OPG (3∼4-fold) and RANKL (over 10-fold) in MG63. Fenoterol, (beta2 agonist) at 10⁻⁷M-10⁻⁵M dose-dependently up-regulated OPG mRNA expression (p<0.05) but only 10⁻⁷M fenoterol increased RANKL mRNA expression (p<0.05) in FBC. Phenylephrine (alphal agonist) dose-dependently increased cell replication in both FBC (10⁻⁶∼10⁻⁴M) and MG63 (10⁻⁸∼10⁻⁵M), as did cirazoline in FBC. The beta2 agonist fenoterol suppressed FBC proliferation (10⁻⁶∼10⁻⁵, p<0.001). The maximal effects of cirazoline in FBC and phenylephrine in MG63 at 10⁻⁶M on proliferation were partially suppressed by 10⁻⁴M urapidil (alphal antagonist). These data indicate that alphal adrenergic receptors are present in human bone cells and could play a role in modulation of bone turnover by the sympathetic nervous system in human bone cells.

• 1.
  Takeda, S. et al. (2002), Cell 111: 305–317.
• 2.
  Togari, A. (2002), Microsc Res Tech 58: 77–84.
• 3.
  Takeuchi, T. et al. (2000), Biochem Pharmacol 61: 579–586.

Disclosures: H.H. Huang, None.

T007

An Isoform of Fibronectin Is Responsible for Decreased Bone Formation in Patients with Primary Biliary Cirrhosis and this Effect Is Not Exclusively Mediated by beta1 Integrins.

N. Kawelke*¹, A. Bentmann*¹, N. Hackl*¹, P. Feick*², M. V. Singer*², I. Nakchbandi³. ¹University of Heidelberg, Heidelberg, Germany, ²University of Heidelberg at Mannheim, Mannheim, Germany, ³University of Heidelberg and Max-Planck Institute for Biochemistry, Heidelberg, Germany.

Patients with chronic cholestatic liver disease experience a decrease in bone formation and density associated with an increase in fracture rates. The cause for this bone loss remains elusive. We have reported that oncofetal fibronectin (oFN) is elevated in patients with primary biliary cirrhosis (PBC) and correlates negatively with circulating osteocalcin levels. To determine a causal relationship between the elevation of oFN and bone loss in subjects with PBC we performed in vitro and in vivo experiments in mice. Fibronectin (FN) was isolated from amniotic fluid (aFN) and characterized as containing both the EIIIA domain and the glycosylated variable region domain (oFN). In vitro experiments showed that mineralization by osteoblasts is diminished to the same degree whether aFN contained the EIIIA domain or not (71% vs. 78% decrease in mineralization using 100 ug/ml FN). This suggests that the presence of the glycosylated variable region was responsible for the decrease in mineralization.

Injection of aFN in CD1 mice for 10 days (1 mg/day/mouse) resulted in a 17% drop in trabecular BMD compared to controls (p<0.05) as measured by pQCT. There was a 30% decrease in mineralizing surface (MS/BS= 44.7 ±3.1 in controls vs. 31.2 ± 1.6 % in injected mice, p<0.005), and a decrease of 45% in the number of osteoblasts (Ob.N/mm BS= 18.2 ± 1.4 in controls and 10.0 ± 0.8 in injected mice, p<0.05).

In order to determine whether this effect is mediated by betal integrins we repeated the experiment in mice in which betal integrin was deleted using the collagen alphal(I)-cre promoter (Betal integrin floxed/floxed-Collagen alpha1(I)-cre/+). Similarly to CD1 mice there was a significant decrease of 50% in the number of osteoblasts when aFN was injected over a period of 10 days (Ob.N/mm BS: 25 ± 3 vs. 13 ± 1, p<0.01; n=8 controls and 4 injected mice).

In summary, we have shown that 1) in patients with PBC the glycoslyated variable region of FN as recognized by the FDC-6 antibody correlates negatively with osteocalcin, 2) decreased mineralization by osteoblasts in vitro is caused by the presence of the same domain, and 3) injection of FN containing this domain in mice results in a significant decrease in osteoblasts number that is not exclusively mediated by the presence of betal integrins on the surface of osteoblasts expressing the collagen alpha1(I) promoter. We therefore propose, that oncofetal fibronectin is responsible for decreased bone formation in patients with primary biliary cirrhosis.

Disclosures: N. Kawelke. None.

T008

Osteogenesis of the Human Ligamentum Flavum by Demineralized Bone Matrix.

J. Lee*¹, H. Kim*², C. Lee*¹, M. Nan*¹, K. Lee*², S. Moon*³, J. Park*³, H. Kim*³, J. Jahng³, H. Lee*³, H. Kim*³. ¹Orthopaedic Surgery, BK21 Medical Science Graduate School of Yonsei University, Seoul, Republic of Korea, ²Korea Bone Bank, Seoul, Republic of Korea, ³Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Republic of Korea.

Many attempts have been made to apply demineralized bone matrix for bone regeneration. It has been reported that DBM derived from human tissues induces bone formation and spinal fusion, and that DBM is widely used as a bone graft substitutes in orthopaedic and dental surgery. In addition, Ligamentum flavum (LF) was reported to have osteogenic potential with stimulation of bone morphogenetic protein-2 which is also included in DBM. Although many potential studies for osteogenesis by DBM have been evaluated, osteogenesis of human LF by DBM have not yet been found. In this study, we elucidated the effect of DBM on osteogenesis of human LF. Experimental group (human LF cell culture) was incubated in media solution containing 5mg/ml or 10mg/ml DBM for 2hr, 2day and 7days, and control group was incubated in media which have no DBM. To assess proliferation rate, we performed Alamar blue assay. To assess osteoinductivity, we performed RT-PCR of osteogenic marker gene and von-kossa, alkaline phosphatase (ALP) staining after incubating LF with DBM. DBM, LF and mixture of DBM and LF was implanted to rude mouse. After 28days, H&E staining was performed. Human LF cell cultured with DBM showed increased proliferation without cytotoxicity compared to control in dose-dependent manner. The expression of collagen type I, ostrocalcin, osterix, dlX5, Human MSX2 mRNA demonstrated upregulation of osteogenic phenotype. Also, result of ALP, von-kossa and H&E staining were shown that experimental group, LF by adding the DBM, showed positive osteogenesis compared to control group. We proved that human LF cells cultured with DBM showed increase in proliferation and upregulation of osteogenic phenotypes. Given osteogenic potential of human LF with DBM, autogenous LF can be used for carrier for DBM and bone graft substitute in spinal surgery. The application of mixture DBM and LF will likely be useful in the clinical reconstruction of bone.

Disclosures: J. Lee, None.

T009

Healing of Critical-sized Bone Defects by Endothelial Progenitor Cells.

D. Lewinson¹, N. Rozen*², T. Bick*¹, B. Shemian*¹, A. Yayon*³, M. Soudry*¹. ¹Research Institute for Bone Repair, Orthopaedic Surgery A, Rambam Health Care Campus, Haifa, Israel, ²Research Institute for Bone Repair, Rambam Health Care Campus, Haifa, Israel, ³ProChon BioTech LTD, Ness-Ziona, Israel.

In this study we developed a novel cell therapy method for promoting repair of large bone defects. Our model tested the healing potential of circulating autologous endothelial progenitor cells (EPC) in a critical-sized bone defect created in sheep tibia. Peripheral mononuclear fraction was isolated using Lymphoprep(tm) (Axis-Shield, Oslo, Norway), seeded on fibronectin-coated plates and cultured in EBM-2 media supplemented with EGM-2MV SingleQuote (Clonetics, Cambrex Bio Science, MD, USA). The endothelial nature of adherent colonies was identified by incorporation of Dil-acetylated LDL (Molecular Probes, Oregon, USA), tube formation on Matrigel (BD Labware, MA, USA) and immunostaining for von Willebrand factor (Dako, Glostrup, Denmark). A defect of 3.2 cm was created in the mid-shaft of sheep tibia, partially preserving the periosteum. Two weeks later, a longitudinal wedge was cut along the regenerating tissue filling the gap, 2×10⁷ EPC were transplanted into the formed tunnel and covered with the removed scar tissue. Sham-operated sheep served as controls. Bone regeneration was followed during the next three months by x-rays radiography every two weeks. At the end of the experiment, the operated fragment bone was removed and subjected to a detailed qualitative and quantitative 3-D evaluation at a resolution of 36 micrometers using a micro computed tomography (μCT) imaging system (μCT 40, Scanco Medical, Bassersdorf, Switzerland). No significant new bone formation was observed radiographically in 4 sham-operated sheep. In contrast, 5 out of six EPC-transplanted sheep showed full bridging at 3 months, starting 2 to 4 weeks post EPC transplantation. Data collected showed an increase of 500% of total tissue volume (TV), 700% of bone volume (BV) in EPC-transplanted gaps vs. sham-operated ones. Material density increased 2-folds both in the entire volume and in the mineralized tissue compartment. We conclude that EPC have a promising potential for healing of large bone defects.

Disclosures: D. Lewinson. Prochon BioTech LTD 2. 3.

This study received funding from: Ministry of Industry & Commerce. Government of Israel and ProChon Bio Tech LTD.

T010

How Does Hypoxia Contribute to the Formation of the External Apical Root Resorption In Vitro?

D. Liu, Z. Ou*. Developmental Sciences/Orthodontics, Marquette University, Milwaukee, WI, USA.

Objectives: External apical root resorption (EARR) is commonly seen in orthodontic patients. However, how the EARR is formed under orthodontic force is not known. When a mechanical force is applied to move a tooth, an immediate response is the compression of periodontal ligament, which often leads to occlusion of the capillary blood vessels in it, in turn, forms an ischemia zone within which all the surrounding cells are subjected to hypoxia. Cementoblasts are cementum (first barrier to be attacked during EARR) — forming cells and have been shown to be involved in the repair of EARR. Therefore, we hypothesize that hypoxia plays an active role in the formation of EARR via the regulation of cementoblasts. The purpose of this study is to investigate the effect of hypoxia on the biological responses of cementoblasts in vitro. Methods: OCCM.30 cells — an immortalized murine cementoblastic cell line (from Somerman MJ, U. Washington), were seeded onto glass slides coated with type I collagen (10μg/cm²) and grown in α-MEM with 10% FBS. Upon confluent, OCCM.30 cells were starved for 24 hours then subjected to 1% hypoxia challenge while controls were kept under normal culture condition of 95% air and 5% CO₂ For the early signaling study, after the onset of hypoxia, media were collected at 5 and 15 minutes to determine the release of ATP and PGE₂. For the long-term functional study, the cells were subjected to 1% hypoxia for 0.5, 1, 3, 6, 12 hours to determine the induction of hypoxia-inducible factor 1-α (HIF-1α) and production of cycloxygenase-2 (COX-2) and sclerostin (SOST — a negative regulatory protein for bone formation) by western blot analysis. Paired t test was used to compare hypoxia treated and untreated groups for each parameter with p value being set at 0.05. Results: For the short-term changes, 1% hypoxia induced a drastic release of ATP at 5min followed by increased PGE₂ release at 15min. For the long-term changes, 1% hypoxia significantly induced HIF-1α production after 3 hours, with a peak reached at 12 hours. COX-2 production was significantly increased after 6 and 12 hours of 1% hypoxia. Interestingly, SOST was for the first time found not only expressed in OCCM.30 cementoblasts but also increased after 6–12 hours of 1% hypoxia. Conclusions: Our data indicate that hypoxia modulates both early molecular signaling and late functional responses of cementoblasts, suggesting its potential role in the modulation of cementum remodeling towards resorption. Clinically, mechanical load exceeding the capillary blood pressure leading to an ischemia-associated hypoxia in periodontal ligament should be avoided to reduce the risk of root resorption during orthodontics. This work was supported by AAOF and EOS research grants.

Disclosures: D. Liu, None.

T011

Indian Hedgehog Is Essential for Postnatal Bone.

Y. Maeda*¹, R. G. Erben², E. Schipani*³, B. Lanske¹. ¹Developmental Biology, Harvard School of Dental Medicine, Boston, MA, USA, ²Department of Natural Science, University of Veterinary Medicine, Vienna, Austria, ³Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

Indian hedgehog (Ihh) is essential for chondrocyte proliferation/differentiation and osteoblast differentiation during prenatal endochondral bone formation. Recently we reported the generation and characterization of tamoxifen-inducible conditional Ihh knockout mice (Col2α1-Cre ER*; Ihh d/d), in which the Ihh gene was successfully ablated from chondrocytes after birth. Our results demonstrated for the first time that Ihh expression in postnatal chondrocytes is essential for maintaining a growth plate, and for sustaining trabecular bone, a normal articular surface, and eventual bone growth after birth. We also showed that Wnt signaling was significantly decreased in these mice suggesting that Ihh activates osteoblast differentiation via the Wnt signaling pathway in postnatal life. Loss of trabecular bone in the Col2α1-Cre ER*; Ihh d/d mutants could be secondary to their growth plate abnormalities. To examine the direct role of Ihh on postnatal bone formation, maintenance of a growth plate in the mutants would be required. We, therefore, induced deletion of Ihh in chondrocytes at P14, after a secondary ossification center was formed. At P21, however, the growth plate was completely lost and time course experiments are being performed now to investigate the kinetics of this event.

In order to dissect whether the loss of the growth plate or the lack of Ihh signaling itself in chondrocytes is responsible for the decrease in trabecular bone, we are now attempting to rescue the growth plate of the Col2α1-Cre ER*; Ihh d/d animals by mating them to col2α 1-constitutively active PTH/PTHrP receptor transgenic mice (col2α 1-Jansen). Using such compound mutant mice, we anticipate rescue of the growth plate despite absence of Ihh signaling. This will allow us to study specific actions of chondrocyte-derived Ihh on postnatal bone.

Disclosures: Y. Maeda, None.

T012

Potential Involvement of Ephrin B2 in the Anabolic Action of PTH in Osteoblasts.

E. H. Allan*¹, K. D. Hausler*¹, J. M. W. Ouinn¹, J. Gooi*¹, T. Wei*², J. E. Onyia², N. A. Sims¹, M. T. Gillespie¹, T. J. Martin¹. ¹Bone, Joint & Cancer, St Vincent's Institute, Melbourne, Australia, ²Lilly Research Laboratories, Indianapolis, IN, USA.

The aim was to identify paracrine factors whose production is controlled by PTH and/or PTHrP, using a mouse marrow stromal cell line, Kusa 4b10 that acquires osteoblast features under differentiating conditions. After the appearance of functional PTH1R in Kusa 4b10 cells they were treated with either PTH(1–34) or PTHrP(1–141) for 1, 6 and 24 hours and RNA subjected to Affymetrix whole mouse genome array. The data was analysed ststistically by SAM (significance analysis of micro-arrays). Of the 45101 probes used on the micro-array, 4621 probes or 3231 genes with functional annotations were differentially expressed by at least 1.5 fold with false discovery rate < 0.1. These genes were then functionally categorized into 228 groups according to their Gene Ontology. Among genes belonging to the family of Ephrins and their receptors, Ephrin B2 and Eph B2 were up-regulated in response to PTH and PTHrP. The effects were validated using independently prepared RNA samples from differentiated Kusa 4b10 cells, UMR106 rat osteogenic sarcoma cells, and primary mouse calvarial osteoblasts, as well as in vivo using RNA prepared from metaphyseal bone of 3 week-old rats 1.5 and 4 hrs after a single PTH injection. Stimulation of mRNA for Ephrin B2 by PTH and PTHrP was maximal at 4–6 hrs, with a 3–6-fold response. The effect was dose-dependent and the curve shifted to the left with phosphodiesterase inhibition. Western blotting showed a sustained increase in Ephrin B2 protein following treatment of Kusa 4b10 cells with PTH for 6, 12 and 24 hours. Neither IL-11 nor active vitamin D affected Ephrin B2 production. Whereas Ephrin B2 production was clearly regulated in osteoblasts by PTH/PTHrP through a cAMP/PKA mechanism, its baseline production remained unchanged throughout differentiation. No evidence was obtained for regulation by PTH/PTHrP of Eph B4 production, which also remained unchanged throughout differentiation. Eph B2 mRNA was increased 2-fold and Ephrin A5 decreased 2-fold after 6 hrs PTH or PTHrP treatment.

Recent evidence implicates osteoclast-derived Ephrin B2 acting through its receptor, EphB4 in osteoblasts, to promote osteoblast differentiation, and through reverse signalling via EphB4 and possibly other members of the receptor family to suppress the formation of osteoclast precursors. The present data raise the possibility that ephrinB2 might act in a paracrine or autocrine manner on the osteoblast under the influence of PTH, and provide a local event related to the anabolic action of PTH.

Disclosures: T.J. Martin. None.

T013

Acceleration of Fracture Healing via Enhanced Vasculogenesis and Osteogenesis Through SCF/cKit Pathway in Lnk-Deficient Mice.

T. Matsumoto¹, H. Nishimura*¹, Y. Mifune¹, T. Shoji*¹, R. Kuroda*², A. Kawamoto*¹, A. Oyamada*¹, M. Horii*¹, M. Miwa*², M. Kurosaka*², T. Asahara*¹. ¹Stem Cell Translational Research, Kobe Institute of Biomedical Research and Innovation / RIKEN Center for Developmental Biology, Kobe, Japan, ²Department of Orthopedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.

Lnk, with APS and SH2-B, belongs to an intracellular adaptor protein family and was recently proved an essential inhibitory signal molecule of the stem cell factor (SCF)-cKit signaling pathway for self-renewal of stem cells in the findings demonstrating enhanced hematopoietic reconstitution in Lnk-deficient mice. We previously reported that systemic administration of human hematopoietic/endothelial progenitor cell (HPC/EPC)-enriched population induces osteogenesis as well as vasculogenesis and provides a favorable environment for functional bone healing in non-healing fracture model of immunodeficient rat. Therefore, we investigated the hypothesis that a lack of Lnk signaling enhances the regenerative response via vasculogenesis and osteogenesis in fracture healing in Lnk-deficient mice. In radiological and histological evaluations, reproducible model of closed femoral fracture in 10-week-old mice demonstrated more prompt fracture healing in Lnkknock out (KO) than wild-type (WT) mice (week2: KO, 70%; WT, 35%, p<0.01, week3: KO, 100%; WT, 90%, n=20). Serial FACS analysis of peripheral blood (PB) demonstrated significantly larger number of Scal + Lin-cells, HPC/EPC-enriched fraction (HPC/EPCs), in KO than WT (p<0.05), and significantly increased HPC/EPCs by fracture-stress with the peak at 1 day post-fracture (p<0.05). Among up-regulated gene expressions at the peri-fracture site by cDNA microarry in KO, angiogenesis/vasculogenesis (CD31, VE- cadhenn, KDR) and osteogenesis (Osteocalcin, Collagenl AI, Cbfal/Runx2) related gene expression assessed by real time RT-PCR were significantly enhanced 7 days post-fracture in KO compared to WT (p<0.01). Immunohistochemical staining at the peri-fracture site demonstrated significantly enhanced density of Scal + cell-derived endothelial cells (ECs)(Scal+/CD31+) and osteoblasts (OBs)(Scal +/Osteocalcin+) 7 days post-fracture in KO compared to WT (p<0.05). Moreover, in addition to a significant higher expression of plasma SCF in KO compared to WT by ELISA (p<0.01), SCF stimulation enhanced these mobilization and incorporation of HPC/EPCs even in WT and more in KO, confirmed by analysis of PB FACS (p<0.05), EC (p<0.05), and OB density (p<0.05). Our data suggest the therapeutic potential of negatively controlling the Lnk system via SCF-cKit pathway for promoting an environment conducive to vasculogenesis and osteogenesis so that fractures can heal promptly.

Disclosures: T. Matsumoto, None.

T014

Withdrawn

T015

Bone Plays an Important Role in the Metabolism of Postprandial Lipoproteins in Mice: Impact on Osteoblast Function.

A. Niemeier¹, D. Niedzielska*², R. Secer*², A. Schilling*³, M. Merkel*⁴, C. Enrich*⁵, P. C. Rensen*⁶, J. Heeren*². ¹Orthopaedics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, ²Biochemistry and Molecular Biology II: Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, ³Trauma-Hand- and Reconstructive Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, ⁴Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, ⁵Biologia Cellular, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain, ⁶General Internal Medicine, Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands.

Dietary lipids and lipophilic vitamins are transported by postprandial lipoproteins and are required for bone metabolism. Despite that, it remains unknown whether bone cells are involved in the uptake of circulating postprandial lipoproteins in vivo. The current study was designed to investigate a putative participation of bone in the systemic postprandial lipoprotein metabolism in mice, to identify potentially involved cell type populations and to analyze whether lipoprotein uptake affects bone function in vivo.

As a model for the postprandial state, chylomicron remnants (CR) were injected intravenously into mice. Bone was compared to organs with an established role in CR catabolism. CR organ distribution and cellular uptake was analyzed by immunohistochemistry, confocal laser microscopy and electron microscopy. Radiolabeled CR were used for quantification of organ-specific plasma clearance and uptake into primary osteoblasts and hepatocytes. The effect of vitamin Kl-enriched CR on plasma osteocalcin was determined by radioimmunoassay.

Next to the liver, bone appeared to contain the most active cells for the uptake of radioactive and fluorescent CR particles from the circulation in vivo. Immunostaining for apoE proved that intact CR particles were taken up in both liver and bone, but not in other organs. Uptake of CR by primary murine osteoblasts and hepatocytes was in a similar range in vitro. Localization studies in bone showed strongest primary binding to sinusoidal endothelial cells, while particle uptake was also observed into macrophages and osteoblasts. Enrichment of injected CR with vitamin Kl resulted in an increase of the degree of osteocalcin carboxylation in vivo.

In conclusion, bone is an organ of major importance in the postprandial lipoprotein metabolism. Bone function in vivo appears to be directly influenced by CR uptake, which has an impact on the secretory function of osteoblasts. This study uncovers a close functional relation between the systemic lipoprotein metabolism and bone metabolism.

Disclosures: A. Niemeier, None.

This study received funding from: Deutsche Forschungsgemeinschaft.

T016

Isolation and Immunohistochemical Analysis of Osteomodulin Expressed in Osteoblast.

K. Ninomiya*¹, T. Miyamoto², N. Fujita*³, T. Suzuki*¹, R. Iwasaki*⁴, M. Yagi*³, Y. Tovama*³, T. Suda*⁵. ¹Orthopaedic Surgery and Cell Differentiation, Keio University School of Medicine, Tokyo, Japan, ²Cell Differentiation, Orthopaedic Surgery and Musculoskeltal Reconstruction and Regeneration Surgery, Keio University School of Medicine, Tokyo, Japan, ³Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan, ⁴Dentistry and Oral Surgery and Cell Differentiation, Keio University School of Medicine, Tokyo, Japan, ⁵Cell Differentiation, Keio University School of Medicine, Tokyo, Japan.

The bone volume is regulated with the well-controlled balance between bone-resorbing osteoclast and bone-forming osteoblast, which is called coupling. Although coupling is a well-known phenomenon, coupling factor has not been identified. To isolate the coupling factor, we performed microarray and cluster analysis among rat 13 tissues, and isolated osteomodulin (OMD) as a candidate of coupling factor. OMD is an extracellular matrix keratin sulfate proteoglycan that belongs to small leucine-rich repeat proteoglycans (SLRPs). Though OMD is not expressed in osteolcast, the expression of OMD couples with osteoclast markers such as matrix metalloproteinase-9 (MMP-9) and tartrate-resistant acid phosphatase (TRAP) in bone. SLRPs are known to have diverse functions such as regulation of matrix assembly, cell growth, and cell differentiation. In in vitro, OMD is expressed in primary osteoblasts and is upregulated during osteoblastic differentiation. To investigate the localization of OMD in in vivo, we established an anti-OMD polyclonal antibody. OMD is mainly produced by alkaline phosphatase (ALP) positive osteoblast. The specificity of the anti-OMD antibody was confirmed by the blocked immunoreactivity of the antibody by synthetic peptide. Op/op mice are osteopetrotic because of defective osteoclast formation due to the lack of macrophage colony-stimulating factor (M-CSF) production. Intriguingly, the immunoreactivity of anti-OMD antibody in trabecular bone in op/op mice is weaker than that of wildtype mice. Since osteoblasts do not express c-fms, a M-CSF receptor, the reduced OMD expression in op/op mouse is confirmed based on the lack of coupling due to a defect of osteoclast formation. We also confirmed that expression of OMD in C2C12 cells increased accompanied by an upregulation of ALP activity during osteoblastic differentiation. Because some members of SLRPs inhibit signal of transforming growth factor (TGF), OMD could modulate differentiation of osteoblast and osteoclast by inhibition of TGF signal. Our results suggest that OMD is expressed in osteoblasts especially activated by osteoclast.

Disclosures: K. Ninomiya, None.

T017

Induction of Mineralized Tissues by In Vivo Trasplantation of Cultured Rat Dental Pulp Cells in Rat Calvarial Bone Defects.

H. Ohba, H. Seto, K. Tokunaga, H. Hama, Y. Inagaki, M. Horibe, E. Takeda, T. Nagata. Health Biosciences, Tokushima, Japan.

Dental pulp tissues have a potency to form mineralized tissues. We investigated the ability of dental pulp cells for mineralized tissue induction in vitro and in vivo. Dental pulp tissues were extracted from maxillary incisors of male Wistar rats. The tissues were minced and digested with 0.5% tripsin and 0.02% EGTA. Dissociated cells were cultured in Eagle's MEM. After subconfluency, cells were collected and seeded in multi-well plate with or without 2 mM β-GP and 50 μg/ml ascorbic acid. Alkaline phosphatase (ALP) activity was determined on days 1 and 5 and the von Kossa staining was performed to detect bone nodule (BN) formation on day 20. Also, the association of bone morphogenetic protein (BMP) on such a mineralized tissue formation was determined by ELISA. The subconfluent dental pulp cells were collected and used in the following in vivo study. Bone defects were made in male Wistar rat calvaria (hole shape, 3.8 mm diameter). The collected dental pulp cells were transplanted in the defects with collagen sponges including 1% collagen gel (2.5×10–⁶ cells/ml). After 8 weeks, rat calvariae were taken out and analysed by soft X-ray and histological examinations. Results showed that ALP activity increased and BNs were clearly formed in the dental pulp cell cultures. ELISA assay showed that dental pulp cells produced BMP and the amount of BMP in the condition medium time-dependently increased, showing 3-fold amount of BMP compared to the control on day 15. The soft X-ray and histological examinations revealed that the transplantation of dental pulp cells induced a greater amount of bone-like tissues in the inside of calvarial bone defects than in the controls, showing 4-fold increase of radiopaque area. Hematoxylin and eosin staining demonstrated that there were many osteoblast-like cells around the new-formed tissues. Present findings indicate that bone defects were recovered by the transplantation of dental pulp cells, suggesting that dental pulp cells have a potency to form mineralized tissues in association with BMP production.

Disclosures: H. Ohba. None.

T018

Imatinib Mesylate Inhibits Bone Formation and Decreases Bone Mass In Vivo by Inhibiting PDGFR-Mediated Osteoblast Mitogenesis.

S. O'Sullivan*¹, D. Naot*¹, K.Callon*¹, M. Watson*¹, G. Gamble*¹, M. Karsdal*², M. Ladefoged*², J. Cornish¹, P. Browett*³, A. Grey¹. ¹Medicine, University of Auckland, Auckland, New Zealand, ²Nordic Bioscience, Herlev, Denmark, ³Pathology and Molecular Medicine, University of Auckland, Auckland, New Zealand.

Imatinib mesylate, an orally active inhibitor of the c-abl (including bcr/abl), c-kit and platelet-derived growth factor receptor (PDGFR) tyrosine kinases, is in clinical use for the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal cell tumours (GIST). Genetic interruption of both c-kit and c-abl signaling in mice induces osteopenia, and treatment with PDGF increases bone mass in rodents, suggesting that imatinib might have adverse effects on the skeleton. In vitro, imatinib exerts complex effects in bone cells. It inhibits the proliferation of osteoblast-like cells induced by both low-dose serum and PDGF. Neither addition of SCF, the c-kit ligand, nor siRNA-mediated knockdown of c-abl, alters osteoblast mitogenesis. Imatinib dose-dependently increases osteoblast differentiation, also by inhibiting PDGFR signaling, an observation that might explain the early increase in bone formation markers observed in humans with CML starting therapy with imatinib. In murine bone marrow cultures, imatinib inhibits osteoclastogenesis stimulated by 1,25 dihydroxyvitamin D₃ and partially inhibits osteoclastogenesis induced by treatment with RANKL and M-CSF. Consistent with these findings, imatinib partially inhibits osteoclastogenesis in RANKL-stimulated RAW-264.7 cells. Treatment with imatinib increases the expression of OPG in bone marrow from CML patients, and osteoblastic cells in vitro. In order to elucidate the skeletal effects of imatinib in vivo, we treated 6 month old Wistar rats with vehicle, imatinib 40mg/kg/day or imatinib 70mg/kg/ day, for 5 weeks. Trabecular bone volume, assessed at the proximal tibia by micro-CT, declined by 20% in the imatinib-treated rats (p<0.05 vs vehicle). Serum osteocalcin (Rat-Mid) fell by 40% in response to imatinib therapy (p<0.01 vs baseline). There was no evidence for an anti-resorptive effect of imatinib in vivo (CTX-I). Collectively, these results suggest that the dominant skeletal action of imatinib in vivo is inhibition of osteoblast mitogenesis by interruption of PDGFR signaling, leading to decreased bone formation and a decline in bone mass. Clinical studies should be undertaken to assess the effect of long-term imatinib therapy on bone mass in humans. These results also suggest that PDGFR signaling in osteoblasts may regulate bone mass in vivo.

Disclosures: S. O'Sullivan, None.

T019

Expression of Metalloproteinase-14 Is Pleiotropic on Alkaline Phosphatase Expression in Osteogenic Differentiation.

D. Palmieri*, S. Soldano*, S. Zanotti*, D. Lombardini*, P. Manduca. DiBio, University of Genoa, Genova, Italy.

It is known that modulation of the expression of AP and MMP-14 are developmentally regulated during osteogenesis.

We here report that in ROB (rat tibia-derived osteoblasts), during early and pre-mineralization stages of osteogenesis, the modulation of the expression of MMP-14 affects that of AP which later jointly condition the formation of nodules and the mineralization of the osteogenic population. We show that the level of expression of MMP-14 affects pleiotropically the expression of AP by up or down modulating, at transcriptional or post-transcriptional levels the expression of either gene in ROB and in cloned derivative lines. Changes in MMP-14 were obtained by activating antibodies or inhibitors or observed in selected clones:

• 1
  - Constitutive expression of MMP-14, obtained by exposure to activating antibodies, determines constitutive expression of AP: no nodules form, no mineralization occurs in the population.
• 2
  - Inhibitors of MMP-14 inhibit also the expression of AP: no nodules form, no mineralization occurs in the population.
• 3
  - Clones constitutive for MMP-14 are constitutive also for AP: no nodules form, no mineralization occurs in the population.

Vice versa, modulation of the expression of AP by GF or fluorides or by inhibitors has no effect on the expression of MMP-14:

• 1
  - Over-induction of AP by F compounds, by IGFII and by ET does not affect the expression of MMP-14: enhanced nodule formation and mineralization occur in the population.
• 2
  - Inhibition of AP by Levamisole or by inhibition of endogenous ET, does not affect the expression of MMP-14: nodules form, but their mineralization is reduced.
• 3
  - Inhibition of AP by Levamisole in clones constitutive for MMP-14 and AP, does not affect the expression of MMP-14: no nodules form, no mineralization occurs. In conclusion, at the onset of osteogenesis, MMP-14 expression is pleiotropic on AP, not vice versa. Transient up-modulation of both is required for nodule formation and mineral deposition. Down-modulation of MMP-14 is required for nodule formation. Down modulation of both enzymes is associated with mineralization.

Disclosures: D. Palmieri, None.

This study received funding from: MIUR.

T020

Role of PLF in Remodeling of Bone Following Injury.

S. Rani*¹, M. F. Barbe¹, A. E. Barr*², J. Litvin*¹. ¹Anatomy and Cell Biology, Temple University, Philadelphia, PA, USA, ²College of Health Professions, Temple University, Philadelphia, PA, USA.

Periostin-Like-Factor (PLF) is expressed in developing bone and is up regulated in adult bones that undergo remodeling due to fractures or repeated high force activity which leads to inflammation followed by injury of the bone. PLF over expression by adenovirus in the bone marrow cavity of rats resulted in increased bone formation. Together, these findings suggest that PLF plays an important role in bone formation. To test the hypothesis that PLF promotes bone formation under conditions of remodeling, tissues obtained from the novel model of Upper Extremity-Work-Related Musculoskeletal Disorder (WMSD) in the rat were obtained and subjected to ELISA, immunostaining and Western blot analysis. Twenty two young adult female Sprague Dawley rats were trained to perform a High Repetition High Force (HRHF) voluntary reaching and pulling task, with a food pellet reward. This task entails pulling a lever isometrically at a target rate of 4 reaches/min with 60% max grip force for 2 hrs/day in four 30 min sessions separated by 1.5 hr each, 3days/ week and leads to mechanical loading of bone. Loaded bone responds by remodeling and under extreme situations stress fractures and resorptive spaces develop. Tissues from the reach limbs of performing rats and from randomly selected forelimbs of control rats were obtained at various times and subjected to immunostaining and Western blot analysis. To investigate the expression of pro-inflammatory cytokines ELISA assay was done on the bone of performing and control animals.

PLF protein was markedly up regulated in the bone of the performing limb of animals exposed to high force and repetitive motion, and the spatial pattern of PLF expression varied over the course of remodeling. Its expression peaked at 6 weeks, during the 12 week-work regimen. It was detected in the periosteum which contains mesenchymal pre-osteoblasts of the ulna and radius, in osteocytes and osteoclasts. It was secreted from proliferating and hypertrophied chondrocytes. The pro-inflammatory cytokines TNF-α and IL-1, also peaked in the bone at 6 weeks of task performance, suggesting a link between the PLF up regulation and the inflammation process.

The expression of PLF in bones undergoing remodeling, recovery and repair suggests that PLF plays a significant role in formation and maintenance of bone tissue. Its up regulation concomitant with the presences of high levels of pro-inflammatory mediators also suggests that it has a role in the inflammatory processes. In future experiments the role of PLF in remodeling and inflammation mediated processes will be examined in depth in the WMSD and other fracture repair models.

Disclosures: S. Rani. None.

This study received funding from: NIAMS.

T021

Pitx2 Inhibits Osteoblastic Conversion of Myoblasts Through Interfering the Promoter Activity of Osterix Gene.

M. Havashi*¹, S. Maeda¹, H. Aburatani*², K. Miyazono*³, T. Imamura¹. ¹Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo, Japan, ²Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan, ³Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

Bone morphogenetic protein (BMP) gives osteoblastic phenotype to myoblast C2C12. The osteoblastic conversion is accompanied by induction of Runx2 and Osterix. However, during the differentiation, C2C12 cannot form calcified bone nodules, which suggests osteoblastic maturating process is inhibited. We have found that induction of Osterix by BMP in C2C12 was transient, which decreased after 24 hours after stimulation. We hypothesized that there may be an inhibitory factor to suppress the expression of Osterix in this period. We screened nuclear proteins of repressors, which were induced late after 24 hours of BMP-stimulation in C2C12, by microarray technique. The expression of paired-like homeodomain transcription factor 2 (Pitx2) up-regulated gradually after 24 hours, and peaked at day four, and remained high level over one week. Since this induction was not seen in osteoblasts and other osteoblastic progenitors, the expression of Pitx2 by BMP may be exclusive to myoblasts. The overexpression of Pitx2 in C2C12 inhibited the induction of Osterix, not Runx2, which was followed by suppressed osteoblastic differentiation. C2CI2 cells induced by shRNA for Pitx2, or MEF isolated from Pitx2 null mice showed enhanced induction of Osterix by BMP with elevated expression of ALP, bone sialoprotein and osteocalcin, while no difference on Runx2 was observed. Finally, we found some putative Pitx2 binding elements (PBE) in the promoter of osterix gene, one of which was shown to be functional target for Pitx2 by gel shift and ChIP assays. These results suggest that Pitx2 play a unique role in myoblast to suppress osteoblastic conversion through inhibiting the expression of Osterix. Thus, Pitx2 may be the first therapeutic target for muscle ossifying diseases, such as fibrodysplasia ossificans progressiva, in which cells BMP signaling is tend to be accelerated.

Disclosures: M. Havashi. None.

T022

TRP Channels Are Both Present and Functional in Human Osteoblast-like Cells.

N. C. Henney*¹, B. A. J. Evans², A. K. Campbell*³, K. T. Wann*¹. ¹Welsh School of Pharmacy, Cardiff University, Cardiff, United Kingdom, ²Department of Child Health, Cardiff University, Cardiff, United Kingdom, ³Department of Medical Biochemistry & Immunology, Cardiff University, Cardiff, United Kingdom.

Transient receptor potential (TRP) cation channels are currently drawing a lot of interest in many tissues, but relatively little interest in bone. TRP channels are mostly non-selective and are generally widely expressed with increased expression in some malignancies¹, and TRPV1 and TRPM8 ligands, for example, may have potential uses as anti-cancer agents² in prostate malignancy. As prostate cancer cells metastasize to and cohabit with bone cells, this area of research is potentially important and deserves attention. The putative roles of TRP channels include growth, repair, metastasis, apoptosis and cell calcium entry — all of which are relevant to osteoblasts. We aim to show that TRP channels are present and active in osteoblast-like cells.

RT-PCR was carried out using intron-spanning primer pairs for TRPV1, TRPV5, TRPV6, TRPM7 and TRPM8 subunits in MG63 and SaOS-2 osteoblast-like cells. Cell proliferation assays were performed over 72 hours to test the effects of known TRP ligands on cell number, using trypan blue exclusion testing and MTS-conversion assay. Patch-clamp analysis was performed on MG63 and SaOS-2 cells using calcium-free and/or sodium gluconate solutions to minimise the confounding influence of other channel subtypes.

RT-PCR has revealed appropriate sized bands for TRPV1, TRPV5, TRPV6, TRPM7 and TRPM8 in MG63 and SaOS-2 cell lines. In cell proliferation assays, > 10 μM capsaicin or capsazepine significantly reduced MG63 cell number (P < 0.05), but 1 μM capsazepine antagonises the effects of > 10 μM capsaicin. In addition, resiniferatoxin (TRPV1 agonist), SB366791 (TRPV1 blocker) and anandamide (cannabinoid and TRPV1 modulator) all decreased MG63 cell numbers. Patch-clamp analysis has revealed small conductance channels of 18 — 30 pS in excised inside-out patches, and slightly larger conductance channels in cell-attached and inside-out patches at depolarised and hyperpolarised potentials in MG63 and LNCaP (prostate cancer) cells.

Given our preliminary data, we propose that five members of the TRP channel family are expressed in osteoblast-like cell lines, and we show electrophysiological and pharmacological data which lead us to suggest that at least one TRP channel type is active and functional in bone. TRP channels could therefore be new targets in the battle against bone disorders, and some known TRP channel ligands could be included in that armoury.

• 1:
  Tsavaler L et al. (2001). Cancer Res 61(9):3760–9
• 2:
  Athanasiou A et al. (2007). BBRC 354:50–55

Disclosures: N. C. Henney, None.

T023

A Lymphoid Enhancer Binding Factor (Lef) 1 Isoform Regulates Osteoblast Maturation.

L. H. Hoeppner¹, E. D. Jensen*², J. J. Westendorf³. ¹Graduate Program in Microbiology, Immunology and Cancer Biology, University of Minnesota, Minneapolis, MN, USA, ²Universiry of Minnesota, Minneapolis, MN, USA, ³Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA.

Lymphoid Enhancer Binding Factor (Lef) 1 is a transcription factor linked with the Wnt/LRP5/β-catenin signaling cascade, which regulates osteoblast proliferation and survival, bone density and skeletal strength. We previously demonstrated that suppression of Lefl promotes osteoblast mineralization while Lefl overexpression blocks osteoblast maturation. We also showed that Lefl blocks Runx2-dependent activation of the osteocalcin promoter. In this study we demonstrate that Lefl blocks activation from the neighboring Runx2 binding site, even when the sequence is changed from a Runx binding element to a GAL4 DNA binding element. A truncated form of Lefl (LeflΔN) that lacks the β-catenin binding domain does not repress osteocalcin expression as well as full-length Lefl, even though it retains a functional DNA binding domain. LeflΔN is transcribed from a promoter in the intron between exons 2 and 3 in humans (exons 3 and 4 in mice). Runx2 binds a consensus Runx binding element in this promoter and activates transcription of the LeflΔN transcript. Northern blot analysis indicates that the 2.3 kB LeflΔN transcript is not present in undifferentiated MC3T3-E1 preosteoblasts but becomes detectable after six days of osteogenic stimulation. These data suggest that premature expression of LeftΔN in osteoblast progenitors might block osteoblast differentiation. To test this hypothesis, we stably overexpressed LeflΔN in the murine myo-osteoblast progenitor C2C12 cell line and induced differentiation in osteogenic medium containing ascorbic acid, β-glycerol phosphate, and BMP2. LeflΔN overexpression decreased the rate of alkaline phosphatase production compared to control cells. Together these data suggest that LeflΔN is a Runx2 target gene and an important regulator of osteoblast differentiation. Our model predicts that LeflΔN is normally expressed during the maturation process where it competes with endogenous Lefl for binding elements and thereby mitigates the Wnt signaling pathway. If expressed earlier, LeflΔN blocks preosteoblast maturation.

Disclosures: L.H. Hoeppner. None.

T024

Orphan Nuclear Receptor Small Heterodimer Partner (SHP) Promotes Osteoblast Differentiation by Regulation of Runx2 Activity.

B. Jeong*, L. Bae*, I. Kim*, J. Koh.. Dental Science Research Institution and Brain Korea 21 project, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea.

Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily, regulating transcription of target genes without a conventional DNA binding domain. This study was undertaken to elucidate roles of SHP in osteoblast differentiation. Primary osteoblasts, mesenchymal progenitor cell lines, and C2C12 were cultured with or without BMP2 (300 ng/ml), and SHP expression was determined by RT-PCR analyses. For evaluation of osteoblast differentiation, alkaline phosphatase (ALP) activity, osteocalcin (OC) production, and Alizalin Red staining assays were done with adenoviral infection of BMP2 and/or siRNA-SHP. To examine transcriptional regulatory actions of SHP, 6XOSE- or OG2 promoter was transfected with Runx2 plasmid. During the BMP2-induced osteoblast differentiation of C2C12 cells, SHP mRNA increased. Transient transfection of SHP significantly promoted the Runx2-dependent stimulation of OG2 or OSE promoter. Knocking down SHP expression by adenovirus siRNA-SHP inhibited BMP-2 induction of ALP activity, OC production, and mineralization with down-regulations of ALP, OC and bone sialoprotein mRNA. These results indicate that orphan nuclear receptor SHP may act as a positive regulator of osteoblast differentiation and mineralization.

Disclosures: B. Jeong, None.

This study received funding from: Brain Korea 21 Project for Dental School.

T025

Withdrawn.

T026

Opposing Effects of Glucocorticoids and Wnt Signaling on Krox20 and Mineral Deposition in Osteoblast Cultures.

N. Leclerc*¹, T. Noh*¹, J. Cogan*¹, D. B. Samarawickrama*¹, E. Smith*², B. Frenkel¹. ¹Biochemistry and Molecular Biology, University of Southern California, Los Angeles, CA, USA, ²Orthopaedic Surgery, University of Southern California, Los Angeles, CA, USA.

The beneficial anti-inflammatory properties of glucocorticoids (GCs) are associated with inhibition of osteoblast-mediated bone formation, leading to osteoporosis. We previously discovered Krox20 as the transcription factor most strongly repressed by GCs in osteoblast cultures, and showed that GC-inhibition of Osteocalcin (OC) transcription was mediated via suppression of an OC Krox20-binding Enhancer (OKE) located immediately upstream of the Runx2-binding OSE2 site. Because GCs also inhibit Wnt signaling, a major regulator of bone mass, we pursued a possible linkage between GCs, Wnt signaling and Krox20 in osteoblasts.

We first characterized the expression pattern of Krox20 in MC3T3-E1 and primary calvarial osteoblast cultures treated or not with 1 μM dexamethasone (DEX). RT-qPCR analysis showed that Krox20 expression was the highest during late stages of osteoblast differentiation. DEX decreased Krox20 mRNA to 50 % of control levels within 15 minutes, suggesting a direct effect. Long-term repression of Krox20 by DEX, however, required new protein synthesis, as it was alleviated after a 4 hours cycloheximide treatment. We previously showed that activity of the OKE was absent in undifferentiated osteoblasts and that fibroblasts acquired OKE activity upon forced expression of Runx2. Here, we ruled out the requirement for Runx2 binding in cis -the OKE remained active after the OSE2 site was mutated. We then examined the possibility that Krox20 is regulated by the Wnt signaling pathway. Indeed, Wnt3A stimulated both Krox20 expression and activity of the OKE, whereas two other bone anabolic agents, BMP2 and PTH( 1 −34), failed to do so. We next addressed the involvement of Wnt signaling in the DEX-repression of Krox20. DEX-inhibition of Wnt signaling was only partially responsible for the 6-fold repression of Krox20 by DEX, as DKK-1 did not completely abolish it but rather reduced it down to 3.7-fold. Finally we asked whether treatment with Wnt3A could antagonize the inhibitory effects of GCs on both Krox20 and the osteoblast phenotype. Wnt3A partially rescued Krox20 expression in DEX-arrested osteoblast cultures and this was associated with rescue of mineralization. However, mineralized nodules only formed when Wnt3A was administered briefly, not chronically, and they were morphologically different from those formed in control cultures.

Our findings are consistent with a role for Krox20 in osteoblast function and suggest that it may contribute to the opposing effects of GCs and Wnt signaling on bone formation.

Disclosures: N. Leclerc, None.

This study received funding from: Arthritis Foundation, National Institutes of Health (AR047052), and J. Harold and Edna L. LaBriola Chair in Genetic Orthopaedic Research at the University of Southern California.

T027

Vimentin Negatively Regulates Osteoblast Differentiation by Inhibiting the Transactivation Activity of Atf4.

N. Lian*, X. Yang.. Center for Bone Biology, Department of Pharmacology, Vanderbilt University, Nashville, TN, USA.

Our previous work has identified Atf4 as an osteoblast-enriched transcription factor that is required for osteoblast terminal differentiation and function (Yang et al. 2004; Yang and Karsenty, 2004). Atf4 transactivates gene transcription of osteocalcin, an osteoblast-specific and mature osteoblast marker gene, by binding directly to its osteoblast-specific cis-acting element, OSE1. The transactivation activity of Atf4 is positively regulated by phosphorylation by RSK2, a protein kinase whose mutation causes Coffin-Lowry syndrome (CLS). CLS is a characterized by mental retardation and skeletal manifestations. In this study we found, by chromatography and mass spectrometry using purified Atf4 protein and osteoblast nuclear extracts, that Atf4 interacts with vimentin, a member of type III intermediate filament proteins. Interaction between Atf4 and vimentin was confirmed by a reciprocal co-immunoprecipitation in COS1 cells that overexpress both Atf4 and vimentin. Vimentin may play a specific role in osteoblast biology since our RT-PCR and Northern hybridization assay results showed that its mRNA expression is enriched specifically in bone and lung. Consistent with published studies vimentin is localized in both cytosol and nucleus as demonstrated by expressing a fusion protein of green florescent protein and vimentin in COS1 cells. To determine whether vimentin affects Atf4's transactivation activity, we performed co-transfection assays using osteocalcin promoter-luciferase reporter constructs and expression vectors encoding Atf4 and/or vimentin. Our results showed that vimentin decreased 80% of the activation of the osteocalcin reporter gene by Atf4. This inhibition is specific because vimentin did not affect the transactivation of a reporter gene by FosB, a transcription factor of the AP1 family. Gel retardation assays showed that this inhibition was caused by the fact that vimentin directly interferes with the binding of Atf4 to OSEI. To further determine the function of vimentin in the osteoblast differentiation process, we have established cell lines that stably overexpress Atf4, vimentin or both. Our results revealed that vimentin affects in osteoblast differentiation and function as shown by measurements of alkaline phosphatase activity, mineralization rate, and expression patterns of osteoblastic marker genes. Together our data suggest that vimentin is not only a structural protein that provides mechanical support for the plasma membrane but also acts as a negative regulator of osteoblast differentiation via its interaction with Atf4.

Disclosures: N. Lian, None.

T028

The microRNA miR-26a Targets the SMAD1 Protein During of Human Adipose Tissue-Derived Stem Cells.

E. Luzi*, F. Marini*, S. Carbonell Sala*, I. Tognarini*, G Galli*, A. Falchetti, M. Brandi. Internal Medicine, University of Florence, Florence, Italy.

Human Adipose Tissue-Derived Stem cells (hADSCs) are a cell population capable to differentiate in adipogenic, osteogenic, chondrogenic, myogenic, and neurogenic lineages. Although different studies suggest a high osteogenic potential for hADSCs, the molecular mechanisms that regulate hADSCs differentiation towards osteogenic precursors and subsequent bone-forming osteoblasts is unknown. MicroRNAs (miRNAs) are a family of naturally occurring, evolutionary conserved, small (approximately 19–23 nucleotides), non-protein-coding RNA molecules that negatively regulate post-transcriptional gene expression. miRNAs are estimated to account for >3% of all human genes and to control expression of thousands of target mRNAs, with multiple miRNAs targeting each mRNA. A role for miRNAs has been identified in both physiological and pathological conditions, encompassing metabolism, proliferation, cell death, differentiation, development, and insulin secretion from pancreatic beta cells, viral infection, and cancer. With few exceptions, physiological targets for miRNAs remain to be identified. Understanding miRNAs function is critically dependent on the identification of its targets. Nucleotides 2- 8 (referred to as “seed”) of the miRNAs are crucial in determining target specificity. Using osteoblast precursors obtained by subcutaneous human adipose tissue we were able to demonstrate that miR-26a levels increased in differentiating osteoblasts and that the inhibition of miR-26a, by 2'-O-methyl-antisense RNA, increased protein levels of the mothers against decapentaplegic homolog 1 (SMADI) transcription factor in treated osteoblasts, up-regulating bone marker genes and, thus, enhancing osteoblast differentiation. Our data showed that combined expression and functional data suggest a role for miR-26a in the differentiation of hADSCs towards the osteogenic lineage by targeting its predicted target, the SMADI protein.

Disclosures: E. Luzi, None.

T029

Osteoblastic Master Regulator Osterix Mediates the Feedback Regulation of BMP-2 Auto-Expression via PI 3 Kinase/Akt Signaling.

C. C. Mandal¹, G. Ghosh-Choudhury*², H. Drissi³, N. Ghosh-Choudhury⁴. ¹Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, ²Medicine, UTHSCSA, GRECC, STVHCS, San Antonio, TX, USA, ³Orthopedics, University of Rochester, Rochester, NY, USA, ⁴Pathology, UTHSCSA, STVHCS, San Antonio, TX, USA.

We have recently reported the requirement of phosphatidylinositol 3 kinase (PI3K)/Akt signal transduction in BMP-2-induced osteoblast differentiation and mature bone nodule formation. BMP-2-inducible Zn-finger transcription factor, osterix (Osx), is required for bone formation during mouse skeletalogenesis. The mechanism by which BMP-2 increases Osx expression is not known. BMP-2 increased expression of Osx protein in a time-dependent manner in the cytosol and nucleus of C2C12 cells, concomitant with Osx mRNA expression. PI3K inhibitor, Ly294002, significantly inhibited BMP-2-induced Osx mRNA and protein abundance. Infection of C2C12 cells with adenovirus vectors expressing dominant negative PI3K or PTEN, a negative regulator of PI3K signaling, blocked expression of Osx mRNA and protein in response to BMP-2. Adenoviral-mediated expression of dominant negative Akt kinase (AdDNAkt), a target of PI3K, inhibited BMP- 2-induced Osx expression. These data for the first time indicate a critical role of PI3K/Akt signaling in osteogenic transcription factor Osx expression during osteoblast differentiation. To investigate the mechanism of Osx expression, we investigated its transcription using reporter construct in which 5'-flanking sequence of Osx gene drives firefly luciferase cDNA (Osx-Luc). Transient transfection of Osx-Luc into C2C12 cells showed increased Osx transcription in response to BMP-2. Analysis of the progressive 5' deletions of the Osx promoter revealed the presence of BMP-2 responsive sequence between −800 bp and −500 bp with respect to the transcription start site. Inhibition of PI3K and Akt kinase activity by Ly294002 or by expression of dominant negative lipid kinase, PTEN and AdDNAkt respectively, abolished BMP-2-induced transcription of Osx. BMP- 2-induced BMP-2 expression is necessary for sustained osteoblast differentiation. The mechanism is not known. We show that expression of Osx increased the transcription of BMP-2 gene, resulting in BMP-2 protein and alkaline phosphatase protein expression in osteoblasts. Together these results for the first time demonstrate that PI3K/Akt signaling regulates the expression of osteogenic master transcription factor Osx in response to BMP- 2. Furthermore, we provide the first evidence that Osx represents the master regulator for BMP-2-induced BMP-2 expression to regulate osteoblast differentiation.

Disclosures: C.C. Mandal, None.

This study received funding from: NIAMS. VA.

T030

Responses of Bone in ANA Knock-out Mice to Ovariectomy.

K. Miyai*¹, M. Yoneda*², F. Mizoguchi¹, T. Havata¹, Y. Ezura¹, K. Nakashima¹, T. Yamamoto*², M. Noda¹. ¹Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan, ²Division of Oncology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Tob, a member of BTG/APRO/TOB family protein, has been reported as a negative regulator of BMP signaling on bone. We have identified that ANA, another member of this family, is expressed in skeletal system, including Meckel's cartilage and cartilages of ribs at E12.5 mice suggesting that ANA plays a role in the proliferation and/or chondrocyte differentiation of the cartilage cells(Yoshida et al. 1998 Oncogene). ANA has been reported to interact with Cafl, whose absence did augment the response of bone to BMP similarly to the observation reported in Tob knock out mice. In fact, Tob knock-out mice revealed alteration in the levels of bone mass after ovariectomy. Based on these observations, we examined whether ANA would be involved in the regulation of bone metabolism. To analyze this point, we examined mice deficient in ANA gene. By crossing heterozygous female and male mutants, progeny pups in each genotype were obtained at normal Mendelian frequency. The growth and appearance of knock-out mice were similar to those in wild type littermates. We assumed that this observation was brought about by compensatory mechanisms covering the expected influence, and hypothesized that certain stress on mouse might reveal its role on bone.

It has been reported that Tob knock-out mice are resistant to reduction of bone volume / tissue volume (BV/TV) in femur after ovariectomy, we therefore examined the effect of ovariectomy on bone mass changes in ANA knock-out mice. BMD levels of femur were similar between ANA knock-out mice and wild type littermates. BV/TV levels in femur were tended to decrease in ANA knock-out mice after ovariectomy, and such trends were similar to those in wild type. In conclusion, unlike Tob konck-out mice, ANA deficient mice responded to ovariectomy similarly to wild type mice.

Disclosures: K. Miyai, None.

T031

Investigation of KCl Cotransport Expression In Canine Osteosarcoma Cell Lines.

H. Ochiai. Research Institute of Biosciences, Azabu University, Sagamihara Kanagawa, Japan.

K-Cl cotransport, defined originally in erythrocytes as Cl-dependent, ouabain-insensitive bidirectional K transport, encoded by four KCC genes, is recognized as a functional and structural reality in various types of cells. As a functional system, KCC is necessary for ionic and volume homeostasis. Since its discovery by swelling erythrocytes in hyposmotic solutions, KCC has been recognized as one of the key K transport membrane proteins affecting regulatory volume decrease (RVD). Although much information on erythrocytes has been reported, little is known about KCC in ostosarcoma cells. In this study, we investigated KCC of two canine osteosarcoma cell lines, parent canine osteosarcoma cell lines (OS) and highly lung metastasizing canine osteosarcoma which was established from five subcutaneous implantation cycles of lung tumor deposits using parent OS (HM-OS). There was no difference in K-Cl cotransport activity in the physiological condition, while NEM revealed significantly higher Rb (K) uptake in HMOS than OS. Western blot analysis indicated KCC1, KCC3 and KCC4 were expressed in both cells, and KCC3 was especially abundant in HM-OS compared with OS. Insulin-like growth factor I (IGF-1) increased KCC activity and mRNA expression in a dose- and time-dependent manner of the three KCCs. This activation was diminished by addition of DIOA, the specific inhibitor of KCC, along with the inhibitory effect of cell growth in both cells. Retinoid, the vitamin A derivative, induced morphologic differentiation in both canine osteosarcoma cells and enhanced cell flattening and spreading, as well as reduction in cell overlapping. Alkaline phosphatase (ALP) activity and ALP staining were decreased and induced an increase in production of type I collagen. Retinoid-treated HM-OS indicated KCC3 expression was reduced to level of OS.

Disclosures: H. Ochiai. None.

T032

Characterization of Regions Upstream of Exon II of the Human CYP19 (aromatase) Gene that Mediate Regulation by cAMP in Murine Osteoblasts.

O. K. Oz¹, T. Hasegawa*², Y. Zhao*¹. ¹Radiology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA, ²Pediatrics, Keio University School of Medicine, Tokyo, Japan.

Estrogen is important for the regulation of bone growth and metabolism in humans. Aromatase P450 (aromatase), the product of the CYP19 gene, catalyzes conversion of androgens to estrogens. Aromatase is expressed in human osteoblasts and cultured fetal osteoblast-like cells. In humans, tissue-specific expression of aromatase is regulated in part, by tissue-specific promoters and by alternative splicing mechanisms. In mice, only two promoters, one specific for brain and one for gonads have been described. Little is known regarding the expression and regulation of aromatase expression in murine bone. Thus, we first wanted to determine if aromatase is expressed in murine long bone. Aromatase transcripts were present in whole bone as determined by RT-PCR. Aromatase protein was detected both in osteoblasts and chondrocytes by immunohistochemistry. To ascertain how aromatase expression is regulated in mouse bone, various amounts (-945/-16 bp to −100/-16 bp) of 5-flanking DNA and promoter of the human CYP19 gene, upstream of exon II (CYP19₁₁) were linked upstream of the luciferase cDNA (LUC) for transient transfection studies utilizing MC3T3-E1 osteoblast cells. Forskolin-induced reporter activity was greatest with the −517/-16 LUC construct and the response to cAMP was lost upon deletion to −140 bp. Mutation of cAMP-responsive element-like sequence (CLS) and steroidogenic factor-1 (SF-1) element sequence sites markedly decreased the response to forskolin. A probe containing the CLS was used in electromobility shift assays; two complexes were formed with MC3T3-E1 nuclear extracts (NE), and a cAMP-responsive element binding protein (CREB) antibody was able to compete formation of these complexes. A probe specific for the SF-1 element formed a complex with MC3T3-E1 NE and this could be competed through the use of an SF-1 antibody. The results indicate that aromatase is expressed in mouse bone and the 5-flanking DNA and Promoter II of the human CYPI9 gene is capable of driving expression of aromatase in the MC3T3-E1 osteoblast cells. These results suggest a role for SF-1 in bone homeostasis and that the osteoblast is an extraglandular source of local estrogen that may play an important role in bone metabolism.

Disclosures: O.K. Oz, None.

T033

Snail, a Transcription Factor, Inhibits the Differentiation of Mesenchymal Stem Cells to Osteoblasts.

S. Park*¹, J. Yook*², J. Byun*¹, Y. Rhee¹, H. Ryoo³, E. Lee*¹, S. Lim¹. ¹Internal Medicine, Yonsei University, Seoul, Republic of Korea, ²Oral Pathology, Yonsei University, Seoul, Republic of Korea, ³Cell and Developmental Biology, Seoul National University, Seoul, Republic of Korea.

Mesenchymal stem cells are able to differentiate into several highly specialized cell types, including osteoblasts, adipocytes, myocytes and chondrocytes. Several key transcription factors play a crucial role in commitment process thereby directing the cells along a particular lineage and suppressing the alternative pathways. However, how the key transcription factors in order to specify several distinct lineages are regulated remains a pivotal question. In this study, we examined whether snail, a zinc-finger transcription factor, regulates the differentiation of mesenchymal stem cells. Snail-overexpression decreased the expression of osteoblast marker genes including type I collagen and ALP in C3H10T1/2 mesenchymal cells. And the expression of Runx2, the osteoblast-specific transcription factor, was significantly reduced by Snail-overexpression. The activity of Runx2 was also suppressed by 2 folds after Snail-overexpression in C3H10T1/2 cells. In addition, siRNA against Snail enhanced the activity of Runx2 in C3H10T1/2 cells. Interestingly, osteohlastogenesis was dramatically stimulated after suppression of endogenous Snail expression by siRNA in osteoblastic MC3T3-E1 cells. These results suggested that Snail plays a crucial role in developmental process from the mesenchymal stem cells to osteoblasts.

Disclosures: S. Park, None.

T034

Role of Nuclear Factor I-X Transcription Factor in Regulation of IGFBP-5 and Osteocalcin Expression in Mouse Osteoblasts.

L. A. Pérez-Casellas¹, K. E. Govoni², K. D. Howard*², G. R. Linares³, C. A. Strohbach*¹, T. A. Linkhart*², D. D. Strong². ¹Microbiology, Loma Linda University, Loma Linda, CA, USA, ²Research, Loma Linda VAMC, Loma Linda, CA, USA, ³Physiology, Loma Linda University, Loma Linda, CA, USA.

The Nuclear Factor I (NFI) gene family plays wide reaching roles in viral DNA replication, regulation of gene transcription and development. Gene knockout studies of NFI have shown that nfi-a -/- mice display mainly neuroanatomical defects, nfi-b -/- mice show lung and brain defects, nfi-c -/- caused primarily a disruption of tooth root and underlying mandibular bone development, and nfi-x -/- mice have brain malformation and also develop a deformation of the spine, due to impaired endochondral ossification and decreased mineralization. We previously reported that the Nuclear Factor I gene family was an important regulator of IGFBP-5 expression in human osteoblast-like cells. Specifically, the isoforms NFI-B and NFI-X significantly activated the IGFBP-5 promoter and siRNA knockdown of NFI-B expression decreased IGFBP-5 mRNA and protein expression. To determine the role of the four NFI isoforms in mouse osteoblast differentiation we evaluated mRNA expression levels in mouse bone marrow stromal cells and mouse osteoblast MC3T3-E1 cells at different stages of differentiation in vitro (αMEM, FBS, Ascorbic Acid and β-glycerophosphate). We found that all four genes were expressed and their expression levels increased at least 10 fold between day 14 and 21 during differentiation. Nfl-x expression was the most abundant in bone marrow stromal cells and differentiated MC3T3-E1 cells and increased the most during differentiation. The increased nfl expression between 14 and 21 days corresponds to previous observations that igfbp-5 expression decreased in MC3T3-E1 cells as the cells fully differentiated and osteocalcin expression increased¹ To determine the role of nfl-x in regulating igfbp-5 expression and osteoblast differentiation, mouse nfl-x expression was inhibited with specific siRNA oligonucleotides in MC3T3- El cells. Nfl-x mRNA levels were decreased at 48 hrs by 80% and mouse osteocalcin mRNA levels were decreased by 65% compared to non targeting control siRNA. However, nfl-x knockdown caused a 2 fold increase in igfbp-5 mRNA levels. In contrast to stimulating IGFBP-5 promoter activity in human osteoblasts (from an IGFBP-5 luciferase reporter plasmid), nfl-x overexpression in transient transfection experiments did not stimulate promoter activity in MC3T3-E1 osteoblasts. These results suggest an important role for NFI-X in regulating osteoblast differentiation, and that NFI regulation of IGFBP-5 expression may occur by different mechanisms in mouse and human. (1) Thrailkill et al. Bone 17:307–313 (1995).

Disclosures: L.A. Pérez-Casellas, None.

T035

Zinc Finger Protein 64 Is a Downstream Target of Runx2 and Regulates Myogenic and Osteogenic Differentiation as a Coactivator of Notchl.

K. Sakamoto*, A. Yamaguchi. Section of Oral Pathology, Tokyo Medical and Dental University, Tokyo, Japan.

Runx2 is a crucial transcription factor for osteoblast differentiation. Notch signaling is required for multiple aspects of mesenchymal cell differentiation and it recently has been recognized as an essential regulator of osteogenesis. However, the mechanism to coordinate these signaling pathways is still poorly understood. In this paper, we identified zinc finger protein 64 isoform D (Zfp64) as a novel coactivator of Notchl. Zfp64 was associated with the carboxyl terminal PEST-containing region of Notchl and was recruited to the promoters of the Notch target genes Hesl and Heyl, which transactivates these Notch downstream genes. Zfp64 expression, which is under the control of Runx2, was upregulated by direct transactivation of its promoter. During embryo- and organogenesis, Zfp64 is expressed mainly in the mesenchymal cells, including osteoblasts and chondroblasts, but not in myocytes. Zfp64 suppressed the myogenic differentiation of C2C12 cells and promoted their osteoblastic differentiation. Our data demonstrates that Zfp64 is a downstream target of Runx2 and regulates differentiation of osteoblasts and myoblasts as a coactivator of Notchl by intermediating two signaling pathways between Runx2 and Notch.

Disclosures: K. Sakamoto, None.

T036

Runx2 Transcriptional Activation Domain 3 Supports Synergistic Runx2-MINT Activation of the Osteocalcin FGF Response Element via A DMAT-Sensitive Kinase.

O. L. Sierra, S. L. Cheng, D. A. Towler. Bone & Mineral Diseases, Washington Univ Sch Med, St Louis, MO, USA.

MINT, the Msx2-interacting nuclear matrix target protein, is an abundant component of the osteoblastic nuclear matrix. MINT functionally interacts with Runx2 to enhance osteocalcin promoter activity in C3H10T1/2 mesenchymal cells. To study Runx2-MINT interaction in greater detail, we concatemerized the 42-bp element (rat osteocalcin promoter FGF response element; OCFRE) that compasses Runx2 binding sites upstream of the unresponsive RSV minimal promoter (LUC reporter). In co-transfection assays, MINT enhanced Runx2- dependent activation of the OCFRE by 3–4 fold, both in the present and absence of exogenous FGF2. However, the related family member Runx1 exhibited only 10% the activity of Runx2 in this assay. Deletion of Runx2 N-terminal Activation Domains (AD) AD1 and AD2 did not affect Runx2-MINT interaction. By contrast, Runx2 (1–312) – lacking C-terminal residues 313 to 528 — had significantly reduced activity, indicating that interaction interface with MINT must reside in the C-terminus. A systematic series of Runx1-Runx2 chimeras were generated to functionally map Runx2-MINT interactions. Runx2 AD1 and AD2 did not convey MINT- dependent activation to Runx1. However, when Runx2 residues 315–402 or 365–402 replaced equivalent domains in the Runx1 C-terminus, both of these Runx1-Runx2 chimeras were capable of augmenting OCFRE activity in the presence of MINT. This region of Runx2, residues 315–402, encodes activation domain 3 (AD3). Inspection of the protein sequence identified multiple potential phosphorylation sites, including cognates for proline-directed protein kinases (PDKs; S319, T326, T407), casein kinase-2 (CK2; S347, S388), and protein kinase C (S330, S364). Alanine scanning mutagenesis was performed in the context of the Runx1-Runx2 chimera. Disruption of S319 and T326 phosphorylation cognates significantly reduced functional Runx2-MINT interactions, indicating that a PDK contributes to transactivation. Other Ala substitutions had no effect. We next examined effects of Ser/Thr protein kinase inhibitors on Runx2-MINT transactivation. Most, including PD98059, had little if any activity. However, the CK2 / Dyrk1 kinase inhibitor 2-dimethylamino-4,5,6,7- tetrabromo-1H-benzimidazole (DMAT) profoundly and dose-dependently abrogated MINT- Runx2 activation and Runx2-transactivation, with an ID50 of 5 uM. By contrast, DMAT did not affect TCF/LEF-dependent transcription. Other CK2 antagonists, such as emodin and apigenin, lacked inhibition. Thus, Runx2 Transcriptional AD3 confers Synergistic Runx2-MiNT Activation on the minimally responsive Runx1 protein. MINT-stimulated transcription of AD3 requires the activity of a DMAT sensitive kinase.

Disclosures: O.L. Sierra, None.

This study received funding from: National Institutees of Health/NIAMS.

T037

LMP-1 Accelerates MC3T3-E1 Osteoblast Differentiation in Part Through Modulation of the IGF System.

C. A. Strohbach¹, S. T. Chen*², S. J. Kleinman*², T. A. Linkhart², D. D. Strong². ¹Loma Linda University, Loma Linda, CA, USA, ²J.L. Pettis VAMC, Loma Linda, CA, USA.

LIM Mineralization Protein (LMP)-1 is a novel intracellular protein that is believed to be a critical regulator of the osteoblast differentiation program. LMP-1 has been shown to promote bone formation in spinal fusion models and enhance fracture repair. LMP-1 has been proposed to act through the induction of osteogenic factors such as BMPs and Runx2. However, the exact mechanism of action of LMP-1 is still unknown. Previously we found that IGFBP6 (BP6) binds intracellular LMP-1, suggesting that LMP-1 interacting with BP6 may affect osteoblast differentiation. The purpose of this study was to determine the effect of LMP-1 overexpression on MC3T3-E1 osteoblast differentiation, and to further elucidate mechanisms of LMP-1 action focusing on the IGF system. MC3T3-E1 osteoblasts were transduced with an MLV-based retroviral LMP-1 or GFP control vector. Expression of LMP-1 was confirmed by Western blot. After transduction, cells were seeded into 6-well plates and stimulated to differentiate by addition of osteogenic media containing ascorbic acid and beta-glycerolphosphate. The effect of LMP-1 on MC3T3-E1 osteoblast differentiation was determined by Alizarin Red staining and real-time PCR analysis of osteoblast marker gene and IGF system component mRNA levels. At 14 days, Alizarin Red staining demonstrated increased nodule formation in cells transduced with LMP-1 compared to GFP. Real-time PCR analysis revealed that LMP-1 significantly increased the mRNA levels of osteocalcin (7-fold), osterix (4-fold), bone sialoprotein (19-fold), alkaline phosphatase (7-fold), and Runx2 (4-fold) compared to control. Furthermore at 14 days, LMP-1 significantly decreased mRNA levels of components of the IGF system including IGF-1 (36-fold), IGFBP4 (4-fold), IGFBP5 (14-fold), and BP6 (5-fold). Since LMP-1 is an intracellular molecule, we investigated whether LMP-1 effected transcription using pGL3-promoter reporter constructs. Transient co-transfection revealed that LMP-1 dose-dependently inhibited BP6 promoter activity. Furthermore, we found that retroviral overexpression of BP6 in MC3T3-E1 cells inhibited differentiation and nodule formation normally observed after 21 days of culture in osteogenic media. These results suggest that 1) LMP-1 stimulates the production of osteoblast specific proteins and transcription factors, 2) LMP-1 is involved in regulation of the IGF system, and 3) LMP-l may have a direct effect on BP6 transcription.

Disclosures: C.A. Strohbach, None.

This study received funding from: Department of Veterans' Aggairs.

T038

An In Vitro Assay to Assess Secreted Osteogenic Factors from Genetically Engineered Celts.

M. Viggeswarapu, M. Bargouti*, C. Rogers*, L. Titus, S. D. Boden. Orthopedics, Emory University, Decatur, GA, USA.

LIM Mineralization Protein-1 (LMP-1) is known to induce secretion of osteogenic factors. Optimization of gene transfer protocols, promoter selection, isoform choice and other details have required resource-intensive in-vivo assays to date. The goal of this investigation was to validate an in-vitro assay to measure the osteogenic potential of secreted proteins from genetically engineered cells. To isolate the paracrine effect of secreted osteogenic proteins we chose the non-osteogenic mouse fibroblast (Nor10) cell line as the genetically engineered donor cells. We tested conditioned media (CM) from those cells for their osteogenic potential on freshly isolated rat calvarial osteobalsts (ROBs) and C2C12 pluripotent cells.

Nor10 cells were seeded at 2.6×10⁴/cm² in DMEM containing 20% FBS. LMP-1 cDNA was delivered using either a plasmid (pCMV-LMP-1) or Adenovirus (Ad5LMP-1) and grown for 72hrs. CM was collected on day 3 and frozen at −70° until used. The CM was thawed, concentrated 10 fold using 10,000 M_r cut off Centriprep/Centricon concentrators, and sterile filtered. Concentrates were resuspended in fresh complete medium to the original concentration and applied to recipient cells (C2C12 or ROB). C2C12 cells were seeded at 2.1×10⁴/cm² in DMEM containing 10% FBS. Cells were grown for 72 hrs and RNA was harvested. Gene expression was determined by reverse transcription followed by real-time TaqMan PCR analysis. Treatment of C2C12 cells with the CM from NorlO cells treated with Ad5LMP-1 or pCMV-LMP1 increased BMP2, BMP7 and osteocalcin mRNA levels. The C2C12 cells, while convenient, do not readily make mineralized bone. Thus, we also tested the osteogenic potential of Nor10 CM on confluent secondary ROB cultures by measuring mineralized nodule formation. Confluent secondary ROB cultures were treated with the CM from LMP-1 overexpressing Nor10 cells and grown for another 14 days. Treatment of ROBs with the CM from Nor-10 cells treated with AdLMP-1 or pCM V- LMP1 induced a 5 to 8 fold increase in mineralized bone nodule formation compared with CM from control Nor-10 cultures transduced/transfected with vector alone. In conclusion, these data demonstrate that we can measure the paracrine effects of cells genetically engineered to produce osteogenic factors using a two cell transfer assay. Intermediate markers of osteoblast differentiation (BMPs) and/or mineralized nodule endpoints can be demonstrated.

Disclosures: L. Titus, None.

This study received funding from: VA, NIH and Medtronic.

T039

Osteoblasts Express CSF-1R and Are a Target for CSF-1.

Y. Wittrant*¹, Y. Gorin*², K. Woodruff*¹, D. Horn*¹, S. Mohan*¹, H. Abboud*², S. Werner¹. ¹Pathology, University of Texas Health Science Center and Audie Murphy VA Hospital, San antonio, TX, USA, ²Nephrology, University of Texas Health Science Center and Audie Murphy VA Hospital, San antonio, TX, USA.

CSF-1, released by osteoblasts, stimulates the proliferation of osteoclast progenitors via the c-fms receptor (CSF-1R) and, in combination with RANKL, leads to the formation of mature osteoclasts. Whether the CSF-1R is expressed by osteoblasts and mediates specific biologic effects in osteoblasts has not been explored. To examine the CSF-1R in osteoblasts, primary calvarial osteoblasts (OB), isolated from wild-type (wt) and CSF-1 deficient op/op mice, were incubated in the presence and absence of recombinant CSF-1 and analyzed for CSF-1R mRNA and protein by RT-PCR and Western blot, respectively. Wt OB cultures were serum starved for 24 hr and the effect of CSF-1 (0–100 ng/ml) on proliferation and gene expression of RANKL, OPG, CSF-1 and osteocalcin (OC) was determined at 48 hr. In wt bone marrow cultures incubated with 1,25(OH)₂D₃(10–⁸M) and dexamethasone (10–⁸M) for 13 days, CSF-1 was tested for its effect on alkaline phosphatase activity (AP), RANKL mRNA and osteoclast formation. Since reactive oxygen species (ROS) influence osteoblast gene expression, studies analyzed the effect of CSF-1 on NADPH oxidase activity, Nox 1 and Nox 4 proteins. Results indicate that wt and op/op OB express CSF-1R mRNA and protein and that CSF-1 dramatically reduces CSF- 1R protein, but not CSF-1R mRNA, levels. In wt OB, CSF-1 decreased RANKL mRNA in a dose and time dependent manner whereas CSF-1 showed no effect on cell proliferation, OPG, OC or CSF-1 mRNA expression. Incubation of marrow cultures with CSF-1 resulted in a significant decrease in AP activity and RANKL expression and this effect was associated with a decline in TRAP activity and CTR expression. In wt OB, NADPH oxidase activity decreased in response to CSF-1 and correlated with a decline in Nox 1 and Nox4 protein levels. Compared to untreated cultures, 1uM DPI (diphenylene iodonium, NADPH oxidase inhibitor) decreased RANKL similar to that observed with CSF-1, and RANKL expression in cultures treated with both CSF-1 and DPI was comparable to each agent alone. These findings provide the first evidence that osteoblasts express CSF-1R and are a target for CSF-1 ligand. CSF-1 may act in an autocrine or paracrine manner to regulate its cognant receptor and osteoblast differentiation. Inhibition of NADPH oxidase activity by CSF-1 likely contributes to the effect of CSF-1 on RANKL. CSF-1-mediated inhibition of RANKL expression on osteoblasts may provide an important mechanism for coupling bone formation/resorption and preventing excessive osteoclastogenesis during normal skeletal growth.

Disclosures: Y. Wittrant, None.

This study received funding from: NIH (AR-42306) and VA's Merit Award.

T040

Non-adherent Circulating Progenitor Cells with Osteoblastic Potential Become Adherent in the Presence of Calcium²⁺ and Are able to Mineralize.

U. I. Moedder, S. Khosla. Mayo Clinic, Rochester, MN, USA.

Circulating progenitor cells with osteoblastic potential may play an important role both in normal bone remodeling and in mediating vascular calcification. In addition, migration and differentiation of these cells into osteoblasts may be regulated by the release of chemokines, such as stromal cell-derived factor-1 (SDF-1), as well as by increased local calcium²⁺ (Ca²⁺) concentrations, which may be as high as 40 mM at sites of active bone resorption and in areas of vascular injury associated with apoptotic cells. Thus, in the present study, we tested whether non-adherent osteoblast progenitors expressed the SDF-1 receptor, CXCR4, and/or became adherent and formed mineral deposits (as assessed by Von Kossa and Alizarin Red staining) in the presence of Ca²⁺.

Osteocalcin positive cells (OCN^pos) were isolated by magnetic activated cell sorting (MACS) and OCN^neg cells and mononuclear cells (MNCs) were used as negative controls. Continuous proliferation of the MNCs and OCN^neg was observed for up to 7 days in vitro following isolation; however, OCN^pos cells failed to proliferate after 24 hours. By flow cytometry, 82% of OCN^pos cells coexpressed CXCR4, the exclusive receptor for SDF-1, which is released at sites of vascular injury or fractures. OCN^pos cells did not adhere to uncoated culture flasks in the presence of regular growth medium for up to 4 weeks. By contrast, if the culture medium contained an additional 2.1 mM Ca²⁺for up to 5 days at the beginning of the culture, the cells started to adhere. Subsequently (and following removal of the additional Ca²⁺), these cells mineralized within 21 days in the presence of osteoblast differentiation medium, as determined by Von Kossa and Alizarin Red staining (Figure). By contrast, MNCs and OCN^neg cells did not mineralize under these culture conditions. Collectively, these findings support the concept that 0CN^pos cells may be recruited to sites of bone remodeling and/or vascular injury via the CXCR4-SDF-1 axis and, in the setting of an increased local concentration of Ca²⁺, be induced to adhere and differentiate into osteoblastic celts.

Disclosures: U.I. Moedder, None.

T041

Human Fetal Bone Cells for Tissue Engineering.

M. Montjovent*¹, C. Scaletta*², L. Mathieu*³, D. Pioletti¹, L. Applegate*². ¹Laboratory of Biomechanical Orthopedics EPFL-HOSR, EPFL, Lausanne, Switzerland, ²Orthopedic Cell Therapy Unit, PAV-03, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland, ³Laboratory of Composite and Polymer Technology, EPFL, Lausanne, Switzerland.

For clinical bone transplantations, tissue engineering techniques based on the delivery of cells to the defect using scaffold materials are currently investigated. Our aim is to evaluate the possible use of human fetal bone cells for tissue repair. Firstly, fetal bone cells were characterized for their osteoblastic phenotype. Then, fetal bone cells were seeded on scaffolds made of poly-L lactic acid (PLA) and reinforced with b-TCP. Proliferation, ALP enzymatic activity were observed, and in vitro mineralization was assessed. Lastly, scaffolds seeded with fetal cells were tested in vivo in critical size defects in the rat cranial model. The cellular doubling time of fetal bone cells was estimated to be 27 hours, whereas this value was significantly higher for adult bone cells and clearly age dependent. Fetal bone cells receiving a differentiation mix showed a strong induction of ALP activity after 4–6 days of treatment and this observation was also verified at high passage numbers. Adult cells showed a lower induction of ALP enzymatic activity. Extracellular matrix analysis demonstrated that fetal bone cells were able to mineralize PLA/b-TCP scaffolds in vitro. Moreover, repair of craniotomy critical size defect was assessed in vivo and we showed that human fetal bone cells possess a high proliferation potential, are able to differentiate into osteoblasts, and they promote bone repair in vivo when seeded on PLA/b-TCP constructs. The use of fetal bone cells, which have the advantage of a high proliferation capacity, for bone tissue engineering could be an alternative to mesenchymal stem cells.

Disclosures: M. Montjovent, None.

This study received funding from: PNR 46 N404640–101114/1.

T042

Modulation of WNT3A Induced Signaling Through Dual-Specificity Phosphatases.

P. V. Nantermet*¹, P. Hodor*², O. Floras*¹, H. Glantschnig*¹. ¹Bone Biology & Osteoporosis, Merck Research Labs, West Point, PA, USA, ²Molecular Profiling, Merck Research Labs, West Point, PA, USA.

There is substantial evidence for a central role of Wnt-signaling in regulating bone mass accrual. Wnt activates a canonical signaling pathway through β-catenin, likely mediated via LRP5 and LRP6. However, other Wnt-mediated non-canonical signaling components like Ca++, protein kinase C and mitogen-activated protein kinases (MAPKs) have also been implicated. Using a mesenchymal pluripotent cell (C3H10T1/2), we observed Wnt3A induced early osteoblastic cell differentiation, which can be blocked by DKK-1. To elucidate the signaling pathways engaged in this cell system, we performed a micro-array analysis in C3H10T1/2 cells treated with WNT3A in the presence or absence of exogenous DKK-1.

Among genes regulated by WNT3A, we identified individual temporal responses of eleven members of the dual-specificity phosphatase (DUSP) family. Despite the amount of information on DUSPs in MAPK signaling, limited information exists on the role of DUSPs in Wnt signaling pathway or on further downstream effects. Within this study we have therefore focused on Dusp 1, 4 and 9 representing early (6 hours), delayed (24 hours) and late (72 hours) responders to WNT3A, respectively.

The WNT3A increased Dusp 1/4/9 expression was blocked only partially by simultaneous addition of DKK1. Experiments using bone marrow derived cells from Lrp5-/- mice revealed that the WNT3A induced increase in Dusp 4/9 transcript levels is independent of LRP5. However, over-expression of a HBM-mutant in HEK293-cells (Lrp5-G171R) seemed to augment WNT3A -induced Dusp 4 transcription. To address DUSP involvement in Wnt/LEF/Tcf signaling, we transiently over-expressed Dusp 4/9 in HEK293 cells. No effect of either phosphatase on Lef/TCF signaling was observed in absence of WNT3A, however, WNT3A induced signaling was further augmented by about 50% and 20% by Dusp 4 and Dusp 9 over-expression, respectively. Furthermore, in-vitro knockdown of Dusp 4 and 9 by siRNA indicated crucial roles of DUSP-4/9 in WNT3A mediated Lef/TCF signaling.

In conclusion, we have identified DUSP family members responding to WNT3A treatment and importantly, in the case of DUSP 4/9, cross-talking with canonical WNT/Lef/TCF signaling pathway. By inference, DUSP mediated control of MAPK's through dephosphorylation, thus might play a role in WNT3A induced C3H10T1/2 osteoblastic cell differentiation.

Disclosures: P. V. Nantermet, None.

T043

Nondestructive Evaluation of Cell Numbers in Bone Marrow Stromal Cells / Biodegradable Scaffold Composites Using Ultrasound.

K. Oe¹, M. Miwa*¹, K. Nagamune*¹, Y. Sakai*¹, R. Kuroda*¹, T. Hasegawa*¹, T. Iwakura*¹, N. Shibanuma*¹, Y. Hata*², M. Kurosaka*¹. ¹Department of Orthopedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan, ²Graduate School of Engineering, University of Hyogo, Himeji, Japan.

Composites of bone marrow stromal cells (BMSCs) and biodegradable scaffold (BS) have been used as a bone graft model. Up to now, the actual number of cells contained in these composites has been difficult to evaluate using nondestructive techniques. In general, ultrasound amplitude through homogeneous material is larger than through heterogeneous material such as BMSCs/BS composites. We developed an ultrasound device to evaluate BMSCs/BS composites.

The ultrasound device has a transmitting probe and a receiving probe attached to the slide gauge frame. Both probes are set to be collinear at opposite sides to propagate ultrasound wave from the transmitting probe to the receiving probe. One of the probes can be moved along the frame to adjust the size of the material to be inserted between them. The ultrasound wave data was recorded by an oscilloscope via an ultrasound pulser/receiver. In this study, we investigated the relationship between the ultrasound amplitude and the actual number of cells, using ultrasound as a nondestructive and quantitative technique. We used BMSCs of S-D rat and examined five experimental groups with different concentrations of BMSCs: 0 (control), 1.0×10⁶, 1.5×10⁶, 2.0×10⁶ and 1.0×10⁷ cells/ml (n=25). Porous ceramic blocks composed of β-tricalcium phosphate were used as scaffolds. All samples were incubated for 24 hours at 37°C and we evaluated them with the ultrasound device. After measurement by ultrasound device, BMSCs/BS composites were detached with 0.25% trypsin during the destruction of the BMSCs/BS composites for 30 minutes. We counted the BMSCs of all BMSCs/BS composites using a Hemacytometer. We repeated this treatment twice, and considered the total cell count numbers to be the actual number of cells.

Ultrasound amplitude was well correlated to the actual number of cells contained in the BS (r=0.779). The estimation equation (y=9879x-33290) was derived from the results. Here, x and y represent the amplitude value and estimated cell number in this study, respectively. The linearity of this estimation equation was validated in the range of seeding cell concentration from 5.8×10⁴ to 19.2×10⁴ of the cell number. The estimated error for the system was 2.591 ×10⁴. These results indicate that the ultrasound device we developed is a valuable, convenient, and nondestructive tool for applications in tissue engineering, especially in the generation of artificial bone tissue.

Disclosures: K. Oe, None.

T044

Liganded versus Unliganded Estrogen Receptor: A Potential Explanation for Paradoxical Regulation of Periosteal Expansion by Estrogens.

M. Ogita, E. Dworakowski*, J. P. Bilezikian, S. Kousteni. Division of Endocrinology, College of Physicians & Surgeons, Department of Medicine, Columbia University Medical Center, New York, NY, USA.

Many studies in rodents, primates and humans indicate that estrogens suppress periosteal expansion. However, in certain situations, such as in men with aromatase deficiency or in the growing skeleton, low estrogen concentrations may promote directly periosteal expansion or, alternatively, be permissive to the stimulatory effects of androgens or mechanical stimulation. Based on evidence that unliganded and liganded ERα regulate the expression of cytokines like TNFα in an opposite manner, we examined the possibility that ERα may also show different, and opposite, functions to regulate periosteal osteoblast differentiation. We have found that 17β-estradiol (E₂) has no effect by itself, but attenuates the stimulatory effect of BMP-2 on the expression or activity of alkaline phosphatase and endogenous targets of BMP-2- and Wnt/β-catenin-mediated transcription in primary periosteal cells. Dihydrotestosterone (DHT) exerts the opposite effects. However, reduction of ERα expression in primary periosteal osteoblast progenitors using siRNA, dose-dependently attenuates BMP-2 — induced osteoblast differentiation, as measured by AP activity. Pre-treatment with 10⁻⁸ M of the specific ER inhibitor ICI 182,780, which causes ER degradation, attenuates the stimulatory effect of BMP-2 on AP activity in primary periosteal cells. BMP-2-induced upregulation of Smad 1/5/8 phosphorylation is not affected by ICI 182,780; but Smad6 (a transcriptional target of BMP-2), osteocalcin and axin2 (a transcriptional target of Wnt/β-catenin) expression, measured at 16 days following treatment with ICI 182,780, is attenuated. These results suggest that ERα is a major cellular switch which can either suppress (in the presence of E₂) or stimulate (in the absence of E₂) periosteal bone formation; perhaps by potentiating the beneficial effects of mechanical loading or androgens. We have also found that low doses of E₂ have no effect on periosteal cell differentiation, but in combination with DHT (1–100 pM), induces AP activity in periosteal osteoblast progenitors. Thus, low dose estrogen exposure may mimic the stimulatory actions of unliganded ERa on periosteal expansion. We propose that the dual action of ERα in periosteal osteoblast differentiation may help to explain the apparent paradox of the dose-dependent biphasic response of the periosteum to estrogens.

Disclosures: M. Ogita, None.

T045

Characterization of Bone Tumors in a Mouse Model for Carney Complex.

E. Pavel*¹, K. Nadella*¹, W. Towns*¹, L. Kirschner*². ¹Human Cancer Genetics, The Ohio State University, Columbus, OH, USA, ¹Internal Medicine, Subdivision of Endocrinology, The Ohio State University, Columbus, OH, USA.

Carney Complex (CNC) is an autosomal dominant neoplasia syndrome caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A (PKA). This genetic defect induces skin pigmentation, endocrine tumors, myxomas, schwannomas and bone tumors termed osteochondromyxomas.

In order to study the link between the PRKAR1A mutations and tumor formation, we generated a mouse model of this condition. Prkar1a^+/− mice develop bone tumors with high frequency, although the associated molecular changes have not been characterized.

Bone tumors observed in Prkar1a^+/− mice were heterogeneous, including elements of myxomatous, cartilaginous and bony differentiation which effaced the normal bone architecture. Immunohistochemical analysis had suggested an osteoblastic origin for the tumor cells which we have confirmed using additional markers. These abnormal cells were typically associated with increased bone remodeling as evidenced by increases in osteoclasts number.

Analysis of primary culture of the tumors confirmed that the tumor cells were derived from the osteoblast lineage; however, fully differentiated functions such as mineralization were impaired and tumor cells exhibited an increase in migratory capability. At the biochemical level, we observed the expected reduction in Prkarla protein and an increase in PKA activity in tumor cells. Moreover, primary tumor cells demonstrated an increased sensitivity to the growth-promoting effects of PKA activation in the cells.

Examination of gene expression in the tumor cells revealed down-regulation of markers of bone differentiation and increased expression of locally acting growth factors, including members of the Wnt signaling pathway. The role of the Wnt signaling pathway has also been confirmed by observing alterations the subcellular distribution of the relevant signaling molecules. In summary, loss of Prkar1a from the mouse leads to the formation of bone tumors analogous to osteochondromyxomas observed in human patients. Dysregulation of PKA leads to the formation of osteoblast-derived bone tumors through dysregulation of differentiation and upregulation of locally-acting factors involved in cell growth and differentiation.

Disclosures: E. Pavel, None.

T046

Effects of PDE7 and PDE8A Inhibition on the Differentiation of Human Mesenchymal Stem Cell-Derived Osteoblasts.

M. H. Pekkinen¹, M. E. B. Ahlström*¹, U. Riehle*², M. M. Huttunen¹, C. J. E. Lamberg-Allardt¹. ¹Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland, ²Institute for Biological Chemistry and Nutrition, University of Hohenheim, Stuttgart, Germany.

Cyclic adenosine monophosphate (cAMP) is an important second messenger involved in a variety of cellular responses to extracellular agents such as hormones and neurotransmitter, i.e. in PTH signaling via cAMP. cAMP is known to be inactivated by the cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 known families; designated PDE 1–11. PDEs determine the amplitudes of cyclic nucleotide mediated hormonal responses and modulate the duration of the signal. Osteoporosis, bone loss, typically reflects an imbalance in skeletal turnover so that bone resorption exceeds bone formation. It is important to understand the role of the PDEs in bone cells, because the bone metabolism, and especially bone formation, could be affected via PDEs. The aim of this study was to investigate the influence of PDE7 and PDE8A inhibition in the differentiation and function of human osteoblasts.

Osteoblasts differentiated from human mesenchymal stem cells (hMSC) were cultured and treated for 48 h with small interfering RNAs (si-RNAs) generated from PDE7 and PDE8 PCR products. Total RNA was isolated from the cells and gene expression was assayed with cDNA microatTay. Quantitative Real-Time PCR was used to confirm the specific PDE inhibition and the cDNA microarray results. bALP measurements were assayed during differentiation and mineralization was determined by quantitative Alizarin red S staining. On the level of mRNA, PDE7 and PDE8 inhibition by RNA interference (RNAi) decreased the gene expression of PDE7A by 60%, PDE7B by 40% and PDE8A by 30%. The silencing of PDE7 increased the expression of several osteogenic genes, such as β-catenin, osteocalcin, caspase 8, and CREB-5 genes and decreased the expression of the 1,25- dihydroxyvitamin D3 receptor gene. The silencing of PDE8A significantly decreased caspase-8, osteoglycin, BMP-1. Mineralization was increased up to threefold in PDE7 siRNA treated cells compared to control and treatment with the PDE7-selective PDE- inhibitor BRL-50481 also increased the mineralization. Silencing with PDE8A siRNAs had no effect on mineralization.

Our results show that specific gene silencing with the RNAi method is a useful tool for inhibiting the gene expression of specific PDEs and that PDE7 upregulates several osteogenic genes and increases mineralization. PDE7 may play an important role in the regulation of osteoblastic differentiation.

Disclosures: M.H. Pekkinen, None.

T047

Conjugated Linoleic Acid Regulates Osteoblast and Adipocyte Differentiation from Mesenchymal Stem Cells.

I. D. Platt*¹, L. G. Rao*², A. El-Sohemy*¹. ¹Nutritional Sciences, University of Toronto, Toronto, ON, Canada, ²Medicine, University of Toronto, St. Michael's Hospital, Calcium Research Laboratory, Toronto, ON, Canada.

Conjugated linoleic acid (CLA) refers to a group of geometric and positional isomers of linoleic acid. The major isomers are 9cis, 11 trans (9,11) and 10trans, 12cis (10,12) CLA, which are both bioactive and often have different physiological effects. CLA has variable effects on bone formation and on adiposity in vivo and in vitro. Osteoblasts and adipocytes are both derived from mesenchymal stem cells (MSCs), and factors that promote the differentiation of one cell type may inhibit the differentiation of the other type. CLA may be one such factor and the effects of CLA on MSCs are not known. The purpose of this study was to determine whether 9,11 and 10,12 CLA affect markers of osteoblast (alkaline phosphatase (ALP), osteocalcin) and adipocyte (lipid droplet) differentiation from human bone marrow-derived MSCs and alter the expression of genes regulating osteoblast (Wnt10b) and adipocyte (C/EBPα) differentiation from these cells. To determine the effects of CLA on osteoblast differentiation, MSCs were seeded at 3×10³ cells/cm², and treated 4 days later with 0–50 μM 9,11 or 10,12 CLA in osteogenic media. Cells were harvested after 11 days in culture for mRNA analysis (Wnt10b, osteocalcin), and for the measurement of ALP activity. Calcium deposition was measured after 18 days in culture. To determine the effects of CLA on adipocyte differentiation, MSCs were seeded at 2×10⁴ cells/cm², and treated 4 days later with 0–50 μM 9,11 or 10,12 CLA in adipogenic media. After 11 days in culture, cells were stained for lipid droplet formation using oil red O, and cells were harvested for mRNA analysis (C/EBPα). Gene expression was determined by real-time RT-PCR and normalized to the expression of β-2-microglobulin. At 12.5 μM, 10,12 CLA increased Wnt10b expression (∼70%), whereas 9,11 CLA decreased its expression (∼33%). At 50 μM, 10,12 CLA increased osteocalcin expression (∼85%), calcium deposition (∼32%), and ALP activity (∼85%), but 9,11 CLA had no effect. At 50 μM, 10,12 CLA decreased the expression of C/EBPα (∼35%), but 9,11 CLA had no effect. However, 9,11 CLA increased oil red O staining at all concentrations and maximally at 50 μM (∼70%). Our results demonstrate that 10,12 CLA increases osteoblast differentiation and decreases adipocyte differentiation from MSCs suggesting that this isomer may prevent bone loss and decrease bone marrow adiposity.

Disclosures: I.D. Platt, None.

T048

Activation of the Non-canonical Wnt Pathway During Human Mesenchymal Stem Ceil Osteogenesis: Microarray Expression Analyses to Examine the Effects of Dexamethasone on Cell Differentiation.

K. T. Rousche*, A. Derfoul*, J. Helm*, R. S. Tuan*. Cartilage Biology and Orthopaedics Branch, NIH/NIAMS, Bethesda, MD, USA.

Non-canonical Wnts enhance osteogenic differentiation in adult human mesenchymal stem cells (hMSCs). The molecular mechanisms involved in activation of this Wnt pathway during osteoblastic differentiation of hMSCs have yet to be been defined. In vitro, glucocorticoids (GCs) are known to maintain differentiation of osteoblasts and to promote osteogenic differentiation of hMSCs. However, administration of high doses of GCs has been shown to suppress growth in children and lead to low bone mineral density and increased fracture risk in adults. To gain insight in the mechanisms of GC action on the differentiation of hMSCs into osteoblasts, we examined the effects of various doses of the GC analog, dexamethasone (DEX), on osteogenic differentiation of hMSCs. The purpose of this study was to identify genes involved in osteogenic differentiation of hMSCs at physiological doses of GCs, as well as genes associated with the inhibition of osteoblast function by therapeutic doses of GCs. We performed DNA microarray analyses of hMSCs cultured in osteogenic conditions in the presence or absence of 10 nM or 1 uM DEX. Human bone marrow was obtained from the femoral head of patients undergoing hip arthroplasty. hMSCs were cultured in monolayer for 12 days in osteogenic medium in the presence or absence of 10 nM to 1uM DEX. Microarray analyses of hMSCs grown in the presence of 10 nM or luM DEX were performed using Affymetrics human Genome U133 Plus 2.0 chips. Results were analyzed using biodata mining RAM and MAS softwares. Data suggest that GCs alter expression of non-canonical Wnts, Wnt receptors and Wnt modulators during hMSC osteogenic differentiation. Wnt5b, FRZB (sFRP3), Twist2, and FRZ6, and sFRP2 were differentially expressed during GC-mediated hMSC osteogenesis. Taken together, these data support a distinct role for non-canonical Wnts in MSC osteogenic differentiation. siRNA and functional studies are underway to further support this data.

Disclosures: K.T. Rousche, None.

This study received funding from: Intramural Research Program of NIAMS, NIH.

T049

Cytoplasmic Interaction of p21 with Runx2 During Osteogenic Differentiation of Mesenchymal Stem Cells.

P. Muller*¹, C. Bergemann*¹, U. Bulnheim*¹, A. Liebold*², G. Steinhoff*², J. Rychly¹. ¹Laboratory of Cell Biology, University of Rostock, Rostock, Germany, ²Department of Heart Surgery, University of Rostock, Rostock, Germany.

Regulating the switch between proliferation and osteogenic differentiation of mesenchymal stem cells is critical for the development of normal bone tissue and more over is of considerable interest in bone tissue engineering using mesenchymal stem cells as vehicles for cell based skeletal therapies. Recent investigations revealed that the protein p21 exhibits multiple functions in addition to its role in cell cycle inhibition. These functions appear to rely on the different cellular localizations and molecular targets. To assess a possible role of p21 in the differentiation of mesenchymal stem cells, we tested the cellular localization of p21 during the osteogenic differentiation of mesenchymal stem cells. Bone marrow derived human mesenchymal stem cells were stimulated to differentiate into osteoblasts by addition of osteogenic differentiation medium (ODM) containing ascorbic acid, glycerolphosphate and dexamethasone. In p21-GFP transfected cells we observed a translocation of p21 from the nucleus to the cytoplasm within 2 hours after addition of ODM. This nuclear export was mediated by CRM1, the major nuclear protein export receptor, because leptomycin B completely blocked the translocation of p21. These experiments further indicated the significance of the CRM1 mediated nuclear export of proteins for the osteogenic differentiation, because inhibition of the export reduced the expression of osteogenic marker proteins and alkaline phosphatase activity. To further assess whether the translocation of p21 is related to known molecules of the cellular machinery that control the osteogenic differentiation we addressed the interaction of p21 with Runx2, the principal osteogenic transcription factor. Coimmunoprecipitation experiments with both nuclear and cytoplasmic fractions revealed that p21 interacts with Runx2 in the cytoplasm during osteogenic differentiation. Blocking the nuclear export of proteins revealed an association of both proteins in the nucleus. We further found that inhibition of the export by leptomycin B with the consequence of blocking osteogenic differentiation did not reduce but rather increased the RNA expression of Runx2. In conclusion, the interaction of p21 with Runx2 in the cytoplasm during osteogenic differentiation suggests that p21 plays a role in the osteogenic differentiation. However, the function of the cytoplasmic interaction of these proteins is not known. We speculate that p21 may act as assembly factor for Runx2 in the cytoplasm to promote or control a nuclear import of Runx2.

Disclosures: J. Rychly, None.

T050

Osteogenesis and Adipogenesis Are Differentially Modulated by PTH and the N- and C-Terminal Fragments of PTH-Related Protein.

R. Santiago*¹, A. Casado*², I. Herrera*³, A. Torres*³, P. Esbrit*⁴, G. Dorado*⁵, J. M. Ouesada*¹. ¹UMM, Hospital U. Reina Sofia, Córdoba, Spain, ²Sanyres (Grupo PRASA), Hospital U. Reina Sofia, Córdoba, Spain, ³Servicio Hematología, Hospital U. Reina Sofia, Córdoba, Spain, ⁴Laboratorio de Metabolismo Mineral y Oseo, Fundación Jimenez Diaz, Madrid, Spain, ⁵Dpto. Bioq y Biol Mol, Universidad de Córdoba, Córdoba, Spain.

Chronic or intermittent parathormone (PTH) administration plays different roles on bone. We evaluated the effects of different patterns of exposure to either PTH (1–34), parathyroid hormone-related protein (PTHrP) (1–36), or PTHrP (107–139) on adipogenesis and osteoblastogenesis in vitro. Bone marrow stromal cells were differentiated to osteoblasts (dexamethasone, ascorbic acid and glycerolphosphate) or adipocytes (dexamethasone, indomethacin and isobutylmethylxanthine), in the presence of PTH or the PTHrP peptides (100 nM), added at the beginning or at day 6 up to the end of the study period. Differentiating cell cultures were sampled at 6, 18, and 24 days. The differentiating process was evaluated by measuring alkaline phosphatase (AP) activity and gene expression (by real time PCR) of osteogenic (AP, runx2, type I collagen, osteocalcin, osteoprotegerin, and RANKL) and adipogenic (PPARγ2 and LPL) markers. Osteoblast mineralization was assessed by alizarin red staining; and adipocyte by oil red staining. Expression of all osteoblastic factors tested decreased with both PTHrP (1–36) and PTH at day 6. However, addition of these peptides, but not PTHrP (107–139), from this day through the end of the culture period increased the expression of these factors at 18 and 24 days, respectively. This was associated with a decrease in PPARγ2 and LPL gene expression induced by either PTHrP (1–36) or PTH within the same time period. On the other hand, continuous treatment with PTHrP (107–139) from the start of cell culture induced adipogenesis, as shown by increased PPARγ2 and LPL gene expression at day 18, and decreased the expression of various osteoblastic products at day 24. Moreover, at the latter time period, the number of adipocytes was 20 % higher in cultures supplemented with PTHrP (107–139), but 20 % lower in those treated with either PTHrP (1–36) or PTH from day 6, compared with that in untreated cells. In conclusion, our findings suggest that the effects of PTH and these PTHrP peptides on bone marrow stromal cells depend on their differentiation stage. Thus, PTH and, to a lesser extent, the N-terminal PTHrP fragment induce osteogenesis in cells that are already differentiating. On the other hand, PTHrP (107–139) was not found to be osteogenic, but it enhanced adipogenesis, in our experimental setting.

Supported by: Sanyres (Grupo PRASA), CM0010/05, PAI CTS-413, and SAF2005–05254.

Disclosures: R. Santiago, None.

T051

Effect of 1,25-Dihydroxyvitamin D₃, Retinoic Acid, and Dexamethosone on the Osteoblastic Differentiation of Human Adipose-Derived Stem Cells.

M. J. Saran*¹, P. A. Zuk*², W. Huang*³, C. K. Huang*⁴, M. Hakimi*¹, D. S. Bischoff*³, G. H. Rudkin*⁵, D. T. Yamaguchi³, T. A. Miller*³. ¹David Geffen School of Medicine, UCLA, Los Angeles, CA, USA, ²Regenerative Bioengineering and Repair Lab, UCLA, Los Angeles, CA, USA, ³Research Service, VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA, ⁴Head & Neck Surgery, UCLA, Los Angeles, CA, USA, ⁵Plastic Surgery, UCLA, Los Angeles, CA, USA.

Human adipose-derived stem cells (ASC) are a promising source of cells for bone tissue engineering; however, the factors that stimulate osteogenic differentiation in human ASC have not been well characterized. Most studies have either been done in animal models or in 2- dimensional (2-D) cultures systems. However, human ASC have been shown to respond differently to osteogenic supplements than animal ASC. Also, 3-D cultures, as compared to 2-D cultures, are a closer approximation to the 3-D matrix that cells encounter in vivo. The purpose of this study was to investigate the supplements that are commonly used to induce osteogenic differentiation in human ASC, and to compare their actions in 2-D versus 3-D cultures. One million human ASC cells were seeded onto 3-D poly(L-lactide-co-glycolide) (PLGA) scaffolds or 2-D PLGA films. Cells were cultured in osteogenic media in the presence or absence of each of the following supplements: 1,25-Dihydroxyvitamin D₃ (VD) [10nM or 100nM], Retinoic Acid (RA) [1 μM], or Dexamethasone (Dex) [100nM], Cells were harvested at 7, 10, and 14 days for RNA extraction. Quantitative real-time RT-PCR was performed to measure mRNA expression of the following osteogenic marker genes: Alkaline Phosphotase (ALP), Osteocalcin (OCN), Bone Sialoprotein (BSP), and Collagen I (Col I). VD increased the expression of all 4 osteogenic marker genes in 3-D cultures compared to control. ALP increased 5-fold on day 14. OCN and BSP increased 2-fold on day 14. Col I increased 3-fold on day 14. 2-D cultures treated with VD followed a similar expression pattern. However, treatment with Dex or RA adversely effected expression of these osteogenic markers in both 2-D and 3-D cultures. On day 7, RA treatment resulted in a 4-fold decrease in ALP expression, an 11-fold decrease in BSP expression, and no change in the levels of OCN mRNA. Similarly, Dex caused a 7-fold decrease in BSP expression and no change in ALP or OCN expression compared to control on day 7. In conclusion, we found that for both 2-D and 3-D cultures, 1,25-Dihydroxyvitamin D₃ promotes the expression of osteogenic marker genes; whereas, Retinoic Acid and Dexamethasone treatment, have no effect or can be detrimental to ASC cell osteogenic differentiation. This is an important step to understanding the best supplements to use to promote human ASC osteogenic differentiation in bone tissue engineering.

Disclosures: M.J. Saran, None.

T052

Microgravity and Bone: Effects of Modeled Microgravity on Osteoblast and Osteoclast Differentiation.

R. Saxena*¹, G. Pan*², J. M. McDonald². ¹Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA, ²Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

Prolonged microgravity experienced by astronauts on long term space missions is associated with a decrease in bone mass and an increase in risk of fracture. Studies on astronauts during spaceflight indicate that increased bone resorption by osteoclasts and decreased osteoblast activity, are jointly responsible for bone loss. To investigate the effect of microgravity on differentiation of osteoclasts and osteoblasts from precursor cells in space, we used the Rotary Cell Culture System (RCCS) which is a ground based system that is able to simulate microgravity conditions. Using the RCCS, we have demonstrated that modeled microgravity inhibited the expression of osteoblast differentiation genes and increased adipocyte genes in human mesenchymal stem cells incubated under osteogenic conditions. This transformation involves reduced RhoA activity and cofilin phosphorylation, disruption of F-actin stress fibers, and decreased integrin signaling through focal adhesion kinase We have recently established a unique system to investigate the effects of modeled microgravity on osteoclastogenesis. The mouse macrophage cell line, RAW264.7 was cultured with microcarrier beads in DMEM supplemented with 10% FBS. Cells attached to beads were placed in the RCCS (microgravity treatment) and rotated at 9 rpm for 8 hours and 24 hours. Bead-attached cells placed simultaneously in culture plates were the gravity controls. After incubations, the cells were detached from beads and stimulated with RANKL (Receptor Activator of NFkB Ligand, 20ng/ml) and allowed to differentiate for 4 days. Differentiated multinucleated osteoclasts were observed under the microscope followed by TRAcP (Tartrate Resistant Acid Phosphatase) staining. Our results show that multinucleated TRAcP-positive osteoclasts derived from microgravity treated cells were increased nearly two-fold compared to that from gravity-treated cells after treatment with RANKL. RT-PCR further demonstrated that TRAcP expression in MMG-treated cells was increased compared to gravity-treated cells. In conclusion, these data indicate that modeled microgravity directly stimulates osteoclastogenesis and inhibits osteoblastogenesis, consistent with the studies of bone loss in humans in microgravity.

Disclosures: R. Saxena, None.

T053

Mesenchymal Stem Cells from Human Adipose Tissue and Bone Marrow: Osteogenic Differentiation and Interaction with Ti6A14V.

I. Tognarini*¹, S. Sorace*¹, R. Zonefrati*¹, G Galli*¹, A. Gozzini*¹, S. Carbonell Sala*¹, Zappoli Thvrion*¹, A. Carossino*¹, S. Ciuffi*, A. Tanini¹, F. Sbaiz*², A. Facchini*², M. Brandi¹. ¹Internal Medicine, University of Florence, Florence, Italy, ²LIMA SpA Medical System, San Daniele del Friuli, Udine, Italy.

In bone tissue engineering and surgery there is a growing need to test the ability of hard biomaterials to control the differentiation of mesenchymal stem cells (MSCs) in order to gain direct bone apposition to implant materials. MSCs, classically used to test implanted biomaterial as Ti6A14V alloy, are isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue derived stromal cells may be a better source of MSCs according to their abundance and accessibility. The aim of this study was to evaluate the osteogenic differentiation of adipose tissue and bone marrow-derived MSCs (PA1 and BMC1, respectively) and to compare their response to Ti6A14V.

The expression of the osteoblastic phenotype was evaluated by monitoring alkaline phosphatase (ALP) activity, expression of osteoblastic markers by quantitative RT-PCR and immunocytochemistry, and calcium mineralization by Calcein staining and Alizarin Red S assay. Cell growth, cytoskeleton morphology and adhesion of differentiated cells on Ti6A14V were evaluated by cell counting and Laser Scanning Confocal Microscopy analysis (actin, tubulin and vinculin). Human osteoblast-like cells (SaOS-2) were used as control.

Thirty days after the osteogenic induction, near 50% of PA1 cells showed an osteoblastic-like phenotype, whereas almost 100% of BMC1 cells resulted entirely differentiated. BMC1 cells cultured on Ti6A14V exhibited an increase of ALP activity that was similar to that seen on culture polystyrene (PS), whereas PA1 cells showed a statistically significant increment of this activity on Ti6A14V compared to that observed on PS. A comparable enhancement of transcript levels of genes involved in osteogenic differentiation was observed in both cell lines on PS and on Ti6A14V during differentiation. Osteoblastic activity was confirmed at the protein level by osteopontin and osteocalcin immunostaining and at the mineral level by the production of calcium deposits. A good adhesion, cytoskeleton organization, and cell growth on Ti6A14V were observed in every cell line, but interestingly only PA1 cells showed a higher level of adhesion to Ti6A14V compared to the control.

In conclusion, the results of this study confirmed that PA1 cells are capable of adhering, proliferating, and differentiating on Ti6A14V as well as BMC1 cells and support the concept that adipose tissue may represent a valuable alternate source of MSCs to bone marrow for hard tissue regeneration.

Disclosures: I. Tognarini, None.

T054

Identification of Human Circulating Side Population (SP) Cells Co-expressing the Mesenchymal Stromal Cell Marker, Stro-1.

A. H. Undale, U. I. L. Moedder, S. Khosla. Mayo Clinic, Rochester, MN, USA.

Stem cells that stain differentially with vital dyes such as Hoechst 33342 are described as side population (SP) cells, which can be distinguished by their capacity to efflux the Hoechst 33342 dye efficiently and by their unique profiles in Hoechst red versus Hoechst blue bivariate fluorescent-activated cell sorting dot plots. SP cells have been identified in a wide variety of mammalian tissues, including murine and human bone marrow and human peripheral blood. These cells can not only regenerate the cells of the hematopoietic lineage but have also been shown to regenerate several mesenchymal lineages, suggesting their pluripotent potential. Studies conducted by Aubin et al (Bone 2006, 38:662) showed that SP cells isolated from fetal rat calvaria were enriched for osteoprogenitor cells.

We undertook the current study to investigate whether SP cells in human peripheral blood expressed the mesenchymal stromal cell marker, Stro-1, which identifies cells with osteogenic potential in human bone marrow. We examined the SP cell population in human peripheral blood mononuclear cells (MNCs) obtained by Ficoll-gradient centrifugation. The SP cell population (mean ± SEM, n=4) was identified by flow cytometry based on the ability of these cells to efflux Hoechst 33342 and their typical Hoechst 33342 profile. Flow cytometric analyses identified 0.14 ± 0.01 % of MNCs as SP cells. Further phenotypic characterization of the SP cell population was done using a monoclonal Ab to Stro-1. We found that 1.6 ± 0.4 % of the SP cell population expressed Stro-1, so that the total percentage of Stro-1^pos/SP^pos cells in MNCs was 0.002%. These results thus demonstrate for the first time the presence of the stromal cell marker Stro-1 on a subset of human peripheral blood SP cells. The circulating Stro-1^pos/SP^pos cells may represent an early mesenchymal stem cell population that could potentially be expanded and have utility in the treatment of metabolic bone diseases.

Disclosures: A.H. Undale, None.

T055

βTrcpl Ubiquitin Complex Regulates Ihh-promoted Osteoblast Differentiation Through Gli2 Ubiquitination.

M. Wada*¹, T. Matsubara¹, K. Hata*¹, T. Imamura*², R. Nishimura¹, T. Yoneda¹. ¹Osaka University, Osaka, Japan, ²JFCR Cancer Institute, Tokyo, Japan.

Accumulating data indicate that Indian hedgehog (Ihh) promotes the differentiation of mesenchymal stem cells into osteoblasts. It is also suggested that the transcription factor, Gli2, which is activated by Ihh, plays a role in this differentiation process. Recent studies have reported that an ubiquitin ligase complex consisting of βTrcpl regulates Gli2 expression, suggesting an involvement of βTrcpl in the osteoblast differentiation. However, the precise role of βTrcpl in Ihh/Gli2-promoted osteoblastogenesis is currently unknown. Furthermore, it is also unclear whether and how Ihh signaling regulates ubiquitination of Gli2 in conjunction with βTrcpl. In the present study, we investigated the role of βTrcpl in the osteoblast differentiation stimulated by Ihh signaling and the relationship between Ihh and βTrcpl ubiquitination system. Overexpression of βTrcpl together with protein kinase A (PKA) and glycogen synthesis kinase 3 (GSK3β), both of which are shown to be involved in controlling βTrcpl activity during Gli2 ubiquitination, truncated Gli2 into the repressor form of 78KD. Overexpression of βTrcpl/PKA/GSK3β also suppressed transcriptional activity of Gli2, suggesting a negative regulatory role of the βTrcpl ubiquitin ligase complex in Ihh/Gli2 signaling. We next examined the effects of a mutant βTrcpl (βTrcpl-F-box), which lacks the F-box domain necessary for ubiquitin ligase complex formation on osteoblast differentiation using the mesenchymal cell line C3H10T1/2. Treatment with Ihh or overexpression of Gli2 promoted osteoblast differentiation in C3H10T1/2 cells. Of note, overexpression of βTrcpl-F-box markedly stimulated the Gli2-induced osteoblast differentiation. However, of interest, βTrcpl-F- box had little effects on the Ihh-induced osteoblast differentiation, raising a possibility that Ihh functions through influencing ubiquitination system upstream of βTrcpl. To investigate this, we focused on GSK3β that functions upstream of βTrcpl. Since GSK3β activity is known to be negatively-regulated by phosphorylation, we examined the effects of Ihh on GSK3β phosphorylation and found that Ihh phosphorylated GSK3β. Moreover, Fused, the serine/threonine kinase which is one of the essential components of hedgehog signaling, also phosphorylated GSK3β. Collectively, our results suggest that the βTrcpl ubiquitin complex modulates the Ihh-promoted osteoblast differentiation by regulating Gli2 ubiquitination and that Ihh down-regulates the βTrcpl ubiquitination system through inactivation of GSK3β as a consequence of its phosphorylation by Fused.

Disclosures: M. Wada, None.

T056

Ectoderm Neural Cortex 1, a Wnt Target Gene, Is Expressed in Chondrocytic and Osteoblastic Cells.

L. E. Worton¹, Y. Shi*², E. J. Smith*², D. G. Little*³, J. P. Whitehead*¹, E. M. Gardiner¹. ¹Diamantina Institute for Cancer, Immunology and Metabolic Medicine, Brisbane, Australia, ²Garvan Institute of Medical Research, Sydney, Australia, ³Children's Hospital, Westmead, Sydney, Australia.

The canonical Wnt pathway has been implicated in bone mass regulation; however, few Wnt target genes affecting this process have been elucidated. Ectoderm Neural Cortex 1 (ENC1) is a reported Wnt target gene found to be involved in adipocyte differentiation. ENC1 has also been reported to interact directly with retinoblastoma protein (p 110 Rb) and with filamentous actin in various cell lines. Therefore, this study was undertaken to establish a role for ENC1 in bone biology. Transcript profiles of fracture callus tissues collected at early stages in a rabbit model of tibial distraction osteogenesis were generated using human ResGen cDNA arrays. ENC1 expression was up-regulated 40-fold between 2 and 4 or 6 weeks post distraction, correlating with a marked change in callus cellular composition. In 2-week callus, fibroblastic and cartilaginous cells predominated. At 4- and 6-weeks, mature osteoblasts and osteocytes were more abundant, coinciding with a notable increase in bone forming activity. ENC1 transcripts were localized to osteoblastic cells and remnant chondrocytes by in situ hybridization of rabbit 4-week fracture callus sections. Furthermore, in normal mouse bone, ENC1 was expressed in growth plate and articular chondrocytes and in mature periosteal osteoblasts. Quantitative RT-PCR analysis confirmed the expression of ENC1 during the differentiation of primary osteoblastic cultures, and of cultured MC3T3-E1 and Kusa-O 4b10 osteoblastic cells. Expression of ENC1 was also confirmed in osteosarcoma and chondrocytic cell lines. Transient expression of epitope tagged ENC1 in MG63 and SaOS2 osteosarcoma cells and stable over-expression in HEK293 and CHO cell lines showed ENC1 to have a cytoplasmic distribution with staining of a subset of nuclei in MG63 cells. There was no evidence of co-localization with filamentous actin. These results indicate that ENC1 is expressed in chondrocytic and osteoblastic cells and may play a role in Wnt stimulated osteoblastic differentiation.

Disclosures: L.E. Worton, None.

T057

Glucocorticoid-induced Leucine-Zipper (GILZ) Enchances Osteoblast Differentiation of Bone Marrow Mesenchymal Stem Cell by Inhibiting Adipocyte Differentiation.

W. Zhang*¹, X. Shi². ¹Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China, ²Institute of Molecular Medicine & Genetics, Medical College of Georgia, Augusta, GA, USA.

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages including osteoblasts and adipocytes. A recently identified transcription factor, glucocorticoid-induced lecine zipper (GILZ), was shown to inhibit the expression of peroxisome proliferator-activated receptor gamma-2 (PPARγ2) and blocks adipocyte differentiation. To investigate the role of GILZ in osteoblast differentiation of MSCs, we isolated bone marrow MSCs from C57BL/6 mice using negative-immuno-depletion and positive-immuno-selection approaches, and overexpressed GILZ in MSCs using retrovirus-mediated gene delivery approach. Our results show that overexpression of GILZ in MSCs increased alkaline phosphatase (ALP) activity and enhanced mineralized bone nodule formation. Consistent to this phenotypic conversion, real-time RT-PCR analysis showed that both basal and differentiation-induced transcripts of the lineage commitment gene Runx2/Cbfal, as well as osteoblast differentiation marker genes including ALP, type I collagen, and osteocalcin, were all increased in GILZ-expressing cells. In contrast, the mRNA levels of adipogenic PPARγ2 and C/EBPα were significantly reduced in GILZ-expressing cells under both osteogenic and adipogenic conditions. In conclusion, results of this study show that GILZ functions as a modulator of MSC differentiation and that GILZ can shift the balance between MSC osteoblast and adipocyte differentiation. These data suggest that GILZ may have therapeutic value for fracture repair and osteoporosis therapy.

Disclosures: W. Zhang, None.

This study received funding from: AHA, Arthritis Foundation.

T058

Partial Rescue of the Vitamin D Deficient Phenotype of 1α-hydroxylase Gene Knockout Mice by Transplantation of Wild-Type Non-Adherent Bone Marrow-Derived Mesenchymal Stem Cells.

Z. Zhang¹, J. Tong*¹, D. Goltzman², D. Miao³. ¹Department of Public Health, Suzhou University, Suzhou, China, ²Medicine, McGill University, Montreal, PQ, Canada, ³Institute of Dental Research, Nanjing Medical University, Nanjing, China.

We previously demonstrated that there are non-adherent mesenchymal stem cells (MSCs) in adult bone marrow and that transplantation of non-adherent bone marrow-derived MSCs (NA-BM-MSCs) can rescue lethal-dose irradiated VDR^−/− mice by reconstructing a hematopoietic system and repairing damaged tissues. To determine whether the transplantation of NA-BM-MSCs can rescue the phenotype of 1α(OH)ase^−/− mice^−/− mice. 2 × 10⁶ NA-BM-MSCs from wild-type mice or vehicle (as a control) were transplanted into lethal-dose irradiated female 1α(OH)ase^−/− mice by tail vein injection. Our results show that all control mice irradiated at a 10 Gy dose died within 2 weeks, however, following transplantation of NA-BM-MSCs, 75% and 70% of lethal-dose irradiated mice were viable at 1 and 2 months, respectively. The phenotype of viable 1α(OH)ase^−/− recipients was compared with that of age-matched, non-irradiated wild-type and 1α(OH)ase-/- mice. 1α(OH)ase mRNA in kidney was undetectable by RT-PCR in non-irradiated 1α(OH)ase^−/− mice but was detected in viable irradiated 1α(OH)ase^−/− recipients, although levels were lower when compared to levels in non-irradiated wild-type kidney. Serum 1,25(OH)₂D₃ levels were undetectable in non-irradiated 1α(OH)ase^−/− mice and rose to 24% of non-irradiated wild-type mice (96±11.3ng/L) in viable irradiated 1α(OH)ase^−/− transplant recipients (23±8.5ng/L). Serum calcium levels rose to 2.08±0.25mmol/L in viable 1α(OH)ase^−/− recipients from 1.6±0.18mmol/L in non-irradiated 1α(OH)ase^−/− mice. Skeletal mineralization improved, but did not reach normal levels in viable 1α(OH)ase^−/− recipients as demonstrated by BMD measurement, micro-CT analysis and von Kossa staining of undecalcified sections. In lethal dose irradiated and non-transplanted control 1α(OH)ase^−/− mice, osteoblasts in the endosteum, periosteum and metaphysis were severely damaged, whereas chondrocytes and osteocytes sustained less injury. Osteoblasts were regenerated in viable 1α(OH)ase^−/− recipients. The number of WBC in the peripheral blood and CD4 and CD8 positive cells in spleen were decreased in non-irradiated control 1α(OH)ase^−/− mice but were normalized in viable 1α(OH)ase^−/− recipients. This study indicates that donor NA-BM-MSCs cells can relocate to multiple tissues where they synthesize 1α(OH)ase and produce 1,25(OH)₂D₃ that contributes to the improvement of serum calcium and skeletal mineralization and where they produce immune cells that contribute to the viability of irradiated 1α(OH)ase^−/− recipients.

Disclosures: Z. Zhang, None.

T059

Tension Stress/Stain Induced Osteogenesis in Canine Leg Lengthening Procedures.

J. J. Zhao, J. A. Jacobson*, Y. Jiang. Osteoporosis and Arthritis Lab, Department of Radiology, University of Michigan, Ann Arbor, MI, USA.

We evaluated distraction osteogenesis under stimulation of mechanical tension stress/strain, and ability of various imaging modalities for detection and monitor its progress, since the rate of distraction depends on the successful production of new bone in the distraction gap. Early evaluation of new bone production is important. Overly fast distraction will inhibit new bone formation and mineralization while overly slow distraction will lead to premature consolidation.

Tibial and fibular midshaft osteotomy was performed in 25 adult canines, distracted at the 7th day post-surgery using Ilizarov's equipment at 1 mm daily till reaching 3 cm lengthening, and examined weekly by ultrasound and radiography. Osteotomy of the left tibia without distraction was severed as control. They were euthanized at 3, 5, 7, 9, and 11 weeks post-surgery. The tibias were examined using DXA, CT, MRI, and processed for histomorphometry.

Ultrasound and DXA detected new bone formation within the distraction site as echogenic foci at 2–3 weeks post-surgery. Serial follow-up sonograms showed that echogenicity increased progressively in number and size till coalesced as echodense bone. Radiographs showed linear streaks of new bone formation in the distracted gap at 3–4 weeks, and a bone bridge formed at about 7 weeks post-surgery. CT showed only bony structure while MRI clearly delineated fibrous, cartilaginous, and bony tissues. Histomorphometry showed location-dependent bone formation and remodeling activity. Both intramembranous and endochondral but mainly intramembranous bone formed linearly oriented columns of interconnecting trabecular plates of woven and lamellar types. Bone formation rate, bone volume, and percentage of bone surface covered cuboidal osteoblast were significantly increased within the distraction and osteotomy callus regions compared with controls. At the origingal cortical bone, periosteal bone formation and increased bone resorption were found. Imaging modalities were highly correlated with histomorphometric measures of cartilage and newly formed bone, and measures of cortical porosity and increased osteoclasts in the original cortical bone (r² = 0.81–0.88).

In conclusion, tension stress/stain induced distraction osteogenesis represents an oriented continuum of primary gap healing, associated with enhanced bone formation likely resulting from rapid recruitment and activation of bone forming cells and increased surface area available for matrix deposition and mineralization. Ultrasound and DXA can detect bone formation and monitor mineralization process earlier than radiograph in leg lengthening procedures. The imaging findings are strongly correlated with histomorphometric measurement.

Disclosures: J.J. Zhao, None.

T060

PTH Enhances ATP Induced ERK1/2 Activation via a Competitive Mechanism Involving G-protein Coupled Receptor Kinase 2 in Osteoblastic Cells.

Y. Gu*, Y. Xing*, H. J. Donahue, J. You. Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, The Pennsylvania State University College of Medicine, Hershey, PA, USA.

In vivo studies show that parathyroid hormone (PTH) enhances mechanically induced bone formation. Previously we and other investigators found that osteoblastic cells responded to mechanical loading induced fluid flow by releasing ATP and activating the P2Y purinergic signaling pathway. In addition, in vitro studies demonstrated that co-activation of P2Y and PTH receptors leads to a profound synergism. However, the molecular mechanism of the synergistic effect of ATP and PTH in osteoblastic cells is unclear. P2Y and PTH receptors are both G-protein coupled receptors (GPCR), which are presumably desensitized by G-protein coupled receptor kinase (GRK). Thus, we hypothesized that PTH enhances ATP induced osteoblastic responses by inhibiting the desensitization of P2Y receptors via a competitive mechanism involving GRK. To test our hypothesis, we first employed osteoblastic MC3T3-E1 cells to examine ERK1/2 activation in response to ATP stimulation in the presence of PTH. Our results indicated that, after pretreatment with 50nM PTH, ATP not only increased the peak activation of ERK1/2 at 5 min, but also extended the activation to 10 min and 15 min compared to those without PTH, demonstrating the synergistic effect of ATP and PTH. Secondly, we over-expressed GRK2 in MC3T3-E1 cells and found that ERK1/2 activation at 5 min was significantly decreased compared with that of MC3T3-E1 cells over-expressing an empty vector (control) in the presence of PTH. In contrast, when we over-expressed a GRK2 dominant negative mutant, GRK2/K220R, in osteoblastic cells, the enhancement of ERK1/2 activation at 5 min was increased further compared with that of control MC3T3-E1 cells in the presence of PTH, suggesting that GRK2 is involved in the synergistic effect of ATP and PTH. Finally, PTH has been shown to induce ERK1/2 activation via PKA signaling pathways. Here we tested whether PKA signal pathways are involved in the synergistic effect by using a PKA activator, forskoline, instead of PTH. Our results showed that activating PKA signaling pathway by forskoline did not enhance ATP induced ERK1/2 activation. Furthermore, we demonstrated that PKA inhibitor did not suppress the synergistic effect of ATP and PTH in osteoblastic cells, suggesting that PKA activation is not required for the synergism. Taken together, our results demonstrate that it is not the PTH downstream signaling pathway (PKA), rather the regulation of PTH and P2Y receptors by GRK2 that is responsible for the synergism, suggesting a competitive mechanism involving GRK2 in MC3T3-E1 cells.

Disclosures: Y. Gu, None.

T061

Phosphorylation of Caveolin-1 in Response to Fluid Shear in Osteoblasts Is Mediated by P2×7 Activation.

S. Majumdar, K. Czymmek*, E. J. McLaughlin*, R. L. Duncan. Biological Sciences, University of Delaware, Newark, DE, USA.

The anabolic responses of bone to mechanical stimuli are triggered by cellular signaling events that occur within seconds and minutes of initiation of load. One of these early signaling events is the rapid release of ATP, and perhaps other nucleotides, that can bind to purinergic receptors that are essential for mechanotransduction. We have recently shown that the P2×7 receptor is central to the response of bone to exogenous loading, in vivo. We have further shown that binding of the P2×7 receptor activates an entry mechanism capable of cellular uptake of large solutes (>600 Da). Since P2×7 receptors have been associated with lipid rafts in the membrane, we postulated that nucleotide binding to the P2×7 receptor could induce endocytosis via caveolin activation. When 12 dynes/cm2 laminar shear was applied to MC3T3-E1 osteoblasts, we found that YO-PRO1, a 629 Da dye, was taken up by the cell within 5 min of the onset of shear, with peak uptake observed at 30 min. Extended application of shear demonstrated that YO-PRO1 uptake subsequently decreased at 45 min. When P2×7 receptors were activated in MC3T3-E1 osteoblasts by the addition of BzATP, the pharmacologic agonist of P2×7, a similar time course of YO-PRO1 uptake was observed. Western blot analysis of MC3T3 cells showed that static controls demonstrated little phosphorylation of caveolin-1 while application of fluid shear stress greatly increased the phosphorylation of caveolin-1. Time course studies demonstrated that phosphorylation of caveolin-1 peaked at 30 min and subsequently decreased at 60 min, which is correlates with our dye uptake experiments. Addition of BzATP also increased caveolin-1 phosphorylation implicating the activation of the P2×7 receptor in the phosphorylation of caveolin. Amino acid sequence analysis of the P2×7 receptor indicates a region which is specific for caveolin binding or interaction, further suggesting an interaction between caveolin and P2×7.

These data suggest that caveolin phosphorylation may be a direct function of P2×7 activation and that caveolin phosphorylation triggers an entry mechanism that is large enough to take in large molecules as seen by the dye uptake experiments. Caveolin has been suggested as a scaffold for protein-protein interactions for downstream signaling events. Thus, caveolin phosphorylation may be central to the cellular transduction of a mechanical signal.

Disclosures: S. Majumdar, None.

This study received funding from: NIAMS: AR051901.

T062

Glucocorticoids Stimulate Wnt Expression in Mature Osteoblasts.

W. Mak, H. Zhou, C. R. Dunstan, M. J. Seibel. Bone Research Program, ANZAC Research Institute, University of Sydney, Australia.

Differentiation of mesenchymal cells towards the osteoblastic lineage is governed by an array of different factors. Glucocorticoids (GC) have been shown to promote osteoblastic differentiation while Wnt signalling can promote osteoblastogenesis and inhibit adipogenesis. However, whether there is a direct relationship between these signalling pathways has not been fully established.

Transgenic (tg) overexpression of 11 beta-hydroxysteroid dehydrogenase type 2 (HSD2), a GC inactivating enzyme, under the control of a 2.3kb collagen type I promoter (Col2.3-HSD2) abrogates intracellular GC signalling in mature osteoblasts. We have previously demonstrated that calvarial cell cultures derived from Col2.3-HSD2 tg mice exhibit reduced osteoblastogenesis and retarded Wnt/ β-catenin signalling. Here, we have bred Col2.3-GFP mice with Col2.3-11ßHSD2 tg mice to generate Col2.3-HSD2-GFP tg and Col2.3-GFP WT mice. We investigated the GC-dependent regulation of Wnts in mature osteoblasts by isolating GFP positive cells using FACS to yield a population of mature osteoblasts. GFP-positive cells were subsequently grown in serum free media. Cultures were treated with a single-dose of 10–8M corticosterone for 0.5, 2, 4, 6 hours.

In GFP positive cell cultures derived from WT mice, Wnt7b mRNA expression was up-regulated 4-fold and 3.5-fold following 2 and 4 hrs of treatment, respectively, using real time PCR analysis. Wnt 10b mRNA expression was up-regulated 2.5-fold at 2 hrs, and 3-fold at 4 hrs, compared to controls. Up-regulation of Wnt7b and Wnt10b expression was blocked by the addition of cycloheximide, suggesting the requirement for de novo protein synthesis. 10–8M corticosterone had no effect on GFP-positive cells derived from Col2.3-GFP-HSD2 tg mice, with no change in the expression of either Wnt protein. In contrast to mature osteoblasts, enriched precursor cells population (first enzymatic release of the calvaria) derived from either Col2.3-GFP WT or Col2.3-GFP-HSD2 tg calvaria Wnt7b was not detected, while there was only low expression of Wnt10b.

We conclude that intact GC signalling in mature osteoblasts, in association with transcriptional regulatory complexes, is essential to stimulate Wnt protein expression and hence osteoblastic differentiation.

Disclosures: W. Mak, None.

This study received funding from: NHMRC.

T063

Prostaglandin E₂ Acts Through EP2/EP4 Receptors to Upregulate RGS2 Expression in Osteoblasts.

C. Nunn*, S. J. Dixon, P. Chidiac*. Physiology and Pharmacology, University of Western Ontario, London, ON, Canada.

Prostaglandin E₂ (PGE₂) can stimulate bone formation in vivo and in vitro. Osteoblasts express multiple subtypes of prostaglandin E₂ (PGE₂) receptors, which are coupled to Gq (EP1), Gs (EP2 and EP4) and Gi/o (EP3). RGS (regulators of G protein signaling) proteins suppress signaling via G protein-coupled receptors (GPCRs). Upregulation of RGS2 expression in osteoblasts mediates cross desensitization between receptors coupled to Gs and those coupled to Gq (J Biol. Chem. 281: 32684, 2006). Thus, RGS2 may play an important role in regulating PGE₂ signaling through its different receptor subtypes. The aims of this study were to determine the effects of PGE₂ on RGS2 expression in osteoblasts, and the receptor subtypes which mediate these effects. Primary mouse calvarial osteoblasts were treated with PGE₂ at different concentrations for multiple time points before extraction and quantification of mRNA using real-time RT-PCR. PGE₂ increased RGS2 expression in a time-dependent manner, which was maximal at 1 h (6 fold increase over control) and decreased to control levels within 6 h. This effect of PGE₂ was concentration-dependent, with an EC₅₀ of 2 μM and maximal effect at 10 μM. To determine the receptor subtypes involved, cells were treated with EP receptor subtype-selective agonists (10 μM) for 1 h. Agonists for EP2/EP4 receptor subtypes (butaprost, 11-deoxy-PGE₂) stimulated RGS2 expression to a similar level as PGE₂, whereas agonists selective for EP1 (17-Ph-ω-trinor-PGE₂) and EP3 (sulprostone) receptor subtypes had little effect on RGS2 expression. Furthermore, isoproterenol (which acts through Gs) dramatically increased RGS2 expression, whereas PGF_2α (which acts through Gq) had no effect. Taken together, these data establish that RGS2 is upregulated in osteoblasts by PGE₂, acting through its Gs-coupled receptors. PGE₂ has been shown previously to stimulate bone formation via both Gs-coupled and Gq-coupled receptor subtypes. Our findings suggest a mechanism for cross desensitization between Gs- and Gq-coupled receptors stimulated by the same agonist (PGE₂), in which activation of Gs leads to increased expression of RGS2, which in turn suppresses signaling through Gq.

Disclosures: C. Nunn, None.

This study received funding from: Canadian Institutes of Health Research.

T064

ATP Modulation of Mitogen-Activated Protein Kinases (MAPKs) and c-fos Expression in Osteoblastic and Breast Cancer Cells.

P. Scodelaro Bilbao*, S. Katz*, R. Boland, A. Russo de Boland*, G. Santillán*. Biologia, Bioquimica & Farmacia, Universidad Nacional del Sur, Bahia Blanca, Argentina.

We previously showed that ATP and UTP increase intracellular calcium concentration ([Ca²⁺]i) in MCF-7 breast cancer and osteoblastic ROS 17/2.8 osteosarcoma cells. The elevation of [Ca²⁺]i was due to Ca²⁺ release from inner stores through activation of the PI-PLC/IP₃ pathway. In addition, mechanical stimulation after ATP or UTP but not ADP induced a transient Ca²⁺ influx in both cell lines, suggesting that P2Y_2/4 receptor activation is required for this mechanical stress-activated Ca²⁺ (SAC) influx. Moreover, ATP-dependent SAC influx mediated the phosphorylation of mitogen-activated protein kinases (MAPKs) ERK1/2, p38 and JNK1 only in ROS 17/2.8 cells. In this work, we investigated the expression of P2Y_(1,2,4) subtype purinoceptors and the participation of PKC and Src family kinases in the activation of MAPKs by ATP in both cell lines. The involvement of ATP dependent SAC influx in the regulation of c-fos expression was examined. RT-PCR studies supported the expression of P2Y₂ subtype receptor in the osteoblastic and breast cells in the osteoblastic and breast cells, while P2Y₁ was not detected neither in MCF-7 nor ROS17/2.8 cells, and P2Y₄ was weakly expressed only in MCF-7 cells. The use of Ro 318220, a PKC inhibitor, showed that PKC participates in the phosphorylation of MAPKs by ATP in both cell lines. Otherwise, the use of PP1 or PP2, Src family kinases inhibitors, involved the participation of such kinases in the phosphorylation of MAPKs by ATP only in ROS 17/2.8 cells, although phosphorylation of Src (Tyr 416) was also observed in MCF-7 cells. Maximum levels of c-fos induction were observed after 30 min treatment with ATP in both cell lines, and the use of Gd³⁺, a SAC influx inhibitor, reduced its expression in ROS17/2.8 but not in MCF-7cells. These results show that P2Y₂ is the main receptor subtype involved in ATP-dependent SAC influx in osteoblasts and breast cells. Unlike MCF-7 cells, Src family kinases and SAC influx, respectively, participate in MAPKs activation and c-fos expression induced by ATP only in ROS17/2.8 cells.

Disclosures: A. Russo de Boland, None.

T065

Ubc9 Promotes Stability of Smad4 and Regulates BMP Signaling Pathway in Osteoblastic Saos-2 Cells.

K. Shimada¹, N. Suzuki², M. Maeno³, M. Eda*¹, K. Yamada*¹, K. Ito*¹. ¹Department of Periodontology, Nihon University School of Dentistry, Tokyo, Japan, ²Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan, ³Department of Oral Health Science, Nihon University School of Dentistry, Tokyo, Japan.

Bone morphogenetic proteins (BMPs) play a specific role in osteoblastic differentiation and maturation. BMPs induce receptor-regulated Smads (R-Smads). The R-Smads form complexes with the common-partner Smad, which is Smad4 in mammals. These complexes translocate and accumulate in the nucleus and regulate the transcription of various target genes. However, the function of Smad4 remains unclear. Here, we performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library from human bone marrow to identify the proteins interacting with Smad4. For this screen, full-length human Smad4 was cloned in pGBKT7 vector to express it as a fusion with the GAL4-binding domain, and the cDNA library was screened. Two cDNA clones of full-length Ubc9 were identified. To analyze the function of Ubc9 in the BMP signaling pathway, endogenous Ubc9 was disrupted in human osteoblastic cell line Saos-2 using a 19-nucleotide siRNA and examined its effectiveness in silencing Ubc9 expression. The gene expression of BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Osterix, was examined using real-time reverse-transcribed PCR and the protein levels of Smad4, Smad1 and phospho-Smad1 were determined with SDS-PAGE and Western blotting. Moreover, to observe whether Smad4 was sumoylated or not, Smad4 was overexpressed using pcDNA3.1 vector in Saos-2 cells and the sumoylated-Smad4 was observed with SDS-PAGE and Western blotting using anti-SUMO1 antibody, because Ubc9 was SUMO-conjugating enzymes E2. The efficiency of silencing Ubc9 mRNA level was approximately 70%. The expression of Runx2, Dlx5, Msx2, and Osterix mRNA was markedly inhibited when Ubc9 was silenced. The protein levels of Smad4 and phospho-Smad1 decreased dose-dependently and the level of Smad1 did not change by siRNA for Ubc9. The sumoylated-Smad4 was detected when Smad4 was overexpressed, and the sumoylated-Smad4 was inhibited by the addition of Senp2, SUMO-protease, was added. In conclusion, it is suggested that Ubc9 promotes stability of Smad4 and Smad4 is sumoylated. Ubc9 should be required the upregulation of BMP signaling pathway because the gene expression of the transcriptional factors and the protein levels of phospho-Smad1 inhibit by siRNA for Ubc9.

Disclosures: K. Shimada, None.

T066

Effect of Prostaglandin D₂ on Na-Dependent Phosphate Transport Activity in Osteoblast-like Cells.

A. Shogo*, S. Sekiguchi, A. Yokoyama*, K. Inagaki*, H. Kakizawa*, N. Hayakawa*, N. Oda*, A. Suzuki, M. Itoh*. Department of Internal Medicine, Fujita Health University, Aichi, Japan.

Prostaglandins (PGs) are well known to act as autocrine and paracrine factors in microenvironment including bone metabolism. Among them, PGD₂ has been reported to be produced by osteoblasts, and stimulates their proliferation, decreases both osteoprotegerin and RANKL and to induce osteoblast chemotaxis, suggesting the anabolic role of PGD₂ in bone metabolism. We have recently shown that Phosphate (Pi) transporter Pit-1 plays a critical role in the mechanism of the development of the extracellular mineralization of osteoblasts, and Pit-1 has been shown to be expressed in a subpopulation of hypertrophic chondrocyte. The aim of the present study is to investigate the effect of PGD₂ on Pi transport and its intracellular signaling mechanism in mouse calvaria-derived osteoblast-like MC3T3-E1 cells. PGD₂ time- and dose-dependently stimulated Pi transport in MC3T3-E1 cells. A protein kinase C (PKC) inhibitor calphostin C partially suppressed the stimulatory effect of PGD₂ on Pi transport. The selective inhibitors of mitogen-activated protein (MAP) kinases, such as ERK and p38, did not affect PGD₂-induced Pi transport. On the other hand, JUNK inhibitor, SP600125, markedly suppressed the enhancement of Pi transport by PGD₂. Both LY294002, a phosphatidylinositol (PI) 3-kinase inhibitors, attenuated PGD₂-induced Pi transport. A selective inhibitor of S₆ kinase, rapamycin, reduced this effect of PGD₂, while Akt kinase inhibitor did not. In summary, these results suggest that PGD₂ stimulates Na-dependent Pi transport activity in osteoblast-like cells. The mechanism responsible for this effect is not mediated by p38 MAP kinase or Akt kinase, but involves activation of PKC, Jun kinase, PI 3-kinase and S₆ kinase.

Disclosures: A. Shogo, None.

T067

AKT's Effects on β-catenin Regulate Osteoblasts' Responses to Mechanical Strain and PTH.

A. Sunters*, M. Muzylak*, G. Zaman*, V. J. Armstrong*, L. E. Lanyon, J. S. Price. Veterinary Basic Science, Royal Veterinary College, London, United Kingdom.

Two major influences controlling bone mass and architecture are mechanical loading and PTH. One of the pathways implicated in bones' adaptive responses to load is the canonical Wnt pathway. In vitro we have shown that exposing osteoblastic cells to dynamic strain inhibits GSK3β phosphorylation of β-catenin (βCat), thereby allowing its translocation to the nucleus where it regulates transcription. Since regulation of βCat via GSK3β inhibition can be controlled by AKT we hypothesised that this effect of strain may be mediated through AKT.

To explore this hypothesis the plastic strips onto which monolayer cultures of UMRI06 rat osteoblast like cells had been seeded were subjected to a short period of dynamic 4-point bending engendering levels of strain sufficient to stimulate proliferation. Western blotting demonstrated that this exposure to strain stimulated a transient increase in the expression of phosphorylated AKT (^pAKT) and phosphorylated GSK3β (inactive) as well as active (de-phosphorylated) βCat (^aβCat). Western blots of nuclear/cytoplasmic fractions showed that the strain-mediated nuclear translocation and activation of βCat were inhibited by pre-treatment with the phosphatidylinositol 3 (PI3)-kinase inhibitor LY294002. Both LY294002 and the AKT specific inhibitor Tricirebene also inhibited strain-related proliferation.

Treatment of UMR cells with high continuous concentrations of PTH (100nM) inhibited strain induced proliferation and reduced both basal and strain-induced levels of ^pAKT. PTH also induced the expression of the phosphatase MAP kinase phosphatase-1 (MKP-1) Since PTH signaling acts in part through protein kinase A (PKa), we treated UMR cells with the PKa inhibitor H89 alone, or in combination with PTH and/or strain. H89 treatment increased basal levels of ^pAKT and inhibited MKP-1 induction by PTH. H89 also resulted in a decrease in levels of total βCat. In strained cells treated with H89 did not influence the increase in ^pAKT but reduced total βCat levels, and thus failed to increase levels of activated βCat. However, a combination of H89, PTH and strain resulted in AKT phosphorylation as well βCat activation with no decline in total βCat levels. Taken together, these data suggest that PI3K/AKT signaling is a key mediator of the responses to at least two key influences on osteoblasts. Acting through regulation of βCat, AKT is integral to osteoblasts' responses to mechanical strain associated with bone formation. The responses of osteoblasts to high levels of PTH associated in vivo with resorption may be the result of PTH inhibiting AKT activation.

Disclosures: A. Sunters, None.

This study received funding from: Wellcome Trust.

T068

Regulation of RANKL Expression by Glucocorticoid in Human Osteoblast-like Cells.

F. Hirano, N. Maruyama*, K. Komura*, K. Okamoto*, Y. Makino*, M. Haneda*. Medicine, Asahikawa Medical College, Asahikawa, Japan.

Glucocorticoid-induced osteoporosis remains the most common secondary form of metabolic bone diseases. The soluble and transmembrane cytokines, RANKL, are produced by the osteoblast under hormonal and cytokine control and are essential for osteoclastogenesis and bone resorption. In contrast, osteoprotegerin is known to bind to RANKL and to block the interaction between RANKL and its receptor RANK. Glucocorticoid has been reported to induce RANKL mRNA expression in osteoblasts, although circulating soluble RANKL is reduced by glucocorticoid. The reasons of this discrepancy are still unknown. The purpose of this study was to clarify the regulation of RANKL expression by glucocorticoid in human osteoblast-like cells. We used human osteoblast-like cell line MG-63 and examined effect of glucocorticoid on RANKL expression. Quantitative real-time RT-PCR and ELISA methods revealed that 100 nM dexamethasone (DEX) significantly increased RANKL mRNA expression (15-fold, p<0.05) in MG-63 cells for a time-dependent manner and decreased soluble RANKL protein in supernatant, as expected. In addition, we found that DEX did not influence RANKL transcriptional activity by reporter gene assay using human RANKL promoter. Moreover, Treatment with actinomycin D and DEX markedly prolonged the half-life of RANKL mRNA in MG-63 cells, as compared to treatment with actinomycin D alone (T1/2 = over 24h v.s. 10h), presumably indicating that DEX-induced RANKL mRNA expression is due to the stabilization of RANKL mRNA. Next, we investigated effect of glucocorticoid on the expression of TNF converting enzyme (TACE), a known RANKL sheddase. The activity of TACE was dose-dependently reduced by DEX. Moreover, 100 nM DEX significantly decreased both mRNA- and protein-expression of TACE by quantitative real-time RT-PCR and ELISA, respectively. Furthermore, 100 nM DEX clearly induced transmembrane RANKL protein in MG-63 cells by flow cytometry and Western blot analysis, suggesting that DEX-reduced soluble RANKL protein expression in supernatant is associated with the decreases of TACE activity and protein expression in MG-63 cells. In conclusion, we presented glucocorticoid up-regulated RANKL mRNA expression via the stabilization of RANKL mRNA and reduced soluble RANKL expression via the decrease of TACE expression, possibly indicating that glucocorticoid-induced transmembrane RANKL expression in osteoblasts mainly played a pivotal role in osteoclastogenesis.

Disclosures: F. Hirano, None.

T069

Calcitriol Inhibits Marrow Stromal Cell Differentiation into Osteoblasts in a Mannner Cooperative with but Independent of TGF-beta.

D. Inoue, H. Ochiai*, R. Okazaki. Third Department of Medicine, University of Teikyo School of Medicine, Ichihara-shi, Chiba, Japan.

Vitamin D is essential to skeletal homeostasis, as impaired action of vitamin D, caused by either vitamin D deficiency or loss-of-function mutations of the vitamin D receptor (VDR), leads to rickets/osteomalacia. However, its direct effect on bone cells has not been fully understood. Recent in vivo studies in mice have shown that the absence of VDR has an anabolic effect on bone, suggesting that vitamin D, at least under physiological conditions, is a negative regulator of bone formation. In the present study, we examined effect of vitamin D on early mesenchymal cell differentiation in our previously established culture system in which a bipotential mouse marrow stromal cell line, ST-2, was induced by BMP-2 to differentiate into osteoblasts and adipocytes.

We found that calcitriol or 1,25-dihydroxyvitaminD3 (1,25D) inhibited the BMP-2-induced ALP activity in a dose-dependent manner with the maximal effect being an almost complete inhibition at 10 nM. A physiological level (0.1 nM) of 1,25D readily caused a significant ALP reduction. Interestingly, in the same culture system, 1,25D also inhibited the BMP-2-induced adipocytic differentiation as assessed by Oil-Red O-positive cell counts. These results indicate that 1,25D has a negative effect on the early phase of osteoblastic differentiation and that this effect is not a result of reciprocally enhanced adipogenesis.

Given the fact that TGF-beta counteracts the BMP stimulation of osteoblastogenesis and is able to modulate the VDR-dependent 1,25D action, we then tested a hypothesis that TGF-beta lies downstream of the anti-osteoblastogenic effect of 1,25D. As expected, TGF-beta inhibited the BMP-2-induced ALP activity in a dose-dependent manner. Suboptimal concentrations of 1,25D and TGF-beta showed additive effects. SB431542, a TGF-beta receptor kinase inhibitor, completely reversed the suppressive effect of TGF-beta, but showed virtually no effect on the effect of 1,25D. Luciferase assays using Id-1 promoter constructs revealed that TGF-beta, but not 1,25D, inhibited BMP-induced Smad-dependent transcription. We therefore conclude that 1,25D inhibits BMP-2-induced early osteoblastic differentiation in a manner cooperative with TGF-beta through a distinct mechanism. Elucidation of the molecular pathway by which 1,25D inhibits the BMP-induced mesenchymal cell differentiation into osteoblasts and adipocytes will not only help understand its physiological role in skeletal homeostasis but may also provide a theoretical basis for the development of novel selective VDR modulators with bone anabolic effects.

Disclosures: D. Inoue, None.

T070

CCN3/NOV Inhibits BMP-2-induced Osteoblast Differentiation by Interacting with BMP and Notch Signaling Pathways

T. Minamizato*¹, K. Sakamoto*¹, S. Nakamura*², A. Yamaguchi¹. ¹Section of Oral Pathology, Graduate School of Tokyo Medical and Dental University, Tokyo, Japan, ²Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Scienc, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan.

Several lines of evidence suggest that the CCN family of proteins is involved in regulation of mesenchymal cell differentiation. We demonstrated that CCN3/NOV associated with the Notch1 extracellular domain and inhibited myoblast differentiation through Notch signaling pathway. Further, we reported that Notch signaling is involved in BMP-2-induced osteoblast differentiation. These results prompted us to investigate the role of CCN3 in osteoblast differentiation and its involvement in the BMP and Notch signaling pathways. To explore whether CCN3 participates in regulation of osteoblast differentiation, we first overexpressed CCN3 and/or BMP-2 in MC3T3-E1 cells by adenoviruses. Transduction with CCN3 alone induced no apparent changes in the expression of osteoblast-related markers, whereas cotransduction with BMP-2 and CCN3 significantly inhibited the BMP-2-induced mRNA expression of Runx2, osterix, ALP, and osteocalcin. Immunoprecipitation-western analysis revealed that CCN3 associated with BMP-2. Compared to transduction with BMP-2 alone, cotransduction with BMP-2 and CCN3 attenuated the expression of phosphorylated Smad1/5/8 and the mRNA for Id1, Id2, and Id3, which are target molecules involved in the downstream of BMP signaling. Meanwhile, transduction with CCN3 activated Notch signaling pathway by increasing the expression of cleaved Notch1, an activated form of Notch1, and the mRNA expression of Hes1 and Hey1, the target genes of Notch in MC3T3-E1 cells. Overexpression of CCN3 also increased the promoter activities of Hes1 and Hey1 in C2C12 cells. Since Hey1/Hesr1 is reported to inhibit Runx2 promoter activity, we investigated the effects of CCN3 on osteoblast differentiation using Hey1-deficient cells isolated from calvariae of Hey1 knockout mice. The inhibitory effects of CCN3 on the expression of BMP-2-induced osteoblast-related markers were nullified in these cells. Finally, we explored the expression levels of mRNA for CCN3 and proteins for phosphorylated Smad1/5/8 and cleaved Notch1 during bone regeneration in mice. All of these were highly upregulated during bone regeneration. Collectively, the present study indicate that CCN3 exerts inhibitory effects on BMP-2-induced osteoblast differentiation by interacting with the BMP and Notch signaling pathways, and these interaction might be involved in the process of bone regeneration.

Disclosures: T. Minamizato, None.

T071

High Mobility Group Proteins (HMGs) Coupled with TNF_α Production Are Essential for Osteoclastogenesis.

A. Brebene*¹, K. Yamoah*¹, A. Arabi*¹, H. Amano², G. Dolios*³, R. Wang*³, R. Yanagisawa*¹, E. Abe¹. ¹Endoctinology, Mt. Sinai School of Medicine, New York, NY, USA, ²Pharmacology, School of Dentistry, Showa University, Tokyo, Japan, ³Genetics & Genomics Sciences, Mt. Sinai School of Medicine, New York, NY, USA.

We previously have shown that TSHR (Thyroid stimulating hormone receptor) null mice exhibit osteoporosis due to increased osteoclast formation stemming from TNF_α overproduction in osteoclast progenitors. Since the enhanced osteoclast formation in the TSHR null mice was normalized in TSHR and TNF_α double null mice, TNF_α is concluded to be a crucial factor for osteoporosis in TSHR deficiency. We next examined in detail the regulatory mechanisms of TNF_α transcription by identifying the responsive sequence of the murine TNF_α promoter and the associated transcription factor. HMG2 (high mobility group 2) was identified by mass spectrometry to bind to a specific sequence of the TNF_α promoter and its expression level was increased by IL-1/TNF_α, or by RANKL treatment in which TNF_α expression was also stimulated. HMG2 in addition to HMG1 not only localized in the nucleus but was also secreted, and stimulated cytokine expression (IL-1, IL-6 and TNF_α) and RANKL-induced osteoclast formation in a positive feedback manner. As HMG1 or HMG2 expression was suppressed by siRNA, TNF_α and TRAP expression were significantly reduced. We also confirmed the parallel expression of HMGs and TNF_α in osteoclast progenitors from TSHR^−/− and TSHR^+/− mice, which overexpress TNF_α, and TNF_α^−/− mice, which underexpress it. These results suggest that the newly identified HMGs are crucial factors in the regulation of TNF_α expression and osteoclast formation under pathogenic conditions.

Disclosures: A. Brebene, None.

This study received funding from: NIH.

T072

The Suppressive Effect of IL-27 in Osteoclastogenesis.

M. Furukawa¹, H. Takaishi*¹, S. Sakai*¹, M. Yoda*¹, H. Takayanagi*², H. Yoshida*³, Toyama*¹. ¹Orthopaedic, Keio university, Tokyo, Japan, ²Department of Cellular Physiological Chemistry, Tokyo Medical Dental University, Tokyo, Japan, ³Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga, Japan.

In recent years identification of cytokine of interleukin-12 family advanced, and it became clear to have different point of action depending on a differentiation stage of Th1. Among them, IL-27 is produced from antigen presenting cells and is viewed as inducing initial-phase Th1 differentiation by mediating receptor WSX-1/GP130 as EBI-3 and p28 heterodimers. Bone metabolism and the immune system have a correlative relationship, and both are controlled by various common cytokines in excessive osteoclastogenesis caused by inflammatory diseases, such as autoimmune arthritis. We examined the osteoclastogenesis and participation to Th17 for the purpose of clarifying a role of IL-27 in the inflammatory bone destruction. CD4+T cells were isolated by magnetic cell sorting from mouse spleen cells and were induced to Th17 using the anti-IL-4, anti-IL-12, anti-IFN_-γ antibody, and IL-23 after stimulation with immobilized anti-CD3, anti-CD28 antibody. IL-17 expression of Th17 markedly increased by PMA+Ionomycin stimulation, and the stimulation of IL-27 restrained production of IL-17 in induction process from Naive CD4+T cells to Th17. With human and mouse bone marrow macrophages, the expression of p28 was strongly induced by LPS (100ng/ml) stimulation, but no changes were observed in WSX-1. In the mouse primary osteoblasts, IL-27 did not affect for the expression of RANKL, which was induced by D3+ PGE2 or IL-17 stimulation. Human CFU-GM was collected by incubating bone marrow obtained through prosthetic joint replacement surgery in a methylcellulose culture containing GM-CSF and IL-3. In the differentiation process induced by M-CSF+RANKL, IL-27 strongly inhibited the osteoclastogenesis of human CFU-GM in a dose dependent manner. (100ng/ml, 85%). Our study revealed that IL-27 directly inhibited osteoclastogenesis of human CFU-GM. The suppressive effect of IL-27, however, is not selective for IL-17 production in the induction process of Th17, but appears to cover a broad group of proinflammatory cytokines. Therefore, we assume that IL-27 can be used to respond temporally or spatially to the clinical state of an autoimmune disorder, and thus to react with versatility to inflammatory bone destruction.

Disclosures: M. Furukawa, None.

T073

Cascade of Gene Expression Indicates Chemokine MCP-1 Is Essential for Human Osteoclast Formation.

R. M. S. Granfar*, A. S. Stephens*, S. R. J. Stephens*, C. J. Day*, N. A. Morrison.. Health Science, Griffith University, Gold Coast, Australia.

The chemokine MCP-1 (CCL2) is essential, in human and mouse, for the formation of foreign body giant cells (FBGC), a multinucleated macrophage cell type similar to osteoclasts. We tested the hypothesis that MCP-1 is necessary for human osteoclast differentiation. On comparing chemokine induction by RANKL with that elicited by LPS or TNF_-α, where numerous chemokines are strongly induced, it appeared that RANKL induced a restricted repertoire of chemokines in osteoclasts. MCP-1 and MIP-β (CCL4) were strongly induced by RANKL in human osteoclast progenitors within 24 hours (1200 fold and 40 fold respectively). Other chemokines (MIP_-α, RANTES and CCL1) peaked later or were not as potently induced. MCP-1 receptor, CCR2b, was also strongly induced by RANKL (500 fold). As CCR2b expression increased through time, MCP-1 mRNA expression decreased, suggesting a feedback autocrine loop in osteoclast precursors.

A truncated form of MCP-1 with 7 amino acids removed (7ND) acts as dominant negative and blocks FBGC formation in mouse and human. Inhibition of MCP-1 autocrine loop signalling using both neutralising antibody and dominant negative MCP-1 (7ND) dramatically suppressed RANKL mediated osteoclast formation. Neutralising antibody to MCP-1 (4μg/mL) reduced osteoclast formation from 240±16 per cm² (control) to 188±17 (p=0.01) while 7ND (50ng/mL) suppressed osteoclast number to 21±1 per cm² (p=4.8×10⁻⁸). Not only did 7ND reagent inhibit osteoclast differentiation, it also suppressed bone resorption activity of purified mature human osteoclasts, reducing dentine resorption from 81±3 to 18±3 pits per dentine slice (p=0.0002). The presence of 7ND prevented early RANKL mediated induction of JUN, suggesting that the MCP-1 autocrine loop is necessary for osteoclast transcription factor induction. Following the early 24 hour peak of induction of MCP-1 induction, calmodulin-1 was maximally induced at three days exposure to RANKL. Transcription factors NFAT1 and NFAT2 (thought to be essential for osteoclast formation) reached peak induction six days after MCP-1 induction.

The fact that 7ND blocked mature osteoclast bone resorption indicates the surprising fact that chemokine signalling may be required for on-going bone resorption. Indeed, treating mature osteoclasts with MCP-1 enhanced bone resorption activity. Taken together, the data suggest that a highly specific assemblage of inflammatory chemokines is necessary for osteoclast differentiation and that continuous chemokine signalling is required for bone resorption.

Disclosures: R.M.S. Granfar, None.

T074

G-CSF Enhances Osteoclast Differentiation and Function.

R. M. S. Granfar*, C. J. Day*, N. A. Morrison.. School of Medical Science, Griffith University, Southport, Australia.

Osteoclasts (OCs) are critical protagonists for the mobilization of the endosteal niche of hematopoietic stem cells. Granulocyte Colony Stimulating Factor (G-CSF) is a widely applied stem-cell mobilization treatment with clinically documented risk of mild to severe bone loss. If OC activity is required for stem cell mobilization, then G-CSF and potentially other stem cell mobilizers should enhance OC differentiation and bone resorption.

The standard human mononuclear cell model of OC formation was used. Cells were cultured in MEM supplemented with 25ng/ml M-CSF. In these conditions, RANKL (20ng/ml) treatment results in OC formation in 14 days. OCs differentiated on collagen coated plates were purified over serum gradients to select for giant cells and then this fraction plated onto sperm whale dentine for bone resorption assays. Recombinant G-CSF (Peprotech) was used at doses up to 25 ng/ml. Markers of OC maturity such as dentine-resorption, multi-nucleation, as well as gene expression and protein presence of tartrate resistant acid phosphatase (TRAP), were analysed.

G-CSF treatment had strong effects on OC biology. As well as stimulating bone resorption activity in mature OCs, G-CSF had an early strong effect on OC size and number. Treatment with RANKL and G-CSF resulted in significantly larger OCs by 7 days when compared to RANKL treated cells (p=0.0004). Once RANKL treated OCs had matured (at 14 days) they were almost the same size as OCs treated with RANKL and G-CSF, suggesting that exogenous G-CSF accelerates early OC formation. RANKL and G-CSF increased the TRAP+ multi-nuclear cell count when compared to RANKL treated cells (7d p=0.00005 and 14d p=0.013). Using real time PCR analysis, TRAP mRNA content was significantly greater were in G-CSF treated OCs, consistent with the increased number of TRAP positive cells compared to control treatment with RANKL (p=0.007). Continual treatment with RANKL and G-CSF over 21 days significantly increased bone resorption activity measured by pits on dentine slices relative to OCs treated with RANKL alone (p=0.012). Similarly, addition of G-CSF significantly stimulated bone resorption in mature purified OCs that had no prior treatment with G-CSF, suggesting that G-CSF receptor is present on mature OCs. Gene array showed RANKL induced G-CSF receptor in OCs (12 fold), consistent with the hypothesis that OCs respond to G-CSF as a result of RANKL mediated induction of G-CSF receptor.

In summary, G-CSF increases osteoclast bone resorption, TRAP expression, cell-size and multinucleation. The osteopenic effect of G-CSF therapy and endosteal hematopoietic stem cell mobilization may be regulated by the same phenomenon; G-CSF stimulation of osteoclast differentiation and bone resorption.

Disclosures: R.M.S. Granfar, None.

This study received funding from: Griffith University.

T075

M-CSF Stimulates Bone Resorbing Activity of Mature Human Osteoclasts Via Increased Activation of AP-1 and NFκB.

J. M. Hodge*, M. A. Kirkland*, G. C. Nicholson. Clinical and Biomedical Sciences: Barwon Health, University of Melbourne, Geelong, Australia.

The critical role of M-CSF in the differentiation of osteoclasts (OC) is well established. In mature OC, M-CSF has been reported to regulate survival and motility, but no clear role in resorptive function has been established. In this study we investigated the role of M-CSF on resorbing activity of mature human OC.

Human OC were generated by treatment of CFU-GM-derived precursors with soluble hRANKL (125ng/mL) and hM-CSF (25ng/mL) for 14–21d. To quantify resorbing activity, mature OC were dissociated from the plastic substrate using a non-enzymatic buffer, settled onto dentine slices and cultured for 72h. Activation of NFκB and AP-1 was assessed using a sandwich ELISA technique. IκBα and ERK were assessed by Western analysis.

Neither M-CSF nor RANKL were required for mature OC survival over 72h. However, resorption function was absolutely dependent on the addition of RANKL. Co-treatment with M-CSF had a concentration-dependent biphasic action, greatly augmenting RANKL-induced resorbing activity (+181%) and actin ring formation (+131%) at 25ng/mL. RANKL alone activated NFκB (+554%). M-CSF had no effect on NFκB but potentiated RANKL-induced activation (+42%). In contrast, RANKL alone had no effect on AP-1 activation whereas M-CSF stimulated weakly (+63%) and the combination produced a synergistic effect (+171%). M-CSF activated phosphorylation of ERK 1/2, peaking at 5 min, whereas RANKL had no effect alone or in combination with M-CSF. M-CSF had no effect on IκBα phosphorylation whereas RANKL alone stimulated, with no additional effect in combination with M-CSF. Time-lapse video-microscopy showed no apparent effect of M-CSF on the motility of OC co-treated with RANKL.

We have demonstrated that M-CSF is not required for survival of mature human OC, but it does potently modulate RANKL-induced resorbing activity via NFκB and AP-1 activation. The molecular mechanism of this effect remains to be determined although we propose a convergence of RANK and fms signaling (“cross-talk”) upstream of the critical OC transcription factors NFκB and AP-1. These findings have important implications for the clinical management of diseases associated with excess bone resorption.

Disclosures: J.M. Hodge, None.

T076

The Effects of IL-12 Related Cytokines, IL-23 and IL-27, on Osteoclast Formation and T Cell Properties.

S. Kamiya*¹, T. Fukawa*¹, C. Nakamura*², T. Yoshimoto*³, S. Wada¹. ¹Clinical Sciences, Josai International University, Chiba, Japan, ²Bio-analytical Chemistry, Josai International University, Chiba, Japan, ³Intractable Immune System Disease Research Center, Tokyo Medical University, Tokyo, Japan.

A number of evidence has shown that bone remodeling process was affected by host immune reactions. Above all, cytokines which could modulate T cell functions were much focused in this area. Interleukin (IL) −23 and IL-27, IL-12-related cytokines, have been shown to be involved in chronic inflammatory diseases by modulating T cell function and to differentiate naive CD4+ T cells to type 1 helper T. Here, we examined the effects of these cytokines on osteoclast (OC) formation induced by M-CSF/RANKL in the presence of T cells. Mouse Bbone marrow macrophage like cells (BMMs) were prepared through incubating bone marrow cells with M-CSF, and were cocultured with activated T cells, which were partially purified from isolated splenocytes by adhesion properties and were stimulated by anti-CD3 antibody (Ab).

When T cells were activated by 0.2 μg/ml anti-CD3 Ab, and were cocultured with BMMs in the presence of M-CSF/sRANKL, the number of OC was reduced compared with that of co-cultures with innate T cells. The addition of IL-23 and IL-27 in this culture much strongly and significantly inhibited OC formation. Treatment with these cytokines alone also reduced OC formation minimally even in the absence of activated T cells. To understand this phenomenon more precisely, we explored possible factors which could be produced by the activated T cells and modulate OC formation. Although the production of TNF_α, IL-4, and IFN_γ, detected by ELISA, was not changed in T cells treated with IL-23 or IL-27, the production of IL-10 was increased by ∼200% in T cells treated with IL-27 these interleukins. This was not the case for IL-23. In this process, we found that Ttreatment with IL-27 also significantly reduced sRANKL production in activated T cells. Furthermore, through FACS analysis revealedwe found that treatment with IL-27 suppressed the expression of membrane-bound RANKL on the T cells. These effects of IL-27 could not be found in osteoblasts when stimulated by IL-6. The effects of IL-23 are now under investigation.

In this study, we found that IL-23 and IL-27 inhibited OC formation when cocultured with activated T cells. The effects of these interleukinsIL-27 would be associated with modified cytokine-expression profile of the T cells. The findings observed in this study would suggest that IL-12 related cytokines may play crucial roles for OC formation in autoimmune-related skeletal diseases.

Disclosures: S. Kamiya, None.

T077

Endogenous TNF_α in Osteoclast Precursor Cells Promotes Osteoclastogenesis Via c-Fos and NFATcl Activation.

A. Nakao¹, H. Fukushima¹, H. Kajiya¹, F. Okamoto*¹, S. Ozeki*², K. Okabe¹. ¹Physiological Science & Molecular Biology, Fukuoka Dental College, Fukuoka, Japan, ²Oral & Maxillofacial Surgery, Fukuoka Dental College, Fukuoka, Japan.

Although TNF_α is well known to be an important factor for bone resorption, particularly in inflammatory bone diseases, the relevance of RANKL and TNF_α in osteoclastogenesis remains unclear. In this study we examined the mechanism of TNF_α induced osteoclastogenesis and its downstream signaling using RT-PCR, ELISA, Western blotting and luciferase activity assay. We show that osteoclastogenesis is suppressed by anti-TNF_α- and anti-TNF receptor type I (TNFRI)-antibodies and in TNF_α- and TNFRI-deficient mice using in vitro culture systems: (1) co-culture of mouse spleen derived osteoclast precursor cells (pOCs) with osteoblasts, (2) pure pOC culture and (3) RAW264.7 cells in presence of RANKL. In co-culture with TNF_α- or TNFRI-deficient osteoblasts and wild type (WT) mouse pOCs, the number of osteoclasts was always reduced compared to that in combination with WT osteoblasts and WT pOCs. When pure pOCs without osteoblasts or RAW 264.7 cells were cultured in the presence of RANKL both TNF_α and TNFRI neutralizing antibodies significantly inhibited RANKL-induced osteoclastogenesis. In contrast, addition of TNF_α increased RANKL-induced osteoclastogenesis in both culture system. Furthermore, fewer osteoclasts were formed in co-culture with WT osteoblasts and pOCs derived from TNF_α- and TNFRI-deficient mice. The number of osteoclasts was recovered by addition of TNF_α into cultures of TNF_α-deficient pOCs, but not TNFRI-deficient pOCs. We also found that RANKL directly induced TNF_α expression and secretion in pOCs and RAW264.7 cells in a time-dependent manner. Endogenous TNF_α in pOCs induced c-Fos and NFATcl. Expression rates of NFATc1 and c-Fos were significantly delayed and decreased in TNF_α- and TNFRI-deficient pOCs compared to WT pOCs during osteoclastogenesis. These results indicate that TNF_α is induced by RANKL in pOCs and serves as an autocrine factor promoting osteoclastogenesis through c-Fos and NFATc1 activation.

Disclosures: A. Nakao, None.

T078

IL-23-Enhanced Osteoclastogenesis Are Mediated by STAT3 and NF-κB Via Upregulation of RANKL from CD4+T cells.

S. Park*, J. Ju*, K. Kang*, I. Kim*, H. Kim*. Internal Medicine, Division of Rheumatology, Collage of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.

Object. IL-23 is a new proinflammatory cytokine which stimulates T cells to subsequently produce inflammatory molecules, resulting in inflammatory arthritis. This study was undertaken to explore the role of IL-23 in CD4T cells on RANKL expression and osteoclastogenic activity.

Method. Joint cells, CD4⁺T cells and Fibroblast like synoviocytes (FLSs) were isolated from IL-1Ra-/-mice. The level of RANKL mRNA were measured with realtime PCR after these cells were treated with various cytokines including IL-23, in combination with inhibitors of the intracellular signal pathway. Osteoclasts precursor cells were cocultured with CD4⁺T cells in the presence of IL-23 and subsequently stained for tartate-resistant acid phosphatase (TRAP) activity. IL-1 Ra^−/− mice were injected intra-articularly with a recombinant adenovirus expressing mouse IL-23.

Results. Expressions of IL-23, IL-17 and IL-1β were increased in arthritic joints of IL-1Ra-/- mice. Coexpression of IL-23, CD4⁺T cells and RANKL in pannus of arthritic joints was revealed by immunohistochemical staining. IL-23 increased expression of both RANKL in the mixed joint cells of IL-1Ra-/-mice. IL-23 enhances the expression of RANKL in CD4⁺T cells. TRAP-positive osteoclastogenesis was enhanced by IL-23-stimulated CD4⁺T cells. Interestingly, IL-23 had the potential to induce osteoclastogenesis in itself without the help of CD4⁺T cells. In vivo, intraarticular injection of adenoviral vector carrying IL-23 accelerated the severity of arthritis in IL-1Ra-/-mice and effected high expression of RANKL at inflammatory joints.

Conclusion. IL-23 induces not only joint inflammation but also bone destruction via RANKL expression in arthritic CD4⁺T cells. The various effects of IL-23 in CD4⁺T cells have an important clinical impact in the view point of joint destruction. The interactions among IL-23, CD4⁺T cells, osteoclasts and RANKL expression may be a potential therapeutic approaches in treating bone destruction in inflammatory diseases.

Disclosures: S. Park, None.

T079

Regulation of Human Osteoclast Precursor Generation by Type II Cytokines and STATs.

K. Park-Min*¹, M. Humphrey*², L. B. Ivashkiy*¹. ¹Arthritis and Tissue Degeneration program, Hospital for Special Surgery, New York, NY, USA, ²VAMC, Oklahoma City, OK, USA.

The first step in osteoclastogenesis is M-CSF-mediated generation of osteoclast precursors (pOC) that are responsive to RANKL stimulation from bone marrow progenitors or monocytes. The purpose of this study was to determine if type II cytokines that suppress osteoclastogenesis (IFN_-γ, IFN_α/β, IL-10) inhibit human pOC generation and to explore underlying mechanisms. pOC were generated from human monocytes (and in some experiments from murine bone marrow cells) using M-SCF in the presence or absence of IFN_-α, IFN_-γ, or IL-10, and stimulated with RANKL. OC formation was analyzed by staining for TRAP positive multinucleated cells, OC gene expression was analyzed using real time PCR, and RANK-induced signal transduction was analyzed using immunoblotting. RNA interference and retroviral transduction were used to study the function of OC genes, and the role of Stat1 and Stat3 were investigated using cells from knockout mice. The generation of human pOC was associated with striking increases in expression of RANK and the key costimulatory receptor TREM2. Subsequent RANKL-induced osteoclastogenesis was strongly suppressed by IL-10, IFN_-γ, and IFN_-α, with concomitant suppression of OC-specific genes such as TRAP and cathepsin K. TREM2 expression in pOC and OC was essentially completely abrogated by IL-10 and IFN_-γ; IFN_-γ and IFN_-aL also suppressed RANK expression. The effects of IL-10 were partially reversed by forced expression of TREM2. IL-10 also suppressed the proximal RANK signaling pathway. Type II cytokines suppress pOC generation by inhibiting expression of TREM2 and RANK. In addition, IL-10 suppresses pOC function by inhibiting signaling responses to RANKL by a mechanism that differs from the previously reported degradation of TRAF6 by IFN_-γ. These results yield insights into the molecular regulation of initial steps of osteoclastogenesis by type II cytokines.

Disclosures: K. Park-Min. None.

T080

Skeletal Muscle-specific Overexpression Pregnancy-associated Plasma Protein-A (PAPP-A) Increases Bone Acretion in Mice.

J. Wergedal¹, M. Rehage*², D. Phang*², W. Xing², B. Bonafede*², D. Hou*², S. Mohan¹, X. Oin*¹. ¹Department of Medicine, Loma Linda University, Loma Linda, CA, USA, ²Musculoskeletal Disease Center, J. L. Pettis Veterans Affairs Medical Center, Loma Linda, CA, USA.

We have recently reported that osteoblast-specific overexpression of PAPP-A, a protease for IGF binding protein-2, −4 and −5, increased bone size and bone formation rate in mice. On the other hand, somatic growth was not significantly enhanced, which is consistent with the lack of PAPP-A transgene expression in other non-skeletal tissues and the lack of detectable PAPP-A in circulation. In this study we tested the hypothesis that an increase in PAPP-A concentration in blood, as occurring during human pregnancy, would exert an anabolic effect on bone. Overexpression of PAPP-A in skeletal muscle using the human skeletal muscle alpha actin promoter significantly increased skeletal muscle mass at an age of 10 weeks. The PAPP-A transgene was highly expressed in muscle, leading to a significant increase in PAPP-A concentration and IGFBP-4 proteolysis in the blood. Conversely, PAPP-A transgene mRNA was not detected in bone. Compared with the wild-type littermates, transgenic mice exhibited a significant increase (P<0.01) in femur total bone content (26% +/-4), cortical bone content (28.1+/-6), and cortical bone area (24%+/-5) determined by pQCT, calvarial BMD (29%+/-6) measured by DEXA, and calvaria thickness (39%+/-6). In conclusion, 1) an increase in PAPP-A concentration in circulation can enhance bone accretion, 2) systemic administration of PAPP-A may be of clinical interest for the treatment of osteoporosis.

Disclosures: X. Qin, None.

This study received funding from: VA.

T081

IL-33 Inhibits Osteoclast Formation Indirectly Through Osteoblastic Cells.

H. Saleh*, J. M. W. Ouinn, T. J. Martin, M. T. Gillespie.. Bone, Joint and Cancer Group, St. Vincent's Institute, Fitzroy, Australia.

IL-33 is a proinflammatory factor that induces Th2 cytokine production and is produced by endothelial cells in rheumatoid arthritis. Its receptor is ST2, and a natural soluble form of ST2 has anti-inflammatory actions. Since IL-33 is related to IL-18, an inhibitor of osteoclast (OC) formation, we investigated the actions of IL-33 on osteoclastogenesis in vitro.

We studied IL-33 effects on murine OC formation from the following cell populations that contain haemopoietic progenitors: bone marrow cells, M-CSF dependent non-adherent bone marrow macrophages (BMM), spleen cells and RAW264.7 cells. OC formation from these cells was stimulated either by treatment with RANKL (100ng/ml) plus M-CSF (30ng/ml), or by co-culture with 1,25 dihydroxyvitamin D3-stimulated osteoblastic cells. In RANKL/M-CSF stimulated cultures, IL-33 dose dependently inhibited OC formation from bone marrow cells and spleen cells, with a maximal effect at 20ng/ml. However, these populations contain large numbers of non-hematopoietic cell types such as stromal cells and lymphocytes. OC formation in cultures of RAW264.7 cells or BMM was not inhibited by IL-33, suggesting that IL-33 does not act directly on these two types of immature myelomonocytic cells to block differentiation. In co-cultures of bone marrow, spleen and BMM populations with osteoblastic cells (primary calvarial osteoblasts or Kusa O osteoblastic cell line), dose dependent inhibition of OC formation by IL-33 was observed with a maximal effect also at 20ng/ml. Since OC formation from RANKL-stimulated BMM was not inhibited by IL-33, this suggests an indirect inhibitory action through the mediation of osteoblastic cells. To investigate whether this involved the induction of a soluble osteoblast-derived inhibitor we co-cultured Kusa O cells and BMM separated by a porous membrane in the presence of 1,25 dihydroxyvitamin D3. IL-33 inhibited OC formation in these cultures even when exogenous RANKL and M-CSF were also added suggesting the inhibition is not mediated by osteoprotegerin (OPG).

Our results indicate that IL-33 is a novel OC inhibitor that acts on osteoblastic stromal cells to induce a soluble inhibitor of OC formation that is not OPG. This points to a significant role for IL-33 and, by extension, its decoy receptor ST2 in bone metabolism.

Disclosures: H. Saleh, None.

T082

Sonic Hedgehog in Fracture Repair and Osteoclast Formation.

T. Shimo*¹, T. Yuasa², S. Isowa*¹, S. Ibaraki*¹, M. Enomoto-Iwamoto*², A, Sasaki*¹, A. Yamaguchi*¹, M. Pacifici², M. Iwamoto². ¹Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan, ²Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA, USA.

Sonic hedgehog (Shh) was originally identified as a critical factor in antero-posterior skeletal patterning in developing limb and has since been found to influence other essential developmental processes. In previous studies we found that Shh and other hedgehog proteins are transiently expressed in experimental bone fractures, indicating that the factors may be important in skeletal healing and remodeling. To test this hypothesis, we first monitored hedgehog gene expression in a mouse costal bone fracture model and then tested hedgehog protein functions. RT-PCR analysis showed that gene expression of Shh, hedgehog receptor Patched (Ptch) and its signal transducer Gli1 was strongly and coordinately up-regulated within 12h after fracture and lasted up to 48h. Up-regulation of family member Indian hedgehog (Ihh) was detected after 72h. In situ hybridization revealed that Shh transcripts were broadly distributed within the fracture site field by 6h, indicating that Shh expression is part of an acute response mechanism mounted by several local cell types. To gain insight into the nature of the cells responding to Shh, we created rib fractures in hedgehog reporter mice that harbor β-galactosidase linked to the promoter regions of the Ptch gene, a direct target of hedgehogs. Reporter activity was prominent and well distributed throughout the bone marrow of the fractured bone by 24h. Interestingly, reporter activity was particularly intense in many TRAP-positive and multinucleated cells, suggesting that hedgehog signaling is activated during formation and/or function of osteoclasts. To test this possibility directly, bone marrow-derived osteoclast progenitor cell populations were isolated from 5 week old mice and cultured in the presence or absence of exogenous recombinant Shh and/or RANKL. While Shh itself did not induce osteoclast formation in the absence of RANKL, it markedly enhanced osteoclast formation in RANKL-treated cultures as determined by TRAP staining and dentin pit formation assay. The stimulatory effects of Shh on RANKL-induced osteoclast formation were also observed in CD11b(+) and RAW264.7 cell cultures, were inhibited by treatment with hedgehog pharmacological inhibitor cyclopamine, and failed to occur in primary cultures of bone marrow cells lacking the hedgehog signaling receptor Smoothened. The results of the study provide clear evidence that hedgehog signaling is rapidly activated during bone fracture repair and could be part of mechanisms enhancing bone marrow progenitor cell function and, in particular, osteoclast differentiation and action.

Disclosures: T. Shimo, None.