ASBMR 29th Annual Meeting

T478

Diagnosis of Fibrodysplasia Ossificans Progressiva (FOP) Prior to the Onset of Heterotopic Ossification. M. Xu*¹, A. Ganguly*², F. S. Kaplan¹, E. M. Shore¹, ¹Orthopaedics, University of Pennsylvania, Philadelphia, PA, USA, ²Genetics, University of Pennsylvania, Philadelphia, PA, USA.

Fibrodysplasia ossificans progressiva (FOP) is a rare and disabling genetic condition characterized by congenital malformation of the great toes and by progressive heterotopic ossification in specific anatomic patterns. Most patients with FOP are misdiagnosed early in life prior to the appearance of heterotopic ossification and undergo diagnostic procedures that can cause lifelong disability. The genetic cause of FOP was recently identified and genetic testing for FOP is now available. We recently evaluated seven children for diagnosis of FOP prior to the onset of heterotopic ossification. A medical history, physical examination, and skeletal survey were obtained on all patients, as well as clinical genetic testing for the canonical FOP mutation. All seven children (four females, three males; ages three months to six years) had congenital malformations of the great toes, but none had radiographic evidence of heterotopic ossification at the time of evaluation. Five of the seven children had soft tissue lesions of the neck and back suggestive of early FOP flareups, three of whom had undergone invasive diagnostic procedures that exacerbated their condition. Two children had no history or signs of soft tissue swelling or flareups. DNA sequence analysis found that all seven children had the recurrent FOP missense mutation, a single nucleotide substitution (c.617G>A) at codon 206 in the glycine-serine activation domain of activin receptor IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor. This study demonstrates that clinical suspicion of FOP early in life on the basis of malformed great toes can lead to early clinical diagnosis, confirmatory diagnostic genetic testing, and the avoidance of further harmful diagnostic and treatment procedures.

Disclosures: E.M. Shore, None.

T479

Teriparatide Therapy in Hajdu-Cheney Syndrome. P. J. Tebben, R. D. Tiegs, B. L. Clarke.. Endocrinology, Mayo Clinic, Rochester, MN, USA.

To describe the response of two patients with Hajdu-Cheney Syndrome (HCS) to therapy with teriparatide.

The clinical, biochemical, and radiographic data of two patients with HCS are provided. Case 1: A 55 year-old female was referred for evaluation and management of osteoporosis and HCS. Clinical findings included progressive acro-osteolysis, short stature, delayed closure of several cranial sutures, enlarged sella turcica, premature loss of teeth, low bone density, and fractures. During a 2 year course of conjugated equine estrogen (0.625 mg/day) and alendronate (70 mg/week) her bone mineral density (BMD) at the spine (L2–L4) declined by 8.2% and at the left hip declined by 3.5%. Serum calcium, phosphorus, creatinine, PTH, and alkaline phosphatase were within the reference range.

Due to declining BMD and vertebral fractures, teriparatide 20 mcg daily for 2 years was substituted for the alendronate. A 12.4% and 4.2% improvement was seen in the spine and left hip, respectively, over the initial 6 months of treatment followed by subsequent decrements in BMD at both sites. She also sustained a left tibial fracture after one year of teriparatide. Acro-osteolysis in her hands progressed while on teriparatide, a process that continued after substituting intravenous zoledronic acid (ZA) for teriparatide. Her BMD continued to decline on ZA.

Case 2: A 50 year old male was referred for evaluation of progressive acro-osteolysis affecting his left hand that was first apparent as a teenager. Although the disease was quiescent for several years, he developed similar findings in his right hand at age 35 while working as an artist. Features consistent with HCS included progressive acro-osteolysis, low BMD, and fractures. Teriparatide therapy had been initiated prior to his referral and after three years of alendronate. Serum calcium and total alkaline phosphatase were mildly elevated presumably due to teriparatide. Serum phosphorus and creatinine were within the reference range.

BMD increased by 7.3% and 10.2% at the spine and left hip, respectively, while on alendronate. BMD increased by 5% at the spine, but declined by 6.2% at the hip while on teriparatide. It was also felt that the rate of progression of acro-osteolysis had increased while on teriparatide.

HCS is an autosomal dominant condition characterized by a slowly progressive skeletal dysplasia including acro-osteolysis. Low BMD, fractures, short stature, and typical facial features are frequently described. The molecular pathogenesis is unknown. Beneficial effects of bisphosphonates have been reported in patients with HCS. Our two cases suggest that teriparatide does not effectively improve bone density or slow acro-osteolysis and that bisphosphonate therapy may be superior.

Disclosures: P.J. Tebben, None.

T480

Expression of GCMB, the Master Switch for Parathyroid Development, Is Normal in Parathyroid Neoplasia. A. Hoschar*¹, C. Ding², M. Milas*³, A. Siperstein*³, M. A. Levine², ¹Anatomic Pathology, Cleveland Clinic, Cleveland, OH, USA, ²Pediatric Endocrinology, Cleveland Clinic Children's Hospital, Cleveland, OH, USA, ³General Surgery, Cleveland Clinic, Cleveland, OH, USA.

Parathyroid cell development requires the expression of the GCMB gene (6p24.2) in specific cells of the third and fourth branchial pouchs during early embryogenesis. GCMB encodes a member of a small family of conserved transcription factors that share a unique DNA binding motif, and is expressed exclusively in parathyroid glands. Although mice and humans that lack functional GCMB fail to develop parathyroid glands, it is unclear whether GCMB is required postnatally to maintain PTH expression and to allow physiological proliferation and neoplastic expansion of parathyroid glands. We therefore studied expression of GCMB in normal (n = 9) and neoplastic parathyroid tissue from subjects with a single parathyroid adenoma who were less than 26 years (n = 9), between 26–79 years (n = 23), and over 80 years (n = 6) as well as from patients with parathyroid carcinoma (n = 5) or multi-gland disease due to MEN 1 (n = 4) or non-MEN (n = 9). Formalin-fixed, paraffin-embedded tissue sections were stained with affinity-purified anti-human GCMB rabbit antiserum using an automated Ventana BenchMark XT staining system. Antibody binding was detected by biotin-streptavidin immunoperoxidase with the iVIEW DAB kit. Slides were examined by light microscopy and 400-cell count was performed on each case. Each cell was categorized with respect to the intensity of nuclear reactivity, and percentages of each of the four categories were then multiplied by a number as follows: “none” × 0, “weak” × 1, “moderate” × 2, and “strong” × 3. This generates a score that ranges from 0 to 300. All staining was nuclear and GCMB staining was detected in all samples. Scores ranged from 120 for MEN1 to 169 for normal glands, with no significant differences detected between any of the groups by ANOVA. In addition, we isolated genomic DNA from 10 solitary parathyroid adenomas and analyzed the nucleotide sequence of the GCMB genes. No mutations were identified but 3 adenomas were heterozygous (C/T) at SNP rs 16870746 located −44 from ATG Because neoplastic parathyroid tissue often exhibits fractional allelic loss or gene silencing, we conclude that the robust expression of GCMB in normal and all neoplastic parathyroid glands is consistent with an important post-natal role for this protein in maintaining the parathyroid phenotype.

Disclosures: M.A. Levine, None.

This study received funding from: Lerner Research Institute.

T481

Screening for Asymptomatic Normocalcemic Hyperparathyroidism in the United States. B. Misra*, S. J. Silverberg, J. P. Bilezikian.. Dept. of Endocrinology, College of Physicians and Surgeons, Columbia University, New York, NY, USA.

Little is known about the epidemiology of normocalcemic hyperparathyroidism (NPHPT) in the United States. Most of the published data come from referral centers in which a selection bias identifies individuals who already have signs or symptoms of primary hyperparathyroidism (PHPT). A better way to establish the incidence of asymptomatic NPHPT is to screen an asymptomatic population that represents “normal” free living individuals by measuring both the calcium and parathyroid hormone (PTH) concentration. In 2003–2004, the National Health and Nutrition Examination Survey (NHANES), which provides epidemiologic data to monitor health trends in the civilian, non-institutionalized US population, measured PTH and calcium levels in over 6000 individuals. Sample weighting to correct for over- or under- sampling permits calculation of national average estimates. Using a sampled NHANES database of 1909 people age ≥ 45 with a creatinine clearance of ≥60cc/min (to eliminate those with renal-based secondary HPT), we screened for an elevated PTH (≥65pg/mL) and a mid-range, normal, corrected serum calcium (9–10mg/dL). Using these screening criteria, 248 individuals were identified (60% female) representing 15% of women and 11% of men. The mean PTH in this group was 83±20pg/mL. Most subjects in this sample were 45–54 years old and Caucasian. National estimates using sample weighting confirm the above demographic data, and suggest that 11% of the American population age≥45 with creatinine clearance≥60 cc/min fit the above criteria. Clearly, this figure overestimates the incidence of NPHPT, because the calcium and PTH levels were not verified by repeat testing, and the incidence of vitamin D deficiency (40–57% in NHANES III, data available soon for NHANES 2003–2004) and other causes of secondary HPT are not known. Significant adjustments consistent with lower percentages of the population with NPHPT are likely. Nevertheless, this initial analysis demonstrates a method whereby using available unselected free-living cohorts, it will be possible to verify initial normocalcemic and elevated PTH values, rule out vitamin D deficiency and other causes of secondary HPT, and thus gain accurate estimates of the prevalence of NPHPT. Future studies will yield more certain estimates and allow a direct test of the hypothesis that these individuals represent the true forerunner of asymptomatic PHPT.

Disclosures: B. Misra, None.

T482

PTH(7–84) Inhibits PTH(1–34)-induced Production of 1,25-(OH)2D3 in Primary Cultured Murine Renal Tubules. K. Nakajima*¹, K. Nohtomi*², K. Takano*¹, K. Sato¹, ¹Insitute of Clinical Endocrinology, Tokyo Women's Medical University, Tokyo, Japan, ²Department of Internal Medicine, Showa University, Tokyo, Japan.

The serum level of 1,25-(OH)₂D is generally increased in patients with primary hyperparathyroidism, but is occasionally not increased, or even decreased, when hypercalcemia is very severe. Recently, we have seen a 65-year-old male patient with primary hyperparathyroidism accompanied by markedly increased serum levels of intact PTH (4085 pg/ml; normal range 10–65 pg/ml) and whole PTH (842 pg/ml; normal range 9.0–39.0 pg/ml). Serum levels of calcium (12.3 mg/dl) and creatinine (3.5 mg/dl) were moderately increased. Despite the markedly increased PTH level, serum 1,25-(OH)₂D was undetectable (<3 pg/ml). After resection of a huge right parathyroid adenoma (37×20×50 mm), the serum calcium level was normalized. To clarify whether PTH(7–84), a degradation product of whole PTH (1–84) that inhibits bone resorption in skeletal tissue, also exerts an antagonistic effect on the kidney, we studied the effect of PTH(7–84) on PTH(1–34)-induced 1α-hydroxlase activity in renal tubules.

Neonatal mouse renal tubules were cultured in DMEM/F-12(1:1) supplemented with 0.2% BSA, insulin (5μg/ml) and transferrin (5μg/ml) for 7 days in the presence or absence of PTH(1–34) and/or PTH(7–84). After changing the medium, 25-OHD, was added to a final concentration of 10⁻⁶ M, and 1.25-(OH)₂D₃ was determined after 3 h of culture. Furthermore, effects of PTH(1–34) and/or PTH(7–84) on mRNA expression were investigated by real-time RT-PCR.

PTH(1–34) dose-dependently increased the level of 1,25-(OH)₂D₃ in the conditioned medium, and this PTH(1–34)-induced production of 1,25-(OH)₂D₃ at 10 ng/ml was dose-dependently inhibited by PTH (7–84). Real-time RT-PCR analysis also confirmed that PTH(1–34) increased the expression of 1α-hydroxylase mRNA. Effect of PTH(7–84) on 1α- and 24-hydroxylase mRNA in the presence of 25-OHD₃ was not constant and is under investigation in the absence of 25-OHD₃.

These in vitro findings suggest that PTH(7–84) inhibits not only bone resorption in skeletal tissue but also 1α-hydroxylase in renal tubules. Although hypercalcemia per se inhibits 1α-hydroxylase activity, non-N-terminal PTH fragments may be also partly involved in the normal or decreased serum level of 1,25-(OH)₂D in severely hypercalcemic patients with primary hyperparathyroidism. This in vitro observation may also account for the decreased serum level of 1,25-(OH)₂D in patients with secondary hyperparathyroidism, in whom the ratio of whole PTH/intact PTH is decreased.

Disclosures: K. Nakajima, None.

T483

In Multiple Endocrine Neoplasia Type 1 (MEN1) the Estimated Ratio of Mutated p27 Versus Mutated MEN1 Genes Is Below 1:100. A. Ozawa*¹, S. K. Agarwal*¹, C. M. Mateo*¹, A. L. Burns*¹, T. S. Rice*¹, P. A. Kennedy*¹, C. M. Quigley*¹, W. F. Simonds¹, L. S. Weinstein¹, S. C. Chandrasekharappa*², F. S. Collins*², A. M. Spiegel¹, S. J. Marx¹, ¹NIDDK, National Institutes of Health, Bethesda, MD, USA, ²NHGRI, National Institutes of Health, Bethesda, MD, USA.

p27 (CDKN1B, cyclin-dependent kinase inhibitor 1B; the alternate name for p27 is KIP1) belongs to one of two classes of cyclin dependent kinase inhibitor. Mice with homozygous knockout of p27 develop endocrine tumors but only in the pituitary intermediate lobe. Rats with a homozygous inactivating mutation of p27 have tumors with features of MEN1 and MEN2.

One variant of MEN1 shows sporadic tumors of both the parathyroids and pituitary. The prevalence of identified MEN1 gene mutations in this variant is far lower than in familial MEN1 (7% versus 90%, p<0.0001), suggesting different causes. Recently one index case of this variant had a germline mutation of p27 (Pellegata et al; PNAS 2006).

Our purpose was to estimate the frequency of p27 mutations in MEN1 and MEN1-like states. We sequenced germline DNA from 34 cases and control cases for the p27 gene by PCR of the protein coding exons and intron/exon boundaries. No identifiable MEN1 mutation was a required criterion for inclusion.

We tested 16 MEN1 cases with sporadic tumor of both parathyroid and pituitary. In these 16 cases, parathyroid tumors were single in 2 cases and multiple in 14. Six pituitary tumors over-secreted GH, 6 over-secreted PRL, 2 over-secreted ACTH, and 2 were nonfunctioning. Eighteen additional index cases with closely related familial tumor phenotypes were also evaluated. There were 11 index cases for families with tumors of the parathyroids and pituitary, 5 index cases of familial isolated tumors of pituitary and 2 index cases of familial hyperparathyroidism with renal angiomyolipoma. One p27 germline single nucleotide change was identified. This predicted a silent substitution of Thr142Thr. Furthermore, there was a normal prevalence of heterozygosity (35%) for a common p27 polymorphism (Val109Gly; GTC to GGC), making a large p27 deletion unlikely in all or most of these cases. Thus there was no pathologic p27 mutation in any of 34 cases. We can estimate the frequency of p27 mutations in MEN1. Assume 1000 cases of MEN1 and MEN1-like variants, about 700 will have MEN1 mutations. Of the remaining 300, we estimate less than 6 p27 mutations. The estimated ratio of p27 versus MEN1 mutation in MEN1 is therefore below 1 to 100. p27 is thus a rare cause of MEN1, but it has implications for understanding endocrine tumors that are disproportionate to its rarity.

Disclosures: A. Ozawa, None.

T484

Long Term Morbidity & Mortality in Untreated Mild Primary Hyperparathyroidism. J. Iskander*, D. Rao.. Bone & Mineral Metabolism, Henry Ford Health System, Detroit, MI, USA.

Optimal management of mild asymptomatic primary hyperparathyroidism (PHPT) continues to be debated despite recent revised guidelines. A small number of prospective studies of untreated PHPT suggested very few disease specific complications, but others differ. Therefore, it is unclear if untreated PHPT is associated with significant longer term morbidity and mortality. Accordingly, we determined long term outcomes in carefully followed patients with untreated PHPT

We prospectively studied 50 patients (8 men and 42 women, and 21 white and 29 black, with a mean age of 66 ± 8y), with mild PHPT to determine longer term effects of chronic PTH excess in the absence of parathyroidectomy (PTX). Patients were evaluated every 6-12 months for up to 18y.

There was a small significant rise in serum creatinine, but none of the values were > 1.5 mg/dl during the first 10y of follow up. Serum PTH and calcium did not change. There was a small but significant decline in forearm absolute BMD with consequent decline in T-score but not in Z-scores. Absolute BMDs and T- and Z-scores did not change either at the spine or at the hip. Quality of life scores, by SF-36, generally remained stable over time. Relevant morbidity and mortality data are in Table

During follow-up, 2 patients started hemodialysis (one 10y after PTX), most likely related to diabetes and hypertension, and 2 patients had kidney stones, one of whom had PTX before the kidney stone incident.

Eleven patients sustained 14 fractures: one each of humerus, ischium, glenoid cavity and femur, 3 – 7y after PTX. Fractures 1–4y before PTX were: 3 foot, 2 finger, and one each of fibula, forearm, femur, ankle, and tibia due to trauma or automobile accident. None of the fractures were considered due to PHPT. Eight patients died at a mean age of 76.3 ± 7.2y from causes unrelated to PHPT (2 cancers, 3 cardiac, one each in hospice, ruptured aneurysm, and unknown; all unrelated to PHPT). Seven of the 9 patients had PTX because of the formal conclusion of the study in concert with existing guidelines.

We conclude that mild untreated PHPT is not associated with continued bone loss or worsening disease severity. There were the usual age and lifestyle related morbidity and mortality, but no disease specific complications. 

Table  .  

Disclosures: D. Rao, None.

T485

Use of Parathyroid Hormone in Hypoparathyroidism. M. R. Rubin, S. J. Silverberg, J. Sliney*, J. P. Bilezikian.. Columbia University College of P&S, New York, NY, USA.

Hypoparathyroidism (HypoPT) is a disorder in which PTH is absent from the circulation. Without PTH, calcium homeostasis is abnormal and bone mineral density (BMD) by DXA is markedly elevated. Calcium and vitamin D supplementation are required but control can be a clinical challenge. PTH seems to be an ideal agent because it replaces what is missing in these individuals. Accordingly, we studied the effects of PTH replacement therapy in HypoPT on biochemical and densitometric abnormalities. We studied 30 subjects (48.9 ± 13 yr; 21 female) with HypoPT (duration 18 ± 13 yrs; 17 postsurgical, 11 autoimmune, 2 DiGeorge). Baseline biochemistries were: serum calcium, 8.7 ± 1 mg/dl (nl 8.5–10.4); PTH, 7.1 ± 6 pg/ml (nl 10–65); urinary calcium, 261 ± 123 mg/l; serum phosphorus, 4.4 ± 0.8 mg/dl (nl 2.5–4.5); total alkaline phosphatase, 66 ± 20 IU/I (nl 33–96); 25-hydroxyvitamin D, 63 ± 76 ng/ml (nl 9–52); 1,25-dihydroxyvitamin D, 33 ± 15 pg/ml (nl 15–60). Baseline calcium supplementation ranged from 0–9 g/d (av,3 g/d); baseline calcitriol doses ranged from 0 to 3 ug/d (av, 0.75 ug/d); baseline parent vitamin D doses (n = 9) ranged from 4000–100,000 IU/d (av, 10,000 IU). Baseline DXA-based BMD was by T-scores at the lumbar spine, +1.7 ± 2.3 (cohort range: −1.60 to +7.90), total hip, +0.57 ± 1.3 (cohort range: −2.10 to + 4.30) and 1/3 distal radius, −0.34± 1.06 (cohort range: −2.6 to +1.5). PTH(1–84) was administered at 100 ug every other day for up to 24 months (range of time: 1–24 months). Calcium supplementation decreased by 33% at 1 month (p<0.01); calcitriol supplementation decreased by 27% at 3 months (p<0.05); parent vitamin D supplementation trended downwards by 31% at 3 months. Serum calcium remained within the normal range, with the greatest increase occuring at 2 months, to 9.3 ± 1 mg/dl (p <0.01). Urinary calcium decreased at 12 months to 164 ± 102 mg/dl (p = 0.02). BMD decreased at the distal 1/3 radius (-2.9 ± 2% at 18 months; p = 0.02), but increased at the lumbar spine (2.9 ± 4% at 12 months; p = 0.02) and did not change at the total hip. We conclude that the administration of PTH improves calcium homeostasis in HypoPT and allows serum calcium levels to be maintained while permitting a reduction in the calcium and vitamin D regimen. It appears that there are differential effects of PTH replacement on BMD by DXA, as a function of skeletal site and composition. Whether other changes in bone quality occur with PTH replacement are currently under investigation.

Disclosures: M.R. Rubin, None.

T486

Bone Quality in Mild Primary Hyperparathyroidism. R. Zoehrer*¹, D. W. Dempster², J. P. Bilezikian², H. Zhou³, S. J. Silverberg⁴, E. Shane⁴, P. Fratzl*⁵, E. Paschalis¹, P. Roschger¹, K. Klaushofer¹, ¹Ludwig Boltzmann Institute of Osteology, Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Med. Dept. Hanusch Hospital, Vienna, Austria, ²Regional Bone Center, Helen Hayes Hospital, & College of Phys & Surg, Columbia University, New York, NY, USA, ³Regional Bone Center, Helen Hayes Hospital, New York, NY, USA, ⁴College of Phys & Surg, Columbia University, New York, NY, USA, ⁵Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany.

Mild primary hyperparathyroidism (PHPT) is a common endocrine disorder characterized by excessive secretion of parathyroid hormone (PTH) and hypercalcemia. The present study examines bone quality in PHPT by applying two imaging technologies: quantitative backscattered electron imaging(qBEI) and Fourier transform infrared imaging analysis (FTIRI). qBEI permits determination of bone mineral density distribution (BMDD, expressed as CaMean, CaPeak, CaWidth and CaLow), and FTIR1 describes the extent of collagen maturity expressed as the ratio of 2 of the major bone type I collagen cross-links (pyridinoline/dehydro-dihydroxylysinonorleucine;collx). Percutaneous transiliac bone biopsies were obtained from 51 unselected subjects with mild PHPT (16 men, aged 28–68; 35 women, aged 26–74). Forty of the biopsies were from untreated patients. Seven biopsies were from subjects biopsied after an average of 6 years after parathyroidectomy (PTX). All biopsies were fixed and dehydrated in ethanol, and embedded in polymethylmethacrylate. By qBEI analysis, PHPT differed from healthy controls in that they had markedly higher CaWidth (+15.7%, p<0.0001) and CaLow (+44.7%, p<0001), and somewhat lower CaMean (-2.5%, p<0.0001). These differences were reversed in the 7 patients who underwent PTX. For FTIRI analysis, spectral images were acquired of 3 areas (each 400 μm × 400 μm) on separate trabeculae devoid of resorption pits and with primary mineralization evident on at least one surface (based on qBEI images). The collx in the PHPT patients was significantly lower than in normal controls (p<0.001), while those studied after PTX was normal. The difference in collx in PHPT was age-independent but gender-dependent: with women exhibiting a lower ratio than men. Both qBEI and FTIRI outcomes were strongly correlated with bone formation rate and mineralizing surface parameters as determined by histomorphometry. These results are consistent with previous observations of increased bone turnover in this disease, and consequently, reduced mean age of bone tissue. Reduced mineralization density and collx in patients with PHPT would be expected to reduce the stiffness of bone tissue. These observations are relevant to considerations of fracture risk in PHPT.

Disclosures: R. Zoehrer, None.

T487

The Relationship between iPTH and Bone Metabolic Markers. Y. Kodama*, Y. Kato*, K. Kanaya*, K. Wada*, S. Shimamoto*, C. Ishi*., Orthopedics, Tokyo Women's Medical University, Tokyo, Japan.

Introduction: Currently, instead of a bone biopsy, bone metabolic markers are often used to evaluate bone quality, since this is non-invasive test. However, in HD patients, the diagnosis and prognosis based on bone metabolic marker levels were sometimes difficult. The pathological histology of renal osteodystrophy (ROD) is well-known to be related to the level of iPTH. We investigated the relationship between iPTH and bone metabolic markers to evaluate bone quality in HD patients.

Patients: iPTH and bone metabolic markers had been measured in our department. The data from 1200 patients, whose background characteristics were available, was analyzed in this study. The patients were categorized into the following 10 groups, HD group, metastatic bone tumor (meta) group and so on.

Methods: 1. In each disease group, iPTH and bone metabolic markers were assessed using multiple comparisons. 2. In HD group, the correlations between iPTH and bone metabolic markers were examined. 3. For each marker, the patients were divided into two groups based on their iPTH levels: low, iPTH <65 pg/mL; and high, iPTH ≥65 pg/mL. Their correlations were then examined.

Results: 1. HD group showed higher iPTH than other groups. ICTP, NTX, and OC had a similar distribution to iPTH. The meta group showed a significantly higher BAP than other groups. 2. sNTX, OC and BAP had statistically significant correlation with iPTH, but ICTP had none. 3. In “high” group, all bone metabolic markers were strongly correlated with iPTH. In “low” group, however, none of the bone metabolic markers were correlated with iPTH.

Discussion: 1. Two hypotheses could explain the results of multiple comparisons. Bone metabolic markers may simply accumulate due to impaired kidney function and due to the differences in the dialyzer membrane's permeability. Alternatively, the bone metabolic markers truly reflect accelerated bone turnover due to secondary hyperparathyroidism. To confirm these hypotheses, we investigated the correlations between iPTH and the various bone metabolic markers. 2. OC had the highest correlation coefficient with iPTH. In HD patients, OC appears to have the best contribution ratio to iPTH. 3. When iPTH is lower than 65 pg/mL, the histopathology of ROD shows aplastic bone disease (ABD) with 100% specificity. To exclude the influence of ABD, the patients were divided into two groups based on their iPTH levels. In this study, all bone metabolic markers were correlated with iPTH in “high” group. Therefore bone metabolic markers can be used to evaluate the bone quality of ROD in patients without ABD.

Conclusions: ICTP, OC, and NTX had similar distribution to iPTH. In HD group, OC had the highest correlation coefficient with iPTH. In HD patients with iPTH ≥ 65 pg/mL, all bone metabolic markers were correlated with iPTH.

Disclosures: Y. kodama, None.

T488

Non-invasive Study of Bone and Cardiovascular Disease in Chronic Hemodialysis Subjects. L. Kooienga*¹, A. Furniss*², A. Bellasi*³, P. Raggi*⁴, G. Block². ¹Division of Renal Disease and Hypertension, University of Colorado Health Sciences Center, Denver, CO, USA, ²Clinical Research Division, Denver Nephrology, Denver, CO, USA, ³Nephrology, University of Milano, San Paolo Hospital, Milan, Italy, ⁴Division of Cardiology, Emory University, Atlanta, GA, USA.

To assess the relationship between interval changes in bone mineral density (BMD) with changes in arterial health as determined by changes in aortic and coronary artery calcification scores (CACS) and arterial stiffness over a 12 to 18 month follow-up period

This is a single center study of subjects on chronic hemodialysis who underwent medical interviewing, dual energy x-ray absorptiometry, electron beam computed tomography, and pulse wave velocity (PWV) assessment at baseline and at follow-up. In addition, subjects had time-averaged laboratory values obtained from routine dialysis monitoring throughout the follow-up period. Medication exposure was defined as > 3 months of the study period.

59 subjects completed baseline and follow-up evaluations. 3 subjects with a parathyroidectomy during the study were excluded. The mean age was 58.8 (15.2) years, mean vintage was 45.9 (41.9) months, and mean follow-up was 13.6 (1.6) months. 54% of subjects were female, 63% were Caucasian, and 30% were African American. ESRD etiology was from diabetes mellitus in 34% and hypertension in 30%. Mean hip BMD at baseline was 0.813 (0.148) g/cm² and decreased by a mean of −2.6 (4.6)% at follow-up. 17 subjects had an improvement or no change in BMD and 37 subjects had a decrease in BMD. In subjects with improvement/no change in hip BMD vs. those that had a decrease there was no significant difference at follow-up in time-averaged albumin, adjusted calcium, adjusted Ca x P product, LDL cholesterol, PTH, or the % with mean PTH between 150–300 pg/mL. Those with a decrease in hip BMD had a higher% of aspirin use (81 vs 47%; p = 0.02), lower Cinacalcet use (27 vs 65%; p = 0.02), and a trend towards lower calcium binder use (57 vs 88%; p = 0.07) and lower serum phosphorus [4.8 (0.8) md/dL vs. 5.2 (0.7) mg/dL; p = 0.09]. They also had a greater change in mean PWV [1.4 (3.6) m/s vs −0.8 (1.8) m/s; p = 0.01] with a concomitant higher median aortic artery calcium score [2496 (47,927) vs. 784 (9514); p = 0.05]. However, there was no significant difference in CACS, cardiovascular events, or fractures between the groups.

69% of prevalent HD patients experienced a dramatic loss of hip BMD over a mean 13.6 month follow-up. This loss of BMD was independent of the typical parameters of bone mineral metabolism, time on dialysis, or age. Subjects who lost hip BMD had higher median aortic artery calcification scores and demonstrated significant worsening of arterial PWV supporting the proposed hypothesis linking loss of bone mineral with vascular pathology.

Disclosures: L. Kooienga, None.

This study received funding from: Shire Pharmaceutical Inc.

T489

The Mechanism of Skeletal Involvment in Cardiovascular Mortality in Chronic Kidney Disease (CKD). S. Mathew*¹, R. Lund*², K. A. Hruska¹, ¹Pediatrics, Washington University, St.louis, MO, USA, ²Medicine, Creighton University, Omaha, NE, USA.

CKD is complicated by high mortality rates due to cardiovascular events. Hyperphosphatemia and vascular calcification (VC) have been determined in observational studies to be risk factors. In animal models with CKD, we have demonstrated that renal injury impairs osteoblast function and decreases bone formation rates without suppressing osteoclastic resorption, i.e. produces the adynamic bone disorder. In an animal model of type 2 diabetes with atherosclerosis, induction of the adynamic bone disorder by CKD contributes to hyperphosphatemia by excess skeletal resorption, and stimulates vascular calcification. Control of the serum phosphorus by stimulation of bone formation or phosphate binding inhibited VC. These results have been confirmed in dialysis patients treated with phosphate binders, and improved patient survival was found. In atherosclerotic vascular smooth muscle cells, the mechanism of phosphorus action in VC was sought and found to be stimulation of a heterotopic BMP-2 and RUNX2 directed osteoblastic transcription program. Despite the expression of these osteoblastic determinants in basal conditions, matrix mineralization by atherosclerotic aortic smooth muscle cell cultures was not observed until media phosphorus was increased. The action of phosphorus on mineralization was due to stimulated expression of osterix, a critical osteoblast transcription factor. Phosphorus stimulated mineralization in vitro was silenced by inhibition of osterix through BMP-7, and by inhibition of BMP-2 action through addition of a BMP antagonist, noggin.

In our animal model of CKD stimulated VC, we found the BMP-2/MSX2 program including RUNX2 expression to be present in the atherosclerotic aorta prior to CKD and unaffected by induction of CKD, analogous to the atherosclerotic cell cultures. CKD stimulated aortic osterix expression. Stimulation of bone formation and control of hyperphosphatemia using BMP-7 or phosphate binding agents, decreased aortic osterix expression, osteocalcin expression and aortic mineralization. These studies demonstrate a multiorgan system of the kidney, skeleton, and the cardiovasculature, that fails due to CKD involving loss of skeletal anabolism, hyperphosphatemia and heterotopic mineralization representing kidney-bone and bone-vascular signaling.

Disclosures: S. Mathew, Genzyme 2; Shire 2.

This study received funding from: NIH.

T490

Bone Microarchitecture As Assessed by High Resolution Quantitative CT (HRpQCT) in Chronic Kidney Disease. T. L. Nickolas¹, D. J. McMahon¹, H. Eisenberg*¹, S. Cabral*¹, C. Go*¹, M. Leonard², E. Shane¹, ¹Medicine, Columbia University, New York, NY, USA, ²Pediatrics, University of Pennsylvania, Philadelphia, PA, USA.

Moderate chronic kidney disease (CKD) and end-stage kidney disease (ESKD) are associated with increased susceptibility to fracture, due in part to the catabolic effects of secondary hyperparathyroidism on cortical bone. However, the effects of moderate CKD on trabecular (Tb) and cortical (Ct) microarchitecture have not been addressed.

High resolution quantitative CT (HRpQCT) (XtremeCT, Scanco Medical AG) is a new imaging technology (resolution <100 microns) that distinguishes Ct from Tb bone and yields noninvasive measurements of Tb and Ct architecture that are more sensitive markers of bone fragility. To assess effects of impaired kidney function on bone architecture, we performed HRpQCT scans in 31 subjects (18 men), aged 51–88 yr, across a range of kidney function. The MDRD formula was used to estimate GFR.

Overall, 5 subjects were on dialysis and 9 had normal eGFR. Of the remainder, the median (range) eGFR was 40 ml/min (11–64). HRpQCT variables that were not normally distributed were log-transformed.

Univariate analyses demonstrated that Tb deficits were significantly associated with lower eGFR and female gender. Ct deficits were significantly associated with lower eGFR and older age. Linear regression was used to investigate the relationship between HRpQCT microarchitectural parameters and eGFR, adjusted for age and gender. Ct density, but not thickness, was directly related to eGFR. Similarly, Tb parameters were worse in patients with lower eGFR, with decreased Tb density, BV/TV and TbN, increased TbSp and heterogeneity of the Tb network (TbSpSD). By multivariate regression analysis adjusted for gender and age, Tb changes were independently associated with eGFR (Table 1).

This preliminary investigation confirms that lower eGFR was associated with both Ct and Tb deterioration that was independent of gender and age. These alterations in Ct and Tb microstructure may contribute to the increased risk of fracture in patients with CKD and ESKD. Further studies are required to elucidate the pathogenesis of the microarchitectural deterioration and its relationship to bone mechanical competence. 

Table  .  

*Gender and Age were not significantly associated either with cortical or trabecular parameters in multivariate linear regression

Disclosures: T.L. Nickolas, Hoffman- La Roche 5; Abbott 8; Gilead 8.

T491

Etidronate Inhibits Progression of Vascular Calcification and Decrease in Bone Mineral Density in Chronic Dialysis Patients. Y. Okada, O. Hashimoto*, T. Tanikawa*, Y. Tanaka.. First Department of Internal Medicine, University of Occupationala and Environmental Health Japan, Kitakyushu, Japan.

Severe atherosclerosis/calcinosis is common in patients on long-term dialysis. No therapy is currently available to suppress the progression of vascular calcification. In the present study, we determined the effect of etidronate (EHDP), a first generation bisphosphonate, on vascular calcification in chronic dialysis patients. 81 patients undergoing chronic hemodialysis were divided into EHDP-treated group (n = 37) and control group (n = 44). EHDP was administered in a single dose (400 mg/day) just before sleep each day for two weeks followed by suspension for 10 weeks, and a repeat of the cycle every 12 weeks for one year. The area of calcification affecting the abdominal aorta (ACA) was measured with X-ray computed tomography. ACA was measured by an image analysis device and the mean ACA of six slices was calculated. ACA was measured at the start and end of the study (12 months) in each patient. Bone mineral density (BMD) was measured by DXA at the start and end of the study (12 months). Scans were performed for the forearm, at one-third radius without A-V fistula. In the EHDP-treated group, no significant changes in BMD were noted throughout the study. However, BMD decreased significantly in the control group from 0.57±0.17 (mean±SD) at enrolment to 0.55 ± 0.18 g/cm² at 12 months (P<0.01). ACA increased significantly in the control group from 71.3±58.6 at enrolment to 83.1±63.6 mm² at 12 months (P<0.01). In contrast, ACA changed in the EHDP-treated group from 71.3±63.3 at enrolment to 73.2±63.5 mm² at 12 months statistically insignificant. The delta increase in ACA in EHDP-treated group was 1.9±18.6 mm² (95%CI 1.33–5.60) which was significantly lower than that of the control group (11.8±19.2 mm², 95%CI 8.19–14.55). The independent association between changes in ACA and various parameters in multivariate linear regression analysis indicated that EHDP was an independent factor for the observed changes in ACA. Our findings suggest that EHDP is potentially useful for prevention of vascular calcification and might improve the prognosis of chronic dialysis patients.

Disclosures: Y. Okada, None.

T492

TRAP5b Profiles in Children with Chronic Kidney Disease (CKD). H. E. Price, C. B. Langman*, Pediatric Kidney Diseases, Northwestern University, Childrens Memorial Hospital, Chicago, IL, USA.

Serum tartrate-resistant acid phosphatase 5b (TRAP5b) is derived from osteoclast activity. Levels of TRAP5b rise during growth spurts in children, associated with bone modeling. The osteodystrophy of CKD in children is associated with high-turnover disease from 2⁰ hyperparathyroidism (SHPTH), resulting in increased remodeling. The purpose of this cross-sectional study was to measure serum TRAP5b in children with CKD, and we asked whether modeling was reflected by its levels. 36 children (23 males (M) 13 females (F) with CKD stages 1–4 (NKF classification using eGFR) had measures made of hormones and factors related to their osteodystrophy. These included iPTH, 25(OH)D, 1,25(OH)₂D, in addition to TRAP5b [TRAP5b levels were made using an ELISA, Quidel Corporation, San Diego, CA.]. Values were compared to 65 healthy children of the same age distribution. As eGFR decreased in CKD, serum TRAP5b increased. There were correlations with iPTH (r = 0.5) and 1,25(OH)₂D (r = 0.5) in children with CKD, but not in healthy children. There was no correlation between TRAP5b & 25(OH)D. When compared to healthy children matched for age and gender, children with CKD had increased levels of TRAP5b in childhood (pre-pubertal) years (F CKD: 19.7+/-6.3 vs. healthy: 8.1+/-3.8 U/L, p< 0.001; M CKD: 12.2+/-4.6 vs. healthy: 6.6+/-3.6, p<0.01). In early adolescence, healthy children had an increase in TRAP5b, followed by a decrease as they approached late adolescence into adulthood. However, children with CKD had a decrease of TRAP5b in early adolescence followed by a steady decline into adulthood. Thus, the values of TRAP5b in early adolescence were lower when compared to healthy children (F CKD: 13+/-8.2 vs. healthy: 10+/-2.7; M CKD: 7.9+/-5 vs. healthy: 9.9+/-3.3). In conclusion, as kidney function decreases in the childhood years, TRAP5b increases, indicating accelerated bone turnover from SHPTH. However, during a period of expected increased bone modeling, and despite SHPTH, TRAP5b values declined, consistent with reduced linear growth in adolescent CKD. Additional prospective studies underway will further clarify these relationships. Thus, TRAP5b may be an important corollary marker to profile bone modeling, since bone biopsy is not clinically available, and SHPTH reflects bone remodeling. The osteodystrophy of pediatric CKD thus may be able to be better separated into abnormalities of bone modeling, and bone remodeling, with a multi-faceted measurement approach that includes TRAP5b.

Disclosures: H.E. Price, None.

T493

Hyperphosphatemia Mediates Oxidative Stress in Bovine Aortic Endothelial Cells. E. Shuto*¹, Y. Taketani¹, R. Tanaka*¹, A. Tanimura*¹, M. Isshiki*², N. Harada*³, H. Yamamoto¹, E. Takeda¹. ¹Department of Clinical Nutrition, University of Tokushima Graduate School, Tokushima, Japan, ²Department of Nephrology and Endocrinology, University of Tokyo, Tokyo, Japan, ³Department of Nutrition and Metabolism, University of Tokushima Graduate School, Tokushima, Japan.

Hyperphosphatemia causes calcification of vascular smooth muscle cells. The calcification is closely associated with progress of cardiovascular disease and mortality in end-stage renal disease (ESRD) patients. We hypothesized that hyperphosphatemia may also contribute the progress of atherosclerosis on early-stage chronic kidney disease by involving endothelial dysfunction. To prove the hypothesis, we investigate the effect of high inorganic phosphate (Pi) loading on the endothelial function using bovine aortic endothelial cells (BAECs). First, we examined production of reactive oxygen species (ROS) by fluorescence ROS indicator (APF: aminophenyl fluorescein), and found that high Pi (1.5∼3mM) increased production of reactive oxygen species in a dose-dependent manner compared with control (0.9 mM Pi). The increase in ROS was inhibited by diphenylene iodonium (DPI; NAD(P)H oxidase inhibitor) and phosphonoformic acid (PFA; Na/Pi co-transporter inhibitor). Therefore, both Pi influx and NAD(P)H oxidase activation play an important role in the ROS production in response to high Pi loading in endothelial cells. In second, we examined effect of high Pi on the nitric oxide (NO) production in response to bradykinin in the BAECs, and found that pretreatment with high Pi (2 mM) for 3 days decreased the hormone-dependent NO production. One possible mechanism of the inhibition of NO production was due to phosphorylation of endothelial NO synthase (eNOS) by conventional PKC activated by high Pi loading. These results suggest that increase in Pi influx can mediate intracellular signal such as PKC activation and ROS production, and would be involved in oxidative stress and some adverse effect on endothelial function. Oxidative stress caused by hyperphosphatemia in endothelium may be a novel patho-mechanism for atherosclerosis and cardiovascular diseases in ESRD patients.

Disclosures: E. Shuto, None.

This study received funding from: Grants-in-Aid for Young Scientist and Exploratory Research from Ministry of Education, Culture, Sports, Science, and Technology in Japan, Uehara Memorial Foundation, and 21st Century COE Program.

T494

Prospective Controlled Study assessing the Clinical Benefit of Evening Administration of Vitamin D in Hemodialysis Patients. K. Takasawa*¹, C. Takaeda*¹, Y. Takeshita*², K. Kawai*², ¹Renal Division, Public Central Hospital of Matto-Ishikawa, Hakusan, Japan, ²Internal Medicine, Public Central Hospital of Matto-Ishikawa, Hakusan, Japan.

Control of serum calcium (sCa) and phosphorus (P) levels is important in improving the survival of hemodialysis patients. We assessed whether sCa is decreased in hemodialysis patients by administering a Vitamin D(VitD) at evening instead of in the morning. We selected 24 patients having a serum Ca level of ≥9.5 mg/dl (Alb-adjusted) from 72 hemodialysis patients at our institution. Concerning the 24 patients above, an informed consent was obtained based on the Helsinki declaration. The 24 patients (the average age is 63.4±14.8 years, the mean duratuion of dialysis is 10.6±8.4 years), who were undergoing oral VitD administration in the morning, were randomized into two groups for morning administration and evening administration of VitD (n = 12 per group) and were prospectively followed-up for 6 months. During the follow-up period, the patients were monitored for the sCa levels, iPTH, osteocalcin, bone alkaline phosphatase and the serum P while receiving a fixed dose of VitD as a rule. However, the dose of iPTH was adjusted as appropriate in compliance with the Japanese guidelines and K/DOQI that recommend a dose of iPTH not exceed 180 pg/mL. Out of the 12 patients treated in the morning, one who suffered from cerebral hemorrhage and 5 who received a reduced dose of VitD were excluded, and the remaining 6 patients (2 treated with alfacalcidol and 4 with falecalcitriol) were evaluated. I have excluded the 5 patients in the evening group because that one had renal transplantation, one died of infection and 3 whose VitD dosage had been reduced, and the remaining 7 patients (4 treated with alfacalcidol and 3 with falecalcitriol) were evaluated. The sCa levels were no difference between two groups at the begining. In the morning group, the sCa level was 10.3±1.0 mg/dl at baseline and 10.4±0.8 mg/dl after 6 months. In the evening group, the sCa level was 9.9±0.6 mg/dl at baseline and was significantly decreased to 9.4±0.6 mg/dl after 6 months (p<0.05). The serum iPTH level was 181.7±133.5 pg/ml at baseline and 208.5±165.4 pg/ml after 6 months in the morning group versus 121.4±139.6 pg/ml at baseline and 142.1±94.9 pg/ml after 6 months in the evening group, showing no pronounced change in the both groups. The osteocalcin levels of the evening group were reduced from 46.6±45.3 to 29.5±22.3 ng/ml, significantly. These data suggest that oral VitD was administered at evening in patients on hemodialysis to address the diurnal rhythm of bone metabolism, and the serum Ca level was decreased as a result in these patients. Evening administration of VitD is more beneficial effect for patients on hemodialysis.

Disclosures: K. Takasawa, None.

T495

Impact of Cinacalcet HCL on Bone Remodeling and Minimodeling in Patients with Secondary Hyperparathyroidism. A. Yajima¹, H. E. Takahashi², T. Akizawa*¹, K. Study Group*⁴. ¹Nephrology, Towa Hospital, Tokyo, Japan, ²Niigata University of Health and Welfare, Niigata, Japan, ³Nephrology, Showa University, School of Medicine, Tokyo, Japan, ⁴KRN 1493 Study Group, Tokyo, Japan.

Purpose; Impact of cinacalcet HCL on hyperparathyroid bone disease was investigated.

Methods; Iliac bone biopsy was taken before and after the treatment with cinacalcet HCL for one year in the three hemodialysis patients with secondary hyperparathyroidism, and histomorphometric parameters including remodeling parameters and minimodeling volume were measured in cancellous bone.

Results; Serum intact PTH was decreased from 747.7±391.1 to 140.3±80.5 pg/ml, respectively. Oc.S/BS decreased from (8.5, 6.2, 6.2) to (1.2, 0.2, 1.0)%, Fb.V/TV decreased from (2.1, 0.3, 0.2) to (0, 0, 0)%, Ob.S/BS decreased from (45.5, 20.0, 22.5) to (7.8, 1.5, 1.0)%, OS/BS decreased from (58.1, 44.7, 37.3) to (17.1, 8.2, 13.5)%, BFR/BS also decreased from (0.069, 0.023, 0.044) to (0.004, 0.003, 0.004) mm³/mm²/year. Minimodeling volume (Ml.V/BV) increased from (0.49, 0.45, 1.73) to (5.18, 4.95, 4.41)%. In additiom, MAR of remodeling sites were decreased from (0.89, 0.79, 0.65) to (0.4, 0.41, 0.39) mcm/day after the treatment of cinacalcet HCL. And MAR of minimodeling sites were decreased from (0.94, 0.84, 0.64) to (0.44, 0.48, 0.40) mcm/day after the treatment. As a result, MAR values were generally greater in minimodeling sites than in remodeling sites.

Conclusions; Cinacalcet HCL well suppressed bone remodeling and turnover, but effectively increased bone formation due to minimodeling in cancellous bone in these patients.

Disclosures: A. Yajima, None.

T496

Minimodeling Reduces the Rate of Cortical Bone Loss in Patients with Secondary Hyperparathyroidism. A. Yajima¹, M. Mitobe*¹, I. Takahashi*¹, T. Nakayama*², O. Otsubo*¹, M. Inaba*³, Y. Tominaga*⁴, A. Ito*⁵. ¹Nephrology, Towa Hospital, Tokyo, Japan, ²Orthopedic Surgery, Towa Hospital, Tokyo, Japan, ³Metabolism, Endocrinology, and Molecular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan, ⁴Transplant Surgery, Nagoya Second Red Cross Hospital, Nagoya, Japan, ⁵Ito Bone Histomorphometry Institute, Niigata, Japan.

Purpose: Secondary hyperparathyroidism causes progressive cortical thinning due to increased bone resorption at the endocortical surface, and increases cortical porosity due to increased resorption at the intracortical surface. However, patients with severe hyperparathyroidism do not always suffer from bone fractures, perhaps because of the increased minimodeling occurring in cortical bone. Because bone formation by minimodeling has not yet been reported in cortical bone, we investigated the effects of cortical minimodeling on the reduction of the rate of bone loss.

Methods: Thirty-five patients with secondary hyperparathyroidism were enrolled. Remodeling and minimodeling parameters at the endocortical and periosteal surfaces as well as at the intracortical surface were measured. The relationships between remodeling parameters and minimodeling parameters at each surface were investigated by linear regression analysis. Cortical bone specimens were classified into three groups according to the values of cortical width and cortical porosity. The relationships of minimodeling parameters at the endocortical surface with cortical width and at the intracortical surface with cortical porosity were investigated.

Results: Some minimodeling parameters showed positive correlations with serum parathyroid hormone levels and remodeling parameters. Minimodeling bone volume at the endocortical surface was greater in the narrow cortical width group than in the wide cortical width group, possibly slowing the progression of cortical thinning. Minimodeling volume at the intracortical surface was greater in the high porosity group than in the low porosity group, possibly slowing the progression of enlargement of the intracortical resorption spaces. Minimodeling of the periosteal surface was found in one specimen.

Conclusions: The results revealed enhanced cortical minimodeling in patients with secondary hyperparathyroidism, possibly representing the reduction of the rate of cortical bone loss.

Disclosures: A. Yajima, None.

T497

Bone Loss Following Small Bowel Transplantation. K. S. Awan*¹, J. M. Wagner*¹, D. Martin*², D. L. Medich*¹, S. Perera*³, K. Abu Elmagd*², S. L. Greenspan¹. ¹Medicine, University of Pittsburgh, Pittsburgh, PA, USA, ²Department of Surgery, UPMC Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA, ³Medicine/ Biostatistics, University of Pittsburgh, Pittsburgh, PA, USA.

Little is known about the impact of small bowel transplantation (SBTx) on skeletal integrity. We compared bone mineral density (BMD, Hologic Discovery A) in 81 patients who had undergone SBTx (43±2 years old, average SBTx 2.2 years before, 65% women) with 51 small bowel controls (SBCon), with similar small bowel diseases (47±13 years old) awaiting transplantation matched for age and gender. In addition to SBTx, surgery could include transplantation of the liver, pancreas, and stomach. Following SBTx, patients receive tacrolimus, supplementary calcium and vitamin D and bisphosphonates if appropriate. BMI was similar for the 2 groups (SBTx 22.7 ± 0.5 kg/m² vs. SBCon 23.1 ± 0.8 kg/m²). Compared with the SBCon, the SBTx group had significantly lower absolute BMD and Z-scores at the hip and femoral neck without differences noted at the spine (Table). 25 hydroxy vitamin D levels were similar in SBTx patients compared to SBCon (17.4 ± 1.5 vs. 21.1 ± 1.9 ng/dl) as were levels of serum calcium (8.9 ± 0.1 vs. 8.9 ± 0.1 mg/dl) and parathyroid hormone (60.5 ± 9.5 vs. 81.6 ± 11.0 pg/ml) Twenty three patients were followed with BMD's prospectively; nine patients before and after transplantation (Pre to Post: mean 1.3 years follow-up); fourteen patients after transplant (Post: mean 1.8 years average follow-up). Bone loss (percent change in g/cm²) was greatest in the patients who were followed through transplantation surgery (Pre to Post), with an average bone loss of 15% at the hip, compared to patients followed after transplantation (Post). There were no significant changes in levels of 25 hydroxy vitamin D, calcium, or parathyroid hormone.

We conclude that patients undergoing SBTx have significant bone loss, primarily at the hip, occurring within the first year post transplant despite preventive measures with calcium, vitamin D and bisphosphonates. 

Table  .  

*p<0.05, **p<0.01 difference from zero for cross-sectional, change from baseline for longitudinal

Disclosures: K.S. Awan, None.

This study received funding from: NIH/K24.

T498

Interest of Platelet-derived Products for Bone Reparation. L. Begot*¹, C. Doucet*², I. Ernou*², F. X. Gunepin*³, C. Laplace-buihle*⁴, J. J. Lataillade*², X. Holy¹, ¹Histology and Tissular Reparation, IMASSA, Brétigny sur Orge, France, ²Département Recherches, CTSA, Clamart, France, ³Service de Chirurgie Orthopédique, HIA Percy, Clamart, France, ⁴Imagerie, Généthon, Evry, France.

The French Army Hospitals have to treat an high number of patients presenting lesions with large loss of bone substance. The size of defects, rendering the bone auto graft strategy unsuitable, suggests a stem cell therapy approach. Progress in tissue engineering make it possible to conceive the development of macroporous ceramic bone substitutes combined to Mesenchymal Stem Cells (MSC) for filling in bone defects. This approach needs cell cultures and implantation in clinical conditions. As platelets are a source of growth factors, Platelet-rich plasma (PRP) could be used as substitute for Foetal Calf Serum (FCS) in cultures and to prepare a platelet-derived Glue (PG) mixture interesting for its properties of adhesiveness, malleability and haemostasis. In this work, we studied the effect of the proliferation and differentiation of MCS in presence of PRP products.

PRP was obtained by apheresis collection and processed for platelet activation to stimulate growth factor release. First, human marrow-derived MSC were isolated and expanded in a medium with 10% FCS or 10% PRP through a process of subculturing. The ability of PRP to enhance MSC proliferation was investigated by measuring cell numbers at consecutive passages, by characterizing MSC phenotype (CD45⁺, CD90⁺, CD105⁺, CD73⁺, CD13⁺) and by performing in vitro osteogenic evaluation (alkaline phosphatase activity. Von Kossa staining, osteocalcin detection). Second, proliferation, survival differentiation were evaluated in glue. Finally, we compared the biocompatibility and functionality of MSC or osteoblastic precursors loaded on bio-ceramics in a mice orthotopic implantation model in presence or not of PG.

Our results showed that PRP medium was considerably more effective in stimulating MSC proliferation than FCS one without affecting their phenotype. Furthermore, MSC cultured with PRP maintained their differentiation properties. MSC and osteoprogenitors were able to proliferate at the surface and within the bio-ceramic in presence of glue. In vitro and in vivo experiments demonstrated that osteogenic differentiation of MSC was improved when PG was associated with the hybrid implant. Osteoblastic precursors appeared to be better candidates than MSC regarding their long term in vivo survival in mice.

In conclusion, our data show that for ex vivo expansion of human MSC prior to transplantation, PRP could efficiently replace FCS and that the PG could be a powerful tool in bone reparation. We suggest that PRP biotechnology should be useful in combination with MSC for bone reconstructive cellular therapy.

Disclosures: L. Begot, None.

This study received funding from: DGA (Direction Générale pour l'Armement).

T499

Long-term Follow up of Bone Mineral Density, Bone Turnover Markers and 25-hydroxyvitamin D in Bone Marrow Transplant Survivors. B. Buehring*¹, D. Krueger¹, W. Longo*², B. Flynn*², N. Binkley¹, ¹University of Wisconsin Osteoporosis Clinical Research Program, Madison, WI, USA, ²University of Wisconsin Blood and Marrow Transplant Program, Madison, WI, USA.

Increasing data are available showing that long-term bone marrow transplantation (BMT) survivors have deficits in bone mineral density (BMD) with consequent increased fracture risk. As the numbers of BMT recipients are increasing and survival rates improve, the impact of decreased BMD and increased fracture risk on morbidity and mortality also rises. Data defining the skeletal effects of BMT beyond six months post transplantation are limited. As such we report twelve month BMD data as well as six month bone metabolism parameters and 250H vitamin D [25(OH)D] levels in BMT survivors.

A subset of 15 long-term BMT survivors with hematologic malignancies from a prospective trial documenting BMT effects on skeletal status were used to obtain these data. This sample of eight women and seven men, mean age 40.8 ± 1.6 years, had BMD measured at the lumbar spine (LS), total proximal femur (TF), and femur neck (FN) on a Lunar Expert (Madison, WI) densitometer at baseline, 100 days, six months and one year post transplant. 25(OH)D, osteocalcin (Oc), bone specific alkaline phosphatase (BSAP) and n-telopeptide of type 1 collagen (NTx) were measured at similar time points.

BMD decreased (p<0.01) in all measured regions. TF and FN declined by 10% and 8% respectively at three months and were reduced 12% and 9% at one year. LS BMD was 9% lower than baseline after 12 months. At time of BMT bone turnover was elevated, and subsequently at 30 days Oc was numerically but not statistically lower. At six months both BSAP and Oc were increased from baseline. NTx trended upwards (p = 0.07) by 30 days and was not different from baseline after half a year.

Low vitamin D status was extremely common prior to BMT being present in 87% (13/15) and remained suboptimal at 6 months in 67%. No clinical fractures were reported within the first 12 months.

In conclusion, following BMT, despite elevation of bone formation, rapid and pronounced bone loss occurs which persists at one year. 25(OH)D levels were low before transplantation and remained low. Femur BMD results are comparable to other studies, however only minimal loss in LS BMD has been reported elsewhere. Although no clinical fractures were reported in the first year post transplantation, these data suggest BMT patients are likely at increased fracture risk. As such, skeletal status and quality of life assessments in these individuals now 5-7 years post transplantation is planned.

Disclosures: B. Buehring. None.

T500

Severe Hypovitaminosis D, Associated with Hyperparathyroidism and Osteopenia/Osteoporosis in a Cohort of Long-Term Kidney Transplant Recipients with Excellent Kidney Function. R. Hayashi*¹, M. Stratilatova*², M. Markell*¹, ¹Renal Diseases, SUNY Downstate Medical Center, Brooklyn, NY, USA, ²Renal Diseases, Westchester County Medical Center, Valhalla, NY, USA.

Vitamin D deficiency, manifested as decreased 25-OH vitamin D levels, is highly prevalent in hemodialysis patients, and may contribute to hyperparathyroidism and renal osteodystrophy. It is widely believed that successful kidney transplantation, which presumably restores the kidney's ability to activate vitamin D by 1-hydroxylase, will result in restoration of Vitamin D homeostasis, and Vitamin D supplementation is not routinely recommended. We measured 25-OH vitamin D levels in 24 long-term (mean time since transplant - 59.6 ± 11.4 mos) kidney transplant recipients (KTR) with excellent function, mean creat 1.6±0.1 mg/dl. There were 14 women (51%), 80% Black or Hispanic, mean age 48.9±1.8 yrs. 60% were receiving tacrolimus and the rest cyclosporine, 80% were taking prednisolone. The mean 25-OH vitamin D level was 12.9±1.3 ng/ml (range 5-28, normal value >40 ng/ml). Only 1 pt had a value >25 ng/ml and 11 pts (46%) had values <12 ng/ml (severe deficiency). Mean intact PTH level was also elevated (150.6±28.4, range 20-624), with 12 pts (50%) having values >300. There was no relationship between 25-OH Vitamin D level and PTH level. Neither Vitamin D level nor PTH value were related to age, gender, race, time since transplant, creatinine, current calcium or phosphate value, hematocrit, albumin or use of activated vitamin D supplements (8 pts). Bone density scans were available for 6 patients with mean T scores of −2.33 in the L-spine and −1.54 in the femoral neck. We conclude, in our population of primarily minority race KTRs: 1. Vitamin D deficiency is universally prevalent, with severe deficiency occurring in 46% of pts. 2. Persistent hyperparathyroidism is present in over 50% of pts despite the long time since transplant, and excellent kidney function. 3. The degree to which vitamin D deficiency contributes to persistant hyperparathyroidism and osteoporosis, in this population with multiple risk factors, is not known. 4. Hypovitaminosis D is prevalent in a Blacks and Hispanics, in general, and whether this degree of deficiency exists in a Caucasian population of KTR's should be studied. 5. The appropriate dose of Vit D remains to be established, as pts on maintenance active vitamin D preparations in this population had PTH levels that were similar to those not receiving it. It is possible that patients should receive 25-OH Vitamin D as well as the active form is. 6. Long term studies examining repletion of Vitamin D and its effect on the accelerated cardiovascular disease & chronic kidney fibrosis that affects the KTR population are suggested.

Disclosures: R. Hayashi, None.

T501

Effect of Risedronate on Bone Loss Associated with Liver Transplantation. S. Guadalix*¹, M. Gomez-Juaristi*¹, G. Martinez¹, C. Vargas*², J. Meneu*³, A. de Lozar*², S. Azriel*¹, E. Jodar*¹, E. Moreno*³, F. Hawkins¹, ¹Endocrinology Service, University Hospital 12 de Octubre, Madrid, Spain, ²Biochemistry Service, University Hospital 12 de Octubre, Madrid, Spain, ³Digestive Surgery, University Hospital 12 de Octubre, Madrid, Spain.

Background: Liver transplantation (LTx) increases the risk of osteoporosis and osteoporotic fractures. Most of the bone loss usually occurs in the first 6 months after transplantation, but many patients remains with low bone mass in the long term. The best preventive measure in this setting is being investigated. Objective: To analyse the effect of weekly risedronate on bone loss in patients with low bone mass after LTx. Patients and Methods: we show preliminary results of a randomized open-label single center prospective study. Forty-three patients (32 males, 11 females; mean age 54.2 ± 11.2 years) with LTx and low bone mass (lumbar and/or femoral T-score <-1) have been included so far. After transplantation (mean time 31.4 ± 15.4 days) patients were randomly assigned to one of two treatment arms: Group 1 (n = 21) received risedronate (35 mg orally, once weekly) plus calcium (1000 mg/day) and vitamin D (800 IU); Group 2 (n = 22) received calcium and vitamin D at same doses. Primary endpoint is BMD change at lumbar spine (L1-L4) and femoral regions (measured at baseline, 6 and 12 months after LTx; Hologic QDR 4500 densitometer); secondary endpoints include biochemical markers of bone turnover (serum β-CTX and P1NP, urinary D-Pyr), and calcitropic hormones (PTH, 25-OH vitamin D) measured at baseline, 3, 6 and 12 months after LTx. Baseline spine X-rays were obtained in all patients. Results: No differences were found in baseline characteristics between groups. At baseline 46.6% of patients had densitometric criteria of osteoporosis, and 16.2% had prevalent vertebral fractures. At 6 months, spine BMD increased in Group 1 (+2.1 ± 6.8 %), and decreased in Group 2 (-0.6 ± 4.6%). Changes were similar at femoral neck and total hip in both groups. Baseline bone resorption markers were increased in 40.5% (β-CTX) and 56% (D-Pyr) of patients. Ninety-three of patients showed vitamin D insufficiency at entry in the study. In Group 1, a significant decrease of serum β-CTX was observed after 3 months (p = 0.032) and 6 months (p = 0.007). Treatment was well tolerated in both groups. Conclusions: Preliminary data of this prospective trial suggest that risedronate could be effective in preventing the natural course of bone loss associated with liver transplantation.

Disclosures: G. Martinez, None.

This study received funding from: Fundacion Mutua Madrileña (2005-072).

T502

An Experimental Rabbit Model of Lunate-like Osteonecrosis: Is Transplantation of Bone Marrow Effective for Remodeling of Necrotic Bone. T. Ogawa*, T. Ishii*, H. Mishima, N. Ochiai*, Orthopaedic Surgery, University of Tsukuba, Tsukuba, Japan.

Introduction Lunate malacia (Kienbock disease) is a progressive wrist disorder characterized by osteonecrosis of the lunate. We decided to investigate whether transplantation of bone marrow (BM), a less invasive procedure, would be an effective treatment. There was no animal Kienböck disease model; so, we have developed an experimental rabbit model of lunate-like osteonecrosis using a rabbit's 4^th tarsal bone. We investigated the validity of the model and the usefulness of BM transplantation for remodeling of necrotic bone.

Material and Method Thirty six adult Japanese white rabbits were used. The bilateral 4^th tarsal bones were removed and soaked in liquid nitrogen for 5 minutes. After the freezing, the bone was inserted in subcutaneous of the back of the rabbit. The total number of the necrotic bone was seventy two. They were divided into three groups, BM transplantation (T), drilling (D) and control (C). In the group T, three drill holes were made cortical surface of the tarsal bone with Kirschner wire, cartilage surface were remained intact, and after subcutaneous insertion they were filled with BM from iliac crest. In the group D, three drill holes were also made but no BM transplantation. In the group C, the talsal bones were only soaked in liquid nitrogen. Fluorochrone label (Calcein) was injected subcutaneously at 8 days and 2 days before sacrifice. We sacrificed nine rabbits (three from each group), obtaining six specimens of necrotic bone from each group, at 2, 4, 8 and 12 weeks. Bone morphological study (Toluidine-blue), ALP staining and TRAP staining were made, using the undecalcified sections.

Results We compared histological finding of the ratio of the fluorochrome-labeled trabecular bone surface in each group. Group C and D had poor new bone formation from 2 to 12 weeks. In group T, new bone formation, were significantly higher than in the other groups at 4, 8 and 12 weeks (Fig. 1). TRAP-positive multinucleated cells were found in group T at 8 and 12 weeks more than another groups. Qualitative analysis, there were found ALP-positive osteoblast-like cells in group T at 4, 8 and 12 weeks more than another groups.

Discussion This study established a new animal necrosis model similar to human lunate, in terms of avascular necrosis and surrounding with cartilage and cortical bone. Using this model we showed that BM transplanted might be effective in new bone formation.

Disclosures: T. Ogawa, None.

T503

Effects of Immunosuppressants on Bone Metabolism (Bone Mineral Density, Bone Vitamin K Analogs, and Serum Osteocalcin and Calcium) in Rats. H. Wakabayashi¹, J. Kanda*¹, A. Takahashi*², K. Onodera*³, ¹Clinical Pharmacotherapy, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan, ²The Clinics of Dentistry for the Disabled, Tohoku University Dental Hospital, Sendai, Japan, ³Dental Pharmacology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.

This study was carried out to evaluate the effects of short-term administration of corticosteroids (hydrocortisone, prednisolone and dexamethasone) on bone metabolism. Growing rats were administered daily with the equiactive dose of corticosteroid (hydrocortisone: 20 mg/kg; prednisolone: 4 mg/kg; dexamethasone: 0.8 mg/kg) at 9 am for two weeks. No significant differences in bone vitamin K analogs (VKs), bone mineral density (BMD) and serum osteocalcin (OC) were observed both in the hydrocortisone-treated group and prednisolone-treated group compared with the control group, whereas significant decreases were observed in MK-4 (one of the VKs), BMD and serum OC of the dexamethasone-treated group compared with those in the control group. Furthermore, we also investigated about a preventative effect against bone loss caused by short-term injection of dexamethasone, using combined administration of alfacalcidol, risedronate, and menatetrenone with dexamethasone. Combined administration of risedronate and menatetrenone with dexamethasone for two weeks prevented the reduction of BMD induced by dexamethasone, especially the administration of risedronate and menatetrenone at 5 pm was more effective to prevent the bone loss than the administration at 9 am. The degree of bone loss induced by drugs has been considered to be dependent on drug type (corticosteroid type) and dose, duration of treatment, age and sex of recipients. Additionally, the present results suggest that the degree of bone loss induced by drug is dependent on the drug administration time.

Disclosures: H. Wakabayashi, None.

T504

α5 Integrin Association with Cx43 Regulates the Function of Osteocyte Hemichannels in Response to Shear Stress. A. J. Siller-Jackson¹, S. Burra¹, S. E. Harris², L. F. Bonewald³, E. Sprague*⁴, J. X. Jiang¹, ¹Biochemistry, University of Texas Health Science Center, San Antonio, TX, USA, ²Perodontics, University of Texas Health Science Center, San Antonio, TX, USA, ³Oral Biology, School of Dentistry, University of Missouri, kansas City, MO, USA, ⁴Radiology, University of Texas Health Science Center, San Antonio, TX, USA.

Primary osteocytes and MLO-Y4 osteocyte-like cells express large amounts of connexin 43 (Cx43) which forms gap junctions and hemichannels. Our previous studies have shown that fluid flow shear stress (FFSS) induces the opening of hemichannels in MLO-Y4 cells. Analysis by gene microarray of MLO-Y4 cells showed elevation of α5 integrin with FFSS and inhibition with a gap junction/hemichannel inhibitor, a result further confirmed by Northern blot assay. α5 integrin blocking antibody significantly decreased FFSS induced dye uptake mediated by Cx43 hemichannels, suggesting a role for α5 integrin in FFSS regulation of Cx43 hemichannels. Immunostaining showed colocalization of α5 integrin with Cx43 not only on the cell surface but throughout the cell body and processes, becoming more pronounced when exposed to FFSS. Cx43 and α5 integrin were not co-localized with vinculin or paxillin, components of focal adhesions, suggesting that the association between Cx43 and α5 integrin is unrelated to the function of integrins in focal adhesions.

Co-immunoprecipitation analysis showed the association of α5 integrin with Cx43. To determine the involvement of α5 integrin in Cx43 hemichannel function, α5 integrin siRNA was transfected into MLO-Y4 cells, which leads to a 90% reduction of α5 integrin levels. Interestingly, cellular localization was significantly altered in α5 integrin siRNA-treated cells with the disappearance of punctate expression of Cx43 on the cell surface. In fact, Cx43 was found outlining the entire cell surface border. FFSS applied to α5 integrin siRNA transfected MLO-Y4 cells resulted in the localization of Cx43 in punctuate spots on the surface, although less prominent than in untransfected cells. More importantly, α5 integrin siRNA completely abolished hemichannel-mediated dye uptake induced by FFSS. Together, these results suggest that α5 integrin plays an essential role in the function of Cx43-hemichannels and is a likely mechanical sensor or tether that transmits mechanical stress to Cx43 hemichannels.

Disclosures: J.X. Jiang, None.

This study received funding from: National Institutes of Health.

T505

Mechanical Activation of β-catenin Is Enhanced After Caveolin-1 Knockdown. M. Ma*¹, B. Sen*¹, N. Case¹, Z. Xie*¹, H. Jo*², T. Gross³, J. Rubin¹, ¹Medicine, UNC, Chapel Hill, NC, USA, ²Emory/GaTech, Atlanta, GA, USA, ³Orthopaedics, U. Washington, Seattle, WA, USA.

Mechanical loading of bone initiates an anabolic, anti-catabolic pattern of gene response; some of this response may be attributed to induction of β-catenin responsive genes. Caveolin-1, a structural membrane protein, can sequester β-catenin. The skeletal phenotype of the caveolin-1 knock-out mouse is of increased trabecular and cortical bone. This suggested to us that the presence of caveolin-1 might limit the process of osteogenesis; indeed we have shown that delivery of siRNA targeting caveolin-1 (siCav) to stromal cells causes upregulation of Runx2 and osterix and suppression of RANKL. We therefore asked if an interaction of caveolin-1 with β-catenin might constrain mechanical stimulation of the osteoblast phenotype. For these experiments we plated CIMC-4 preosteoblast cells expressing caveolin-1 on BioFlex collagen plates. Application of 2%, 0.17 Hz substrate strain for 2 h followed by 2 h incubation induced a β-catenin gene response: WISP-1 rose to 1.86 ± 0.06 fold (p < 0.05), returning to baseline 6 h later. Other β-catenin target genes, Runx2 and osteoprotegerin, were not increased at 4 h after the strain protocol. Delivery of siCav (50 nM) caused an 80% reduction in cav-1 mRNA and >75% reduction in caveolin-1 protein compared to control siRNA (siSCR). After strain, at 4 h, +siCav cells showed not only an enhanced WISP-1 response, but also significant elevation of osteoprotegerin (+siCav+strain 1.7±0.4 fold above +siCav-unstrained) and Runx2 (1.4 ± 0.15 fold increased). RANKL expression was repressed to <0.4 that of unstrained controls, and was unaltered by siCav, indicating that RANKL repression did not involve caveolin-1. We next explored means of measuring β-catenin activation after mechanical stimulation. Strain caused an increase in cytoplasmic β-catenin levels as assayed by Western after 3 h. Though the TopFlash β-catenin reporter was activated by LiCl in CIMC-4 cells (3.1 ± 1.7 fold above untreated cells), we have not yet shown that strain induces TopFlash activity. However, siCav treated cells showed increases in basal cytoplasmic β-catenin, and basal and strain stimulated TopFlash activity were increased by 2.14 and 2.5 fold respectively. Our data indicates that a reduction in caveolin-1 is accompanied by an increase in the β-catenin pool accessible to exogenous stimuli. This suggests that not only can caveolin-1 limit incoming mechanical signals by sequestering the actionable β-catenin pool, but also that mechanical activation of β-catenin may involve its release from the membrane.

Disclosures: M. Ma. None.

This study received funding from: NIAMS/NIH.

T506

Nmp4/CIZ Contributes to Fluid Shear Stress Induced MMP-13 Gene Induction in Osteoblasts. K. Panyayong¹, J. Yang*², F. M. Pavalko*³, R. Gerard-O'Riley*³, J. P. Bidwell², ¹Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hatyai, Songkhla, Thailand, ²Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA, ³Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN, USA.

Mechanical force or load impacts bone remodeling. The expression of matrix metalloproteinase-13 (MMP-13), involved in bone turnover, is elevated in stretched MC3T3-E1 osteoblast-like cells. Strain-mediated forces on bone move fluid through the canalicular-lacunar network resulting in fluid shear stress (FSS) on the membranes of bone cells initiating remodeling. Although the nuclear events mediating putative FSS-induced changes in osteoblast MMP-13 transcription are unknown, previous studies with osteoblasts suggest an overlap between osteoblast FSS- and PTH-induced signal response pathways. MMP-13 PTH response is regulated by a 110 bp 5′ regulatory region, conserved across the mouse, rat, and human genes, that supports the binding of numerous transcription factors including Runx2, c-fos/c-jun, Ets-1, and nuclear matrix protein 4/cas interacting zinc finger protein (Nmp4/CIZ), a nucleocytoplasmic shuttling trans-acting protein that attenuates PTH-driven transcription. Nmp4/CIZ also binds p130^cas, an adaptor protein implicated in mechanotransduction. Our objective was to determine whether Nmp4/CIZ contributes to FSS-induced changes in MMP-13 transcription. MC3T3-E1 cells were transfected with MMP-13 promoter-reporter or Nmp4/C1Z promoter-reporter constructs, then exposed to FSS (12 dynes/cm², 1-5 hr). MMP-13 mRNA levels and Nmp4/CIZ-DNA binding activity were analyzed by real-time PCR and gel shift assays, respectively. Western analysis of nuclear, membrane, and cytoplasmic fractions was used to assess FSS-induced changes in p130^cas subcellular distribution. We observed that FSS (3-5 hr) increased MMP-13 promoter-reporter activity approximately 2-fold in MC3T3-E1 cells attended by a comparable increase in mRNA expression. This was accompanied by a decrease in Nmp4/CIZ binding to its cis-element within the PTH response region, the mutation of which abrogated FSS response. Interestingly, FSS enhanced Nmp4/CIZ promoter activity. FSS also induced p130^cas nuclear translocation. We conclude that the PTH regulatory region of MMP-13 also contributes to FSS response and that Nmp4/CIZ plays similar but distinct roles in mediating hormone- and FSS-driven induction of MMP-13 in bone cells.

Disclosures: K. Panyayong, None.

T507

The Presence of p120-Catenin in Osteocytes and Other Bone Cells. H. W. Sampson, A. R. Parrish*, A. C. Dearman*, W. E. Zimmer*, Systems Biology and Translational Medicine, TAMHSC College of Medicine, College Station, TX, USA.

P120 is a member of the armadillo supergene family which has been reported to bind cadherins at a juxtamembrane domain and regulate their cell surface trafficking. It can become cytoplasmic regulating activities of Rac, Rho and Cdc42; thus functioning in maintenance of cell shape and enhanced cell motility. It can also enter the nucleus where it is considered to possibly modulate Wnt/β-catenin/TCF signaling in a synergistic manner in the nucleus to activate matrilysin expression. The purpose of this study was to document the presence of p120-catenin in bone cells. We used conventional RT-PCR, Western blot, immunohistochemical and fluorescent immunohistochemistry in conjunction with light and fluorescent microscopy and confocal microscopy to study in situ bone cells and also MLO-Y4 osteocyte-like cells in culture (a generous gift from Dr. Lynda Bonewald, University of Missouri-Kansas City). The p120-catenin antibody used was a monoclonal antibody at 1:100 dilution and the biotinylated secondary antibody, was incubated in conjugated streptavidin horseradish peroxidase followed by Betazoid 3, 3′ Diaminobenzidine. In the case of the fluorescent studies the secondary antibody was Alexa Fluor 488 goat anti-mouse IgG (molecular probes, OR). The in vivo studies were conducted on mouse tibia and all experimental procedures were in compliance with the guiding principles in the “Care and Use of Animals” as published in the Am. J. Physiol., and were approved by the TAMU animal care and oversight committee. All methods used indicated a high concentration of p120 RNA and protein in osteocytes, both in situ and in culture, in osteoblasts, and in chondrocytes of the resting zones and hypertrophic zones of the mouse growth plate. All studies indicated a cytoplasmic or sub-membrane localization. We have demonstrated, for the first time, the presence of p120-catenin in osteocytes and other bone cells. This is an adhesion molecule whose importance in cell signaling is becoming more and more apparent with time and with its ability to move from the plasma membrane, to the microtubule system of the cytoplasm and to the nucleus is positioned to play an important role in cell development and possibly in mechanotransduction.

Disclosures: H. W. Sampson, None.

T508

The Effects of Compressive Loading on Matrix Production by Osteoblastic Cells Cultured in Polyurethane Scaffolds. A. Sittichokechaiwut, A. Scutt, G.C. Reilly. Engineering Materials, University of Sheffield, Sheffield, United Kingdom.

Bone tissue engineering constructs are composed of scaffold materials and matrix synthesizing cells. It is likely that mechanical stimulation of tissue engineered bone will enhance matrix synthesis, however, the underlying mechanisms by which osteoblasts respond to their mechanical environment in 3-D are poorly understood. The aims of this study are to examine the effects of cyclic compressive loading on bone matrix production in 3-D scaffolds over 20 days of culture. 3-D polyurethane scaffolds with an average pore size 150 micrometres were cut into cylinders of 10 mm diameter and 10 mm height. Scaffolds were seeded with MLO-A5 osteoblastic cells (Kato et al. JBMR 2001), kindly donated by Dr. L. Bonewald, at densities of 2.5×10⁵ per scaffold. Cell-seeded scaffolds were dynamically loaded in compression at 1Hz, 5% strain in a biodynamic chamber (BOSE/Electroforce3200). Loading was applied for 2 hours per day at day 5, 10 and 15 of culture. Between loading, scaffolds were cultured in an incubator in standard conditions for up to 20 days. Cell-seeded scaffolds were assayed on days 10, 15 and 20 of culture for scaffold stiffness (Young's modulus of elasticity), cell viability by MTS assay, calcium by alizarin red staining and collagen content by sirius red staining. Cells survived in all loaded samples, final relative cell number was increased at day 10 but slightly lower at days 15 and 20. Calcium content increased in all loaded samples and was significantly higher on day 20 (p<0.01, t-test) compared with non loaded samples. Total collagen also increased significantly at day 15 and 20 in loaded samples compared with non loaded samples (p<0.02). Scaffold stiffness was higher in loaded samples at the end of experiment (∼39 vs 62 Pa, p<0.01). In conclusion, although the number of viable cells was lower in loaded samples after 10 days, the amount of calcium, collagen and scaffold stiffness was greater, indicating the formation of new extracellular matrix within the constructs. Therefore, cyclic compressive loading at 5% strain for 2 hours every 5 days has the potential to produce more bone matrix than static culture conditions.

Disclosures: A. Sittichokechaiwut, None.

This study received funding from: The Royal Society of London.

T509

Calibration of the ZETOS Bone Loading System. S. Garcia*¹, H. L. Ploeg*¹, E. L. Smith², ¹Mechanical Engineering, University of Wisconsin, Madison, WI, USA, ²Population Health Sciences, University of Wisconsin, Madison, WI, USA.

An ex vivo bone culture and loading system, ZETOS, has been developed to study morphological and physiological responses of trabecular bone (10 mm diam., 5 mm ht.). A piezoelectric actuator (PZA) expands, compressing the specimen while measuring expansion and load. Calibration was performed when manufactured, but the system's compliance changes with time, requiring recalibration. The purpose of this study was to develop and validate a recalibration protocol for the ZETOS bone loading system.

Ten aluminum (alloy 7075-T6) reference bodies (RBs) of known stiffness (40 to 2000 MPa, trabecular bone stiffness range) were designed using finite element analysis (FEA) and manufactured. Geometry was verified using a coordinate measuring machine, at 72 points throughout the RB, showing uniform surfaces (SD = 2.34 μm). Calibration of the unloaded PZA expansion transducer was performed with a fiberoptic sensor. All RBs were tested in ZETOS, recording force and expansion. A FEA model duplicated the physical compression setup of the RBs in ZETOS. Stiffness was found for each RB by dividing total reaction force by displacement. FEA was validated with physical testing in an Instron by compressing six RBs on a setup replicating the ZETOS system. RB deformation was measured using a 10x microscope and calibrated digital camera. Percent differences of FEA-determined stiffness were found with respect to stiffness from Instron testing. ZETOS force and expansion, and compression from FEA were integrated in a calibration table. For verification of the calibration table, all RBs were tested in ZETOS, as well as 3 metal springs of unknown stiffness, comparing these results to those from the FEA.

FEA results had a mean percent difference with respect to Instron testing of 5.08%. The results from the Instron measurements were used to validate the FEA. Verification of RB stiffness determined by ZETOS after calibration resulted in an overall mean difference from the FEA of 1.10%. Results from testing of the 3 metal springs found a mean percent difference in stiffness, compared to their FE models, of 2.25%.

Calibration of the ZETOS loading system is important to assure stiffness measurement accuracy. FEA was an essential tool for RB design and calculated stiffness. The combination of physical testing and FEA enabled a precise determination of RB stiffness. Test results validated the FEA, which provided results that could not be physically measured to generate the calibration table. The successful development of a recalibration protocol of the ZETOS assures accuracy of trabecular bone stiffness measurements.

Jones, D.B. et al. European Cells and Materials, 2003, 5, 48-60Smith, E.L. et al. J. Bone and Mineral Research, 2001, 16(Suppl 1), S481

Disclosures: E.L. Smith, None.

This study received funding from: University of WI Graduate School.

T510

Identification of Genes that May Modulate the Musculoskeletal System's Early Response to Unloading. M. E, Squire¹, M. Monaghan*², L. R. Donahue³, A. Dhundale*², C. T. Rubin⁴, S. Judex⁴, ¹Biology, University of Scranton, Scranton, PA, USA, ²Center for Biotechnology, Stony Brook, NY, USA, ³Jackson Laboratories, Bar Harbor, ME, USA, ⁴Biomedical Engineering, Stony Brook University, Stony Brook, NY, USA.

Bone and skeletal muscle are both capable of remodeling their structures to accommodate altered levels of mechanical loading. In the absence of functional loading, musculoskeletal tissue is lost at rates that can exceed 2% per month. Based on the functional relationship between bone and muscle, it is plausible that similar molecular mechanisms may be involved in the early stages of their responses to altered mechanical loading. In this study, we examined gene expression in bone and muscle following short-term hindlimb unloading, with a particular interest in genes that were differentially regulated in both tissues. Total RNA was extracted from the whole proximal tibia and soleus of adult (4 mo) female F1 crossbred (BALB/cByJ x C3H/HeJ) mice following 4d of normal cage activity (control) or tail suspension (disuse) (n = 5 each). Reference samples were hybridized with either a control or disuse sample onto custom oligonucleotide arrays (n = 5 per group) containing 16,442 ESTs (70mer oligos) from the Operon mouse genome library (v2). Following normalization and filtering, 164 (tibia) and 115 (soleus) differentially regulated genes were identified (p<0.05), with only 3 genes common to both lists. Further classification of affected genes based on their “biological function” annotations revealed 17 genes (tibia; 7 up- and 10 down-regulated) and 14 genes (soleus; 6 up- and 8 down-regulated) that belonged to the “cell communication” category. Some of these identified genes have known roles in classic signaling pathways or are known intracellular signal transducers in bone or skeletal muscle. In the “response to stimulus” category, 14 genes (tibia; 6 up- and 8 down-regulated) and 8 genes (soleus; 2 up- and 6 down-regulated) were significantly affected. This list included one gene that was down-regulated 1.2 and 2.0 fold in the tibia and soleus, respectively, following unloading. In addition, one significantly down-regulated gene in the tibia (-1.4 fold) encodes for a receptor protein that has been identified previously as a responder to mechanical stimuli in osteoblasts and osteocytes. Current qPCR studies will validate these findings. Taken together, the results suggest that although some genes may mediate the early response of both tissues to mechanical unloading, the vast majority of genes involved are likely distinct across tissues. Future analyses will be needed to examine whether the lack of common genes is due a difference in molecular mechanisms or a difference in the temporal response in bone vs. muscle.

Disclosures: M.E. Squire, None.

T511

Ultrasound Induces Hypoxia-Inducible Factor-1 Activation and iNOS Expression Through Integrin/ILK/Akt Pathway in Cultured Osteoblasts. C. Tang¹, W. Fu*², D. Lu*³, T. Tan*¹, R. Yang⁴, ¹Pharmacology, China Medical University, Taichung, Taiwan, ²Pharmacology, National Taiwan University, Taiwan, Taiwan, ³Pharmacology, National Taiwan University, Taipei, Taiwan, ⁴Orthopaedics, National Taiwan University & Hospital, Taipei, Taiwan.

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US stimulation increased nitric oxide (NO) formation and the protein level of inducible nitric oxide synthase (iNOS). US-mediated iNOS expression was attenuated by anti-integrin α5β1 or β1 antibodies but not anti-integrin αvβ3 or β3 antibodies or focal adhesion kinase mutant. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) also inhibited the potentiating action of US. US stimulation increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, US stimulation also increased the stability and activity of HIF-1 protein. The binding of HIF-1α to the HRE elements on the iNOS promoter was enhanced by US stimulation. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for US-induced HIF-1α activation and subsequent iNOS up-regulation. Taken together, our results provide evidence that US stimulation up-regulates iNOS expression in osteoblasts cells by a HIF-1α-dependent mechanism involving that activation of ILK/Akt and mTOR pathways via integrin receptor.

Disclosures: C. Tang, None.

T512

Bone Density and Body Composition Values in Healthy Japanese Girls. T. Aizawa*¹, M. Konish*², O. Zhou², ¹Dept of Health and Sport Sciences, Mukogawa Women's University of Letters, Nishinomiya, Kyogo, Japan, ²GE Healthcare Asia, Shanghai, China.

Reference values for bone mineral density (BMD) and soft tissue assessment with dual-energy X-ray absorptiometry (DXA) provide valuable clinical information to help physicians evaluate children with growth disorders and metabolic diseases. As reference values can vary among populations in different geographic regions, it may be advisable to establish region-specific reference values. We measured the total body BMD and body composition in 557 Japanese healthy girls (ages 12 to 19 yrs) with a Lunar DPX-NT (GE Healthcare) densitometer. BMD increased rapidly (4%/year) as did height, bone area (BA), and fat free mass (FFM) between ages 12 and 14 years, presumably approximating the end of puberty. Another period of rapid increase was found at from ages 16 to age 17 years, when both BMD and BA increased at 4%/year, although height remained stable. On average, BMD values increase from age 12 to 18 at 3%/year, while BA and FFM increased by about 3%/year and 4%/year respectively. There was a strong correlation between bone mineral content (BMC) and lean mass (LM) (r = 0.734, p<0.01), as well as between bone area and height (r = 0.786, p<0.01) over the entire age range. Between the ages of 12 and 19, BMD, bone area and FFM increased rapidly in this population. BMD values at age 18 and 19 for these Japanese girls were slightly higher values previously reported for Caucasian girls of that age. 

Table  .  

Disclosures: T. Aizawa, None.

T513

Bone Mineral Density, Body Composition, Body Mass Index and Height in Children Treated for Acute Lymphoblastic Leukemia. M. Ansari*¹, N. Alos², A. Moghrabi*¹, ¹Pediatrics-Hematology-oncology service, CHU Ste-Justine and University of Montreal, Montreal, PQ, Canada, ²Pediatrics-Endocrinology service, CHU Ste-Justine and University of Montreal, Montreal, PQ, Canada.

Short stature, obesity and bone morbidity are commonly reported as late effects of therapy for childhood with acute lymphoblastic leukemia (ALL). We evaluated height, body mass index, body composition and lumbar bone mineral density in children after treatment for ALL.

Patients and Methods: Between January 1997 and August 2002, a cross-sectional study was done in children in remission treatment for ALL, using the Dana-Farber-Cancer-Institute (DFCI) consortium protocol 95-01. Lumbar bone mineral density (BMD) and body composition were measured by dual energy X-ray absorptiometry (DEXA). Height, body mass index (BMI), lumbar BMD (areal and volumetric) and body composition (fat mass and lean mass) were converted to z scores, adjusting for age and sex.

Results: 132 patients were enrolled.; 99 were evaluated using DEXA. Median age at diagnosis was 3.8 years (0.5 – 12.6 years). Mean follow-up from end of therapy was 3.2 years (0.6 to 7.1 yrs). There was no significant difference in population characteristics between risk groups (Standard and High Risk, SR/HR), including age, height, BMI, percentage Body Fat (% Fat) and Lean Body Mass (LMB) Z score. The mean Z score for % Fat was −0.715 ± 1.25 SD and the mean Z score for LBM was 1.36 ± 1.06 SD. There was no bone fracture or back pain reported. The mean volumetric lumbar spine BMD Z-score was 0.31 ± 0.97 SD for all patients; 0.36 ± 1.0 SD for HR (n = 39) and 0.28 ± 0.95 SD for SR patients (n = 60) (p = 0.69). No patients had a volumetric lumbar spine BMD (g/cm³) Z-score of less than −2 SD.

Conclusions: Children with ALL treated with DFCI protocol 95-01 in our institution have little side ffects related to growth, body composition and BMD, at a median interval of 3.2 years post end of therapy. Longer follow up is needed to confirm these results as well as comparison to cohorts using different protocol therapies.

Disclosures: N. Alos. None.

T514

Bone Ultrasonography in the Longitudinal Monitoring of Bone Status in Patient with Rett's Syndrome. S. Gonnelli¹, C. Caffarelli*¹, J. Hayek*², A. Montagnani*¹, A. Cadirni*¹, L. Tanzilli*¹, B. Franci*¹, B. Lucani*¹, R. Nuti¹, ¹Institute of Internal Medicine, University of Siena, Siena, Italy, ²Department of Child Neurology and Psychiatry, University of Siena, Siena, Italy.

Rett syndrome, an X-linked neurodevelopmental disorder primarily affecting girls, is frequently characterized by osteopenia with a consequent increased risk of fragility fractures.

The aim of the study was to evaluate the usefulness of Quantitative Ultrasound (QUS) at phalanxes in the assessment and monitoring of bone status in Rett patients.

In 109 Rett girls (10.1 ± 6.1 yrs) and in 101 age-matched controls, serum calcium, bone alkaline phosphatase, parathyroid hormone (PTH), 25-hydroxyvitamin D (25OHD) and QUS parameters at phalanxes by Bone Profiler-IGEA (amplitude dependent speed of sound: AD-SoS and bone transmission time: BTT) were measured at baseline and then yearly for 3 years.

At baseline both QUS parameters and 25OHD levels were significantly lower in Rett patients than in controls. Serum 25OHD was inversely correlated with serum PTH (r = −0.38; p<0.05) and BTT Z-score (r = 0.37 p<0.01). BTT Z-score was significantly lower (p<0.05) in the patients with 25OHD serum levels ≤ 9 ng/ml (-2.49 vs −1.58). Multiple regression analysis showed that BTT was significantly influenced by 25OHD levels. Eighty-two Rett patients completed the 36 month longitudinal study. During the longitudinal study the patients who at baseline were non-ambulatory presented a worsening of their bone status, and BTT and AD-SoS Z-score presented the tendency to markedly decrease. In contrast the BTT and AD-SoS of patients with severe or mild-moderate ambulatory impairment showed mild changes. At years 2 and 3 the difference in Z-score between the non-ambulatory group and the mild-moderate group was significant for both AD-SoS and BTT. At the end of the study period no-significant differences in AD-SoS or BTT Z-scores were observed between Rett patients of the non-ambulatory group and those with severe ambulatory impairment. The AD-SoS Z-score at the end of the study period resulted significantly influenced by the changes in ambulatory performance and the use of anticonvulsant therapy.

In conclusion this prospective study suggests the usefulness of QUS parameters at phalanxes, namely AD-SoS and BTT, in the monitoring of bone status in Rett patients. More specifically we found that in Rett patients QUS parameters are markedly decreased and that ambulatory impairment and low levels of 25OHD play a key role in the progressive deterioration of bone status.

Disclosures: S. Gonnelli, None.

T515

Prevalence of Osteopenia in Children with Early Juvenile Chronic Arthritis (JCA). L. Kroger*¹, H. Kroger², ¹Paediatrics, Kuopio University Hospital, Kuopio, Finland, ²Orthopaedics, Kuopio University Hospital, Kuopio, Finland.

Children suffering from juvenile chronic arthritis (JCA) are at risk of developing osteoporosis due to inflammation, reduced/ limited physical activity and use of corticosteroids.

Because these children may have been suffering from inflammation a long time before seeking medical advice, we wanted to study whether bone mineral density (BMD) is already decreased at the time of diagnosis of JCA.

The material consisted of 52 consecutive patients, aged 5-15 yrs, admitted to the paediatric outpatient clinic in Kuopio University Hospital, Finland between 2004-6 and fulfilling the criteria of JCA. A total of 40 girls (77%) and 12 boys (23%) were included in the study. BMD was measured using dual X-ray absorptiometry (Lunar DPX-IQ). Results were expressed as bone mineral content (BMC; g) and BMD (BMC/ projected area; g/cm). To minimise the effect of bone size on BMD, apparent volumetric BMD (BMDvol, g/cm³) was calculated using a cylindrical model published earlier by Kroger et al.

The mean age was 10.8 yr for boys and 11.8 yr for girls (p = ns). Thirty-three children (69%) suffered from polyarthritis (PA) and 16 (31%) from oligoarthritis (OA). There were no differences in weight or height between sexes or between different JCA type. BMDvol at the femoral neck was lower in boys as compared to girls (p = 0.025). The girls with PA showed significantly lower volumetric spinal Z-scores (-0.46 vs. + 0.52, p = 0.01) and volumetric femoral neck Z-scores (-0.21 vs. + 0.44, p = 0.04) than girls with OA. In all, 29% of children (PA = 11, OA = 4) showed volumetric BMD Z-score values lower than −1 SD either in lumbar spine or femoral neck. If only areal BMD Z-scores were considered, 13.4% of patients were found osteopenic. Low BMD values were found both in PA and OA cases.

In conclusion, measuring BMD is important in all children suffering from JCA, since BMD is decreased in substantial number of patients already at the time of diagnosis. This group of patients needs special attention, eg. supplementation of calcium and vitamin D, and repeated BMD measurements in the follow-up.

Disclosures: L. Kroger, None.

T516

Evaluation of the Muscle-bone Relationship in the Midthigh of Children with Cerebral Palsy. C. M. Modlesky¹, A. Meyers*¹, E. Hoffmann*¹, J. J. Smith¹, F. Miller*², ¹Health, Nutrition and Exercise Sciences, University of Delaware, Newark, DE, USA, ²Orthopaedics, Al duPont Hospital for Children, Wilmington, DE, USA.

The strong relationship between muscle and bone has led to the proposal of a muscle-bone index in which a measure of bone that reflects its resistance to fracture, such as cortical volume or section modulus (Z), is divided by a measure that reflects muscle strength, such as muscle mass. Understanding the muscle-bone relationship may give us further insight into the mechanisms underlying poor bone development in children. Children with cerebral palsy (CP) who are unable to ambulate independently have poor muscle and bone development in the thigh and a high rate of fracture in the femoral shaft. However, the muscle-bone relationship is poorly studied in children with CP. The purpose of this study was to determine if the muscle-bone relationship in the midthigh is different in children with CP than typically developing children. Nine nonambulatory children with CP (8 to 14 years) and 9 typically developing children within the 10th and 90th age-based percentiles for height, weight and BMI were studied. Magnetic resonance images of the thigh (1 cm thick and 0.5 cm apart) were collected along the entire length of the more involved femur using a torso PA coil (GE 1.5 T; TR = 750, TE = 14, FOV = 16, 1 NEX, Phase = 512; Frequency = 512). Images at the level of the middle third of the femur were identified and measures of bone size, composition and strength [i.e., total and cortical volume, and anterior-posterior and medial-lateral section modulus (Z_ap and Z_ml, respectively)] and muscle mass were estimated using custom software developed with Interactive Data Language (IDL; Research Systems, Inc, Boulder CO). Indexes that reflect the muscle-bone relationship were determined by dividing total bone volume, cortical bone volume, Z_ap and Z_ml by muscle mass. There were no group differences in age. Tanner stage or BMI; however, height (1.26 ± 0.12 vs. 1.44 ± 0.08 m) and weight (27.9 ± 10.8 vs. 36.3 ± 4.5 kg) were lower in children with CP than controls (P < 0.05). Children with CP also had 55% lower total bone volume (19.8 ± 6.5 vs. 44.4 ± 11.2 cm³), 55% lower cortical volume (12.1 ± 4.2 vs. 27.6 ± 7.3 cm³) 61% lower Z_ap (0.31 ± 0.11 vs. 0.80 ± 0.23 cm³) and 60% lower Z_ml (0.29 ± 0.10 vs. 0.73 ± 0.21 cm³) in the midfemur and 58% lower muscle mass (0.436 ± 0.144 vs. 1.030 ± 0.175 kg) in the midthigh (P < 0.001) than controls. However, none of the indexes that reflect the muscle-bone relationship were different between groups (P > 0.05). The findings suggest that children with CP who are unable to ambulate independently have a muscle-bone relationship in the midthigh that is not different from typically developing children.

Disclosures: C.M. Modlesky, None.

This study received funding from: National Institutes of Health and the National Osteoporosis Foundation.

T517

Poor Bone Growth in Children with Cerebral Palsy Is Underestimtated by Dual-energy X-ray Absorptiometry. J. J. Smith¹, F. Miller*², C. M. Modlesky¹, ¹Health, Nutrition and Exercise Sciences, University of Delaware, Newark, DE, USA, ²Orthopaedics, A.I. duPont Hospital for Children, Wilmington, DE, USA.

Femoral fracture incidence is elevated in children with cerebral palsy (CP). Prior fracture occurrence increases subsequent fracture risk which increases treatment costs and decreases their quality of life. Therefore, decreasing fracture incidence in this population is important. Size is a major determinant of a bone's resistance to fracture as wider bones are more resistant to fracture than thinner bones. There is evidence that children with CP have considerably thinner bones at the femoral shaft compared to typically developing children; however, the size of the distal femur, a primary fracture site, has not been evaluated. The purpose of this study was to determine if the distal femur is smaller in children with CP who are unable to ambulate independently compared to typically developing children. Nine nonambulatory children with CP (8-14 years) and 9 typically developing children between the 10th and 90th age-based percentiles for height, weight and BMI participated in the study. Bone area of the distal femur (1.4 cm region above the growth plate) was determined using dual-energy X-ray absorptiometry (DXA; Delphi W, Hologic Inc.). Trabecular bone cross-sectional area of the lateral side of the distal femur (approximating the region evaluated using DXA) was determined using magnetic resonance imaging (MRI; GE 1.5 T, 3D fast gradient echo sequence, reconstructed spatial resolution = 175 × 175 × 700 μm3). There were no group differences in age, pubertal status or BMI; however, children with CP had lower height (1.25 ± 0.04 vs. 1.43 ± 0.03 m) and weight (26.7 ± 3.7 vs. 35.6 ± 1.2 kg) compared to controls (P < 0.05). Although bone area determined by DXA was ∼ 13% lower in children with CP than controls, this difference was not statistically significant (P = 0.07). On the other hand, trabecular bone cross-sectional area determined by MRI was ∼ 36% lower in children with CP than controls and the difference was statistically significant (P< 0.01). The findings suggest that the distal femur is substantially smaller in nonambulatory children with CP than typically developing children and the degree of the discrepancy is underestimated by DXA.

Disclosures: J.J. Smith, None.

This study received funding from: NIH and National Osteoporosis Foundation.

T518

Bone Age Measurements by Ultrasound Are Equivalent to Radiological Assessments. C. Wuester, M. Hartmann*, W. Omran*, U. Cordes*, Clinic for Endocrinology, Mainz, Germany.

The purpose of the study was to show that the measurements of bone age (BA) by ultrasound at the wrist correlates with radiological BA measurements on conventional hand X-rays.

103 patients (age range 9.5 – 22.5 years, mean 14.4) were included. Thirty-one patients had delayed puberty, 17 patients had constitutional short stature. 5 patients GH deficiency and the other were normals. The ultrasound method to determine bone age at the wrist (BonAge^TM, Sunlight Medical Ltd., Tel Aviv, Israel; arewus, Mainz, Germany) evaluates the relationship between the velocity of the wave (speed of sound) passing thorough the distal radial and ulna epiphysis and growth, using gender- and ethnicity-based algorithms. Three experienced investigators (one radiologist, two endocrinologists) analysed conventional X-rays of the left hand and assigned bone age scores according to the Greulich & Pyle atlas (G&P). The investigators were blinded to the chronological age (CA) and the BonAge^TM results. DXA was measured at the spine and hip using Osteocore 3 by MediLink/arewus, Mainz/Germany.

Correlation coefficients of BA ultrasound with the 3 X-ray investigators were r = 0,80, 0,82 and 0,88 respectively. The correlation between the three X-ray investigators were r = 0.92, 0.92 and 0.96 respectively among each others and not significant different from the correlation with the ultrasound results. BMC rather then BMD correlated best with ultrasonic BA. 

Figure Figure 1.. shows the mean BA of the ultrasound-based system and the three radiological investigations. There was no statistical significant difference between the four evaluations. We conclude from the data that BonAge^TM ultrasound system to measure bone age is precise and accurate. The results show high correlation with radiological assessments. The system has the advantage of being radiation-free which is important in a target group of still growing patients.

Disclosures: C. Wuester, None.

T519

Low Doses of Pamidronate for Children with Metabolic and Genetic Diseases. C. Bowles*¹, R. Kreikemeier*², S. Coughlin*², H. Plotkin¹, ¹Pediatrics, UNMC, Omaha, NE, USA, ²Metabolics, Children's Hospital, Omaha, NE, USA.

Retention of calcified cartilage is a well-known dose-related side effect of pamidronate treatment in children and adolescents with OI. Low doses of pamidronate are effective to treat pediatric patients with cerebral palsy and osteogenesis imperfecta.

IV pamidronate (0.75 mg/kg/day for two days) was administered every 4 months to 13 non-ambulatory children and adolescents (age range 1.5 – 12 y.o) with metabolic and genetic diseases. Diagnosis included mitochondrial complex I deficiency, fumarase deficiency, pediatric neurotransmitter disease, myotonic dystrophy, Micro syndrome, Rett syndrome, and undefined genetic syndromes. On the first day of treatment patients received half dose to minimize the acute phase reaction, and acetaminophen was administered every 6 hours during the first 2-day infusion. Lumbar spine (LS) and femoral neck (FN) BMD (DEXA, Hologic Delphi) Z-scores were assessed at baseline, 4, 12 and 24 months. Calcium, phosphorus, magnesium, alkaline phosphatase, osteocalcin, NTx, 25 and 1,25 vitamin D levels in serum, were also assessed. All patients had adequate calcium and vitamin D intakes for age. The study was approved by Children's Hospital IRB.

BMD measurement z-scores at the femoral neck (FN) (N = 11) and the lumbar spine (LS) (N = 13) showed statistical evidence of an increase in bone mineral density at both the femoral neck and the lumbar spine with pamidronate treatment at 24 months. The rate of increase appears to have been initially less at the femoral neck than the lumbar spine (increases at 4 months: FN: 0.38; LS: 0.85. The increase at 1 year of follow-up at the two sites was similar (FN: 1.08; LS: 1.18). Further improvement in BMD was not seen at the 2 year follow-up. 

Table  .  

With low doses of pamidronate, there is no significant change over 2-year for serum 1,25 vitamin D, 25 vitamin D, total calcium, ionized calcium, sNTx, or osteocalcin measures. BMD in both LS and FM increase for the first year and remains stable during the second year of treatment.

Disclosures: C. Bowles, None.

T520

Effects of Risedronate on Bone Change at Children with Osteogenesis Imperfecta. C. Galesanu¹, C. Ciubotariu*¹, M. R. Galesanu*², D. Raileanu*¹, C. Tache*¹, T. Bostaca*³, ¹Endocrinology, University of Medicine and Pharmacy, IASI, Romania, ²Osteodensitometry, Centre of Imaging and Radiologic Diagnosis, IASI, Romania, ³Radiology, University of Medicine and Pharmacy, IASI, Romania.

Osteogenesis imperfecta (OI) is a disturbance in the synthesis of type I collagen, predominant protein of the extracelular matrix of most tissues. In bone, this defect causes osteoporosis, which leads to on increased tendency to fracture. Type 1 collagen is also a constituent of dentin, sclerae, blood vessels, ligaments and skin; individuals with OI may have also abnormalities of these structures. OI type IA is without dentinogenesis imperfecta. The OI type I B is a rare form with tooth abdormalities. The major sings of OI type I include blue sclerae, hearing loss, mild bone fragility.

The aim of our study was to verify the efficacy and safety of risedronate treatment in children with OI type IA.

Eight children, six girls and two boys were treated 12 months with risedronate. The children weighing under 30 kg received 35 mg risedronate at 14 days and children weighing more 30 kg received 35 mg risedronate weekly.

Three girls were declared with history of fractures (between 8 and 16). BMD was assessed at lumbar spine (LS) using Hologic DXA scanning at time 0 and 12 months. The results at time 0 by DXA-BMD were compared with reference data for bone density published by I.M. von der Sluis in 2002 (Children's Bone Health) Z-score at time 0 (DXA-BMD) was in all cases more −2.5.

At baseline mean BMD at LS was 0.562 g/cm2 for girls vs. mean BMD reference data which is 0.896 g/cm2; it was a deficit on 38%. After a year of risedronate therapy the mean BMD at LS was 0.619 g/cm2, a gain of 10%. For the boys the baseline mean BMD at LS was 0. 790 g/cm2 vs. mean BMD reference data withch is 1.096 g/cm2; it was a deficit on 28%. After a year of risedronate, the mean BMD at LS was 0.916 g/cm2, which is a gain of 15%. No adverse effects regarding gastrointestinal tract under risedronate therapy. No new fractures. The treatment and the following of children going on.

Risedronate therapy is accompanied by a important increase of LS-BMD after a year. Children with OI type IA respond to oral bisphosphonates at standard doses for osteoporosis. Risedronate 35 mg weekly seems to be safe and effective in children with O.I.

Disclosures: C. Galesanu, None.

T521

Vitamin D Status of Apparently Healthy Schoolgirls from two Different Socioeconomic Strata in Delhi: Relation to Nutrition and Lifestyle. R. Marwaha*, Department of Endocrinology and Thyroid research Centre, Institute of Nuclear Medicine and Alllied Sciences, Delhi, India.

Forty to fifty percent of total skeletal mass is accumulated during childhood and adolescence, which is influenced, by sunlight exposure, physical activity, lifestyle, endocrine status, nutrition and gender. In view of scarce data on the association of nutrition and lifestyle with hypovitaminosis D in Indian children and adolescents, an in-depth study on 3127 apparently healthy (6-18 years) schoolgirls from the lower (LSES = 1477) and upper socioeconomic strata (USES = 1650) in Delhi was carried out. These girls were subjected to anthropometry and clinical examination for hypovitaminosis D. Randomly selected 404 (LSES = 193, USES = 211) girls from the two strata underwent detailed lifestyle, dietary, biochemical and hormonal assessment. Clinical features of vitamin D deficiency were noted in 11.5% girls (12.4% LSES and 10.7% USES,). USES girls had significantly higher Body Mass Index (BMI) than LSES counterparts. Prevalence of biochemical hypovitaminosis D (serum 25-OHD <50nmol/1) was seen in 90.8% girls (LSES = 89.6% and USES = 91.9%). Mean intake of energy, protein, fat and calcium, as well as milk intake of USES girls was significantly higher across all ages. Among the LSES group, the intake of carbohydrate, fibre, phytate was higher. Physical activity and sun exposure in terms of duration and percentage of body surface area exposed was significantly greater in LSES. In conclusion, in the absence of fortification of food products with vitamin D in India, diet alone appears to have an insignificant role; however lifestyle factors may contribute to the vitamin D status of apparently healthy school subjects.

Disclosures: R. Marwaha, None.

W001

Effect of Energy Restriction on Bone Turnover in Physically Active Females. C. Sale¹, J. P. Greeves¹, J. P. R. Scott¹, W. D. Fraser², ¹QinetiQ, Farnborough, United Kingdom, ²University of Liverpool, Liverpool, United Kingdom.

Initial military training can result in a negative energy balance in female recruits, which may alter bone turnover and potentially increase the risk of stress fracture injury. The aim of the present study was to investigate the acute effect of reduced energy intake on bone turnover in females and to examine the interaction with daily training.

Seven habitually active females (mean (ISD) age: 26 (5) y; height: 1.67 (0.30) m; body mass: 66.7 (9.5) kg) completed all aspects of the study. This study was approved by the QinetiQ Research Ethics Committee. Subjects completed two conditions in a counterbalanced crossover design. Each condition comprised 5 d of energy restriction (50% of normal daily intake), which were separated by a minimum of 7 d. In one condition, subjects completed a 1.5-mile self-paced run on the afternoon of each day (EX) and in the other condition subjects remained sedentary (SED). Energy intake on the exercising days was increased to compensate for increased energy expenditure so that the level of energy restriction was the same in both conditions. Blood and second-void urine samples were obtained after an overnight fast on days 0 (baseline, [B]), 3 (mid-restriction, [D3]), 6 (post-restriction, [D6]) and 7 (recovery, following 24 h with no restriction, [D7]). Blood was assayed for markers of bone resorption (β-CTx), bone formation (P1NP, Bone ALP) and regulatory hormone concentrations (T₃, IGF-1, leptin and cortisol). Urine was assayed for markers of bone resorption (UfPYD and UfDPD).

Five days of energy restriction resulted in a significant (p<0.01) loss of body mass in both EX (66.4 (9.8) kg to 65.1 (9.5) kg) and SED (66.8 (9.2) kg to 65.0 (8.8) kg). P1NP concentrations declined significantly (p<0.05) from B (44.29 (11.88) μg·L⁻¹) to D3 (39.43 (12.49) μg·L⁻¹) and D6 (38.71 (12.22) μg·L⁻¹) in EX, and from baseline (46.14 (15.91) μg·L⁻¹) to D6 (37.43 (11.33) μg·L⁻¹) in SED. PINP concentrations remained significantly lower than B (p<0.05) on D7 in SED (40.29 (12.65) μg·L⁻¹). Bone ALP and markers of bone resorption (β-CTx, UfPYD and UfDPD) remained unchanged in both EX and SED conditions. There were no differences in markers of bone turnover between EX and SED. Energy restriction resulted in a significant (p<0.05) reduction in leptin concentrations in both EX (between B and D6 and D7) and SED (between B and D3, D6 and D7), whilst IGF-1 and T₃ concentrations were reduced in EX only (IGF-1 between B and D6 and D7; T₃ between B and D3). No changes in cortisol concentrations were observed.

Short-term energy restriction resulted in a reduction in bone formation, as shown by a decrease in PINP, which occurred independently of moderate exercise training.

Disclosures: C. Sale, None.

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

W002

Osteoblast Response to Titanium Microtopography Is Dependent on Integrin Alpha-2. R. Olivares-Navarrete*¹, P. Raz*¹, R. A. Chaudhri*¹, J. Chen*¹, M. Wieland*², B. D. Boyan¹, Z. Schwartz¹, ¹Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA, ²Institut Straumann AG, Basel, Switzerland.

Titanium surface microtopography modulates phenotypic maturation of osteoblast-like cells, production of local regulatory factors, and response of osteoblasts to bone anabolic agents such as 1,25 dihydroxyvitamin D3 [1,25(OH)₂D₃]. Changes in cell morphology suggest that integrin-mediated signaling may play a role. Alpha-2 and beta-1 integrins are increased in osteoblasts grown on Ti surfaces with rough microtopographies and 1,25(OH)₂D₃ modulates expression of beta-1 on these same substrates, but alpha-5 is not affected. We showed that silencing beta-1 alters osteoblast response to substrate microtopography and 1,25(OH)₂D₃. Here we determined if integrin alpha-2 was specifically responsible for the surface-dependent changes in osteoblast response. Alpha-2 siRNA targeted 21 bases starting at base-3406 of the alpha-2 gene (NM-002203.3). Double-strand oligonucleotides were constructed in a pSupressorNeo vector containing a U6 promotor. A permanent MG63 cell line exhibiting 70% reduction in alpha-2 protein was established. Normal and silenced MG63 cells were grown on tissue culture polystyrene (plastic) or on Ti substrates with different surface microtopographies and surface energy. Confluent cultures were treated with 1,25(OH)₂D₃ for 24h. Real-time PCR showed that alpha-2 and beta-1, but not alpha-5, alpha-v, or beta-3 integrin subunits were increased on surfaces with rough microtopographies and further enhanced on surfaces with high surface energy, as were mRNAs for alkaline phosphatase, osteocalcin, OPG and TGF-β1. Alpha-2 siRNA did not alter levels of alpha-5 or beta-1 protein. However, effects of surface topography and surface energy on alkaline phosphatase activity, osteocalcin, and levels of PGE2, OPG and TGF-β1 in the conditioned media were abolished. Moreover, response to 1,25(OH)₂D₃ was reduced, even when cells were cultured on plastic or smooth Ti substrates. The results indicate that the stimulatory effects of surface microtopography and surface energy on osteoblastic differentiation depend on alpha-2 and suggest that alpha-2 signaling is involved in osteoblast-dependent regulation of bone remodeling. Silencing also reduced effects of 1,25(OH)₂D₃ supporting our previous studies showing that the synergistic effect of surface roughness and 1,25(OH)₂D₃ is mediated by the same signaling pathways. Silenced MG63 cells responded to microrough surfaces in a comparable manner, whether or not surface energy was high, suggesting that the role of alpha-2/beta-1 is to detect surface structure rather than surface chemistry.

Disclosures: Z. Schwartz, None.

This study received funding from: Children's Healthcare of Atlanta, NIH AR052102, NSF 9731642, ITI Foundation, Instituit Strauman AG.

W003

Local Transplantation of Human Multipotent Adipose-Derived Stem Cells Improve Fracture Healing. T. Shoji*¹, Y. Mifune*¹, M. Ii*², M. Kurosaka*³, T. Asada*⁴, T. Asahara*¹, ¹Laboratory for Stem Cell Translational Research, Riken Kobe Institute, Kobe, Japan, ²Foundation for Biomedical Research and Innovation, Kobe, Japan, ³Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan, ⁴Stem Cell Sciences KK, Kobe, Japan.

It's well known that human adipose tissue is one of the sources of multipotent stem cells called “adipose-derived stromal cells”¹. Among them, human multipotent adipose-derived stem (hMADS) cells obtained from young donor demonstrating fast adherent cheracteristis in culture, have extensive expansion capacity ex vivo, multilineage differentiation potential and long-term engraftment and tissue regeneration capacity. It has already reported hMADS may have therapeutic potential for muscle diseases². Based on these evidences, we test the hypothesis that hMADS contributes to fracture healing in a rat femur nonunion fracture model.

We separated crude SVF from adipose tissue in human young donors. We selected fast-adherent cells and expanded them. They were treated as hMADS cells. A reproducible model of femoral fracture was created in nude rat (F344/nude rat) with cauterized periostereum which leads to nonunion 8 weeks post-fracture³. The hMADS cells (1 × 10⁵/rat) or PBS was transplanted locally with atelocollagen gel after fracture creation.

Human-specific osteoblast (OB) and endotherial cell (EC) related genes were detected by RT-PCR of tissue RNA isolated from peri-fracture site in the hMADS group at week 2. Differentiated human OBs and ECs were identified in the hMADS group by immunohistochemical staining. They also demonstrated human cell-derived osteogenesis and angiogenesis. Capillary density at peri-fracture site scaled by vascular staining was higher in the hMADS group than the PBS group. Moreover, mean flux ratio of laser doppler perfusion imaging was significantly greater in the hMADS group. 9 of 10 fractures were healed radiographically and histologically at week 8 in the hMADS group. On the other hand, 1 of 10 in the PBS group was observed (Fig.).

hMADS cells transplanted to the fracture site contribute to fracture healing via osteogenesis and angiogenesis in rat non-union fracture model. hMADS the small amount of an easily available tissue source may have a therapeutic potential for fracture healing.

1. Zuk, P.A. et al. Tissue Eng., 7:2, 2001.

2. Anne-Marie, R. et al. J.Exp.Med., 201:9, 2005

3. Kokubu, T. et al. J.Orthop.Res., 21:3, 2003

Disclosures: T. Shoji, None.

This study received funding from: Stem Cell Sciences KK.

W004

Vitamin A Deficiency Delays Healing Process After Cortical Bone and Bone Marrow Injury. K. Tanaka*¹, S. Tanaka¹, A. Sakai¹, Y. Katae*¹, H. Hirasawa*¹, N. Nakura*¹, K. Sabanai*¹, K. Menuki*¹, H. Yamane*¹, Y. Shimizu*¹, Y. Arai*², T. Nakamura¹, ¹Orthopaedic surgery, University of Occupational and Environmental Health, Kitakyusyu, Japan, ²Division of Hard Tissue Research, Institute for Oral Science, Matsumoto Dental University, Matsumoto, Japan.

The necessity of vitamin A in chondrogenesis and generation of limbs has been reported. The role of vitamin A in bone regeneration has not been elucidated. Using three groups of 10-week-old male C57BL/6J mice, we investigated the effects of vitamin A deficiency on healing process after cortical bone and bone marrow injury. One group was vitamin A deficiency (VAD) mice, which were fed the diet without vitamin A from 10 day of gestation to the end of the experiments. Another group was deficiency-sufficiency (VADS) mice, which were fed the diet without vitamin A from 10 day of gestation until weaned and thereafter fed the standard diet. The other was sufficiency (VAS) mice, which were fed only the standard diet. We made drill-hole injury, 1mm in diameter, at the anterior portion of the diaphysis of bilateral femurs. One group was used for sequential analysis of the amount of regenerating bone using in vivo micro CT. The femurs of each group were also harvested at 7, 14, 21 days after operation to do histomorphometry and quantitative mRNA analysis.

Regenerating bone was observed from 7 day in VAS and VADS mice, and appeared to be almost healed until 28 day, while cortical bone defect was still apparent in VAD mice at 28 day. In histomorphometrical analysis, the volume of medullary callus tended to decrease in VAD mice throughout the experimental period and regenerated cortical bone volume of the drill-hole area in the VAD mice was lower than that in the VAS at day 7, 14 and 21. Osteocalcin mRNA expression in VAD mice apparently decreased compared with VAS mice at day 7.

These results clearly demonstrated that vitamin A deficiency suppressed medullary callus formation and cortical bone healing in association with the impairment of osteoblast maturation after cortical bone and bone mallow injury.

Disclosures: K. Tanaka, None.

W005

Interaction of galectin-9 with Lipid Rafts Induces Osteoblast Proliferation Through the c-Src/ERK Signaling Pathway. R. Tanikawa¹, Y. Okada¹, K. Nakano*¹, T. Tanikawa¹, R. Hosokawa*², Y. Tanaka¹, ¹First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan, ²Department of Oral Reconstruction and Rehabilitation, Kyushu Denatal College, Kitakyushu, Japan.

Galectin-9 is a β-galactoside-binding lectin that modulates many biological functions by interacting with particular carbohydrates attached to proteins and lipids. The role of galectin-9 in bone metabolism remains unclear. This study investigated galectin-9 in osteoblasts with respect to proliferation and lipid raft-dependent regulatory mechanisms. We used human osteoblasts purified from metaphyseal trabecular bone in the proximal femur. The effect of galectin-9 on osteoblast proliferation was tested using WST-8 (consists of tetrazolium salt). Protein phosphorylation was assayed by western blotting and confocal microscopy was used to localize lipid rafts. Galectin-9 induced osteoblast proliferation in a dose- and time-dependent manner. Galectin-9 induced the activated form of c-Src and ERK phosphorylation, and PP2, a Src-family tyrosine kinase inhibitor, inhibited both osteoblast proliferation and ERK phosphorylation. And c-Src si RNA transfection inhibited galectin-9 induced osteoblast proliferation. Furthermore, galectin-9 induced clustering of lipid rafts in osteoblasts. Disruption of lipid rafts by β-methylcyclodextrin inhibited galectin-9-induced proliferation, as well as the galectin-9-induced activation of c-Src. Our results indicated that galectin-9 interacts with lipid rafts and induces osteoblast proliferation through the c-Src/ERK signaling pathway. We propose that clustering of lipid rafts induced by galectin-9 might be an important trigger of the c-Src/ERK signaling cascade for osteoblast proliferation.

Disclosures: R. Tanikawa, None.

W006

The Effects of Chlorobenzenes on Bone Formation. Z. Valkusz¹, Q. Nagyeri*¹, M. Radács*¹, B. Hegedus*², A. Juhász*³, A. Petri*⁴, J. Julesz*¹, M. Galfi*², ¹Endocrine Unit, University of Szeged, Szeged, Hungary, ²Environmental Protection Group, University of Szeged, Szeged, Hungary, ³Department of Psychiatry, University of Szeged, Szeged, Hungary, ⁴Department of Surgery, University of Szeged, Szeged, Hungary.

Several chemicals used in the agriculture, industry and community as pesticides and fungicides may induce environmental damages and alterations in the homeostasis of biological organisms. It is known that these pollutants such as chlorobenzenes (CIB) have toxic effects on liver, immune-, nervous- and endocrine systems due to their chemical properties. Our aim was to investigate the effects of chronic expositions to discrete doses (0.1 or 1 μg/kg/day) of CIB on bone structure and activity of liver enzymes in rats. Wistar male rats (150-250 g) were given tri- and hexachlorobenzenes (1:1) dissolved in ethanol (0.05%) through gastric tube for 1 and 3 months. Control animals were untreated rats (absolute control, n = 10), rats treated with tap water (negative control, n = 10 and treated with ethanol (positive control, n = 10). At the end of the experimental period the animal's body weight and organ weight were measured (by gravimetric method); serum and femoral samples were taken. Serum liver enzymes (γGT, SGOT, SGPT) were measured (with colorimetric methods). Electrically-mobilized Ca²⁺ and bone mineral content (BMC) of femur were determined. The bone structure was investigated by histological methods. Data were analyzed with SPSS software.

The activity of liver enzymes, the weight of animals and the main organs did not change significantly applying expositions due to their subtoxic characteristic. Both of the doses, but particularly the 1 μg/kg/day dose could modulate the electrically mobilized Ca²⁺ and BMC of the femur in a dose- and time-dependent manner. The histology of femur was altered only with 1 μg/kg/day doses of CIB.

Our data suggest that 1μg/bw.kg/day doses of CIB, which is permanently present in our environment may be a risk factor for alteration of bone formation. However, a minimal dose (0.1 μg/bw.kg/day) CIB is not likely to disturb the bone structure.

Disclosures: Z. Valkusz, None.

W007

Osteoblast-Mediated Bone Formation Is Suppressed at Sites of Focal Bone Erosion in Inflammatory Arthritis. N. C. Walsh¹, C. A. Manning*¹, K. W. Condon*², J. Ratliff*², K. Iwata*², P. B. Burr², E. M. Gravallese*¹, ¹Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA, ²Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA.

Osteoclast (OC)-mediated focal articular bone erosion is a common feature of rheumatoid arthritis (RA). Despite treatment for RA with attenuation of focal bone erosion, most patients do not repair erosive lesions. Therefore osteoblast (OB)-mediated bone formation may be impaired at these sites. We have used the K/BxN serum transfer arthritis (STA) model to determine the impact of inflammatory arthritis on OB-lineage cells at sites of focal bone erosion. In prior studies we determined that differentiation of OB-lineage cells is compromised at sites of erosion, with a paucity of mature OB-lineage cells at these sites. We now confirm by in situ hybridization that OB-lineage cells expressing mRNA for markers of mature OB-lineage cells (alkaline phosphatase and osteocalcin) are absent from sites of erosion, but are present at sites remote from erosion. In addition, the capacity of OB-lineage cells to form bone at sites of erosion was determined by dynamic bone histomorphometry. STA was induced in 12 week-old male C57B1/6J mice and the fluorochromes, alizarin and calcein, were administered with a 10 day interval. The navicular bone was assessed as a predictable site of erosion. Fluorochrome labeling, indicating newly mineralized bone, was observed at sites remote from invading inflammatory tissue (pannus). In contrast, minimal label incorporation was observed at sites where pannus was in contact with the bone surface. Quantitation of mineralized surface/total bone surface (MS/BS,%) confirmed this observation, with a reduction in the MS/BS (%) at the pannus-bone interface compared to bone surfaces remote from the pannus, and non-arthritic navicular bones. Bone formation parameters quantified in the distal femur (a site remote from inflammation) showed no differences between arthritic and non-arthritic mice. Together our results indicate that OB differentiation and bone formation activity is suppressed at sites of erosion in inflammatory arthritis, contributing to net loss of bone at these sites. Previously we have shown that antagonists of the Wnt signaling pathway, DKK1 and sFRP1, are expressed at erosion sites and in this study, qRT-PCR analysis confirms the induction of DKK1 and sFRP1 mRNA at sites of erosion over time. These factors are candidates for inhibiting OB function at these sites in this model of inflammatory arthritis. As proof of principle, a recent study published by Diarra et al., showed that DKK1 blockade in inflammatory arthritis models resulted in protection from focal bone erosion.

Diarra, D., et al (2007) Nature Med. 13(2): 156–63

Disclosures: N.C. Walsh, None.

W008

A Locus on Mouse Distal Chromosome (Chr) 4 Exerts Genetic Control of Bone Size by Regulating the Differentiated Function of Periosteal Cells. J. E. Wergedal¹, M. H. C. Sheng¹, K. L. Shultz², D. J. Baylink³, S. Mohan¹, W. G. Beamer², ¹Musculoskeletal Disease Center, Jerry L. Pettis VA Medical Center, Loma Linda, CA, USA, ²The Jackson Laboratory, Bar Harbour, ME, USA, ³Medicine, Loma Linda University, Loma Linda, CA, USA.

Genes that regulate bone cross sectional size and strength are important to identify because they are potential therapeutic targets for increasing bone strength in osteoporotics. QTL studies using multiple inbred strain crosses including C3H/HeJ (C3H) × C57BL/6J (B6) have revealed that Chr 4 contains an important locus that regulates bone size. Based on the findings that a region of Chr 4 in C3H, when transferred to B6, increases bone size (Table 1) and that C3H osteoblasts exhibit higher activity than B6 osteoblasts both in vivo and in vitro, we proposed the hypothesis that Chr 4 QTL contains a gene that increases bone size by regulating osteoblast activity. To evaluate this hypothesis, we characterized the processes that contribute to increased bone size by histomorphometry in a B6 congenic strain, B6.C3H-4T. Both periosteal and endosteal bone formation were elevated in the congenic line (Table 1). Furthermore, the increased bone formation was due to increased mineral apposition rate (MAR) rather than increased forming surface (Md.S). These increases in bone formation are consistent with our previous study showing higher osteoblastic activity in C3H mice than in B6 mice and suggest that the genes responsible at least in part reside on Chr 4. To further narrow down the region of the Chr 4 containing the gene of interest, sublines from the B6.C3H-4T have been produced. The subline (B6.C3H-4T-3) demonstrating the largest difference in cross sectional area (19 +/-3% difference, p<.001; Mean+/-SE) from the B6 background strain (B6.C3H-4T-3) covered the region between 50 cM and 80 cM. Summary: 1) This study confirms that the congenic line B6.C3H-4T has increased periosteal bone formation due to osteoblast with increased activity. 2) A subline of the B6.C3H-4T with a smaller segment of Chr 4 from the C3H strain also has increased cross sectional size. These observations are consistent with the presence of a gene(s) on the distal portion of mouse chromosome 4 regulating the differentiated function of periosteal cells.

Table Table 1.. Femur midshaft MAR, bone formation rates (BFR), and mineralizing (forming) surface (MS) at periosteum and endosteum in B6.C3H-4T and B6 control mice (mean±SE).

Disclosures: J.E. Wergedal, None.

W009

Attachment of Osteoblast-like Cell on ALD-derived Hydroxyaptite Surface. L. Xu¹, P. Rahkila*¹, T. Saiavaara*², M. Putkonen*³, H. J. Whitlow*², S. Cheng¹, ¹Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland, ²Department of Physics, University of Jyväskylä, Jyväskylä, Finland, ³Beneq Ltd., Vantaa, Finland.

Focal adhesions contain structural and secondary signaling molecules crucial to cell function. The attachment, adhesion and spreading of osteoblasts are essential events when studying their proliferation and differentiation on biomaterial surfaces. Different techniques have been utilized for the deposition of hydroxyapatite (HA) which offers an interesting selection of biomimetic surface coating material. Here we have used a novel Atomic Layer Deposition (ALD) method to produce thin HA films for osteoblast culture experiments. ALD is a highly controlled and conformal method for thin film deposition especially suitable for preparation of microelectronic and nanoscale materials and coating of three dimensional structures. In this study, ALD was used to obtain stable and biocompatible nanocrystalline HA films on Si (100) substrate. The topography of the films was analyzed by AFM. Heavy ion elastic recoil detection analysis (HI-ERDA) was utilized for the determination of Ca, P, O and H contents in the films. Crystalline films were obtained when amorphous as-deposited films were annealed at 800°C. AFM showed a quite uniform nanocrystalline HA layer interspersed with macrocrystalline HA. MC3T3-El cells were used to test the bioactivity of ALD-derived HA films (ALD-HA). 1 × 10⁵ cells in serum-free a-MEM medium were seeded onto UV sterilized silicon plates (10×10mm²) coated with annealed ALD-HA and unannealed amorphous films. Cells were cultured for 1 hour and 3 hours. Thereafter the cells were fixed in 4% paraformaldehyde. Vinculin was immunostained by alaxa-546 labeled antibody. F-actin was stained by alaxa-488 labeled phalloidin. Hoechst staining was used to show the cell nucleus. Cell behavior on ALD-HA surface was examined under inverted confocal fluorescence microscope. Osteoblasts adhered to ALD-HA surface after 1 hour incubation, presenting numerous filopodias and some focal adhesion sites. But the cells did not spread well and F-actin was not clearly organized. Vinculin appeared as diffuse perinuclear staining. Well-organized cytoskeleton was noticed in the cells incubated for 3 hours. Forming focal adhesion sites were clearly seen at the colocalization of vinculin and actin filaments. When comparing the amorphous and annealed crystalline surfaces the adhesion of the cells as well as the formation of focal adhesion sites are faster on the latter surface. These results suggested that crystalline ALD-HA is a promising surface coating material for the study of osteoblast/osteoclast biology.

Disclosures: L. Xu, None.

This study received funding from: Academy of Finland and Finnish Ministry of Education.

W010

Mechanosensitivity of Osteoblasts Is Regulated by Actin Polymerization Through Activation of RhoA. W. Yang, M. Malik*, K. L. van Golen*, K. Czymmek*, E. Adams*, G. Madden*, A. Kronbergs*, R. L. Duncan. Biological Sciences, University of Delaware, Newark, DE, USA.

While the skeleton is exquisitely sensitive to mechanical stimulation, bone becomes quickly desensitized to continued mechanical loads. We have previously reported that fluid shear rapidly increases the formation of actin stress fibers in osteoblasts and that depolymerization of the actin cytoskeleton increases the activity of the mechanosensitive, cation selective channel and the intracellular calcium response to fluid shear in osteoblasts. Numerous studies from cells derived from a variety of tissues indicate that activation of small GTPases can alter the cellular cytoskeletal integrity. Here, we postulated fluid shear rapidly activates RhoA to increase actin polymerization, thereby regulating the mechanosensitivity of osteoblasts. We found that RhoA was activated within 15 min of application of 12 dynes/cm² fluid shear to MC3T3-E1 osteoblasts. Confocal microscopy demonstrated that this shear-induced activation resulted in phosphorylation of the actin severing protein, cofilin, by LIM kinase II. Cofilin was rapidly translocated to the nucleus. Inhibition of this pathway with Y27623 or C3 transferase prevented shear-induced cofilin phosphorylation and translocation, as well as actin stress fiber formation in osteoblasts. Using Atomic Force Microscopy to determine changes in the elastic modulus of MC3T3 osteoblasts in response to shear, we found that fluid shear produced an 8-fold increase in cellular stiffness that was completely abrogated by inhibition of the RhoA pathway. Using intracellular calcium imaging, we found that inhibition of RhoA did not change the peak calcium response to shear, but did significantly increase the number of cells responding to shear. We have previously observed that MC3T3 osteoblasts rarely respond to a second bout of shear with an increase in intracellular calcium if that challenge was administered within 30 min of the first onset of shear. However with RhoA inhibition, 50% of the cells that responded to the initial application of fluid shear responded to a second shear challenge. These data indicate that RhoA plays an important role in the loss of mechanosensitivity of osteoblasts to continued loading by increasing the actin cytoskeletal organization and stress fiber formation. This change in actin integrity increases cellular stiffness that may reduce the ability of the cellular membrane to deform to additional shear.

Disclosures: W. Yang, NIH/NIANS (Adaptation of osteoblasts to mechanical stimulation. R01 AR043222) 2.

This study received funding from: NIH/NIANS.

W011

Studies on Improving Bone Bonding Ability of Titanium by Hydrothermal Treatment with CaCl₂. L. Zhang*¹, M. Nakagawa*¹, Y. Ayukawa*², R. Z. LeGeros³, S. Matsuya*¹, K. Koyano*², K. Ishikawa*¹, ¹Department of Biomaterials, Faculty of Dental Science, Kyushu University, Fukuoka, Japan, ²Department of Removable Prosthodontics, Faculty of Dental Science, Kyushu University, Fukuoka, Japan, ³Department of Biomaterials & Biomimetics, New York University College of Dentistry, New York, NY, USA.

Titanium has been used extensively as an implant material in dentistry and orthopedics. However, the bone bonding ability of Ti is much lower than those of osteoconductive materials such as hydroxyapatite, characterized by delayed bone formation and fibrous tissue interposition at Ti-bone interface. Alkali treatment is reported to be effective in improving the osteoconductivity of Ti. After such kind of treatment, Ti can bond calcium ions to its surface in vivo and this enhances bone formation. We hypothesized if calcium ions are bonded on Ti surface before it is implanted into the bone, the period of bone formation might be shortened. Therefore the purpose of this study is to find a method to fabricate Ca-bonded Ti and thus to improve the bone bonding ability of Ti implant materials.

In order to attain Ca-bonded Ti (Ca-Ti), we investigated hydrothermal treatment with CaCl₂ solution. From the results of X-ray photoelectron spectroscopy analysis, the Ca-Ti was successfully fabricated by using hydrothermal treatment with 10 mmol/L CaCl₂ at 200°C for 24 h. To reveal the bone cell responses to Ca-Ti, rat bone marrow cell experiments were performed. Results showed that Ca-Ti promoted cell adhesion and proliferation, accelerated the production of osteocalcin and the mineralization of the extracellular matrix. A rodent tibia model was applied to determine the in vivo bone response to Ca-Ti surface. Ca-Ti implant showed osteoblasts adhesion and new bone formation within 1 week. When compared with untreated or alkali treated Ti, Ca-Ti showed higher bone contact ratio from the early stage: 1 week 55%, 2 weeks 88%, 4 weeks 96%. The early, direct bone bonding of Ca-Ti forms a striking contrast to untreated Ti which showed late, fibrous tissue-intervened bone formation [Figure].

We concluded that through hydrothermal treatment with CaCl₂, Ti can be altered into the osteoconductive material. Such surface treatment is a promising alternative for modifying Ti (or Ti alloy) implant surfaces for accelerated osteointegration and skeletal fixation. 

Figure Figure.. Different tissue responses to untreated Ti and Ca-Ti after 2 weeks in rat tibia

Disclosures: L. Zhang, None.

W012

Ciz, a Nucleo-cytoplasmic Shuttling Protein Interacts with Extracellular Matrix Proteins. T. Hayata¹, T. Nakamoto*², Y. Ezura¹, H. Hirai*³, M. Noda¹, ¹Molecular Pharmacology, Medical Research Institute, Tokyo Medical & Dental University, Tokyo, Japan, ²Medicine and Rheumatology, Tokyo Medical & Dental University, Tokyo, Japan, ³Hematology and Oncology, University of Tokyo, Tokyo, Japan.

Ciz is a zinc finger transcription factor with nucleocytoplasmic shuttling activity. We previously showed that Ciz-null mice show high bone mass phenotype, suggesting that Ciz is a negative regulator of bone formation in vivo. However, how Ciz suppresses bone formation through protein-protein interaction remains unclear. In the present study, to better understand molecular mechanism of Ciz function in bone formation, we wished to identify the binding partners of Ciz. We performed a yeast two-hybrid screening for Ciz-interacting protein, after constructing a cDNA library from neonatal mouse calvaria. An N-terminal fragment of Ciz (Ciz-deltaZF, amino acid 1-279) that lacks zinc finger domain and contains a proline-rich domain was used as a bait. The proline-rich domain is thought to bind to SH3 domains of signaling proteins. After the screening of 4.2×10⁵ clones, 150 positive clones were sequenced. While Ciz is an intracellular protein, 47% of the clones encoded genes for extracellular matrix proteins (ECM), including collagen type 1 alpha 2 (Colla2), 57%; *Colla1, 10%; Fibulin 2 (Fbln2), 7%; laminin receptor 1 (Rpsa), 7%. Coimmunoprecipitation experiments using in vitro translated proteins confirmed binding of Ciz-deltaZF to the fibrillar collagens C-terminal domain of Colla1 and Colla2. Association of the transfected Ciz and C-terminal domain of Col1a1 was also confirmed in Cos-7 cells by immunoprecipitation experiments. Although the physiological significance of the interactions is currently under investigation, these results revealed that Ciz interacts with fragments of extracellular matrix proteins at least under the tested condition.

Disclosures: T. Hayata, None.

W013

Hypoxia Accelerates the Transformation of Osteoblasts to Osteocytes. M. Hirao, J. Hashimoto, N. Yamasaki*, W. Ando, H. Tsuboi*, A. Myoui, H. Yoshikawa. Orthopaedics, Osaka University Graduate School of Medicine, Suita, Japan.

Osteocytes are derived from osteoblasts, but reside in the mineralized bone matrix without direct blood flow. It has been reported that MLO-Y4, osteocyte-like cells show much higher expression of ORP150, which is induced by hypoxia, than osteoblast-like cells (27th, 28th ASBMR). Accordingly, we hypothesized that the oxygen tension may regulate the transformation of osteoblasts to osteocytes. MC3T3-E1 cells and calvariae from 4-day-old mice were cultured under normoxic (20% O₂) or hypoxic conditions (5% O₂). To investigate osteoblastic differentiation and tranformation to osteocytes, alkaline phosphatase (ALP) staining and alizarin red staining were done and expression of various factors (osteocalcin, DMP1, MEPE, FGF23, Cx43, ORP150) was assessed by real time RT-PCR, ELISA analysis, western blot analysis, immunocytochemical analysis, and immunohistchemical analysis. Hypoxic culture promoted the increased synthesis of mineralized matrix by MC3T3-E1 cells. ALP activity was increased initially during hypoxic culture, but decreased during osteogenesis. Osteocalcin production was also increased by hypoxic culture, but decreased after mineralization. Hypoxia accelerated the change of ALP activity and osteocalcin production in cultured MC3T3-E1 cells. Furthermore, expression of DMP1, MEPE, and FGF23, and Cx43 which are osteocyte-specific or osteocyte-predominant proteins, by MC3T3-E1 cells was greater under hypoxic than normoxic conditions. In mouse calvarial organ cultures, the number of cells in the bone matrix and cells expressing DMP1 and MEPE were increased by hypoxia. In MC3T3-E1 cell cultures, ORP150 expression was only detected in the mineralizing nodules under normoxic conditions, while its expression was diffuse under hypoxic conditions, suggesting that the nodules were hypoxic zones even in normoxic cultures. Collectively, these findings suggest that a low oxygen tension promotes osteoblast maturation and subsequent transformation to osteocytes.

Disclosures: M. Hirao, None.

W014

Adiponectin Action Influences Osteoblast Related Gene Expression. T. Ikeo¹, A. Kamada¹, I. Tamura*¹, S. Goda*¹, Y. Yoshikawa¹, E. Domae*¹, Y. Takaishi¹, T. Miki², ¹Biochemistry, Osaka Dental University, Hirakata, Japan, ²Geriatric Medicine, Osaka City University, Osaka, Japan.

Adiponectin, which is one of the adipocyte-derived biologically active molecules, is abundantly present as a plasma protein, and exhibits various biological functions, such as regulating energy homeostasis, increasing insulin sensitivity in the liver and skeletal muscle, and protecting the vascular walls from atherosclerosis. Low plasma adiponectin levels are known to cause insulin resistance and metabolic syndrome. Recently, receptors of adiponectin (AdipoR1 and AdipoR2) were cloned and found to be expressed ubiquitously. We previously demonstrated gene expression of the receptors in osteoblast, indicating that adiponectin may affect bone metabolism. In this study, we investigated the expression of osteoblast differentiation markers in osteoblasts which showed suppressed receptor expression due to small interfering RNA (siRNA), to elucidate the influences of the failure of adiponectin action on bone metabolism.

Murine pro-osteoblastic cell line, MC3T3-E1 cells were cultured in alpha-MEM with 10% FBS. Two days after transfecting siRNA against adiponectin receptors (siRNA-AR), the cells were stimulated by ascorbic acid (VC) and beta-glycerophosphate (BGP) to induce osteoblast differentiation. The mRNA expression level was measured using real-time quantitative RT-PCR two days after the addition of VC and BGP.

VC and BGP-dependent induction of bone morphogenetic protein-2 (BMP-2) and osteocalcin were significantly suppressed by siRNA-AR compared with non-silencing siRNA controls (p<0.01). Both mRNA expression levels of BMP-2 and osteocalcin were correlated with that of AdipoR2 (r = 0.895 and r = 0.893, respectively, p<0.001), while there was no correlation of osteoblast differentiation markers with AdipoR1. Suppression of the gene expression of adiponectin receptor in osteoblasts inhibited the induction of BMP-2 and osteocalcin. Hence failure of adiponectin action may suppress the progression of osteoblast differentiation and bone formation.

Disclosures: T. Ikeo, None.

This study received funding from: Grant-in-Aid for Scientific Research (C)(16591877).

W015

Estrogen Receptor-Related Receptor Gamma (ERRγ) Negatively Regulates Osteoblast Differentiation and Mineralization Through Runx2-and Smad-Dependent Pathway. B. Jeong*¹, I. Bae*¹, I. Kim*¹, H. Choi*², J. Koh¹, ¹Dental Science Research Institution and Brain Korea 21 project, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea, ²Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju, Republic of Korea.

Recently the orphan nuclear Estrogen receptor-Related Receptors (ERRα,β,γ) have been cloned and described their expressions in bone development. This study was undertaken to elucidate roles of ERRγ in osteoblast differentiation. Mouse myoblast C2C12 and pre-osteoblast MC3T3-E1 cells were infected with adenovirus ERRγ (AdERRγ) or siERRγ (AdsiERRγ), in the presence or absence of BMP2. Osteoblast differentiation was determined by alkaline phosphatase (ALP) activity and osteocalcin (OC) production. Mineralization was measured by Alizarin Red staining. For the transcriptional regulation of ERRγ, coimmunoprecipitation, glutathione S-transferase (GST)-pull down, GST-competition and reporter gene assays were done. ERRγ was ubiquitously expressed in various mesenchymal cells, and increased by BMP2 treatment in C2C12 cells. Overexpression of ERRγ significantly inhibited BMP2-induced ALP activity, OC production and mineralization with decreases in BMP2 induction of ALP and OC mRNA expression. AdsiERRγ enhanced ALP activity and OC production in C2C12. Transient transfection of ERRγ significantly inhibited both Runx2-dependent stimulation of OC promoter activity and Smad-dependent modulation of SBE promoter activity. Co-immunoprecipitation and GST pull-down assay showed physical interactions between ERRγ and Runx2-p300 complex or Smad-1 and −5. These results suggest that orphan nuclear ERRγ should be a novel negative regulator of osteoblast differentiation through interfering with Runx2 and Smad signaling.

Disclosures: B. Jeong, None.

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

W016

Downstream Signal Transactivation by FGF-4/Osterix Transgene Partially Imitates Changes of BMP-2 Induced Osteogeneic Transcription Factors in NIH3T3 Cells. S. Kuroda, M. Samee*, H. Kondo, S. Kasugai. Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan.

Bone morphogenetic proteins (BMPs) are well known potent inducers of osteogenesis. In the process of bone formation, BMP-2 stimulates the three osteogeneic master transcription factors: Dlx5, Runx2 and Osterix, mediated by the Smad pathways. Fibroblast growth factors (FGFs) are important signal molecules involved in both intramembranous and endochondral ossification. The transactivation of downstream genes such as Runx2 but not Osterix by FGF/FGF receptor signalings is one of the target initial events for osteoblastic differentiation mediated by the PKC pathway. FGF-4 plays important roles in bone development during embryogenesis, and systemic or local application of the recombinant protein demonstrated increase of bone formation in our previous studies. Furthermore, an additional treatment of FGF-4 over BMP-2 enhanced ectopic mineralization in our implant study. However, the mechanisms by which FGF-4 or BMP-2 signaling for osteogenesis are not well understood. In this study, we profiled the signal transactivation of osteogenic transcription factors including Dlx5, Runx2 and Osterix, activated by transgenes in an NIH3T3 cell culture. NIH3T3 cells were seeded in 24-well plates. Each well harbored 1 × 10⁵ cells in 500 ml D-MEM with 10% FBS. The experimental periods were scheduled as day 3, 7 and 14. The cells were given a transfection using a plasmid vector encoding for FGF-4, Osterix, FGF-4/Osterix or BMP-2. A vehicle vector was also applied for control. At each time point, the samples were subjected for RT-PCR, alkaline phosphatase (ALP) activity, and histological stainings for ALP positive cells and mineralized nodules. BMP-2 gene transfer was followed by elevation of Dlx5, Osterix and Smadl. On the other hand, FGF-4 transgene did not affect the changes of these gene expressions. However, the combination of FGF-4/Osterix overcame weak effects of FGF-4 or Osterix alone, resulting in up-regulation of Dlx5 mRNA as high as in the BMP-2 transfection. Increase of ALP activity per DNA, which was prominent in the FGF-4/Osterix and theBMP-2 transgenes, was not seen in the FGF-4 transgene. The histological works did not elicit nodule apposition in any of the treatments. Conclusively, the current study demonstrated that not FGF-4 alone but FGF-4/Osterix combination partially mimicked BMP-2 in this fibroblastic cell culture, regarding the signal transactivation of two osteogeneic transcription factors, Dlx5 and Osterix, although Runx2 mRNA expression was weak over time. Further studies remain to explore the downstream signal pathways of FGF-4 and BMP-2 using other techniques and different cell cultures.

Disclosures: S. Kuroda, None.

This study received funding from: Japan Society for the Promotion of Science.

W017

Gene Networks in Osteoblasts that Are Regulated by the Dlx Gene Family: Requirement for Dlx Transcription Factors in Mesenchymal Commitment to the Osteoblast Lineage. H. Li¹, W. Yang², A. C. Lichtler¹, S. E. Harris², ¹Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA, ²Periodontics, University of Texas Health Science CTR, San Antonio, TX, USA.

Previous studies demonstrated that Dlx genes promote osteoblast differentiation, are expressed very early in the osteoblast lineage and are stimulated dramatically by BMPs. However, evidence suggests that Dlx genes are functionally redundant, making the study of Dlx genes in regulating osteoblast function difficult. We used a dominant negative Dlx homeodomain-engrailed repressor fusion protein (DNDlx) to inhibit Dlx protein induction of target genes. Co-transfection of DNDlx inhibited Dlx2, 3, 5, and 6 induction of a Dlx reporter construct, and infection of osteoblast cell lines with DNDlx suppresses BMP induced genes. Mouse marrow stromal cells (MSC) expressing DNDlx produced fewer alkaline phosphatase positive colonies and mineralized nodules than control cultures. For comparison, we also expressed Dlx5 which greatly accelerated osteogenesis. Full genome expression microarray analysis of the RNA from these cultures at 11 and 16 days was carried out to discover new and altered pathways regulated by Dlx genes. At 11 days, mineralization had just begun and by 16 days, mineralization is almost complete. Affymetrix array data from 430 2.0 with 41,000 probe sets were analyzed with the Limma program, and genes with a 2.6 or more fold change and P < 0.01 were analyzed for functional classification changes using the David program and compared to other gene sets from Gene Expression Omnibus at NCBI. In the 11 day cultures, over 500 genes were specifically inhibited by DNDlx; by Gene Ontology (GO) classification, these included ribosomal proteins, genes associated with osteoblast mineralization and differentiation, such as Osterix and with primary metabolism and mitochondrial function, extracellular matrix and collagen production. Over 120 GO classes are regulated. 398 significant genes were increased at least 2.6 fold by DNDlx, indicating that DNDlx promoted the differentiation of the MSC towards chondrocyte and immunological like phenotypes, probably because DNDlx specifically suppresses the osteoblast lineage. 497 genes were positively regulated by Dlx5 overexpression and many were in the skeletal development and mineralization classes determined by functional clustering with David. We identified 32 poorly annotated genes showing a strong correlation with many osteoblast markers, some induced 10 to 50 fold. Of the 1042 genes downregulated by overexpression of Dlx5, many were characteristic of chondrocytes and immune cells also suggesting that Dlx genes are critical for driving mesenchymal precursors to the osteoblast lineage.

Disclosures: H. Li, None.

This study received funding from: NIH.

W018

Pyrrolidine Dithiocarbamate Inhibits SOD1 Gene and Cell Growth by Activating JNK Pathway in Human Osteosarcoma Cells (U2OS). G. Ngouala*, V. Afonso*, H. Riera*, P. Collin*, A. Lomri.. Lariboisiere Hosp., INSERM U-606 & University Paris VII, Paris 10, France.

It has recently been shown that the alteration of the cell-redox status affects the transcription factor expression and activity. Pyrrolidine dithiocarbamate (PDTC) is a potent antioxidant agent that can switch the expression of genes dependent on the activation of the transcription factors AP-1 and NF B. PDTC is also a metal-chelating compound that acts as antioxidant or pro-oxidant and is widely used to study redox regulation of cell function. In the present study, we investigated effects of PDTC on the antioxidant gene SOD1 and cell growth in human osteosarcoma cells (U2OS), using transfection and cell growth assays. We have previously reported that treatment of cells with TNF-alpha repressed SOD1 gene and protein expression. We now show that PDTC treatment does not protect SOD1 from TNF inhibition, but induced further repression. PDTC effects are not linked to NF-kappaB repression. Transfection experiments of U2OS cells with reporter constructs harboring different promoter regions of SOD1 gene indicated the presence of a functional PDTC-responsive region located between positions −157 and +17 of the promoter. Furthermore, transfection studies of JNK-/- cells with SOD1 promoter did not show any repression by PDTC, indicating that JNK pathway is important for PDTC action on SOD1 gene. In contrast, when JNK pathway was restored in these cells, PDTC was able to repress again SOD1 promoter activity. Furthermore, PDTC induced significant inhibition of osteosarcoma cell growth, probably through SOD1 repression and this effect was not linked to cell toxicity. These results indicate that PDTC-dependent repression of SOD1 and cell growth in U2OS osteosarcoma cells is tightly regulated by JNK pathway and point-out SOD1 as a target gene in the anti-proliferative effects of PDTC in cancer cells

Disclosures: A. Lomri, None.

W019

Endogenous Hesr/Hey Genes Redundantly Support Osteoblast Differentiation. S. Maeda¹, H. Kokubo*², H. Aburatani*³, S. Nomura*⁴, S. Komiya*⁵, K. Miyazono*⁶, T. Imamura⁷, ¹Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo, Japan, ²Division of Mammalian Development, National Institute of Genetics, Mishima, Japan, ³Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan, ⁴Department of Pathology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan, ⁵Department of Neuromusculoskeletal Disorder, Orthopaedic Surgery, Graduate School of Medicine and Dentistry, Kagoshima University, Kagoshima, Japan, ⁶Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan, ⁷Department of Biochemistry, The Cancer Institute of The Japanese Foundation for Cancer Research, Tokyo, Japan.

Bone morphogenetic proteins (BMP) induce osteoblast differentiation of mesenchymal progenitor cells, which event is governed by two master regulators, Runx2 and Osterix. In addition to these transcription factors, other nuclear proteins are also induced by BMP, which roles in osteoblast differentiation are obscure. It has been reported that Hesr1/Heyl is induced synergistically by BMP and Notch signaling in vitro, and osteoblasts express Hesr1/Heyl in vivo. Other group showed in the last ASBMR, that ubiquitous overexpression of Hesr1/Heyl in vivo induced progressive osteopenia in the transgenic mice due to decreased osteoblast differentiation. However, it is still not clear whether endogenous level of Hesr1/Heyl also acts inhibitory to bone formation or not. To answer this question, we analyzed bones of Heyl/Hesr1 knockout mice. Unexpectedly, the bone mineral density and bone morphometric analysis of Hesr1/Heyl -/- mice showed no overt phenotype. We considered this result might be due to redundancy between Hesr/Hey genes. Indeed, we found that Hesr3/HeyL gene is also expressed in bone in vivo. So we generated Hesr1/Heyl:Hesr3/HeyL double knockout (Hesr1-/-:3-/-) mice. Some of Hesr1-/-:3-/- mice died before weaning as recently reported by other group. However, survivors of double null mice showed normal growth, which could be subjected to bone analysis. Contrary to our expectations, the bone mineral density of four-allele mutants was decreased. Analysis of mRNA extracted from bones of these mice revealed that osteocalcin was elevated. As we found in vitro, that Hesr1/Heyl interacts with Runx2 and inhibits its function, this interaction may be, at least in part, responsible for the phenotypes. These datas suggest that endogenous expression level of Hesr/Hey genes plays a supportive role in regulating adult bone formation.

Disclosures: S. Maeda, None.

W020

Osterix Functions as Downstream of Runx2 and Msx2 During BMP2-Regulated Osteoblastogenesis. T. Matsubara¹, K. Kumiko*¹, K. Hata*¹, A. Yamaguchi², H. Aburatani*³, R. Nishimura¹, T. Yoneda¹, ¹Biochemistry, Osaka University graduate school of dentistry, Osaka, Japan, ²Oral Pathol, Tokyo Med Dent Univ, Tokyo, Japan, ³Res Cent Adv Sci Tech, Univ Tokyo, Tokyo, Japan.

Complete absence of bone formation and osteoblasts in null mice indicates an Sp1 family transcription factor Osterix/Sp7 is necessary for bone development. Osterix expression is known to be associated with BMP2 actions. However, the precise mechanism that controls the expression and function of Osterix during BMP2-induced osteoblastogenesis remains elusive. In this study, we attempted to study this. Since a previous study suggests that Osterix is a downstream of Runx2, we first examined the effects of Runx2 overexpression on Osterix expression. We found that overexpression of Runx2 induced Osterix expression in mesenchymal cell lines, C2C12 and C3H10T1/2, and primary mesenchymal cells isolated from Runx2-deficient mice along with an upregulation of ALP and osteocalcin expression, demonstrating that Osterix functions as a downstream of Runx2. However, of interest, BMP2 treatment induced ALP activity and Osterix expression in the Runx2-deficient cells. These results suggest that expression and function of Osterix is regulated by Runx2-independnet mechanism in addition to Runx2-dependnet mechanism. To verify this possibility, we examined whether Smad signaling is involved in this process. Overexpression of Smad1 and Smad4 up-regulated Osterix expression in the Runx2-deficient cells. In contrast, an inhibitory Smad, Smad6 markedly suppressed BMP2-induced Osterix expression in these cells, indicating that Smad signaling is required for Runx2-independent induction of Osterix. Because Msx2 has been shown to regulate BMP2-induced osteoblastogenesis independent of Runx2, we tested whether Msx2 is involved in the Runx2-independent regulation of Osterix. In the Runx2-deficient cells, treatment with BMP2 induced Msx2 expression and overexpression of Msx2 markedly up-regulated Osterix expression and ALP activity. These data indicate an important role of Msx2 in regulating Osterix expression and suggest that Osterix may have distinct roles from Runx2 during osteoblast differentiation. To test this idea, we performed microarray analyses using Runx2-deficient cells introduced with either Runx2 or Osterix, and found that Osterix up-regulated several genes that are distinct from Runx2. In conclusion, our results suggest that BMP2 regulates Osterix expression through Msx2 but independent of Runx2 and that Osterix controls osteoblast differentiation of mesenchymal cells in Runx2-dependent and independent fashions.

Disclosures: T. Matsubara, None.

W021

Retinoblastoma Binding Protein-1 (RBP1), a Novel Factor Critical For Runx2 Expression and Transcriptional Activation. D. G. Monroe¹, J. R. Hawse¹, M. Subramaniam¹, F. J. Secrete¹, S. Khosla², T. C. Spelsberg¹, ¹Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA, ²Endocrine Research Unit, Mayo Clinic, Rochester, MN, USA.

The retinoblastoma binding protein-1 (RBP1) is a broadly expressed transcription factor involved in the suppression of proliferation mediated through the retinoblastoma protein (pRb), a major pathway implicated in the pathogenesis of numerous cancers including osteosarcoma. We have previously demonstrated an estrogen receptor-α-dominant regulation of RBP1 in osteoblasts (OBs), suggesting an important role of RBP1 in OB function. Here we present evidence that RBP1 is involved in Runx2 expression as well as Runx2-dependent transcriptional activation, therefore potentially playing a broader role in general skeletal biology. Overexpression of wild-type RBP1 in the U2OS human osteosarcoma significantly enhances Runx2-dependent activation of a synthetic Runx2-dependent reporter construct (p60SE2-Luc), whereas a mutant RBP1 construct, containing a deletion of the LxCxE motif necessary for RBP1 binding to pRb, fails to enhance Runx2 activity. Similar experiments were conducted in the SAOS2 human osteosarcoma, which lacks pRb activity. Overexpression of Runx2 and/or RBP1 in SAOS2 cells resulted in comparatively weak activation of the p6OSE2-Luc, suggesting that an intact pRb/RBP1 system is necessary for maximal regulation of Runx2-dependent transcription. To further characterize the importance of RBP1 in the transcriptional regulation of bone marker genes, we utilized siRNA against RBP1 in mouse calvarial cell cultures. Reduction of RBP1 resulted in a significant transcriptional inhibition of important bone marker genes such as Runx2, osterix, BMP2, osteocalcin, osteopontin, TIEG and BMP2. Similar siRNA experiments targeting both RBP1 and pRb expression were conducted in the mouse MC3T3-E1 osteoblast cell line. Reduction in the expression level of either RBP1 and/or pRb resulted in an inhibition of Runx2-dependent transcriptional activation of these bone marker genes. These results suggest a transcriptional system where both RBP1 and pRb are necessary for maximal Runx2-dependent transcriptional activation in osteoblasts. These data are consistent with previous studies showing that BMP2-treated Rb-/- mouse embryo fibroblasts fail to form bone in vitro and that the Rb knockout mice are not viable. Current experiments are focused on deciphering the long-term effects of RBP1 depletion using both stable shRNA osteoblast cell lines and examining the skeletal phenotype of RBP1 knockout mouse models.

Disclosures: D.G. Monroe, None.

W022

Identification of Osteoblast-specific Co-Regulator Complex for Vitamin D Receptor (VDR). E. Ochiai*¹, H. Kitagawa*², I. Takada*², Y. Tsushima*³, S. Fujiyama*⁴, S. Sawatsubashi*⁴, M. Kim*⁴, Y. Mezaki*², K. Takagi*³, K. Takeyama*², K. Yamaoka*⁵, S. Kato⁶, T. Kamimura*³, ¹Teijin Institute for Bio-Medical Research/Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan, ²Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan, ³Teijin Institute for Bio-Medical Research, Tokyo, Japan, ⁴ERATO, Japan Science and Technology Agency, Saitama, Japan, ⁵ERATO, Japan Science and Technology Agency, Saitama/ Teijin Institute for Bio-medical Research, Tokyo, Japan, ⁶Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo/ ERATO, Japan Science and Technology Agency, Saitama, Japan.

Vitamin D actions in regulating bone metabolism and calcium homeostasis have been well described. Such actions of vitamin D are believed through nuclear vitamin D receptor (VDR), and gene disruption of VDR in mice caused severe rachitic phenotype. And the benefit of vitamin D for bone formation has been shown. Indeed several VDR ligands are applied for clinical use but are expected to reduce side effects like hypercalcemia. Such actions of vitamin D on bone were well known, but the molecular bases of the skeletal vitamin D action are not elucidated in that VDR cofactor complexes formed in osteoblasts. To address this issue, we used biological approach to identify the osteoblast specific co-regulator complex interacting with VDR. Using GST-fused VDR ligand binding domain (LBD) as a bait, nuclear complexes associated with liganded VDR-LBD were purified from nuclear extract of HOS osteoblastic cell line and HeLa as a control cell line. Together with well-reported DRIP/TRAP complex components, CDP (CCAAT Displacement Protein, also known as CUTL1, a homeodomain transcription factor) has identified as a novel ligand-dependent and osteoblast-specific VDR interactant by MALDI-TOF/MS. Furthermore using glycerol density gradient centrifugation, the CDP complex was distinct from the DRIP/TRAP complex by their molecular weights. By a GST-pull down and co-immunoprecipitation assay, CDP exhibited ligand-dependent interaction with VDR. In a transient expression assay with GAL4-VDR-LBD in HOS cells, CDP co-activated ligand-induced transactivation of VDR. Ectopic CDP expression by adenovirus vector potentiated the expression of the VDR target gene expression in HOS cells and induced osteoblastic differentiation in human osteoblastic SaM-1 cells. From the present findings, it is likely that CDP serves as an osteoblast-specific co-activator complex component for VDR. Currently, recruitment of CDP on the VDR target gene promoters are under investigation and will be presented.

Disclosures: E. Ochiai, None.

W023

Activation of Liver × Receptor Inhibits Hedgehog Signaling and Osteogenic Differentiation of Marrow Stromal Cells. W. Kim, K. Mouillesseaux*, V. Meliton*, C. M. Amantea*, F. Parhami. Medicine, UCLA, Los Angeles, CA, USA.

Liver × receptors (LXRs) are members of the family of nuclear hormone receptors that are activated by specific oxysterols and pharmacological agents such as TO901317 (TO) and GW3965 (GW). LXRs are involved in a variety of physiologic processes including lipid and glucose metabolism, cholesterol homeostasis, and inflammatory signaling. We previously reported that treatment of bone marrow stromal cells (MSC) with TO and GW inhibits spontaneous osteogenic differentiation of these cells, and therefore speculated that LXR activation may inhibit osteoblast differentiation and bone formation. Recently LXRs were found to influence osteoclast differentiation and activation, with LXR^−/− mice demonstrating an improved cortical bone phenotype. We also reported that specific osteogenic oxysterols, including 20S-hydroxycholesterol (20S), induce the osteogenic differentiation of MSC by activating the hedgehog (Hh) signaling pathway. In addition, we found that TO and GW blocked oxysterol-induced osteogenic differentiation of MSC, and therefore hypothesized that LXR activation may interfere with osteogenic differentiation induced by the activation of Hh signaling. In order to test this hypothesis, we examined the effects of TO and GW on sonic hedgehog (Shh)-induced osteogenic differentiation of MSC line, M2-10B4 (M2). Results showed that both TO and GW (1–10 μM) caused a significant inhibition of Shh-induced alkaline phosphatase (ALP) activity and osteocalcin mRNA expression after 3 and 8 days, respectively. Furthermore, treatment of M2 cells with LXR ligands inhibited Shh-induced mRNA expression of Hh target genes. Glil and Patched. siRNA to LXRα and LXRβ caused an 80–90% inhibition of mRNA expression for these genes, as well as inhibition of ligand-induced expression of LXR target genes, ATP-binding cassette (ABC) transporter proteins ABCA1 and ABCG1. The ability of TO and GW to inhibit Shh-induced signaling and ALP activity was blocked in cells transfected with LXRα and LXRβ siRNA, but not in cells transfected with control scrambled siRNA. These results demonstrate for the first time that LXR activation inhibits Hh signaling and osteogenic differentiation of MSC, suggesting a negative role for LXR in bone metabolism. Modulation of Hh signaling through LXR activation may be a novel therapeutic strategy relevant to a variety of indications including age-related osteoporosis and tumorogenesis that is caused by aberrant Hh signaling.

Disclosures: F. Parhami, None.

This study received funding from: NIH/NIAMS.

W024

Expression of Osteoblast-Specific Transcription Factors in Aged Mice. J. M. Patsch¹, M. Rauner*¹, D. Stupphann*¹, K. Tragl*², P. Pietschmann¹, ¹Institute of Pathophysiology, Medical University of Vienna, Vienna, Austria, ²Ludwig-Boltzmann Institute of Aging Research, Vienna, Austria.

Bone mass and bone stability decline during aging. Impaired osteoblast function is an important mechanism of age-related bone loss. The cellular and molecular mechanisms, which mediate age-dependant changes in osteoblast function, are poorly characterized. Therefore, the first aim of our study was to determine the gene expression pattern of the osteoblast-specific transcription factors runx2 and osterix in aged mice. Secondly, we analysed the gene expression of the osteoblast proteins alkaline phosphatase (AL.P), receptor activator of NFkB ligand (RANKL) and osteoprotegerin (OPG).

Bone marrow stromal cells were isolated from 18-months old and 6-weeks old mice. Osteoblast differentiation was induced by adding 5 mM β-glycerophosphate and 50 μM ascorbic acid to the culture medium. After 7, 14, 21 and 28 days total RNA was isolated, and quantitative real-time PCR analysis was performed. The marrow-free bones were homogenized in liquid nitrogen and also analyzed by real-time PCR. In bone marrow cultures from old mice, the mRNA level of ALP was significantly lower at all time points when compared to young mice. Runx2 mRNA levels also decreased during the first 28 days of culture. We did not detect an age-dependant alteration of ex-vivo gene expression of osterix, RANKL or OPG mRNA levels of ALP were significantly lower in the bone tissue samples of old mice (p < 0.05) In direct correlation with our results obtained from cultured cells, the tissue mRNA levels of osterix, RANKL and OPG remained unaffected by age.

Our results demonstrate that not all osteoblast-specific markers decline with aging. In particular, the expression levels of ALP and runx2 mRNA were downregulated in bone tissue and osteoblast cultures from aged mice. As ALP and runx2 are early markers in osteoblast differentiation, we conclude that aging mainly affects early steps of osteoblasts differentiation.

Disclosures: J.M. Patsch, None.

W025

Interactions Between PTH and the gp130 Cytokine Pathway in Differentiating Osteoblasts. J. M. Quach, E. H. Allan, K. D. Hausler*, M. T. Gillespie, T. J. Martin. St. Vincent's Institute, Melbourne, Australia.

Signaling through gp130 is required for normal osteoblast function, and PTH for accrual of maximal bone mass, yet the crosstalk between these pathways is not well understood. This work is aimed at understanding how PTH interacts with the gp130 cytokines in osteoblasts and precursors, by identifying transcriptional regulators responding to both classes of ligand. A gene array was performed on mouse marrow stromal cells, Kusa 4b10, which were differentiated through 16 days of culture to the time the cells developed functional PTH receptor. The cells were treated with PTH (1-34) or with PTHrP (1-141), and RNA used to probe Affymetrix whole mouse genome micro-arrays. Several genes known to be regulated through PTHIR (e.g., RANKL, RGS-2, VDR, LIF, IL-6) were used to validate responses on independent Kusa 4b10 RNA samples. Notably, several of the genes enhanced by PTH or PTHrP were involved in the gp130 pathway. These were IL-6, IL-11, LIF and CLCF, as well as the signal-transducing proteins for these cytokines gp130, LIFR, OSMR, and the signaling molecules STAT3, JAK3, SOCS2 and SOCS3. Each of these genes was validated on independent Kusa 4b10 RNA samples. From twelve transcriptional regulators differentially expressed in response to PTH and PTHrP and confirmed by real time-PCR on independent RNA samples, two were chosen for further investigation because pathway analysis indicated links to the gp130 signaling pathway. These were Btg2 (B-cell translocation gene 2) and Zinc-finger protein-36 (Zfp36), both of which have been implicated in bone cell function. Btg2 mRNA was increased 4-fold by PTH at 1 hr, 70-fold with Oncostatin M (OsM), 5-fold with LIF and 3-fold with cardiotrophin-1. Zfp36, which has been shown to bind to AU-rich elements in mRNA and to decrease mRNA stability, was increased 3-fold by PTH, 70-fold by OsM, 2.5-fold by both LIF and cardiotrophin-1. Additionally, both Btg2 and Zfp36 mRNA were rapidly induced by 3 to 4-fold with PTH and 16 to 25-fold with OsM in primary mouse calvarial osteoblasts. Analysis of these interactions between PTH/PTHrP and gp130 cytokine production and action provides new information on local communicating processes in bone.

Disclosures: J.M. Quach, None.

W026

Identification and Characterization of Runx2 Phosphorylation Sites involved in Matrix Metalloproteinase-13 Promoter Activation. N. Selvamurugan, E. Shimizu, N. C. Partridge. Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA.

Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism. It acts through its G-protein-coupled receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. Matrix metalloproteinase-13 (MMP-13; collagenase-3) plays a critical role in PTH-induced bone resorption and endochondral bone formation. Our earlier studies showed that PTH induced MMP-13 gene transcription in the rat osteoblastic cell line, UMR 106-01. We identified the PTH-response elements in the regulatory region of the MMP-13 gene and the proteins (Runx2 and Fos/Jun) binding to them. PTH acts via protein kinase A (PKA) to phosphorylate and stimulate the transactivation of Runx2 through a PKA consensus site. To further identify PTH-stimulation of Runx2 phosphorylation in vivo, immunoprecipitation of Runx2 and Western blot analysis were performed. We found that PTH stimulated Runx2 phosphorylation mostly on serine residues with a small amount at threonine and tyrosine residues in UMR 106-01 cells. To determine Runx2 phosphorylation residues in vivo, Runx2 protein was metabolically labeled in COS-7 cells and subjected to immunoprecipitation, acid hydrolysis and thin layer chromatography. Runx2 phosphorylation was increased at serine residues after 8-Br-cAMP treatment. Further, the Runx2 phosphorylation sites in vivo were identified by transient transfection of a c-myc-tagged Runx2 construct into COS-7 cells, followed by 8-Br-cAMP treatment, immunoprecipitation and matrix-assisted laser desorption/ionization MS analysis. Runx2 was identified to be phosphorylated on serines 14 and 332, and threonine 325 after 8-Br-cAMP treatment. To determine the role of Runx2 phosphorylation sites for MMP-13 promoter activation, the wild type Runx2 and Runx2 mutated at these phosphorylation sites (at single, double or triple sites) were transiently transfected into either COS-7 or C3H10T1/2 cells along with the MMP-13 promoter construct. In the presence of 8-Br-cAMP, the wild type Runx2 construct stimulated MMP-13 promoter activity; while the Runx2 construct having mutations at all three phosphorylation sites was unable to stimulate MMP-13 promoter activity in these cells. Thus, we have identified for the first time Runx2 phosphorylation sites necessary for PKA stimulated MMP-13 promoter activation and this event may be critical for bone remodeling.

Disclosures: N. Selvamurugan, None.

W027

Connexin43 Amplifies FGF2-Responsiveness in a Runx2/Protein Kinase C delta- dependent Manner. F. Lima, C. Niger, J. P. Stains. Orthopaedics, University of Maryland, Baltimore, MD, USA.

We examined the molecular and biochemical role of the gap junction protein, connexin43 [Cx43], in the transcriptional response of osteocalcin to fibroblast growth factor 2 [FGF-2]. By luciferase reporter assays, we identified that the osteocalcin transcriptional response to FGF2 in MC3T3 cells is markedly increased by overexpression of Cx43. The transcriptional synergy between Cx43 and FGF2 on osteocalcin transcription is dependent on the dose of FGF2, affecting at 5, 10 and 25 ng/ml. The amplification of FGF2 responsive osteocalcin transcription by Cx43 is mediated by runx2, via its OSE2 cognate element, but not by a previously identified connexin-responsive Sp1/Sp3 binding element. Disruption of Cx43 function with siRNA against Cx43 markedly attenuated the response of MC3T3 cells to FGF2 treatment. Similarly, overexpression of connexin45 - a gap junction protein with “dominant negative” properties with respect to Cx43 function - diminished FGF2 induced osteocalcin transcription. To assess the signal transduction machinery responsible for the Cx43-dependent enhanced FGF2 responsiveness, we used chemical inhibitors and/or siRNA to block ERK, p38 and JNK cascades, as well as, protein kinase C delta [PKCd] activity. FGF2 was able to induce the phosphorylation of all of these cascades, as shown by immunoblotting with phospho-specific antibodies. Despite the activation of multiple signal transduction pathways, only the PKCd inhibitor, rottlerin, was able to abrogate the Cx43-dependent enhancement of osteocalcin transcription in response to FGF2. In order to illuminate how Cx43, PKCd and runx2 mediate the osteoblast response to FGF2, we examined the biochemical changes occurring in FGF2 treated cells. We identified by immunoprecipitation that PKCd and Cx43 physically interact. Further, we show by immunocytochemistry that upon treatment with FGF2, PKCd is translocated to the nucleus. By immunoblotting, we show that PKCd and runx2 are phosphorylated in response to FGF2 treatment. The abundance of phosphorylated protein is increased by overexpression of Cx43, suggesting that the degree of activation is enhanced by increased Cx43 levels. In total, these data show that the gap junction protein Cx43 plays a critical role in regulating osteoblast response to FGF2, as both gain and loss of Cx43 function impact the FGF2-induced osteocalcin transcription. This effect is mediated by PKCd, which can physically “dock” with Cx43, and, when activated, translocate to the nucleus where it enhances runx2 transcriptional activity.

Disclosures: J.P. Stains, None.

This study received funding from: NIH grant AR052719.

W028

TGFβ Inducible Early Gene-1 Directly Binds to, and Represses, the OPG Promoter in Osteoblasts Resulting in Decreased Support of Osteoclast Differentiation. M. Subramaniam¹, J. R. Hawse¹, P. G. Monroe¹, A. Sanyal², M. Cicek², M. J. Oursler¹, T. C. Spelsberg¹, ¹Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA, ²Endocrine Research Unit, Mayo Clinic, Rochester, MN, USA.

TGFβ Inducible Early Gene-1 (TIEG) is a member of the Krüppel-like family of transcription factors that plays an important role in TGFβ mediated Smad signaling. To understand the physiological role of TIEG, we generated TIEG-null (TIEG^−/−) mice. Calvarial osteoblasts isolated from these mice have a reduced ability to support osteoclast differentiation in vitro. Gene expression studies revealed decreased RANKL and increased OPG expression in TIEG^−/− osteoblasts suggesting a role for TIEG in regulating the expression of these genes. Since OPG is one of the most important regulators of osteoclast differentiation, and since TIEG^−/− osteoblasts have decreased levels of OPG expression, we sought to determine if TIEG directly regulates OPG expression. A luciferase construct, containing an approximately 1.6 kb fragment of the mouse OPG promoter, was cloned into a pGL3 reporter vector and transiently transfected into TIEG^−/− calvarial osteoblasts with and without a TIEG expression vector. The OPG promoter activity was completely abolished in the presence of TIEG suggesting that TIEG directly regulates the expression of OPG in osteoblasts. In order to determine the region of this promoter through which TIEG acts, sequential 5′-deletion constructs were created. Transient transfection of these constructs revealed that the TIEG binding site(s) resides within a 200 bp region of the OPG promoter. To determine if TIEG protein directly interacts with this region, transient ChIP analyses were performed using a TIEG-specific antibody. The results of these studies revealed that TIEG does bind to this region of the OPG promoter. As a confirmation, real-time PCR analysis of these ChIP assays demonstrated that this 200 bp region is enriched by approximately 8-fold in the presence of TIEG protein relative to vector transfected cells. These studies confirm that TIEG interacts with this specific region of the OPG promoter to inhibit its activity. Since we have previously shown that TIEG regulates target gene expression through Sp-I sites, we scanned this region of the OPG promoter for potential TIEG binding elements. Interestingly, this sequence analysis identified 4 Spl binding sites and suggests that TIEG could be acting through these DNA elements to regulate the expression of the OPG gene. Taken together, these results confirm that TIEG directly binds to and inhibits OPG promoter activity and as expected, TIEG^−/− mouse osteoblasts display a significant inability to support osteoclast differentiation.

Disclosures: M. Subramaniam, None.

W029

C/EBPβ Is a Crucial Partner of ATF4 in Osteoblast Differentiation. H. Tominaga*¹, S. Maeda¹, M. Hayashi*¹, S. Akira*², T. Komori*³, S. Komiya*⁴, T. Imamura¹, ¹Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo, Japan, ²Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Department of Host Defense, and The 21st Century COE, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan, ³Department of Developmental and Reconstructive Medicine, Division of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, ⁴Department of Neuromusculoskeletal Disorder, Orthopaedic Surgery, Graduate School of Medicine and Dentistry, Kagoshima University, Kagoshima, Japan.

ATF4 is a member of CREB/ATF family basic leucine zipper (bZIP) transcription factors, and plays a crucial role in osteoblast differentiation in cooperation with Runx2. ATF4 binds to DNA element OSE1, which sequence exists in osteocalcin gene promoter, to enhance the differentiation. We and other groups have reported that another bZIP protein C/EBPβ also enhance osteoblast differentiation with Runx2 synergistically, through interacting to CEBP-binding element. ATF4 and C/EBPβ are reported to be able to form stable heterodimers. Moreover, OSE1 resembles reported DNA binding sequence for ATF4-C/EBPβ heterodimers. Thus we hypothesized that the heterodimers could act on OSE1. First we confirmed interaction of endogenous ATF4 and C/EBPβ in primary calvarial osteoblasts. We next tested the question by gel shift and luciferase assays. ATF4-C/EBPβ heterodimers bound OSE1 with more strong affinity than homodimers of ATF4. The heterodimers induced more intense signals from luciferase constructs of OSE1 and OC promoter than ATF4-homodimers, which activity was elevated to maximum level with Runx2. We finally studied function of C/EBPβ on bone formation in vivo by analyzing C/EBPβ knockout mice, which phenotype regarding bone has not been studied. C/EBPβ null mice showed delayed bone formation like ATF4 null mice. These results suggest that C/EBPβ plays an indispensable role in forming active complex with ATF4 and Runx2 on OSE1, in addition to CEBP-binding element, during osteoblast differentiation.

Disclosures: H. Tominaga, None.

W030

Osrl Gene Expression Is Regulated by Runx2 and Ikaros Transcription Factors. M. Yamauchi¹, S. Kawai², A. Amano*², T. Ooshima*¹, ¹Department of Pediatric dentistry, Osaka university Graduate School of Dentistry, Suita-Osaka, Japan, ²Department of Oral Frontier Biology, Osaka university Graduate School of Dentistry, Suita-Osaka, Japan.

Odd-skipped gene is one of the pair-rule genes which contribute to Drosophila somite formation. Two mammalian homologues. Odd-skipped related 1 (Osr1) and Osr2, has been shown to be expressed in limb, branchial arches, kidney, eye, and dermis. Osr1 is known to play a critical role in the heart and urogenital development. While, the function of Osr1 in bone formation as well as the regulation of Osr1 gene expression are still unclear. To understand regulatory mechanism of Osr1 gene expression, we cloned mouse Osr1 promoter (4.7kb) and analyzed the promoter activity.

We constructed 7 deleted promoters and cloned them into pGL-3 basic vector. As a result, two regulatory regions which consisted of positive regulatory region (-741bp∼-144bp) and negative regulatory region (-1651bp∼-742bp) were found in Osr1 promoter. GATA1, CP-2, Ikaros, Nkx-2.5, Runx1, and Runx2 binding sites existed around these regulatory regions. Osr1 promoter-luciferase reporter construct and various transcription factors were co-transfected and luciferase activity was measured. As a result, Ikaros and Runx2 decreased Osr1 promoter activity, and it was also found that Osr1 gene expression was dose-dependently downregulated by these factors. Furthermore, point mutation at transcription factor binding site for Ikaros or Runx2 prevented the downregulatory effects by them. We also confirmed the binding of transcription factors to Osr1 promoter with chromatin immunoprecipitation and electrophoretic mobility shift assay. These data indicates that Osr1 gene expression is negatively regulated by at least two transcription factors, Ikaros and Runx2. Ikaros is one of the important regulators of lymphocyte differentiation. Runx2 is a crucial regulator of bone formation. Osr1 is regulated by these transcription factors, suggesting that Osr1 has a functional role in lymphocyte differentiation and bone formation.

Disclosures: M. Yamauchi, None.

W031

Age-Related Intrinsic Changes in Human Bone Marrow-Derived Mesenchymal Stem Cells and Their Differentiation to Osteoblasts. J. Glowacki¹, S. Zhou¹, J. S. Greenberger², M. W. Epperly*², J. P. Goff*², C. Adler*¹, M. S. LeBoff³, ¹Orthopedic Surgery, Brigham and Women's Hospital, Boston, MA, USA, ²Radiation Oncology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA, ³Medicine, Brigham and Women's Hospital, Boston, MA, USA.

A sub-population of marrow-derived stromal cells (MSCs, a.k.a. mesenchymal stem cells) has potential to differentiate into multiple cell types, including osteoblasts. We tested the hypotheses that there are intrinsic effects of age on human MSCs. We measured the effect of age on proliferation, cell cycling, apoptosis, p53 pathway genes, senescence-associated β-galactosidase (SA-β-gal), and osteoblast differentiation in hMSCs that were obtained from marrow discarded from men and women (17-90 years) undergoing orthopedic surgery. Low density mononuclear cells were isolated by centrifugation on Ficoll/Histopaque 1077 and hMSCs were established in monolayer culture with phenol red-free a-MEM, 10% Fetal Bovine Serum-Heat Inactivated, 100 U/ml penicillin, and 100 μg/ml streptomycin. For some experiments, the low-density mononuclear cell suspensions were sorted with FITC-labeled STRO-1 antibody. Viable hMSCs at passage 2 were counted every other day with a hemocytometer. Cell cycle was assessed by FACS. SA-β-gal-positive cells were enumerated cytochemically. Osteoblast differentiation was assayed in basal medium with osteogenic supplements (10 nM dexamethasone, 5 mM b-glycerophosphate, and 170 μM ascorbic phosphate). For most analyses, nonparametric statistical tools were used.

Doubling time of hMSCs was 1.6-fold longer in cells from the older (78.8 ± 13.5 hrs, n = 13) than the younger subjects (48.8 ± 0.7 hrs, n = 3) and was positively correlated with age (r = 0.60, p = 0.0137, n = 16). With age, there was uniform prolongation of the duration of all phases of the cell cycle, but with age, more cells were apoptotic (r = 0.723, p = 0.0248, n = 9). Further, there were age-related increases in expression of p53 and its pathway gene p21 and BAX (RT-PCR. There were 4-fold more hMSCs positive for SA-β-gal in samples from older (8.9 ± 2.4, n = 12) than younger subjects (2.3 ± 1.8, n = 5, p<0.001). With age, there was a significant decrease in generation of osteoblasts both in the STRO-1* cells (r = −0.714, p = 0.047, n = 8) and in adherent MSCs (r = −0.81, p = 0.010, n = 8).

In sum, there are age-dependent decreases in proliferation and osteoblast differentiation and increases in p53 and its pathway genes, SA-β-gal. and apoptosis in hMSCs. The p53 pathway may have a critical role in age-related changes of hMSCs, in light of recent murine data that p53 is a negative regulator of osteoblast differentiation. These findings support the view that there are intrinsic alterations in human MSCs with aging that may contribute to the process of skeletal aging.

Disclosures: J. Glowacki, None.

This study received funding from: NIH.

W032

Wdr5 Is Essential for Osteoblast Differentiation. F. Gori, E. Zhu*, M. B. Demay. Endocrine Unit, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA.

Wdr5, a BMP-2 induced gene, is developmentally expressed in osteoblasts and accelerates osteoblast differentiation in vitro. Transgenic mice overexpressing Wdr5 in maturing osteoblasts display accelerated endochondral bone formation during embryonic development, associated with earlier activation of the canonical Wnt signaling pathway. To address whether Wdr5 is essential for osteoblast differentiation, plasmid-based small interfering RNAs (siRNAs) were used to stably suppress endogenous Wdr5 protein levels in MC3T3E-1 cells. Levels of endogenous Wdr5 protein were assessed by Western analyses after 7 days in culture, when Wdr5 protein levels are maximally expressed. Clones expressing two of the selected siRNAs targeting Wdr5 demonstrated a significant decrease in endogenous Wdr5 protein levels relative to that of the scrambled (siRNA-S) clone. This reduction of endogenous Wdr5 levels markedly inhibited osteoblast differentiation, resulting in a significant decrease in alkaline phosphatase activity, impaired expression of Runx-2 and osteocalcin mRNAs and absence of mineralized matrix formation. Based on the observation that overexpression of Wdr5 enhances canonical Wnt signaling in osteoblasts, the effects of Wdr5 silencing on a key mediator of this pathway, β-catenin, was examined. A decrease in nuclear β-catenin protein levels was observed with Wdr5 knockdown, suggesting that inhibition of canonical Wnt signaling may, in part, underlie the impaired osteoblast differentiation observed when Wdr5 protein levels are suppressed. Analysis of Wnt target genes demonstrated that suppression of Wdr5 expression resulted in a significant decrease in the levels of c-myc, the expression of which is increased by canonical Wnt signaling. Furthermore, the expression of SFRP-2, which is repressed in response to Wnt signaling, was increased in association with Wdr5 knockdown. Although suppression of Wdr5 protein levels decreased the antiapoptotic protein Bcl-2 and increased caspase 9 activation, only a minimal increase in apoptosis was observed in these cells (from 3.2% to 5%). Thus, our investigations demonstrate that Wdr5 not only accelerates osteoblast differentiation when it is overexpressed, but is also essential for osteoblast differentiation. In addition, our findings suggest that Wdr5 is required for optimal canonical Wnt signaling in MC3T3-El cells.

Disclosures: F. Gori, None.

W033

The Role of EphB/ephrin-B Interactions in Cell Attachment and Spreading of Mesenchymal Precursors Derived from Human Bone Marrow and Dental Pulp Tissues. A. Arthur*¹, S. Koblar*², S. Gronthos¹, ¹Division of Haematology, Institute of Medical and Veterinary Science, Adelaide, Australia, ²Faculty of Health Sciences, University of Adelaide, Adelaide, Australia.

The Eph family of receptor tyrosine kinases and their ligands, the ephrin molecules, appear to play an important role in skeletal development during embryogenesis. Our study examined the expression pattern and function of EphB/ephrin-B molecules on mesenchymal stem/precursor cells derived from human bone marrow and dental pulp tissues. Multiple receptors and ligands were identified on dental pulp stem cells (DPSC) and bone marrow stromal stem cells (BMSSC) by real time PCR and immunohistochemistry. The function of EphB/ephrin-B molecules during BMSSC and DPSC attachment and spreading was assessed in the presence of different Eph/ephrin-Fc fusion proteins using an established in vitro cell-spreading assay. In response to either EphB2-Fc or ephrin-B1-Fc, the stem cells formed rounder and smaller cell bodies demonstrated by F-actin distribution, and restricted their ability to spread. Additionally, immature focal adhesions were identified at the edge of the cell membrane, reminiscent of spreading initiation centres (SIC), which have only been observed at early stages of cell spreading. In the presence of Inhibitors, Eph forward signaling through the mitogen activated protein kinase (MAPK) pathway restricted cellular spreading, while reverse signaling through the ephrin ligand was mediated via the phosphorylation of the Src homology (SH2) domain activating downstream signaling cascade. Collectively, these studies indicated that EphB/ephrin-B molecules provide an inhibitory environment for mesenchymal stem/precursor cell attachment, consequently resulting in the lack of spreading and detachment. These results may have implications for calcified skeletal/dental tissue development and regeneration.

Disclosures: S. Gronthos, None.

This study received funding from: NHMRC

W034

PTEN and Osteoblastogenesis. A. R. Guntur¹, M. C. Naski², ¹Biochemistry, UTHSCSA, San Antonio, TX, USA, ²Biochemistry & Pathology, UTHSCSA, San Antonio, TX, USA.

Osteoblastogenesis is marked by increased expression of PTEN, the chief inhibitor of phosphatidylinositol 3-kinase (PI3K) signaling. Based on this observation we hypothesized that PTEN/PI3K signaling regulates osteoblast differentiation. To test this hypothesis we conditionally inactivated PTEN in osteoblast precursors. This caused a dramatic increase in osteoblast differentiation and bone formation. Moreover, precocious differentiation ensued, indicating loss of physiological restraints. Increased numbers of osteoblasts were in part accounted for by enhanced cell proliferation and diminished pathways of feedback inhibition. Accelerated differentiation coincided with increased levels of the hedgehog-dependent transcription factor Gli2. Increased hedgehog signaling was evidenced by up-regulation of patched, a transcriptional target of hedgehog. We conclude that PTEN is required to restrict osteoblast replication and halt precocious differentiation. The results demonstrate that the PI3K signaling pathway is an attractive target for pharmacological regulation to enhance bone mass.

Disclosures: A.R. Guntur, None.

W035

Role of Type III NaPi Transporter(s) in Osteoblastic Differentiation. C. Haldrup*¹, L. B. Nielsen*², J. Lykke*³, L. Pedersen¹, ¹Dept of Molecular Biology and Inst of Clinical Medicine, Aarhus University, Aarhus, Denmark, ²Dept. of Paediatrics, Glostrup University Hospital, Glostrup, Denmark, ³Dept. of Medical Biochemistry and Genetics, University of Copenhagen, Copenhagen, Denmark.

The role of the type III sodium-dependent inorganic phosphate (Pi) transporters in osteoblastic differentiation and mineralization was addressed using the murine preosteoblastic cell line, MC3T3-E1. These cells differentiate over time into mature mineralizing osteoblasts upon addition of ascorbic acid and β-glycerophosphate. To impair the function of the 2 type III transporters, Pit1 and Pit2, cells were infected with retroviruses, which employ Pit1 and/or Pit2 as receptors for infection and thus inhibit their function. Blocking of Pit1 and Pit2 protein function was confirmed by virus interference studies. Mineralization was analyzed with Alizarin red. Expression of Pit1 and osteocalcin were followed by Northern blotting. Pi uptake was analyzed using 32Pi.

We found that infecting MC3T3-E1 cells with a virus impairing both Pit1 and Pit2 severely reduced their ability to mineralize. Infection with this virus only reduced Pi uptake with about 30% suggesting that the 2 type III transporters are not responsible for the bulk Pi uptake in the cells. However, Pit1 has previously been shown to be upregulated during osteoblastic differentiation implying that at least Pit1 has a specialized function in osteoblasts. To further analyze the role of Pit1, we investigated Pi uptake in undifferentiated MC3T3-E1 cells overexpressing epsilon-tagged human Pit1 or Pit2. These results surprisingly showed that although human Pit1 was present in the cell membrane, it did not facilitate an increased Pi uptake compared to wt cells. In contrast, Pi uptake was more than tripled when Pit2 was overexpressed. Thus, Pi transport does not seem to be Pit1′s primary function in undifferentiated MC3T3-E1 cells. Moreover, while osteocalcin could still be upregulated in cells infected with a virus impairing Pit2 function, its expression was undetectable in cells infected with a virus impairing both Pit1 and Pit2 function suggesting that Pit1 has a direct effect on osteoblastic differentiation. In conclusion, the results strongly suggest that Pit1 is necessary for osteoblastic differentiation and, possibly as a consequence of this, also for mineralization. The data do not reveal the exact role of Pit1 in differentiation, however, since overexpressed Pit1 does not appear to facilitate an increase in Pi uptake in undifferentiated MC3T3-E1 cells, we speculate that 1) Pit1 Pi transport function is differently regulated in the membrane in differentiated osteoblasts compared to undifferentiated cells and/or 2) Pit1 may function in signaling.

Disclosures: C. Haldrup, None.

This study received funding from: Danish Medical Research Council.

W036

Heparan Sulfate Proteoglycan (HSPG) Aulfation During In Vitro Osteogenesis. L. M. Haupt*¹, A. van Wijnen², G. S. Stein³, V. Nurcombe*¹, S. M. Cool¹, ¹Stem cell and tissue repair laboratory, IMCB, Singapore, Singapore, ²Department of Medicine, University of Massachusettes, Worcester, MA, USA, ³Medical School, University of Massachusettes, Worcester, MA, USA.

The heparan sulfate proteoglycans (HSPGs) are a family of extracellular matrix (ECM) and cell membrane-associated complexes that mediate a host of soluble, cell:cell, and ECM interactions. They are composed of a core protein to which highly variable glycosaminoglycan (GAG) sugar chains of the heparan sulfate class are attached, and they are capable of interacting with multiple morphogens, including the fibroblast growth factors (FGFs), Wnts, and the bone morphogenic proteins (BMPs), based upon their side chain sulfation patterns. Structural heterogeneity within the HS chain is generated during chain initiation and polymerisation, with the key modifications performed by the specific, coordinated actions of the glycosyltranferases (Ext-1 and −2), the N-deacetylase/N-sulfotransferases (NDST1-4), as well as 2-O-sulfotransferase and 6-O-sulfotransferase enzymes. Under osteogenic conditions in vitro, MC3T3 cells reach confluence between days 6 and 8, exit the cell cycle and commit toward the osteogenic lineage. We have compared the expression profile of the glycosyltransferase and sulfotransferase enzymes under non-osteogenic and maintenance media culture conditions between the proliferative (Day 6) and differentiative (Day 14) phases of cell culture to determine a role for HSPG sulfation activity during in vitro osteogenesis. Under osteogenic conditions, MC3T3 cells demonstrated increased gene expression of osteogenic markers with a coincident decreased gene expression of Ext-1 and −2 genes but increased expression of NDST-3. Significantly, the osteogenic driven cells dramatically increased their syndecan-1 and glypican-3 gene expression profiles as compared to their non-osteogenic counterparts. In addition, these cells alter their FGFR profile, increasing FGFR2, FGFR3 and FGFR4 gene expression levels. Combined this data implicates the production of shorter, highly sulfated HSPGs during in vitro osteogenesis. In particular, glypican-3 and syndecan-1 appear to be central to the in vitro differentiation of MC3T3 pre-osteoblast cells by the mediating growth factor response of the cells.

Disclosures: L.M. Haupt, None.

This study received funding from: A Star Singapore.

W037

Continuous Treatment with PTH Is Anabolic in Cultured Osteoblasts from COX-2 Knockout Mice. H. Huang*, S. Choudhary, L. Raisz, C. Pilbeam. Endocrinology Division, University of Connecticut Health Center, Farmington, CT, USA.

PTH is a potent anabolic agent in vivo, but its anabolic effects in vitro are difficult to demonstrate. Since PTH induces cyclooxygenase (COX)-2 and PGE₂ production in osteoblasts and PGE₂ is anabolic in vitro, we hypothesized that whatever anabolic effects PTH might have in vitro would be diminished in cells from COX-2 knockout (KO) mice compared to cells from wild type (WT) mice. Marrow stromal cells (MSCs) and primary calvarial osteoblasts (POBs) from 6-8 wk old COX-2 KO and WT mice in a CD-1 background were treated with PTH (10⁻⁸ M) or vehicle (control) for up to 21 d. Alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA levels were determined at d 14 and 21, respectively, by real-time PCR and mineralization at d 21 by alizarin red staining. In both MSC and POB cultures, ALP and OCN mRNA expression were 35%-50% lower in KO control cultures than in WT control cultures. PTH had no effect on ALP and OCN mRNA expression in either MSC or POB WT cultures but did increase expression in KO cultures relative to controls. PTH increased ALP and OCN expression 6.5-fold (p < 0.01) and 4.4-fold (p < 0.01), respectively, in KO MSC cultures and 2.1-fold (p<0.05) and 7.0-fold (p<0.01), respectively, in KO POB cultures. PTH had no effect on mineralization in WT MSC or POB cultures but increased mineralization in both KO MSC and POB cultures. Basal cAMP levels in POB cultures, in the presence of the phosphodiesterase inhibitor IBMX, were 2-fold higher in WT compared to KO cells (p<0.05). PTH treatment for 15 min increased cAMP 70-fold in WT and only 40-fold in KO cells. Hence, the increased anabolic effects of PTH in KO cells do not appear to be associated with increased cAMP production. To screen for other factors that might be responsible for the differences in PTH-mediated anabolic effects, POBs from WT and KO mice were cultured for I wk and treated with and without PTH for 1, 3 and 24 h and mRNA expression measured by realtime PCR. No change in BMP-2 mRNA expression was observed with PTH. PTH increased Runx-2 mRNA by 2-fold in both WT and KO cultures at 3 h (p<0.01). PTH increased IGF-1 by 1.7-fold in KO cultures at 24 h (p<0.01) but had no effect in WT cultures. In contrast to the increased anabolic effects of PTH in KO cells, the PTH stimulation of osteoclast differentiation in marrow cultures, measured by counting TRAP positive multinucleated cells at d 7 of culture, was decreased 70% (p <0.01) in COX-2 KO cultures compared to WT cultures as reported previously. We conclude that absence of COX-2 increases the stimulatory effects of PTH on osteoblastic differentiation in vitro but decreases the stimulatory effects of PTH on osteoclastic differentiation in vitro. These data are consistent with the observation that intermittent PTH has greater anabolic effects in COX-2 KO mice than in WT mice.

Disclosures: H. Huang, None.

This study received funding from: NIH DK-48361 and AR-47673.

W038

Bioactive Tripeptides Enhance Osteoblast Function In Vitro. M. M. Huttunen*, M. Pekkinen*, M. E. Ahlström*, C. J. Lamberg-Allardt. Dept. of applied chemistry and microbiology/Nutrition, University of Helsinki, Helsinki, Finland.

The puropse of this in vitro study was to elucidate the short- and long-term effects of bioactive tripeptides Ile-Pro-Pro (IPP), Val.Pro-Pro (VPP) and Leu-Lys-Pro (LKP) on osteoblast proliferation, differentiation and bone formation. Preliminary screening of the peptides on osteoblast proliferation was carried out on rat osteosarcoma UMR-106 cells. Further studies were carried out on human mesenchymal stem cells (Poietics, Cambrex Bio Science) purified from bone marrow and osteoblasts differentiated from them. Cell proliferation experiment was carried out with 5, 50 and 500 μM peptide concentrations and 6-, 24- or 48-h treatment time. DNA synthesis activity was assessed by a Cell Proliferation ELISA kit (Roche Diagnostics GmbH, Germany). The expression profiles of osteoblasts were compared after a 24-h treatment with 50 μM IPP, VPP or LKP with a Hum16-K protocol, consisting of 16 000 human gene probes. Microarray results were confirmed by qRT-PCR (Mx3000P, Stratagene) according to the manufacturer's instructions using a Brilliant SYBRGreen qPCR Master mix kit. IPP's long-term effects on nine genes in hMSC-differentiated osteoblasts was analysed by qPCR on days 13, 17 and 20. Total protein content and bALP activity were assessed on days 3, 7, 12, 14, 17, 21 and 34 of culture. Cell culture mineralization was assessed by Alizarin Red S staining. The results show increased UMR-106 and hMSC proliferation. Microarray data analysis revealed IPP to upregulated 270 genes and downregulated 100 genes. The respective numbers for VPP were 25 and 10 and for LKP 16 and 14. IPP regulated genes associated with cell differentiation, cell growth and cell signal transcription. The upregulation of these genes indicates that IPP enhances osteoblast proliferation and differentiation. Long-term treatment with IPP enhanced osteoblast gene expression in favour of bone formation and increased mineralization, but did not influence bALP activity. Apoptosis related caspase-8 and PTHrP were downregulated. Our results show that bioactive peptides stimulate pathways resulting to increased osteoblast function and bone formation. Agents that increase the number and function of osteoblasts can improve bone mass and structure and decrease fracture risk. Bioactive peptides such as IPP may well conduct some of the positive effects dietary protein has on bone.

Disclosures: M.M. Huttunen, None.

W039

Progenitor Cells in Traumatically Injured Muscle Have Characteristic Similarities to Bone Marrow-Derived Stem Cells. W. M. Jackson*¹, S. M. Koehler*¹, J. R. Giuliani*², R. S. Tuan¹, L. J. Nesti², ¹Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD, USA, ²Department of Orthopaedics and Rehabilitation, Walter Reed Army Medical Center, Washington, DC, USA.

Heterotopic ossification (HO), or the formation of mature bone in soft tissues, is prevalent in patients with severe time-of-war extremity wounds. Although the cellular mechanisms of HO pathogenesis are unclear, it is likely that mesenchymal progenitor cells in the traumatized muscle (tmMPCs) are responsible for HO formation. The relationship between these MPCs and bone marrow-derived mesenchymal stem cells (bmMSCs) is unknown; therefore, the objective of this study was to compare the phenotype and differentiation potential of tmMPCs in traumatically injured muscle to bmMSCs.

With IRB approval at Walter Reed Army Medical Center, muscle debridements were obtained from patients sustaining traumatic orthopaedic injury, and bone marrow was obtained from total hip arthroplasties. The tmMPCs were isolated from the muscle after extensive mincing, collagenase digestion and direct plating onto tissue culture plastic (TCP). bmMSCs were isolated from the marrow by direct plating onto TCP. After culture expansion, the expression of characteristic cell surface markers for MSCs was assayed by flow cytometry. Both groups of cells were cultured in defined differentiation media for osteogenesis, chondrogenesis, adipogenesis and myogenesis. The differentiation potential was assayed with histology (osteogenesis: alizarin red; adipogenesis: oil red O; chondrogenesis: alcian blue; myogenesis: hematoxylin) and by expression of lineage dependent genes (osteogenesis: ALP, Col I, and Runx2; adipogenesis: LPL and PPARg2; chondrogenesis: Col II, Col IX, AGN and Sox 9; myogenesis: MyoD and myosin heavy chain).

Although there were slight morphological differences between tmMPCs and bmMSCs, both populations expressed cell surface markers characteristic for MSCs (CD 73, CD 90 and CD105), and did not express markers absent on MSCs (CD14, CD34 and CD45). After 21 days in differentiation media, the histological staining and genetic expression of tmMPCs and bmMSCs was characteristic for differentiation into osteoblasts, chondrocytes and adipocytes but not into myoblasts.

This study demonstrates that MPCs in traumatized muscle have similar phenotype and differentiation potential as bmMSCs. The tmMPCs appear to be distinct from myogenic progenitors since they do not readily differentiate into myoblasts or form myotubes. Our findings suggest that dysregulation of the tmMPCs by inflammatory and wound response signals associated with trauma would be sufficient to induce ectopic osteogenesis.

Disclosures: W.M. Jackson, None.

This study received funding from: The Military Amputee Research Program. Walter Reed Army Medical Center.

W040

Temporal Regulation of Histogenesis by Mesenchyme During Craniofacial Bone Development. A. H. Jheon*, B. F. Eames*, R. A. Schneider*, Orthopaedic Surgery, UCSF, San Francisco, CA, USA.

Development of bones involves a vast array of molecular and cellular interactions. Although, the roles of mesenchyme and epithelium in the patterning of facial bones have been studied extensively, little is known about their roles in the temporal control of bone histogenesis, which involves distinct and sequential modules including pre-osteoblast specification, osteogenic condensations, osteoid deposition, and mineralization. In the present study, we exploit the different developmental rates between quail and duck (quail hatch in 17 days whereas duck hatch in 28 days) to test the extent to which mesenchyme and epithelium control the initiation and progression of these modules during intramembranous bone development. By transplanting faster-developing quail mesenchyme adjacent to slower-developing duck epithelium (to generate chimeric quck), or conversely, slower-developing duck mesenchyme adjacent to faster-developing quail epithelium (to generate duail), the contribution of mesenchyme or epithelium can be determined. We show that mineralization of intramembranous bones occurs concordant with the developmental times of the donor. Thus, in quck, the first trace of facial bone mineralization occurs 36 hours earlier on the quail-donor side compared to the duck-host side, whereas in duail, mineralization occurs approximately 36 hours later on the duck-donor side. Further observations in quck show that deposition of osteoid and appearance of osteogenic condensations also occur according to the donor time line. These histological observations correlate with the molecular expression profiles of various genes necessary in osteoblast differentiation and mineralization. Preosteoblast-specification, measured by Runx2 expression, arises at a stage far before any evidence of bone and its expression is concomitant with a decrease in cell proliferation and an increase in cell differentiation. Thus, by utilizing the quail-duck chimeric system, we conclude that the temporal control of the various modules of intramembranous bone development is determined by mesenchyme, whereas epithelium appears to play a permissive role.

Disclosures: A.H. Jheon, None.

This study received funding from: CIHR, NIH.

W041

Flow Cytometric Immunophenotyping of Human Periodontal Ligament Cells. N. Kawanabe, K. Murakami*, S. Murata*, T. Yamashiro*, Department of Orthodontics and Dentofacial Orthopedics, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

To characterize its primitive phenotypes analogous to mesenchymal stem cells, we investigated cell surface antigens' expression in cultured human periodontal ligament (PDL) cells. After obtaining approval from the Ethical Committee, intact permanent teeth were collected from healthy patients who were undergoing orthodontic treatment at Okayama University Hospital. The PDL cells isolated from these teeth were cultured. The cells were incubated with antibodies, then subjected to flow cytometric analysis. Primary human PDL cells used in this study showed fibroblastic spindle shape and capillary network formation. Flow cytometric studies demonstrated that PDL cells express mesenchymal stem cell markers, including CD9, CD13, CD29, CD44, CD73, CD90, CD105 and CD166, while they are negative for many hematopoietic markers (CD2, CD3, CD4, CD8a, CD14, CD16, CD19, CD20, CD24, CD33, CD34, CD38, CD41a, CD45, CD66b, CD117, CD133 and CD235a), endothelial cell markers (Flt-1, Flk-1 and von Willebrand factor), and an epithelial cell marker (cytokeratin 5/8). Integrins positive for PDL cells were CD29 (β1), CD41a (α1) and CD51 (αv). Positive cytokine receptors were CD71 (Transferrin R), CD105 (endoglin), CD119 (IFNγ R) and CD120a (TNF RI), and negative were CD117 (c-kit), CD121a (IL-1 R) and CD124 (IL-4 Rα). PDL cells expressed matrix receptors such as CD54 (ICAM-1) and CD166 (ALCAM), but lacked CD31 (PECAM-1), CD56 (NCAM) and CD106 (VCAM-1). Human leukocyte antigen (HLA) expressed in PDL cells was HLA-ABC, however, HLA-DR expression was not detected. No reactions of STRO-1 and CDCP-1 (CUB-domain-containing protein 1) were observed in PDL cells. Thus, the antigens expressed in PDL cells were basically identified with that of bone marrow stromal precursors, suggesting that PDL cells possess primitive characteristics as mesenchymal stem cells.

Disclosures: N. Kawanabe, None.

W042

Effects of BHH9, an Ethanol Extract from Herbs, on Expression of Vascular Endothelial Growth Factor and Their Receptors During Human Osteoblasts Differentiation. D. Kim¹, J. Huh*², Y. Baek*², D. Choi*², D. Park*², J. Lee*², ¹Internal Medicine, Kyung Hee University Hospital, SEOUL, Republic of Korea, ²Acupuncture & Moxibustion, College of Oriental Medicine, Kyung Hee University, SEOUL, Republic of Korea.

Endochondral bone formation is regulated by systemically and locally acting growth factors. Vascular endothelial growth factor (VEGF) is an important regulator of bone formation and osteoblast differentiation. The aim of this study was to identify the BHH9 that may induce the angiogenic factor VEGF and its receptors (VEGFR) in osteoblast from human subchondral bone.

BrdU assay was used to determine the proliferation of human osteoblast cells. RT-PCR and ELISA assay were used to demonstrate the message and protein expression of VEGF, and extracellular matrix proteins including alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN) and collagen I (Coll). Flow cytometry analysis (FACS) was used to confirm the expression of VEGF, VEGFR-1 and VEGFR-2.

BHH9 slightly increased cell proliferation, and dramatically increased ALP activity in human osteoblast cells. Also, the expression of OCN, OPN, and Col I mRNA did not affected by treatment with BHH9 at 3 days and 7 days, but BHH9 was markedly enhanced at 14 days of culture. In addition to, BHH9 stimulated message and protein expression of VEGF and VEGFR-2 expression at 7 days of culture.

These results suggest that BHH9 plays an important role in bone remodeling by stimulating osteoblast differentiation via up-regulation of VEGF expression and possibly lead to the development of bone-forming drug.

Disclosures: D. Kim, Oriental Medicine R&D Project, Ministry of Health & Wellfare, Republic of Korea 2.

This study received funding from: Grant of the Oriental Medicine R&D Project, Ministry of Health & Welfare, Republic of Korea.

W043

Hairy Enhancer of Split 1 (HES-1) Is an Anti-adipogenic Mediator of Osteogenic Oxysterols and Hedgehog Signaling. W. Kim¹, V. Meliton*¹, C. M. Amantea*¹, T. J. Hahn², F. Parhami¹, ¹David Geffen School of Medicine, UCLA, Los Angeles, CA, USA, ²VA Greater Los Angeles Healthcare System and Geriatric Research, Education, and Clinical Center, Los Angeles, CA, USA.

Age-related bone loss is associated with a progressive decrease in bone formation and an increase in adipogenesis in the bone marrow. An increase in adipose tissue volume and a decrease in trabecular bone volume in bone marrow have been observed with aging and in patients with osteoporosis. Growing evidence indicates that PPARγ plays an important role in the regulation of bone metabolism. It has been shown that hedgehog signaling pathway, activated by sonic hedgehog protein, inhibits adipogenic differentiation. Previously, our lab reported that osteogenic oxysterols inhibit PPARγ expression and adipogenic differentiation of pluripotent bone marrow stromal cells (MSC) by activating hedgehog signaling. The present report identifies the molecular mechanism(s) by which oxysterols and hedgehog signaling inhibit PPARγ expression and adipogenic differentiation. Microarray-based gene expression analysis revealed that osteogenic oxysterols caused a 2 fold induction of HES-1 mRNA expression in the pluripotent M2-10B4 (M2) MSC after 48 hours of treatment. Q-RT-PCR analysis of M2 cells showed that HES-1 expression was induced by the osteogenic oxysterol 20S-hydroxycholesterol (20S) and sonic hedgehog (Shh) in a time-dependent manner. HES-1, a target of notch signaling, is a basic helix-loop-Helix DNA binding protein that is down-regulated during adipogenesis, and notch signaling profoundly inhibits adipogenesis through HES-1. After 48 hours, 20S (5μM) or Shh (200ng/mL) completely blocked PPARγ mRNA expression in M2 cells treated with the PPARγ ligand, troglitazone. 20S- and Shh-induced HES-1 expression was completely blocked by the inhibitor of hedgehog signaling pathway, cyclopamine (4μM), suggesting that HES-1 is a downstream target of hedgehog signaling. We also found a HES-1 binding site in the mouse Gli1 promoter using a Genomatix software, suggesting a potential feedback loop between Gli1 and HES-1. To demonstrate that HES −1 is a repressor of adipogenesis in MSC, we transfected M2 cells with 0.5, 1, 2, and 4 μg of pCMV- HES-1 overexpression vector, and the effect of HES-1 overexpression on PPARγ expression was assessed by Q-RT-PCR. Results showed that troglization-induced PPARγ expression was inhibited by HES-1 overexpression in a dose-dependant manner. Altogether, the present study suggests that the anti-adipogenic effects of osteogenic oxysterols and Shh are mediated by a HES-1 dependent mechanism.

Disclosures: W. Kim, None.

This study received funding from: NIH/NIAMS.

W044

Proinflammatory Control of BMP-Smad-driven Transcription and Osteogenesis. O. Korchynskyi¹, P. ten Dijke*², D. D. Patel*³, S. S. Makarov*⁴, ¹Orthopedic Surgery, Virginia Commonwealth University, Richmond, VA, USA, ²Leiden University Medical Center, Leiden, The Netherlands, ³Thurston Arthritis Reserach Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, ⁴Thurston Arthritis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Impaired bone homeostasis contributes to development of osteopenia, osteolysis and joint erosions during the rheumatoid arthritis (RA). Bone morphogenetic proteins (BMP) are crucially important regulators of osteogenesis. Activation of specific BMP receptors (BMPR) leads to activation of the major BMP signaling pathway, namely intracellular Smad proteins, as well as other, Smad-independent, pathways that transduce BMP signals to the nucleus. Using in vitro tissue culture approaches we show that activation of NF-κkB pathway with proinflammatory cytokines IL-1β and TNFα inhibits osteogenic differentiation of pluripotent mesenchymal precursor cells through Smad7-independent inhibition of Smad1/5 transcriptional activity. Immunoblot and EMSA experiments show that correspondingly neither Smad1/5 phosphorylation by BMPR-Is, nor direct Smad1/5 binding to DNA into BMP target genes promoters are affected by the activation of NF-κB pathway with TNFα, or by the overexpression of NF-κB signaling components. Nevertheless, Smad1/5 transactivation and, consequently, transcription of BMP target genes is greatly reduced upon activation of NF-κB signaling. Neither ectopic expression of Smad1/5, nor CBP/p300 can rescue the negative effect of NF-κB pathway activation. We used Real time PCR to analyse BMP and TNFα target genes mRNA induction in the presence of protein synthesis cycloheximide and found that negative effect of NF-κB activation requires new protein synthesis due to the induction of BMP signaling inhibitor expression. Futhermore, we found two distinct TNFα/NF-κB target genes (T/NTG) that are novel potent inhibitors of BMP signaling. Interestingly, overexpression of T/NTG1 induces apoptotic death of osteoblasts. shRNA-mediated knockdown of the expression of each T/NTG results in partial rescue of BMP-Smad-driven transcription from inhibition by TNFα. Thus, our data suggest these T/NTGs as possible central candidates responsible for the development of osteolysis and joint erosions during the RA. We are validating this current hypothesis in vivo using BMP reporter mice and ectopic bone formation in SCID mice models.

Disclosures: O. Korchynskyi, None.

W045

BK Channels in Human Osteoblast-like Cells - Properties and Function. B. Li*¹, N. C. Henney*¹, B. A. J. Evans², P. Reviriego*¹, A. K. Campbell*³, K. T. Wann*¹, ¹Welsh School of Pharmacy, Cardiff University, Cardiff, United Kingdom, ²Child Health, School of Medicine, Cardiff University, Cardiff, United Kingdom, ³Medical Biochemistry & Immunology, Cardiff University, Cardiff, United Kingdom.

Ion channels which are normally associated with excitable cells are also emerging players in the life and death decisions of many other cells. We have focussed on large conductance Ca²⁺-activated K⁺ channels (BK channels) in osteoblast-like cells. BK channels exist as a tetramer of 4 α subunits associated with β subunits, and the type of β subunit, modifies both the voltage-and Ca²⁺-sensitivity and the pharmacological fingerprint of BK channels¹. Here we attempt a pharmacology of the channel, test its role in proliferation and try to specify the subunit composition of BK channels in MG63 cells.

Single channel activity was measured using patch-clamp methods. Determination of numbers of osteoblast-like cells was achieved by use of the MTS assay. RT-PCR was carried out to investigate the expression of α (KCNMA) and β (KCNMB) subunits of the BK channel.

BK channels were observed in cell-attached patches at potentials close to rest. In excised outside-out patches external tetraethylammonium chloride (TEA, 500μM) reduced the open probability, producing reversible flickery block. Intriguingly, both BK-selective blockers, iberiotoxin (IbTX- 5-60nM) and tetrandrine (5-30μM) also blocked the channel in outside-out patches. Internal paxilline (10μM) blocked reversibly. The BK channel opener, isopimaric acid (10μM), also increased channel activity. RT-PCR showed that the α (KCNMA) subunit and the β1-4 (KCNMB1-4) subunits were present. Some blockers had a dual effect on cell numbers. Thus TEA and tetrandrine increased cell numbers at low (1mM and 3μM respectively), and reduced cell numbers at high concentration (>10mM and >10μM). In contrast, IbTX (10 – 300nM) was inactive.

These data are the first systematic description of the pharmacology of the BK channel in bone cells and it turns out to be novel. This has implications for both the subunit composition of the channel and its use in therapeutic intervention. The data also show that BK may have a role in modulating MG63 cell numbers and future investigations are focusing on characterising further the pharmacology of the channel and defining the native subunit composition, and the role of the channel in mineralization and secretion in osteoblast-like cells.

1. Wang YW, Ding JP, Xia XM, Lingle C.J. (2002) Consequences of the stoichiometry of Slo1 alpha and auxiliary beta subunits on functional properties of large-conductance Ca²⁺-activated K⁺ Channels. J Neurosci, 22(5): 1550-61.

Disclosures: B. Li, None.

W046

Application of Bioluminescence in Stem Cell-Based Bone Formation. K. E. de Rooii¹, G. van der Horst*¹, H. C. M. Sips*¹, I. Que*¹, L. J. A. van der Wee-Pals*¹, C. H. F. Bloys*¹, J. Hoogendam*², C. W. G. M. Löwik¹, M. Karperien¹, ¹Endocrinology, LUMC, Leiden, The Netherlands, ²Pediatrics, LUMC, Leiden, The Netherlands.

Current treatments for osteoporosis suppress bone resorption, thereby blocking further deterioration, but do not restore bone. Therefore, therapies stimulating bone formation would greatly enhance treatment of osteoporosis.

The bone forming osteoblasts originate from the multipotent mesenchymal stem cells (MSCs). MSCs have great potential for application in tissue engineering of bone and cartilage. However, differentiation of MSCs in vitro or in vivo as well as interactions with other cells or biomaterials in vivo is poorly understood. Therefore, we have created a stem cell model which, in combination with bioluminescent imaging, allows us to study these processes in intact mice over time.

The murine mesenchymal progenitor cell-like KS483 cell line was genetically modified enabling efficient generation of isogenic stable cell clones by Flp-mediated recombination by integrating 1 copy of a FRT-target site into the cell's genome. After we had tested the efficacy of the FRT-site by the overexpression or knock-down of the RunX2 gene, it was used to insert a luciferase2 gene containing a His tag enabling us to follow cell fate in vivo by bioluminescence after implantation in nude mice and the detection of cells ex vivo by immunohistochemistry using antibodies against the His tag. KSfrt HisLuc2 cells were used to perform a bone marrow ablation assay in which the cells were injected in the marrow cavity. Cell fate was followed by non-invasive bioluminescent imaging. At several time points, mice were sacrificed and both tibias were isolated for analysis. Using an antibody against the His tag, we could identify KSFrt HisLuc2 cells in the callus which was formed following bone marrow ablation. In addition, to study their fate outside the bone forming environment and to study possible adverse effects, cells were also transplanted subcutaneously. We have followed cell fate non-invasively for 20 weeks and afterwards isolated the luciferase expressing tissue for immunohistochemical analysis.

Finally, we used the genomic FRT site for the insertion of gene reporter constructs, which respond to Wnt- or BMP-signaling. Isogenic stable cell lines were obtained that were highly sensitive for Wnt and BMP, respectively, and retained multipotency. In conclusion, genetically modified KSfrt cells provide a simple and fast model to study MSC function. In combination with bioluminescent imaging, we will use this model to evaluate the effects of biomaterials on stem cell function in vitro and in vivo as a first step towards a bone replacement therapy for osteoporosis.

Disclosures: C.W.G.M. Löwik, None.

This study received funding from: Dutch Program for Tissue Engineering (DPTE).

W047

The LIM Protein, LIMD1, Modulates Osteoblast Differentiation and Function. H. F. Luderer¹, S. Bai*², G. Longmore*³, ¹Cell Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA, ²Pathology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA, ³Cell Biology and Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, USA.

Recent studies have shown that LIMD1 contributes to osteoclast (OC) differentiation in vivo, suggesting LIMD1 may be involved in maintaining normal bone homeostasis. To test this hypothesis, bone density measurements of Wild Type (Wt) and Limd1-/- mice were compared. In addition, the capability of primary calvarial cells isolated from Limd1-/- and Wt mice to differentiate, mineralize, and regulate OC differentiation was analyzed to determine if LIMD1 affected osteoblast (OB) function. While Limd1-/- mice lack any gross skeletal abnormalities at steady state, defects in both differentiation and function of primary calvarial cells in culture were found. Limd1-/- calvarial cells display increased mineralization and accelerated differentiation compared to Wt controls despite equivalent rates of proliferation. More specifically, RT-PCR analysis of differentiating calvarial cells demonstrated that Runx2 regulated genes are expressed earlier in Limd1-/- calvarial cells compared to Wt. Intriguingly, LIMD1 interacts with Runx2 by co-immunoprecipitation, suggesting a potential mechanism explaining the observed defects. Furthermore, RT-PCR analysis showed decreased expression of RANKL, a potent inducer of OC differentiation, and increased expression of osteoprotegrin, the inhibitor of RANKL, in Limd1-/- calvarial cells. In agreement with this observation, when co-cultured with Wt primary bone marrow derived macrophages, Limd1-/- calvarial cells supported differentiation of fewer OCs than Wt controls. Since LIMD1 was clearly important for OB differentiation in vitro, mice were treated with an anabolic stimulus to determine if LIMD1 contributed to stress response. Wt and Limd1-/- mice were treated daily with parathyroid hormone (PTH) or vehicle for four weeks. Analysis by both micro-CT and histomorphometry showed no differences in bone density or bone growth between PTH treated groups. Interestingly, Limd1-/- PTH treated mice exhibited increased OB numbers compared to Wt. To determine if this was due to differing osteoprogenitor numbers, total marrow flushed from the long bones of Wt and Limd1-/- mice was cultured under normal, adipogenic or osteogenic conditions. Interestingly, loss of Limd1 resulted in an increased number of osteoprogenitor cells while the number of adipogenic and fibroblastic colonies was unchanged. In conclusion, LIMD1 affects OB differentiation, OB function and contributes to maintaining mesenchymal stem cell lineages.

Disclosures: H.F. Luderer, None.

W048

Local Transplantation of G-CSF Mobilized CD34-positive Cells Contributes to Fracture Healing. Y. Mifune¹, T. Matsumoto², A. Kawamoto*¹, R. Kuroda*², T. Shoji*¹, M. Miwa*², K. Masahiro*², T. Asahara*¹, ¹Stem Cell Translational Research, Kobe Institute of Biomedical Research and Inovation / RIKEN Center for Developmental Biology, Kobe, Japan, ²Department of Orthopedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.

We recently reported that human peripheral blood (PB) CD34+ cells, an endothelial/hematopoietic progenitor enriched cell population, contribute to fracture healing via vasculogenesis and osteogenesis in intravenous transplantation (IT) method. In addition to a confirmation of human PB CD34+ cell differentiation into osteoblasts (OBs) in vitro, we performed next experiments to prove a hypothesis that granulocyte colony stimulating factor mobilized peripheral blood (GM-PB) CD34+ cells, transplanted locally to the non-healing fracture model of immunodeficient rat, dose-dependently contribute to fracture healing. In vitro study, GM-PB CD34+ cells proved their capacity to differentiate into OBs by alizalin red staining and RT-PCR. In vivo study, as a proof of a therapeutic superiority of local transplantation (LT) to IT, the immunostaining for HLA-ABC at the peri-fracture site showed the significantly massive recruitment of the human cells in rats receiving LT compared to IT. RT-PCR and immunohistochemistry demonstrated significantly higher expression of human specific markers for endothelial cells and OBs in animals receiving high (10⁵) compared to middle (10⁴) and low (10³) dose of CD34+ cells and PBS at week 2. As enhancement of intrinsic vascularization and osteogenesis at the peri-fracture site, capillary density was significantly greater in high group compared with the other groups, as well as in middle group than low and PBS groups, and OB density was significantly greater in high group compared with the other groups. The Laser Doppler Perfusion Imager estimating a blood flow at the fracture site indicated the high and middle groups had a significantly higher perfusion value than the other groups at week 1 and 2. Radiograpphic assessment showed all of the high group and half the middle group obtained union at week 8, while all fractures in the other groups failed to union. The degree of fracture healing assessed by Allen's histological classification was significantly higher in high group than the other groups at week 8, as well as middle group than low and PBS groups (high, 3.8±0.13; middle, 2.1±0.16; low, 0.4±0.48; PBS, 0.0±0.00, respectively, P<0.01 for high vs other groups and middle vs low or PBS group). In this study, we suggested GM-PB CD34+ cells have a potential for vasculogenesis and osteogenesis with fracture healing, and more than 1×10⁴ CD34+ cells per rat were necessary and effective for fracture healing in LT method. These findings must be a help to close up to the feasibility for clinical application.

Disclosures: Y. Mifune, None.

W049

In Vitro and In Vivo Effects of sFRP3 Overexpressed by Phi 31 Integrase Strategy. D. Han*¹, S. Park*¹, S. Kim*¹, Y. Kim², Y. Rhee*¹, Y. Chung*³, S. Lim¹, ¹Internal Medicine, Yonsei University, Seoul, Republic of Korea, ²Internal Medicine, NHIC Ilsan Hospital, Seoul, Republic of Korea, ³Internal Medicine, Ajou University, Seoul, Republic of Korea.

Wnt signaling increases bone mass through a number of mechanism. Secreted frizzled related proteins (sFRPs) do not have transmembrane and cytosolic domains, thus they bind and act as decoy receptors for Wnt proteins and antagonize the Wnt/frizzled signaling pathway. However, previous studies reported that sFRP3 decreased osteoblast proliferation and increased osteoblast differentiation. Because Tg or KO models regulating Wnt signaling may alter bone development, It is more desirable to evaluate the effects of sFRP3 in skeletally mature animals with pharmacological activators or inhibitors. In this study, we overexpressed sFRP3 by Phi 31 integrase strategy in 7-20 weeks old ICR mouse (male) to demonstrate the effects of sFRP3 in bone. sFRP3 expression in liver was documented by PCR and confocal microscopy analysis. Serum sFRP3 was detected at ∼ ug range by ELISA. In sFRP3 overexpressed mice, femur BMD measured by PIXImus was increased. Both BV/TV and Trabecular number measured by μCT were also increased, and Tb.Sp (trabecular separation) was decreased. Furthermore, three point bending test revealed increased load max and slope of femurs. To clarify the mechanism of sFRP3 induced positive effects on bone, sFRP3 was overexpressed in MC3T3E1 preosteoblast cells. Overexpression of sFRP3 reduced Wnt3a mediated proliferation, and real time PCR analysis revealed that colla 1a1 expression was increased. In addition, the luciferase assay presented that Wnt-responsive gene activation were increased in sFRP3 treated group. Based on our findings, we propose that constitutive overexpression of sFRP3 increases BMD and bone strength. Furthermore, continuous over activation of Wnt signal might not be beneficial to bone, and coordinated regulated activation of Wnt signal should be necessary for optimal bone remodeling and bone strength.

Disclosures: D. Han, None.

W050

Osteocyte Specific DMP1 and MEPE Expression after Mechanical Stimulation in the Mouse Ulnae Loading Model: Correlation to Strain Fields, Dynamic Bone Formation, and Potential Role in Bone Mineral Quality. S. E. Harris¹, J. Gluhak-Heinrich*², M. A. Harris¹, W. Yang¹, L. F. Bonewald³, D. Riha*⁴, D. P. Nicolella⁴, P. S. N. Rowe⁵, A. G. Robling*⁶, C. H. Turner⁶, ¹Periodontics, U, Texas Health Sci Ctr San Antonio, San Antonio, TX, USA, ²Orthodontics, U, Texas Health Sci Ctr San Antonio, San Antonio, TX, USA, ³Oral Biology, U, of Missouri at Kansas City, Kansas City, MO, USA, ⁴Southwest Research Institute, San Antonio, TX, USA, ⁵Periodontics, Kansas University Medical Center, Kansas City, KS, USA, ⁶Indiana University School of Medicine, Indianapolis, IN, USA.

DMP1 and MEPE are involved in local effects on bone mineralization as well as systemic phosphate metabolism. These two genes are highly expressed in osteocytes and suggest that the osteocyte can alter its local environment as well as act as an endocrine cell. We previously have shown preliminary data that DMP1 and MEPE increased at 24hrs after a single 2.4N load for 30 sec at 2 Hz. We now show a direct highly statistically significant correlation between gene expression and strain field determinations from finite element analysis. This correlation shows osteocyte specific DMP1 and MEPE expression is highest in areas of maximal compressive and tensile strains in cortical bone. DMP1 is an early response gene and its expression is first stimulated at 1hr after loading and continues to increase up to 24hrs. Load induced MEPE expression is a late response gene and does not increase with load until 24hrs. Using a 3D model relating the 3 dimensional strain field with both DMP1 and MEPE expression, the gene expression threshold (Get) for both genes is approximately 1500 μstrain. MEPE protein is cleaved by a cathepsin like protease to a highly phosphorylated C-terminal ASARM peptide. We know that overproduction of MEPE and the ASARM peptide are associated with hypophosphotemia and osteomalacia. DMP1 affects local mineralization by proper maintenance of canaliculi and lacunar walls and, if deleted, may result in the osteocyte being overstimulated. As we previously showed, MEPE and the ASARM peptide are overexpressed in the DMP1 knock-out model, supporting that osteocytes in this model are experiencing higher local strain than control mice under normal cage conditions. The normal role of MEPE and its response to load may be related to the capacity of MEPE and the ASARM peptide to alter local mineral to matrix properties. 

Figure Figure 1.. Correlation of Load Induced MEPE Expression(24hrs) with maximum surface Compressive Strain Determined by Finite Element Model. MS is the midshaft and d1 to d5 are 1mm Intervals distal to MS and p1 and p2 are 1mm Intervals proximal to midshaft

Disclosures: S.E. Harris, None. This study received funding from: NIH.2

W051

Cell-Cell Communication in Living Bone and Its Regulation Factor. Y. Ishihara¹, H. Kamioka*¹, T. Takano-Yamamoto², T. Yamashiro*¹, ¹Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan, ²Division of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate School of Dentistry, Sendai, Japan.

Intercellular network among osteocytes consists of gap junctions. Gap junctional intercellular communication (GJIC) is thought to play an important role in integration and synchronization in bone remodeling. We hypothesized that extracellular pH (pH_o) and extracellular calcium ion ([Ca²⁺]_e), which were dynamically changed by osteoclast while bone remodeling, would affect GJIC among osteocytes. GJIC among osteocytes in chick calvaria was analyzed by using Fluorescence Replacement After Photobleaching (FRAP) analysis. Simultaneously, we examined the effect of PTH on GJIC in osteocytes. Anti-Connexin43 (Cx43) immunolabelling was used to identify gap junction localization in chick calvaria. FRAP analysis was performed to osteocytes in chick calvariae which were exposed to 5.0 μM Calcein-AM. We assessed subsequent recovery of fluorescence intensity into the photobleached cell by the fluorescence replacement from surrounding cells. In each experiment, bone fragments were preculrured with conditioned medium before FRAP analysis. Cx43 immunoreactivity was detected in most of osteocyte processes without any specific distribution. FRAP analysis showed dye-coupling among osteocytes in chick calvariae. Fluorescence intensity recovered to 43.7 ± 2.2% within 5 min after photobleaching. Pretreatment of 18 α-GA, a reversible inhibitor of GJIC, significantly decreased replacement of the fluorescence to 10.7 ±2.2% in a bleached osteocyte. When pHo was decreased from 7.4 to 6.9, replacement of the fluorescence in a bleached osteocyte was significantly decreased from 43.3 ± 2.9% to 19.7 ± 2.3%. Conversely when pH_o was increased from 7.4 to 8.0, it was significantly increased up to 61.9 ± 4.5%. When [Ca²⁺] was altered from 1 mM up to 25 mM, replacement of the fluorescence in a bleached osteocyte was significantly decreased from 47.0 ± 6.1% to 16.1 ± 2.1%. In addition, when bone fragments were exposed from 1 nM to 10 nMrPTH, replacement of the fluorescence in a bleached osteocyte was significantly increased up to 60.7 ± 7.2% after 3 h. These results indicated GJIC among osteocytes in bone are regulated by extracellular environment and hormonal stimulation in bone remodeling.

Disclosures: Y. Ishihara, None.

W052

Greater Sensitivity of Osteocytes to Shear Stress as Compared to Osteoblasts: PGE₂ Production and Wnt/β-catenin Signaling. M. L. Johnson, M. A. Kamel, N. A. Kim-Weroha*, B. Holladay*, S. Kotha.. Oral Biology, UMKC School of Dentistry, Kansas City, MO, USA.

The osteocyte is widely held to be the primary cell in bone responsible for sensing mechanical loads due to its location in bone and it has been shown that primary osteocytes are more sensitive to shear stress than primary osteoblasts. Ironically, the majority of in vitro studies examining mechanoresponsiveness have used primary osteoblast cultures and/or osteoblastic cell lines as surrogates for osteocytes. We compared the sensitivity of osteocyte MLO-Y4 cells to osteoblast 2T3 cells using fluid flow at 2 and 16 dynes/cm². Media was sampled at 5, 15, 30, 60 and 120 minutes for PGE₂ production and at 120 min for nuclear translocation of β-catenin. At 2 dynes/cm², PGE₂ was not detectable in the media from the 2T3 cells (<10 pg/ml). At 16 dynes/cm², PGE₂ was first detectable 15 min after initiation of fluid flow and increased to 90 pg/ml by 2 hours. In contrast, at 2 dynes/cm², MLO-Y4 cells produced 450 pg/ml PGE₂ by 5 minutes which continued to increase for the 2 hours of shear stress to a maximum of 1400 pg/ml. At 16 dynes/cm², PGE₂ production was 500 pg/ml at 5 minutes and reached >2000 pg/ml at 2 hours. At 2 dynes/cm², β-catenin nuclear translocation was detected by immunostaining in MLO-Y4 cells, but not in 2T3 cells. At 16 dynes/cm² both cell lines showed nuclear translocation. We further examined the sensitivity of osteocytes versus osteoblasts in vivo using ulna loading of the TOPGAL mouse. Nuclear translocation of β-catenin, assayed by lacZ activity, was initially observed in osteocytes within bone at 1 hour post load but not until 24 hrs in cells on the bone surface. At 4 hours post loading immunostaining for sclerostin, which binds to Lrp5 and acts as an inhibitor of β-catenin signaling; demonstrated high levels of sclerostin in most osteocytes, except those with active β-catenin signaling, implying a reciprocal relationship. We hypothesize that this initial activation of β-catenin signaling is PGE₂ dependent and thereby able to bypass the inhibitory effects of sclerostin on the Lrp5 co-receptor that regulates Wnt/β-catenin signaling. These observations support the hypothesis that osteocytes are first cells in bone to sense and respond to mechanical load.

Disclosures: M.L. Johnson, None.

W053

Fluid Flow Shear Stress and Prostaglandin E2 Activates β-catenin Signaling in MLO-Y4 Osteocytic and 2T3 Osteoblastic Cells. M. A. Kamel, Y. Kitase, N. A. Kim-Weroha*, B. Holladay*, S. Kotha, L. F. Bonewald, M. L. Johnson.. Oral Biology, UMKC School of Dentistry, Kansas City, MO, USA.

The low-density lipoprotein receptor-related protein 5 (Lrp5) and the Wnt/β-catenin signaling pathway that it regulates are known to be required for new bone formation in response to mechanical loading. Prostaglandins are also known to be rapidly released from bone cells in response to mechanical loading. We have proposed a model in which Prostaglandin E₂ (PGE₂) released by osteocytes and/or osteoblasts, in response to mechanical loading activates β-catenin signaling through a mechanism that is independent of Lrp5 co-receptor regulation of the Wnt/β-catenin pathway. This model accounts for the activation of β-catenin signaling in the presence of inhibitory proteins such as sclerostin that is produced by osteocytes. We hypothesized that this activation occurs through a PGE₂ mediated inhibition of Glycogen Synthase Kinase-3β (GSK-3β), a key regulator of intracellular levels of β-catenin. To test this hypothesis we treated MLO-Y4 osteocytic and 2T3 osteoblastic with 5 × 10⁻⁶ M PGE₂ for 2 hours. We observed a significant increase in phosphorylation of GSK-3β as determined by western blot compared to untreated cells. Interestingly we also observed parallel changes in GSK-3α phosphorylation, which can also regulate intracellular β-catenin levels. Immunostaining of PGE₂ treated cells demonstrated nuclear accumulation of β-catenin compared to non-treated cells. 2T3 osteoblastic and MLO-Y4 osteocytic cells were subjected to 16 dynes/cm² of fluid flow for 2 hours. Fluid flow significantly increased the phosphorylation of GSK-3β and α, and increased nuclear translocation of β-catenin. These data support our hypothesis that PGE₂ released in response to mechanical loading/fluid flow shear stress leads to inhibition of GSK-3β and nuclear translocation of β-catenin. Our data also suggests a possible, previously unsuspected role for GSK-3α in the regulation of β-catenin signaling in bone cells. We propose that this initial activation of β-catenin signaling leads to changes in key target genes, such as sclerostin and Wnts creating a permissive environment for further amplification of the Wnt/β-catenin signaling at the level of Lrp5. Loss of Lrp5 prevents this amplification step, which is required for new bone formation.

Disclosures: M.A. Kamel, None.

W054

The Effects of Mechanical Strain on Osteoytes - Induced Bone Cell Proliferation and Recruitment. S. Ko, J. Lee*, S. Kim*., Dept. of Pharmacology & Dental Therapeutics, Kangnung National University, Gangneung, Republic of Korea.

There are several evidences that osteocyte plays a critical role in bone remodeling. Healthy or apoptotic osteocytes can send signals to other bone surface cells like osteoblasts, osteoclasts, osteoclast precursors and bone lining cells through their networking in canaliculi. And osteocyte which responds to mechanical strain may send signal to other cells. Therefore, to determine the role for osteocytes and mechanical strain in bone remodeling, we examined the effect of mechanical strain and apoptosis of osteocytes on osteoclast precursors and osteoblasts proliferation and recruitment. We used the MLO-Y4 cells as in vitro model for osteocytes, RAW 264.7 cells and MOCP-5 cells as osteoclast precursor, and 2T3 cells as osteoblasts. MLO-Y4 cells conditioned medium (Y4-CM) was collected after 24h culture. For fluid flow experiments, MLO-Y4 cells were exposed to 2 hrs of plusatile fluid flow(PFF) at 2, 4, 8, 16 ± 0.6 dynes/cm² using Flexcell Streamer™ system. For induction of MLO-Y4 cell apoptosis, MLO-Y4 cells were treated with 100 μM etoposide and apoptosis was determined with increase of casapase-3 activity and ratio of trypan blue straining cells. We did proliferation assay of RAW 264.7 and 2T3 cells with control media or 10% Y4-CM at specific time. The migration of RAW 264.7 and 2T3 cells was assayed using transwells with control media or 10, 20, 50, 100% Y4-CM. MLO-Y4-CM increased osteoclast precusor proliferation and migration. Y4-CM decreased 2T3 cell proliferation and migration. After MLO-Y4 cells were exposed to PFF, Y4-CM decreased RAW 267.4 cell proliferation, migration and 2T3 migration compared to control CM (Y4-CM without strain). However Y4-CM exposed PFF had no effect on 2T3 osteoblastic cell proliferation. Apoptotic Y4-CM induced by etoposide increased the proliferation of 2T3 cells compared to control Y4-CM.

These results suggest that osteocytes can regulate the bone remodeling by communication with osteoclast precursors and osteoblasts and that osteocytes can communicate mechanical signals and their apoptosis to other cells.

Disclosures: S. Ko, None. This study received funding from: Korean Research Foundation Grant funded by the Korean Government (R04-2004-000-10146-0).

W055

Ex Vivo Model of Stress Shielding and Osteocyte Viability in Human Trabecular Bone. H. Kroger¹, V. Mann², B. Noble², ¹MTEC and Dept. of Orthopaedics, University of Edinburgh and University of Kuopio, Kuopio, Finland, ²MTEC, University of Edinburgh, Edinburgh, United Kingdom.

Stress shielding associated periprosthetic bone loss is common after total hip and knee arthroplasty (THA, TKA). However the cell/molecular mechanism that drives this bone loss is unknown. Osteocyte death has been hypothesised to initiate localised bone remodelling. Here we test the hypothesis that mechanical unloading of specific regions of human trabecular bone will engender osteocyte death in a site and mechanical strain specific manner.

Excised femoral head material was obtained from an individual patient undergoing THA. Trabecular bone cores 10mm diameter were prepared plan parallel to a height of 5mm (+/-2μm) within 2 hours post surgery under sterile conditions. Regional stress shielding (SS) was accomplished in bone cores (n = 3) through the removal of a portion of load-transmitting bone, such that mechanical stimulation below this area was disrupted. The bone cores were then randomly assigned into 4 groups: Time Zero (T0, immediately flash frozen n = 4), loaded control (LC, n = 3), unloaded control (UC, n = 3) and stress shielded (SS n = 3). These samples were maintained within a bioreactor system for 7 days under conditions of either no mechanical stimulation (UC) or mechanically stimulated (LC, SS). Mechanical stimulation was carried out for 5 minutes on a daily basis using a maximum of 3000 μstrain in a waveform corresponding to physiological jumping exercise. Osteocyte viability was analysed in unfixed/undecalcified bone sections using in situ histochemical analysis of Lactate Dehydrogenase (LDH) activity. Total cell number was determined using DAPI nuclear stain. The results are expressed as the ratio of LDH positive osteocytes to total number of osteocytes corrected for bone area (Viability Index). In the SS samples the viability index was determined separately in the region of bone which experienced mechanical loading (SSLR) and the unloaded region (SSUR) within the same sample.

The viability index of bone maintained in the bioreactor in the absence of mechanical stimulation decreased relative to T0 samples (1.54 UC vs.7.47 T0, p<0.001). Application of mechanical stimulation increased viability relative to unloaded samples (3.10 LC vs 1.54 UC, p<0.001). In the stress shielded samples the viability was reduced in the stress shielded region relative to the loaded region within the same sample (0.96 SSUR vs. 2.74 SSLR, p<0.001). There was no significant difference between the loaded region in the stress shielded sample and the loaded control (2.74 SSLR vs. 3.10 LC, p = 0.857).

In conclusion unloading is associated with a decrease in osteocyte viability. Decreased osteocyte viability may play a significant role in stress shielding induced bone resorption and bone loss.

Disclosures: H. Kroger, None.

W056

Bone Biomarker Responses to Short-Term Training: Influence of Exercise Mode vs. Osteogenic Index. M. E. Lester*¹, R. K. Evans*¹, W. J. Kraemer*², J. S. Staab*¹, J. R. Pierce*¹, D. E. Catrambone*¹, H. M. Isome*¹, M. L. Urso*¹, B. A. Spiering*², D. L. Hatfield*², C. M. Maresh*², B. C. Nindl*¹, ¹Military Performance Division, US Army Research Institute of Environmental Medicine, Natick, MA, USA, ²Dept of Kinesiology, University of Connecticut, Storrs, CT, USA.

The value of monitoring bone turnover biomarkers during short-term exercise training is not established. Further, it is not clear whether these biomarkers are influenced by either exercise mode or the osteogenic index (OI = I * LN(N+1)), where I = mean weekly intensity of activity in bodymass units, and N = number of loading cycles per wk (Robbling and Turner, ESSR 2003). This study examined the influence of three, 9-wk exercise regimens (exercise performed 3 d/wk) on bone turnover responses. Fifty-seven women (21.7 yr) were randomly assigned to a control (CON), aerobic (AER), resistance (RES), or combined aerobic and resistance (COMB) training group. Serum markers of bone formation [BAP and Osteocalcin (OC)] and bone resorption (TRAP5b and CTx) were assessed after 0, 4, and 9 weeks of the exercise training via immunoassays. Changes were analyzed using a 4×3 repeated measures ANOVA with a Fisher's LSD post-hoc where appropriate, and significance was accepted at P ≤ 0.05. The average weekly OI for the groups were: RES group = 14.1, AER = 22.2, and COMB = 40.1. Results for BAP and OC appear below: 

Table  .  

Values are mean±SE; *P< 0.05 vs. week 0; ‡P< 0.05 vs. week 6; #P<0.05 vs. CON group For BAP, interaction effects revealed increases at 4 weeks for all exercise groups, while further increases at 9 weeks were only evident for the RES and COMB groups. For OC concentrations, only RES and COMB increased over the training period. There were no significant changes in CTx during training; however there was a significant decrease in TRAP5b over time (wk 0: 3.7±0.1 > wk4: 3.4±0.1 = wk9: 3.3±0.1 U/L). The greatest increase in biomarkers of bone formation was observed for the RES group, which had the lowest OI. Our results demonstrate 1) biomarkers of bone formation are more responsive to short-term exercise training than are biomarkers of bone resporption, 2) exercise modes that incorporate resistance training increase biomarkers of bone formation independent of OI. While the OI was developed and validated upon structural increases in bone strength, our data indicate that exercise programs with higher OIs do not necessarily contribute to a biochemical milieu reflective of increased bone formation.

Disclosures: M.E. Lester, None.

W057

Nanoprobing Osteocytic Subcellular Compartments by Surface-Enhanced Raman Spectroscopy. I. Pavel*¹, M. Mahmood*¹, K. Vyas*², M. Whitlow*¹, L. I. Plotkin², S. C. Manolagas², A. S. Biris*¹, T. Bellido². ¹Nanotechnology Center / Applied Science Department, Univ Arkansas at Little Rock, Little Rock, AR, USA, ²Center for Osteoporosis and Metabolic Bone Diseases, Univ Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA.

The optical fields provided by single nanoparticles (NPs) or NP aggregates of noble metals can enhance by up to 15 orders of magnitude the Raman spectroscopic signals of neighboring molecules. This technique, called surface-enhanced Raman spectroscopy (SERS), was used herein to study the molecular composition of the nanometer-scaled environment of NPs delivered into living MLO-Y4 osteocytic cells. Gold or silver NPs of 10-20 nm diameter were prepared by citrate- or borohydride-mediated reduction of chloroauric acid or silver nitrate, respectively. Cells were exposed to growing medium without or containing NPs (10⁻¹² or 0.5×10⁻⁹M) for 5 min to 12 h. Medium containing NPs was then removed; and cells cultured in fresh medium for a total of 12 h for all conditions and fixed. NP uptake was followed by light microscopy and their subcellular localization was visualized by transmission election microscopy. NPs were detected in different subcellular compartments depending on their concentration and time of exposure. In cells exposed to 10⁻¹² M, NPs were detected only in the cytoplasm irrespective of the time of exposure. On the other hand, in cells exposed to 0.5×10⁻⁹ M, NPs were found near the plasma membrane and in the cytoplasm after 10 to 30 min; and in cellular organelles and the nucleus after 12 h. SERS spectra were recorded from 1 μm diameter areas of different subcellular compartments using a confocal Raman microscope in a back-scattering geometry. A He-Ne laser of 632.8 nm wavelength and 2 mW power at the sample level was used for excitation. Acquisition time was 20 sec. No signal was detected in cells not exposed to NPs. Moreover, in cells incubated with NPs, SERS signals were detected only in areas near NPs or NP aggregates. The SERS spectra indicate that distinct chemical compositions characterize different subcellular compartments. Thus, spectra recorded from cytoplasmic areas exhibited mostly vibrational modes characteristic of proteins, lipids and carbohydrates; whereas, spectra obtained from nuclear, chromatin-rich areas revealed vibrational modes typical of DNA as well as proteins. To our knowledge, this is the first demonstration that NPs serve as nanosensors by providing SERS signatures of different subcellular compartments, including the nucleus, in bone cells. This approach might be used for detecting with high sensitivity subcellular changes associated with biological processes such as cell replication or apoptosis.

Disclosures: I. Pavel, None. This study received funding from: NIH and Arkansas Nanotechnology Center.

W058

Estimation of Osteocyte Density from Total Lacunar Population. S. Qiu., S. Palnitkar*, D. Rao.. Bone and Mineral, Henry Ford hospital, Detroit, MI, USA.

Many investigators estimate osteocyte density based on measurement of osteocyte lacunae. The prerequisite for this estimation is that the empty lacunae remaining after osteocyte death should disappear subsequently, leading to a decrease in total lacunae. There is evidence for such disappearance of empty lacunae. However, the relationship between osteocyte and total lacunar densities remains unclear. This study attempted to determine whether the decreased total lacunar density can be used to estimate the loss of osteocytes.

From our archived iliac biopsy sections, the cancellous bone in 136 white women (92 healthy and 44 with osteoporotic vertebral fracture) were examined. Ten unbroken areas were randomly selected in each section stained with Goldner's trichrome for determining the numbers of osteocytes (Ot.N), empty lacunae (EL.N) and total lacunae (Tt.L.N) per bone area. Table 1 shows the correlations of osteocyte density with densities of empty lacunae and total lacunae (Pearson test). The correlation between osteocyte and total lacunar densities was excellent in superficial, deep and whole bone (SB, DB and WB) in all subject groups (Table 1). The correlation between osteocyte and empty lacunar densities was significantly weaker in both fracture patients and normal women.

In conclusion, bone biopsy specimens contained well preserved osteocytes, so the iliac biopsy sections were used to estimate the relationship between osteocyte and total lacunar densities. The results indicate that, regardless of the location in bone, >80% variation in osteocyte density can be explained by total lacunar density in either normal subjects or patients with fragility fracture. Therefore, the decrease in total lacunar density can represent the loss of osteocyte, although the rate of osteocyte death will be underestimated due to the time lag needed for lacunar obliteration.

Table Table 1.. Correlation between Ot.N, EL.N and Tt.LN

A: 38 premenopausal women; B: 54 postmenopausal women;

C: 44 postmenopausal women with fragility vertebral fracture Data expressed as r² (p value)

Disclosures: S. Qiu, None.

W059

Histochemical Assessments on the Distribution of Osteocyte-Lacunar Canalicular System. U. Sobhan¹, M. Li², K. Oda*³, T. Maeda*⁴, R. Takagi*⁵, N. Amizuka², ¹Oral Maxillofacial Surgery 2, Niigata Universitty, Niigata, Japan, ²Center for Transdisciplinary Research, Niigata Universitty, Niigata, Japan, ³Biochemistry, Niigata Universitty, Niigata, Japan, ⁴Oral Anatomy, Niigata Universitty, Niigata, Japan, ⁵Oral maxillofacial surgery 2, Niigata Universitty, Niigata, Japan.

The purpose of this study is to histochemically evaluate the arrangement of osteocyte-lacunar canalicular system (OLCS) associated with bone remodeling, i.e., OLCS in sites of rapidly and slowly remodeled bone. Twelve-week-old female ICR mice were fixed with aldehyde solution, and the femora and tibiae were extracted. They were then decalcified with 10% EDTA solution prior to paraffin embedding. In order to envision the distribution of OLCS and the localization of osteoclasts and/or osteoblasts on regular paraffin sections, we performed double and triple stainings of silver impregnation (modified Schoen's method), alkaline phosphatase (ALP) immunohistochemistry and/or tartrate resistant acid phosphatase (TRAP) by Azo dye method. In addition, paraffin sections were also stained for detection of osteopontin or DMP-1 (dentin matrix protein-1) with silver impregnation. When analyzing the double and triple stainings, the bone matrix with ALP-positive plump osteoblasts, indicative of active form of osteoblasts, and TRAP-positive osteoclasts showed an irregularly arranged OLCS. In contrast, the region covered by ALP-positive bone lining cells, which are less active osteoblasts, demonstrated well arranged OLCS. Osteopontin-positive cement lines divided different bone matrices: one showed well-arranged lacunar canaliculi and the other figured haphazardly arranged canaliculi, thereby indicating the possibility that bone remodeling reconstructs the distribution of OLCS. Although DMP-1, specifically expressed in osteocytes, has been postulated to be involved with mechanotransduction in bone, DMP-1- immunoreactive osteocytes were unevenly distributed throughout the cortical bone that showed the regular arrangement of OLCS; the distribution of DMP-1 did not seem to be related to the distribution patterns of OLCS. Statistical analysis showed a higher number of canaliculi for each osteocyte in the areas that has been remodeled, compared to the area with ongoing remodeling. Our findings suggest that bone remodeling would reorganize the OLCS into regularly-arrangement.

Disclosures: U. Sobhan, None.

W060

Modulation of Site Specific Bone Adaptation by Signaling in Osteocytic Networks. B. J. Ausk*, T. S. Gross, S. Srinivasan. Orthopaedics, University of Washington, Seattle, WA, USA.

The osteocytic network in vivo is characterized by non-uniform cellular distributions and heterogeneity in cell-cell connectivity. While the mechanosensory role of osteocytic cells is broadly posited, the functional significance of the cellular network and its complex architecture remains opaque due to its current inaccessibility. In part to explore this process, we previously developed an agent based model (ABM) that simulates how real-time Ca²⁺ signaling induced in osteocytic networks by mechanical stimuli influences osteoid secretion by osteoblastic cells. The ABM accurately simulated whole tissue bone formation induced by cyclic (1-Hz) and 10-s rest-inserted loading protocols at 3 different strain magnitudes and at 2 different loading cycle numbers (error < 15%). Using this ABM, we examined the hypothesis that cell-cell signaling within the osteocytic network influences site-specific bone adaptation induced by mechanical stimuli. The osteocyte network topology was determined via imaging of thin sections obtained from the tibia midshaft of C57BL/6J mice (5 μm, n = 6, 16-wk) and the ABM was used to simulate focal osteoblastic activity around the mid-shaft for baseline loading protocols that were previously found to be osteogenic in vivo. We explored the general hypothesis by examining 3 conditions in which osteoblastic activity was: 1) linearly related to local tissue strains, 2) further modulated by strain related thresholds, or 3) further modulated by signaling within the underlying osteocytic network. We found that activation of osteoblastic cells solely by local tissue strains was poorly correlated with site specific adaptation observed in vivo (r² = 0.17 ± 0.04). Unexpectedly, when strain thresholds further modulated osteoblast activation, the relation was only minimally improved (r² = 0.23 ± 0.05, p = 0.3 vs strain magnitude only). Interestingly, when cell-cell signaling in the osteocytic network activated individual osteoblastic cells, the relation between ABM simulations and site specific osteoblastic activity observed in vivo was significantly improved (r² = 0.41 ± 0.06, p = 0.01 and 0.05 vs conditions 1, 2). This dramatic improvement in ABM predictions when cell-cell signaling is included (2.4 and 1.8 fold, respectively) suggests that interactions between osteoblasts and the osteocytic network are more important in influencing focal osteoblast activity compared with just the distribution of mechanical strains. This study, for the first time to our knowledge, begins to quantify the functional significance of the bone cell network in vivo. Given this relation, knowledge of the cellular topology within a bone is likely to be critical if osteoblastic activity is to be targeted to structural sites most in need of bone accretion.

Disclosures: S. Srinivasan, NIH 2. This study received funding from: NIH.

W061

The Elastic Modulus of Osteoblasts and Osteocytes. Y. Sugawara¹, H. Kamioka*¹, R. Ando*¹, Y. Ishihara¹, S. A. Murshid*², K. Hashimoto*³, N. Kataoka*⁴, K. Tsujioka*³, F. Kajiya*⁴, T. Yamashiro*¹, T. Takano-Yamamoto². ¹Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmacentical Sciences, Okayama, Japan, ²Department of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate School of Dentistry, Sendai, Japan, ³Department of Physiology, Kawasaki Medical School, Okayama, Japan, ⁴Department of Medical Engineering, Kawasaki Medical School, Okayama, Japan.

In general, growth, cell cycle progression, gene expression, and other cell behaviors are sensitive to changes in the mechanical force. Therefore, modulations of cellular mechanical properties exerted by mechanical force are intimately related to physiologically important processes. To understand the various functions of osteoblasts and osteocytes, a physical portrait of the mechanical properties of the osteoblasts and osteocytes is necessary. However, it has been difficult to study their mechanical properties because they are in mineralized tissue. In this study, we analyzed the elastic modulus of isolated osteoblasts and osteocytes. On the other hand, the cellular tension was created by the cytoskeletal components based on focal adhesion sites. Therefore, we also examine the relationship between focal adhesion and the elastic modulus. In order to isolate osteoblasts and osteocytes from embryonic chick calvariae, serial treatment was performed with collagenase type I and EDTA. The elastic modulus of living cells was analyzed with Atomic Force Microscopy. We analyzed the elastic modulus of living cells both on peripheral region of the cell body and nucleus region. Osteoblasts and osteocytes were identified by alkaline phosphatase substrate and by OB 7.3, which was a chick osteocyte-specific antibody, respectively. To examine the implication of focal adhesion formation on the elastic modulus, GRGDS, which is the inhibitor of focal adhesion, was pretreated in the culture and we analyzed elastic modulus of the cells. Elastic modulus of peripheral region was higher than that of nucleus region in both osteoblasts and osteocytes. Furthermore, the elastic modulus of peripheral and nucleus region in osteoblasts were higher than those in osteocytes. After treatment with GRGDS, the elastic modulus of osteoblasts was decreased. On the contrary, osteocyte didn't change their elastic modulus. In summary, the elastic modulus of osteoblasts was higher than that of osteocytes. Osteoblast elastic modulus might be influenced by the cytoskeleton based on cell adhesion.

Disclosures: Y. Sugawara, None.

W062

Effects of 3 Months of Aerobic Exercise on Bone Turnover in Premenopausal Non-obese Women. N. I. Williams*¹, J. J. Sanders*², M. De Souza³, ¹Kinesiology, Penn State University, University Park, PA, USA, ²Physiology, Penn State University, University Park, PA, USA, ³Physical Education and Health, University of Toronto, Toronto, ON, Canada.

Exercise is generally believed to represent an osteogenic stimulus, especially when high impact forces are experienced. Few studies have addressed whether moderate impact aerobic activity representing a significant cardiorespiratory stimulus can favorably impact bone turnover in premenopausal non-obese women. To begin to elucidate whether moderate impact aerobic activity (treadmill running, cycling, and elliptical) can alter bone turnover we assessed the effects of 3 months of aerobic exercise and controlled diet on bone formation (PINP) and bone resorption (serum NTX) in 17 premenopausal eumenorrheic women (age = 21 +/- 1 yrs; weight = 57.7 +/- 5.6 kg). Subjects were randomly assigned to either non-exercise (n = 10) or exercising (n = 7) groups. Food intake kcals in both groups was prescribed and adjusted throughout the intervention such that subjects maintained body weight. For both groups, dietary composition was 55% CHO, 15% PRO, and 30% FAT, and a multivitamin with calcium (40 mg) was provided. Supervised exercise training was 5 days per week 30-90 minutes at 70-85% of maximal heart rate. Daily urine samples for estrone-1-glucuronide (E1G) and pregnanediol glucuronide were collected during a baseline menstrual cycle and each day during the intervention. VO2max (ml/kg/min), increased in both groups, but the increase in exercising subjects was greater (ANOVA time effect P < 0.001; time × group P = 0.07); Non-exercise, Pre = 32.4 +/- 3.1, Post 36.9 +/- 5.1, Exercise, Pre = 36.5 +/- 4.2, Post = 44.3 +/- 4.7. A significant decrease in bone formation over time was observed when both groups were considered together (ANOVA time effect P = 0.004). Exercise produced a trend toward a greater decrease in bone formation (ANOVA time × group effect P = 0.099). Means +/- sd for PINP (μg/1) were non-exercise, Pre = 143 +/- 36, Post = 129 +/- 27; Exercise, Pre = 185 +/- 79, Post = 140 +/- 48. A significant overall increase in bone resorption was observed when both groups are considered (ANOVA time effect; P = 0.013), but no significant effect of exercise was revealed (ANOVA time × group; P = 0.484). Means +/- sd for NTX (nmol/1) were Non-exercise, Pre = 12.8 +/- 0.9, Post = 14.9 +/- 2.8; Exercise, Pre = 14.9 +/- 2.2, Post = 16.1 +/- 2.7). Overall decreases in bone formation, and increases in bone resorption are surprising and may be attributable to changes in diet or other factors. These data do not support favorable changes in bone turnover in response to moderate impact aerobic exercise in previously sedentary premenopausal non-obese women.

Disclosures: N.I. Williams, None.

This study received funding from: NIH-R01 HD 39245-01A1 and M01RR10732.

W063

Knockout of Cathepsin K in Adult Mice Does Not Result in Bone Fragility. S. J. Hoffman¹

V. Shen², X. Liang¹, C. A. Capriotti*¹, G. B. Stroup³, S. Kumar¹, ¹Musculoskeletal Diseases, GlaxoSmithKline, Collegeville, PA, USA, ²MDS Pharma Services, Bothell, WA, USA, ³GlaxoSmithKline, King of Prussia, PA, USA.

Cathepsin K (Cat K) plays an essential role in osteoclast-mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice as well as lack of functional enzyme in the human condition, pycnodysostosis, results in osteopetrosis. However, there is controversial data over the bone fragility of Cat K knockout mice. In the current study, histomorphometry as well as bone mass and strength of the mid-femur were evaluated in Cat K knockout (KO) and wild-type (WT) adult female mice.

WT and KO female mice were sacrificed at 4 and 6 months of age. The left femur was excised and underwent pQCT imaging as well as biomechanical testing of the mid-femur. Histomorphometric analysis was performed on the mid-femur of a different set of mice of the same age and genetic background.

KO mice had greater total and cortical area and moment of inertia (MI) of the mid-femur when compared to WT. In addition, total and cortical bone mineral content (BMC) and density (BMD) were greater for the KO mice at both ages. The increases in area measurements resulted from a combination of greater cortical thickness and periosteal surfaces in the KO mice. In the KO mice, cortical area, BMC, BMD, thickness and MI significantly increased from 4 to 6 months of age whereas these changes were not observed in the WT mice.

Histomorphometric analysis showed that the KO had greater total area and reduced marrow area which resulted in greater cortical area and width compared to WT mice. No difference in periosteal bone formation rate (BFR) was observed between mice but decreased with age for both genotypes. On the endosteal surface, BFR was increased and eroded perimeter decreased in the KO mice compared to WT at 6 months of age. Intracortical remodeling spaces were observed only in a small percentage of KO mice and very little woven bone was observed.

The bending test determined that maximum load and stiffness were increased 27% and 56%, respectively, in the 6 month old KO mice compared to the WT. Energy (work to failure) increased with age in both genotypes, however, no difference was detected between the KO and WT mice at either age. Post-yield displacement tended to decrease (39 and 24% reductions in the 4 and 6 month mice, respectively) in the KO compared to the WT mouse. When the size and geometry of the bone is taken into account, no differences in ultimate strength and elastic modulus between WT and KO were observed.

These data suggest that KO mice have different bone content and geometry than WT mice and it changes with age. These data suggest that the material present in bones of both genotypes is of similar quality although this may be due to different contributing factors.

Disclosures: S.J. Hoffman, GlaxoSmithKline 3.

W064

Conditional Inactivation of the Cathepsin K (Ctsk) Gene in Adult Mice Results in Osteopetrosis without Activation of Compensatory Mechanisms for Impaired Bone Resorption. R. Kiviranta¹, I. Koskivirta*¹, J. Morko*¹, J. Heikkinen*¹, E. Bergman*¹, S. L. Alatalo*², J. Risteli*³, E. Vuorio*¹, ¹Dept. of Medical Biochemistry and Molecular Biology, University of Turku, Turku, Finland, ²Dept. of Anatomy, University of Turku, Turku, Finland, ³Dept. of Clinical Chemistry, University of Oulu, Oulu, Finland.

Cathepsin K is the major protease responsible for matrix degradation in osteoclasts. We recently reported that Ctsk-deficient mice are osteopetrotic but retain some degree of bone turnover due to several compensatory mechanisms for the impaired bone resorption, including enhanced osteoclastogenesis and elevated expression of other proteases (MMP-9, MMP-13, MMP-14). The purpose of this study was to investigate the effects of inactivation of the Ctsk gene in adult mice and to determine whether the same compensatory mechanisms would arise when the gene is inactivated after development.

For this purpose, we generated mice harboring a modified Ctsk allele where exon 5 was flanked by loxP sites and transgenic mice with ubiquitous expression of tamoxifen-controlled Cre recombinase, which were crossed to produce mice homozygous for the floxed Ctsk allele and heterozygous for the tamoxifen-contolled Cre (CKLP^{loxP/loxPCre+}). Animals were treated with either tamoxifen or vehicle at the age of 2 months and given booster injections at 3 months. Samples were collected at the age of 4 months.

Northern and pQCT analyses verified that CKLP^loxP/loxP mice were normal. Treatment of CKLP^{loxP/loxPCre+} mice with tamoxifen for 7 days efficiently induced recombination in the Ctsk allele and thus production of truncated Ctsk mRNA. pQCT measurements revealed that inactivation of the Ctsk gene at the age of 2 months resulted in osteopetrosis by 4 months of age. The phenotype appeared more striking than in conventional Ctsk knockout mice. Furthermore, the tamoxifen-treated animals exhibited some features of pycnodysostosis, such as decreased bone size, that we did not observe in our conventional Ctsk knockout animals. Decreased levels of serum resorption markers CTX and ICTP verified the impaired bone resorption in the tamoxifen-treated animals. Serum TRACP activity was not changed, suggesting no significant change in osteoclast numbers. Molecular biological analyses of bone samples demonstrated decreased expression of MMP-14 and cathepsin L in tamoxifen-treated mice, but no changes in the expression of TRACP, MMP-9 or MMP-13, all of which were upregulated in our conventional knockout mice. In conclusion, inactivation of the Ctsk gene in adult mice results in ostepetrosis that appears more pronounced than in conventional Ctsk knockouts. This may be due to the lack of compensatory mechanisms observed in conventional Ctsk knockout mice.

Disclosures: R. Kiviranta, None.

W065

Regulation of Osteoclast Activity by Cathepsin K-Generated Type I Collagen Fragments. S. R. Wilson*¹, C. Peters*², P. Saftig*³, D. Bromme*¹, ¹Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada, ²Albert-Ludwigs-Universitaet Freiburg, Freiburg, Germany, ³Christian-Albrechts-Universitaet Kiel, Kiel, Germany.

Cathepsin K (catK) is responsible for the degradation of type 1 collagen in osteoclast mediated bone resorption. As collagen fragments are known to be biologically active, this study investigates their potential regulatory effect on mature murine osteoclasts.

Soluble type I collagen, type II collagen, and murine long bone powder were subject to degradation reactions by 200-400nM catK, L and 50nM MMP-1. Soluble collagen degradation products or a GRGDS peptide were added to isolated neonatal mature murine osteoclasts seeded on type I collagen substrate. Alternatively, the collagen I substrate was predigested with catK and L. Wild type, catK and catL deficient osteoclasts were also treated with cysteine proteinase inhibitor LHVS (5μM) and MMP inhibitor GM6001 (5μM). After 24h osteoclasts were stained with FITC-phalloidin and actin rings were counted as a percentage of total osteoclast number.

Mature murine osteoclasts seeded on type I collagen substrate treated with catK degraded type 1 collagen or bone demonstrated a lower percentage of actin rings. This was demonstrated in wild type, catK deficient and catL deficient osteoclasts. However, this was shown with neither undegraded collagen, nor collagen degraded by MMP-1 or catL. This inhibition could be partially abrogated by the presence of a vitronectin receptor blocking antibody. CatK deficient osteoclasts and wild type osteoclasts treated with LHVS were found to have a lower basal level of active osteoclasts compared to untreated wild type cells. The number of actin rings was increased by seeding catK deficient osteoclasts on catK pre-digested type 1 collagen but not catL pre-digested type I collagen. MMP inhibition had no effect on actin ring percentage on any cell type studied.

CatK is known to be the most efficient mammalian collagenase. These studies suggest it may release collagen fragments containing RGD sequences, which are not normally exposed. These soluble fragments may then interact with osteoclast integrin receptors disrupting the actin ring. This is a novel regulatory role for catK and collagen fragments in bone resorption.

Disclosures: S.R. Wilson, None.

W066

PIAS3 Modulates Osteoclastogenesis by Down-Regulation of NFATcl and OSCAR. K. Kim*, J. Lee*, J. Kim*, H. Jin*, N. Kim. Research Institute of Medical Sciences and Medical Research Center for Gene Regulation, Chonnam National University Medical School, Gwangju, Republic of Korea.

Protein inhibitor of activated STAT3 (PIAS3) has been shown to regulate the activity of various transcription factors. Here, we show that overexpression of PIAS3 in bone marrow-derived monocyte/macrophage lineage cells attenuates osteoclast formation and down-regulates the expression of NFATcl and osteoclast-associated receptor (OSCAR), which are important modulators in osteoclastogenesis. PIAS3 has been shown to associate with histone deacetylase 1 (HDAC1) as well as with transcription factors, including microphthalmia transcription factor, NFATcl, and c-Fos. Moreover, overexpression of PIAS3 inhibits transactivation of target genes such as NFATcl and OSCAR. This inhibitory effect of PIAS3 is possibly mediated by HDAC1 recruitment to the promoter regions of NFATcl and OSCAR. Furthermore, silencing of PIAS3 by RNA interference in osteoclast precursors enhances osteoclast formation as well as gene expression of NFATcl and OSCAR. Taken together, our results reveal that PIAS3 acts as a modulator in osteoclastogenesis

Disclosures: K. Kim, None.

W067

The POZ-Zn Transcriptional Regulator OCZF/LRF Is Induced by RANKL and Increases c-Fos Expression in Osteoclastogenesis. A. Kukita¹, T. Shobuike*¹, M. Asagiri*², H. Takayanagi², F. Pessler*³, K. Matsuo⁴, T. Kukita⁵, ¹Pathology and Biodefense Medicine, Saga University, Saga, Japan, ²Cell Signaling and COE program, Tokyo Medical and Dental University, Tokyo, Japan, ³Rheumatology Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA, ⁴Keio University School of Medicine, Tokyo, Japan, ⁵Oral Biological Sciences, Kyushu University, Fukuoka, Japan.

Osteoclast-derived zinc finger (OCZF, also known as ZBTB7, FBI-1, LRF and Pokemon) is a member of the BTB/POZ zinc finger family of transcriptional regulators that have been implicated in cell differentiation and oncogenesis. We have reported that OCZF is highly expressed in rat osteoclasts in vivo and plays a role in osteoclastonegesis in bone marrow culture. In the present study, we investigated the role of OCZF and its mouse homologue LRF in RANKL-induced osteoclastogenesis. Expression of LRF mRNA and protein was specifically induced by RANKL and M-CSF but not M-CSF alone in mouse bone marrow macrophages and the macrophage cell line, RAW-D. LRF protein was localized in the nuclei of osteoclast precursor cells and multinucleated osteoclasts. Targeted inhibition of LRF by small interfering RNAs markedly suppressed the formation of TRAP-positive multinucleated osteoclasts but not mononuclear osteoclast precursor cells in the differentiation from RAW-D cells. We then expressed OCZF under the control of the cathepsin K promoter (Ctsk-OCZF) in RAW-D cell. Expression of OCZF from the cathepsin K promoter did not induce osteoclast differentiation in the absence of RANKL, but stimulated RANKL-induced osteoclastogenesis. In addition, Ctsk-OCZF expression markedly increased the protein levels rather than mRNA levels of c-Fos and NFATcl in the presence of RANKL. Importantly, Ctsk-OCZF induced c-Fos protein in the absence of RANKL. Furthermore, immunofluorescence studies by using confocal microscopy demonstrated that LRF is co-localized with c-Fos in the nuclei of osteoclasts. Moreover, retroviral expression of OCZF did not rescue the osteoclastogenesis of c-Fos KO mice, while LRF protein levels were extremely low in the coculture of osteoblasts and spleen of c-Fos KO mice. Together, these data suggest that OCZF/LRF promotes osteoclastogenesis by positively cooperating with c-Fos protein in the presence of RANKL.

Disclosures: A. Kukita, None.

W068

Annexin VIII Is a Critical Bone Matrix-dependent Osteoclast Gene Transcriptionally Regulated by RANKL and NFATcl. K. P. McHugh¹, Z. Shen¹, R. P. O'Sullivan*¹, T. N. Crotti¹, M. R. Flannery*¹, S. R. Goldring², ¹Rheumatology, Beth Israel Deaconess Medical Center, Boston, MA, USA, ²Hospital for Special Surgery, New York, NY, USA.

Analysis of tissues from sites of bone erosion in rheumatoid arthritis (RA) and periprosthetic osteolysis revealed that cells expressing the full morphological and functional properties of mature osteoclasts (Ocs) are restricted to the immediate bone surface. Therefore, in addition to cytokines, we hypothesize that adhesion to bone matrix plays a role in determining the genetic profile and functional properties of fully differentiated resorbing Ocs.

Through expression profiling of mouse Oc cells differentiated on bone surfaces vs plastic, we have previously identified a cluster of bone matrix-dependent Oc genes, with annexin VIII (AnxA8) as a prominent member. The annexins are a family of calcium-dependent phospholipid binding proteins, some members of which bind F-actin. Annexin family members perform diverse cellular functions with many mediating vesicular trafficking and targeting. AnxA8 message was found, by QPCR, to increase temporally during Oc formation with kinetics similar to that of CTR and the beta-3 integrin. AnxA8 expression was confirmed at the protein level in bone-adherent Ocs in peri-implant tissues and at sites of bone erosions in RA. SiRNA-mediated gene knockdown in RAW264.7 cells was utilized to determine the function and requirement for AnxA8 in Ocs. Consistent with a key role in Oc formation, siRNA knockdown blocked Oc spreading and actin ring formation, and was shown to result in a >50% inhibition of resorption of mineralized matrix.

We have further characterized transcriptional regulation of the AnxA8 gene in Ocs by cloning the mouse AnxA8 promoter and generating deletion reporter constructs. The mouse AnxA8 promoter displays significant homology with its human homologue in a proximal region and in a separate region near −4kb. RANKL induced the AnxA8 promoter up to 10 fold following transient transfection into RAW264.7 cells. We next tested for NFATcl trans-activation since the AnxA8 promoter responds to RANKL and the evolutionarily conserved regions contain consensus NFAT binding sites. Consistent with an Oc-specific and Ca⁺⁺ regulated gene, we find that the mouse AnxA8 promoter is trans-activated by NFATcl. Using an in vitro model of bone-matrix-dependent Oc differentiation, we have identified a novel matrix-dependent and required Oc gene. We show here that the promoter region contains functional Oc regulatory elements. The AnxA8 gene plays a key role in Oc formation and we speculate that AnxA8 represents a potential new target for anti-Oc therapy in osteolytic and metabolic bone diseases.

Disclosures: K.P. McHugh, None.

This study received funding from: NIH: NIAMS.

W069

Myosin IIA Regulates Precursor Cell Fusion and Osteoclast Motility. B. K. McMichael¹, R. B. Wysolmerski*², B. S. Lee¹, ¹Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA, ²Neurobiology and Anatomy, West Virginia University, Morgantown, WV, USA.

Osteoclasts, large multinucleated cells formed by fusion of monocyte/macrophage precursors, differentiate by stimulation of precursors with macrophage colony stimulating factor (M-CSF) and receptor for activation of nuclear factor kappa B ligand (RANKL). M-CSF and RANKL stimulation leads to altered expression of many genes. In this work, expression of the nonmuscle myosin IIA (myoIIA) during osteoclastogenesis was examined, as was its role in regulating cell differentiation and osteoclast activity. Class II nonmuscle myosins are ATP-driven motors that contain a motor domain that binds actin and a tail that forms a coiled-coil structure. Myosin IIA has been implicated in many cellular functions including cytokinesis, maintenance of cortical tension, and cell sperading. However, its role in osteoclast function has not yet been well examined.

Our previous studies showed nonmuscle myoIIA to be distributed within podosomes and the actin ring of polarized osteoclasts, suggesting that myoIIA may play a role in osteoclast motility and bone resorption. During a normal seven day differentiation period of both mouse RAW 264.7 and primary marrow cells, myoIIA protein levels temporarily diminished on days 3-4, corresponding to the time at which fusion of osteoclasts precursors is initiated. During this same time course, myoIIA mRNA levels did not change indicating the loss of expression to be a post-transcriptionally mediated process. Pulse-chase analyses demonstrated that this temporary loss of myoIIA expression was due to increased degradation of the protein during days 3-4. The timing of this alteration in expression suggests a potential role for myoIIA in osteoclast differentiation. Therefore, RNA interference was used to suppress expression of the myoIIA heavy chain during the latter stages of osteoclastogenesis in both RAW 264.7 and mouse marrow cells. This suppression led to the generation of very large osteoclasts that were a result of increased precursor fusion, as demonstrated by elevated numbers of nuclei per cell. These large osteoclasts showed decreased cell motility in addition to a failure to reattach to substrate following dissociation. On bone, these cells formed either very large actin rings or multiple rings per cell. While the large cells were capable of resorbing bone to a similar extent as normal-sized controls, their diminished motility was apparent from a clustered patterning of resorption pits. These results suggest that nonmuscle myosin IIA is required during osteoclastogenesis to limit the extent of precursor fusion and to promote osteoclast motility.

Disclosures: B.K. McMichael, None.

W070

Diverging Pathways of Calmodulin Regulation of Osteoclastogenesis. K. J. Micoli, A. Zayzafoon*, S. Singhal*, J. M. McDonald. Pathology, UAB, Birmingham, AL, USA.

Osteoporosis results from an imbalance in bone remodeling that favors resorption, and increased osteoclastogenesis is one mechanism important in development of low bone mass.

The purpose of these studies was to determine the role of calmodulin (CaM) in regulating osteoclastogenesis via calcineurin (CaN)/NFAT and CaMKII. The studies utilized in vitro differentiation of RAW 264.7 cells, Western blotting, TRAcP staining, IF, CaN and CaMKII assays, PCR, and siRNA or shRNA transfection to determine the CaM/CaMKII-dependent events in the RANK pathway.

RANKL treatment of RAW 264.7 cells induces rapid phosphorylation of numerous signaling molecules, including ERK, JNK, p38, and Akt, and upregulation of c-fos. Inhibition of CaMKII with 5 μM KN93 prevented RANKL-induced phosphorylation of ERK, JNK and Akt, c-fos upregulation, and blocked osteoclast formation without preventing NFAT nuclear translocation. CaMKII is a complex holoenzyme, composed of α, β, γ and δ isoforms, all of which are expressed in RAW cells during differentiation. RAW cells expressing CaMKIIα shRNA did not form osteoclasts in response to RANKL treatment, and siRNA constructs against β, γ and δ also resulted in marked inhibition of osteoclastogenesis. Interestingly, RAW cells were also found to express a non-kinase form of CaMKIIα, αKAP, which is upregulated during osteoclastogenesis and may play a role in redistributing CaMKII during later stages of differentiation. We show that CaMKII activity increases rapidly (peaking at 10-15 min) following RANKL treatment, and peaks again after 72 h, consistent with a role in regulating both early and late RANK signaling. RANKL is known to induce nuclear translocation of NFAT, presumably through activation of calcineurin. Our data show that, in fact, RANKL activation leads to an approximate two-fold increase in CaN activity after 10-15 min, but activity drops to 50% of basal after 72h, which is when peak nuclear NFAT is detected. This apparent paradox is partly explained by data showing that RANKL treatment downregulates casein kinase 1 α, which phosphorylates NFAT. Thus, despite low levels of CaN activity, NFAT remains in the nucleus because the phosphorylation pathway is shut down to an equal extent.

These data are the first to describe actual enzyme activity for CaMKII and CaN in this system and are consistent with a model in which RANK signaling leads to rapid increases in intracellular calcium, activation of calmodulin, and CaMKII, which regulate osteoclastogenesis at early and late time points.

Together, these studies provide evidence that CaMKII is the primary target of CaM signaling in the RANK pathway, and represents a novel NFAT-independent target for inhibiting osteoclastogenesis.

Disclosures: K.J. Micoli, None.

W071

Osteoclasts Generated in Ectopic Bone Are Derived from Postmitotic Osteoclast Precursors (pOCPs). A. Muto*¹, T. Mizoguchi*², T. Noguchi*¹, N. Udagawa³, N. Takahashi², ¹Department of Periodontology, Aichi-gakuin University school of Dentistry, Nagoya, Japan, ²Institute for Oral Science, Matsumoto Dental University, Shiojiri, Japan, ³Department of Biochemistry, Matsumoto Dental University, Shiojiri, Japan.

We have reported that when collagen pellets containing BMP-2 (BMP-pellets) were implanted into mice, osteoclasts as well as osteoblasts appeared simultaneously in the BMP-pellets (Endocrinology 147:3366, 2006). We also showed that cell cycle progression and subsequent cell cycle arrest in osteoclast precursors are required for their differentiation into osteoclasts. The quiescent osteoclast precursor cells were named “postmitotic osteoclast precursors” (pOCPs) (JBMR 20 Suppl 1: M244, 2005). Here, we examined whether pOCPs are present in the blood, using a system of the BMP-induced ectopic bone formation. BMP-pellets were implanted into dorsal muscular pouches of mice. To discriminate between proliferating and quiescent cells, mice were given 5′-bromo-2′-deoxyuridine (BrdU) in drinking water during an implantation period of 2 weeks. Then, BMP-pellets and tibiae were recovered from the mice, and subjected to TRAP and BrdU staining. The results obtained from this work are as follows: (1) In tibial sections, BrdU-positive nuclei were detected in bone marrow cells, osteoblasts, chondrocytes and mesenchymal cells. In contrast, none of the nuclei in TRAP-positive osteoclasts incorporated BrdU. (2) In BMP-pellet sections, many TRAP-positive osteoclasts were found to be in close contact with ALP-positive osteoblasts. A large number of mesenchymal cells including osteoblasts possessed BrdU-positive nuclei. Some of the macrophage-like cells also had BrdU-positive nuclei. In contrast, all the nuclei in TRAP-positive osteoclasts observed in the BMP-pellets were negative for BrdU. We have reported that osteoblasts prepare the osteoclast niche, which supports a long-term survival of pOCP. Osteoclasts were differentiated from pOCPs in the osteoclast niche in response to several stimuli including the intraperitoneal administration of RANKL. In the present study, osteoclasts in ectopic bone are also differentiated from pOCPs without cell proliferation. These results suggest that pOCPs are present in the blood flow and osteoblasts may play a role in specific homing of pOCPs to the precise sites of action of osteoclasts.

Disclosures: A. Muto, None.

W072

M-CSF Independent Mechanisms for Osteoclastogenesis. Y. Nakamichi¹, N. Udagawa², H. Yasuda³, M. Nakamura², Y. Kobayashi*¹, N. Takahashi¹, ¹Institute for Oral Science, Matsumoto Dental Univ., Shiojiri, Japan, ²Dept. of Biochemistry, Matsumoto Dental Univ., Shiojiri, Japan, ³Nagahama Institute for Biochemical Science, Oriental Yeast Co., Ltd., Nagahama, Japan.

M-CSF and RANKL are osteoblast-derived cytokines essential for differentiation of monocytes (MOs)/macrophages (Mϕs) into osteoclasts (OCs). M-CSF-deficient op/op mice exhibit severe osteopetrosis due to deficiencies of OCs. It is known that the defect is prominent especially in young op/op mice and OCs gradually appear with aging. However, the molecular mechanisms for OC formation in the absence of M-CSF have remained elusive. To elucidate M-CSF independent mechanisms for OC formation, we performed four experiments as follows: (1) We tried to induce OC formation by administering an 1α,25(OH)₂-vitamin D₃ analog (2MD) or GST-RANKL (kindly provided by Oriental Yeast Co., Ltd.) intraperitoneally in young (3-week-old) op/op mice in which OCs are totally absent. When op/op mice were administered 2MD (30 pmol/day) for 2 days, many TRAP-positive OCs were formed at the surface of trabecular bone. Similarly, when GST-RANKL (40 μg/day) or M-CSF (10⁵IU/day) was administered into op/op mice for 4 days, many OCs appeared in bone tissues as if they had existed in WT mice. Concomitantly, serum TRAP5b activity, an indicator of osteoclastic bone resorption, was increased by the administration of these agents. (2) To assess the requirement of M-CSF for OC formation in normal animal, WT mice were pretreated with M-CSFR (c-Fms)-neutralizing antibody (AFS98) for a month and followed by simultaneous treatment with both 2MD and AFS98 for 4 days. 2MD effectively induced TRAP-positive cells and hypercalcemia under the conditions where the c-Fms signal was suppressed. (3) To find a substitute for M-CSF, we examined whether M-CSF-related growth factors (PDGF, SCF, VEGF, FLT3L) could replace M-CSF in the in vitro differentiation of Mϕs into OCs. None of these factors supported in vitro OC formation. (4) To investigate whether cell adhesion molecules such as N-cadherin and ICAM can support OC differentiation, we tested in vitro OC formation on the plates precoated with adhesion molecules. N-cadherin but not ICAM induced mononuclear TRAP-positive cells even in the absence of M-CSF. These findings suggest that M-CSF is not absolutely required for in vivo OC formation in WT mice as well as op/op mice. In culture conditions, TRAP-positive cells were induced in N-cadherin-coated plates in the absence of M-CSF, suggesting that the adhesion-mediated signal may play a role in OC differentiation in vivo.

Disclosures: Y. Nakamichi, None.

W073

Expression of Angiotensin II Receptor in Osteoclasts and Its Roles on Differentiation and Survival. C. Nakamura¹, S. Kamiya*², T. Fukawa*², N. Nimura*¹, S. Wada², ¹Bio-Analytical Chemistry, Josai International University, Chiba, Japan, ²Clinical Sciences, Josai International University, Chiba, Japan.

Angiotensin (Ang) II is well known for its action on vasoconstricion, vascular cell proliferation, and remodeling of myocardium. In this study, we examined whether Ang II could be involved in bone remodeling process, since it was found recently that bone marrow cells expressed high levels of Ang II receptor.

Bone marrow macrophages (BMMs) were prepared from bone marrow cells (BMCs) through incubating with M-CSF, and then treated with M-CSF/sRANKL. Ang II receptor (AT_1A, AT_1B and AT₂) expression was examined by RT-PCR and Western blot analysis through osteoclasts (OCs) development from precursors.

AT_1A mRNA was highly expressed and was stable throughout culture period in BMCs, BMMs and OCs, whereas AT_1B mRNA was increased parallel to OC differentiation. Expression of AT₁ which could not be discriminated as protein levels of AT_1A and AT_1B was not changed in these cells. AT₁ mRNA and protein expression was much lower than AT₁ but the levels were increased in either BMMs or OCs. To examine possible roles of Ang II on OC development, we studied the effects of AT₁ agonist (L-162,313, 10⁻⁵M) in the cells. Treatment with AT₁ agonist completely suppressed OC formation at the early culture period (day 0-3) and even late culture period (day 3-7). Treatment with AT₁ agonist on mature OCs 24-48h reduced the number of viable OCs. AT₁ antagonist (Losartan), AT₂ agonist (CGP42112A) and AT₂ antagonist (PD123319), however, did not affect OCs differentiation and their survival. The mechanism of these inhibitory effects of AT₁ agonist remains to be clarified, but we are currently focusing on down-stream signaling and changeable cytokine expression related to OC differentiation.

In this study, we found that AT_1B and AT₂ receptor expression are increased parallel to OC differentiation and that AT₁ agonist inhibits OC differentiation and survival, which may indicate possible roles of Ang II-AT₁ pathway on OC formation and bone remodeling process.

Disclosures: C. Nakamura, None.

W074

The TREM2 Pathway Is Involved in Osteoclastogenesis Induced By Nucleoside Reverse Transcriptase Inhibitors in AIDS. G. Pan*, J. M. McDonald.. Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

An increased incidence of osteopenia and osteoporosis in HIV infected patients receiving antiretroviral therapy has now been reported in several studies. Our previous studies show that antiviral nucleoside reverse transcriptase inhibitors (NRTIs) stimulate osteoclast differentiation in vitro and produce osteopenia in mice. Moreover, NRTI-enhanced osteoclastogenesis is mediated through the RANK pathway. The underlying molecular mechanisms responsible for activating the RANK pathway by NRTIs have not been elucidated.

We analyzed osteoclastogenesis-associated gene expression in nucleoside-stimulated osteoclastogenesis and found that antiviral NRTIs up-regulated the expression of triggering receptor expressed on myeloid cells 2 (TREM2) in RAW264.7 cells and mouse bone marrow macrophages. To further determine the role of TREM2 in NRTI-stimulated osteoclastogenesis, antibodies against TREM2 were used to determine whether NRTI-stimulated osteoclastogenesis can be inhibited. The elevated osteoclastogenesis (TRACP) induced by RANKL plus AZT was reduced by TREM2 antibody approximately to the level of RANKL treatment alone, whereas antagonists of the purinergic receptor failed to inhibit NRTI-stimulated osteoclastogenesis. Then, we determined whether NRTI-enhanced RANKL-mediated osteoclastogenesis is associated with TREM2 expression. Our results indicated that RAW264.7 cells with high TREM2 expression were sensitive, whereas cells with low TREM2 expression were resistant to NRTI-stimulated differentiation of osteoclasts. Finally, we determined the function of the TREM2 pathway in NRTI-stimulated osteoclastogenesis. NRTIs stimulated the promoter activity of NFATcl, increased the translocation of NFATcl into nuclei, and accelerated actin formation, all down-stream events of TREM2 signaling. In conclusion, osteoclast differentiation induced by NRTI not only requires RANK signaling, but also requires the costimulatory TREM2 pathway. NRTIs activate the TREM2 pathway, which interact with the RANK pathway enhancing osteoclastogenesis. The TREM2 pathway is largely responsible for increased osteoclastogenesis induced by NRTIs during treatment of AIDS which induces osteopenia/osteoporosis.

Disclosures: G Pan, None.

W075

Cobalt Incorporation into Calcium Phosphate Layers Stimulates Osteoclast Differentiation and Activation via Hypoxia Inducible Factor Pathway. S. Patntirapong*¹, P. Habibovic*², P. V. Hauschka¹, ¹Orthopaedic Surgery, Children's Hospital, Boston, MA, USA, ²Tissue Regeneration, University of Twente, Enschede, Netherlands Antilles.

One of the major etiologies of implant failure is activation of macrophages by particulate wear debris, resulting in inflammatory stimulation of osteolysis. Recent studies have shown an increase of metal ions released from wear particles and metallic prostheses due to mechanical stress. Furthermore, these metal ions seem to accumulate in body fluids, peri-implant soft tissues, and bone mineral. We hypothesized that metallic ions could directly activate osteolysis, particularly cobalt (II), which is a known “hypoxia mimic” activator of hypoxic gene expression and a component of alloys used for prostheses. To address this question, we prepared calcium phosphate (CaP) coatings with traces of cobalt ions to examine the effects on osteoclast formation and resorptive activation. Treatments were divided into 2 conditions with various cobalt concentrations either incorporated into the CaP layer, or added to culture media. Scanning electron microscopy (SEM) and inductively coupled plasma mass spectrometry (ICP-MS) were used to visualize the CaP crystallization, and to measure Co²⁺ concentrations in the CaP and in the culture medium, respectively. SEM demonstrated that incorporated Co²⁺ did not modify the nucleation and growth mechanism of the CaP crystallizing system. Measurement of Co²⁺ by ICP-MS indicated the release of Co²⁺ from the CaP layers during equilibration with culture medium. Low concentrations of cobalt delivered to developing osteoclast precursors by both routes increased osteoclast differentiation as well as resorptive function. A 1.7 to 1.75-fold increase in osteoclast numbers and a 2.3 to 2.7-fold more resorptive activity of the CaP layer were observed for 0.1 – 20 μM Co²⁺, which is within the range found in peri-implant tissues in vivo. In addition, we investigated the stabilization of hypoxia inducible factor alpha (HIF-α), an important transcription factor of the hypoxic pathway, and its target genes. Immunoblotting showed that HIF-1α and HIF-2α were stabilized in osteoclastic cells exposed to Co²⁺. Quantitative RT-PCR demonstrates upregulated mRNA levels of glucose transporter-1 (Glut-1), phosphofructokinase muscle (Pfkm) and vascular endothelial growth factor (Vegf) by Co²⁺ treatment, indicating the osteoclastic switch to glycolytic metabolism. This direct effect of Co²⁺ on osteoclasts through the HIF pathway may contribute to osteolysis and aseptic implant loosening independently of the inflammation-mediated pathways.

Disclosures: S. Patntirapong, None.

This study received funding from: DOD grants BC044721 and BC045539.

W076

Inhibition of Osteoclastogenesis by Prosthetic Wear Debris Involves Suppression of RANK Expression. E. Purdue*, K. Ly*, L. Ivashkiv*, B. Nestor*, T. Sculco*, S. Goldring.. Osteolysis Research Laboratory, Hospital for Special Surgery, New York, NY, USA.

Osteoclasts, multinucleated foreign body giant cells, and mononuclear phagocytic macrophages are derived from a common myeloid precursor. Multiple factors control the commitment of these precursors to each of the cell types, including cytokines, cell-cell and cell-matrix interactions. There remains controversy, however, regarding the effect of activation of phagocytic pathways on myeloid cell fate. The present studies were undertaken to define the effect of phagocytic activation on osteoclast differentiation in order to gain insight into the mechanisms associated with prosthetic wear debris-induced osteolysis, the major cause of implant failure after total joint replacement. Human monocytes were cultured with MCSF in the presence or absence of polymethylmethacrylate (PMMA) bone cement or titanium wear particles followed by treatment with or without RANKL. Osteoclastogenesis was assessed by identification of TRAP-positive multinucleated cells. Real-time RT-PCR and Microarray analysis were used to determine the effects of PMMA and titanium particles on the pattern of gene expression. At doses of 30 particles per cell, both PMMA and titanium completely inhibited RANKL-induced osteoclast formation. At lower numbers, osteoclastogenesis was reduced but not obliterated. Interestingly, the osteoclasts formed in the presence of reduced particle number appeared largely devoid of particles. PMMA and titanium also inhibited RANKL-induced expression of the osteoclasts markers cathepsin K and TRAP. Most strikingly, PMMA and titanium very strongly inhibited expression of RANK, both in the presence or absence of RANKL. Our data suggest a molecular explanation for the observation, first recorded over fifteen years ago, that osteoclasts within the osteolytic tissues associated with periprosthetic osteolysis do not contain particles (Willert et al, Clin Orthop Relat Res. 1990 258:108–21). Although it has been reported that PMMA can induce osteoclastogenesis and that particle-laden macrophages can differentiate into osteoclasts, our results indicate that phagocytic activation inhibits RANKL-induced osteoclast formation, and that this affect can be attributed to suppression of RANK, which is essential for RANKL-induced osteoclast formation. Taken together with the well-known ability of wear debris to activate pro-inflammatory cytokine signaling in myeloid cells, and the more recent observations that wear debris can promote alternative, non-inflammatory, macrophage activation, these results highlight the multiple ways in which wear debris can influence myeloid cell fate in periprosthetic osteolysis.

Disclosures: E. Purdue, None.

W077

Functional Role for Spinal Muscular Atrophy (SMA) Gene Expression in Bone Remodeling. S. Shanmugarajan¹, K. J. Swoboda*², S. T. Iannacone*³, W. L. Ries¹, B. L. Maria*¹, S. V. Reddy¹. ¹Charles P. Darby Children's Research Institute, Medical University of South Carolina, Charleston, SC, USA, ²University of Utah School of Medicine, Salt Lake City, UT, USA, ³University of Texas Southwestern Medical Center, Dallas, TX, USA.

Spinal muscular atrophy (SMA) is the second most common fatal childhood disorder which occurs 1 in 8000 newborns. Although the core clinical features of SMA include muscle weakness caused by degenerating lower motor neurons, patients also have a high incidence of bone fractures. SMA determining gene encoding protein, SMN levels are significantly reduced due to deletions/mutations in the telomeric SMN1 gene in these patients. However, a centromeric SMN2 gene copy can not compensate for the deficiency due to an aberrant splicing of the exon 7 region. SMN expression is ubiquitous. We have recently reported SMN expression and differential splicing in bone resorbing osteoclasts. Also, we have shown SMN interaction with the osteoclast stimulatory factor (OSF). It is our hypothesis that SMN plays an important role in the osteoclast (OCL) formation/bone resorption activity. SMA bone disease involves OCL development/abnormalities due to SMN deficiency which leads to bone fractures. In the present study, we utilized the Smn -/ - SMN2 mouse model of SMA to determine the functional role for SMN in bone remodeling. DEXA analysis demonstrated a significant decrease in total bone area and poorly developed caudal vertebra in Smn-/-; SMN2 mice. Interestingly, these mice also showed pelvic bone fractures similar to patients with SMA. Histological analysis demonstrated a thin porous cortex of cortical bone and thin trabeculae at the proximal end of the growth plate in the caudal vertebrae of Smn deficient mice compared to wild-type mice. Further, histochemical staining of caudal vertebra in the Smn-/-;SMN2 mice portrays the presence of activated osteoclasts on the sparse trabeculae and in the inner endosteal surface of the thin cortex. In contrast, OCL are mostly confined to the base of the growth plate region in wild-type mice. Histomorphometic analysis further confirmed an increased OCL number and a decreased number of osteoblast cells on bone surfaces in Smn deficient mice. Also, urine N-telopeptides of type I collagen (NTx) levels are significantly increased (13.6 fold) in these mice indicating excess bone resorption by osteoclasts. Furthermore, Smn deficient mouse bone marrow cultures demonstrated a significant increase (54%) in OCL formation and bone resorption capacity compared to wild-type mice. Taken together, our results implicate SMN function in bone remodeling. Therefore, understanding bone remodeling in SMA may lead to novel therapeutic approaches to prevent bone fractures and enhancing the quality of life in SMA children.

Disclosures: S. Shanmugarajan, None.

W078

Adenosine Receptors in the Osteoclast. S. Stephens*¹, J. Auchampach*², A. Blangy³, R. Rose'Meyer*¹, J. Headricks*¹, N. Morrison¹. ¹Medical Sciences, Griffith University, Gold Coast, Australia, ²Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA, ³CRBM-CNRS, Montpellier, France.

The osteoclast is a cAMP-sensitive cell whose formation is stimulated by the decrease in cAMP via follicle-stimulating hormone but whose function is inhibited by the increase in cAMP via the calcitonin receptor. Adenosine receptors (ARs) are PI G-protein-coupled receptors composed of four subtypes AR₁, AR₂A, AR₂B and AR₃. These receptors have diverse effectors but all regulate the levels of intracellular cAMP via modulation of adenylyl cyclase (AR₁/AR₃ decreases cAMP and AR₂A/AR₂B increases cAMP). We found all four ARs expressed in human osteoclasts with AR₁ being the most abundant and upregulated (∼15 fold compared to non-RANKL-treated control). Later experiments confirmed the presence of all four ARs in murine osteoclasts with AR₁ being the only osteoclast-specific AR, albeit the least expressed. In this study, we set out to establish whether ARs when stimulated, could affect RANKL-induced osteoclast differentiation. Using adenosine (1–10μM), the endogenous agonist for all adenosine receptors, we showed a significant increase in osteoclastogenesis. This effect was not blocked by P2 receptor antagonists confirming that this effect is PI-selective and not via the ATP-specific P2 receptors whose role in augmenting osteoclastogenesis is already confirmed. Next, to evaluate which receptor(s) is/are responsible for increased osteoclast formation, the agonists CCPA, CGS1280, NECA and Cl-IB MECA were used in place of adenosine for the specific activation of AR₁, AR₂A, AR₂B and AR₃ respectively. At specific concentrations (1-10nM), the agonists showed that all ARs can mediate an increase in osteoclast formation with up to 7 times more osteoclasts compared to RANKL-only treated cells. Moreover, the combination of all agonists did not further increase osteoclastogenesis (ie non additive actions) implying that the four ARs signal through the same pathway or converge through a common signalling gateway. A common pathway is possible through the IP3 signalling cascade but not via cAMP. IP3 signalling is directly linked to Ca2+ extrusion from the endoplasmic reticulum which can activate NFATc via calmodulin/calcineurin and potentially explains the pathway exploited by ARs in the osteoclast.

Disclosures: S. Stephens, None.

This study received funding from: Griffith University.

W079

RANKL Is Produced by Adipocytes and Stimulates Osteoclastogenesis. S. Takeshita, K. Ikeda.. Bone and Joint Disease, National Center for Geriatrics and Gerontology, Obu, Japan.

RANKL is an osteoclastogenic cytokine that is induced on osteoblastic/stromal cells in response to osteotrophic hormones, such as 1α,25(OH)₂D₃ or PTH. However, it remains to be determined which cell types actually express RANKL in vivo in response to types of cues. We have found that long-term cultures of mouse whole bone marrow cells, under adipogenic induction with Dexamethasone and IBMX, exhibit strongly induced osteoclastogenesis without the addition of any osteotrophic hormones or RANKL. In fact, RANKL expression was induced in marrow stromal cell lines, ST2 and MC3T3-G2/PA6, following adipogenic induction. RANKL was induced transiently during adipogenesis, with concomitant down-regulation of OPG, but not at a later stage with the accumulation of lipid droplets, suggesting that adipocytes at a certain stage of differentiation express RANKL and support osteoclastogenesis. In order to test this hypothesis, we used an aged model in which bone marrow is full of adipose cells. Bone marrow cells from 2-year-old C57BL/6 male mice cultured under osteogenic conditions (β-glycerophosphate and ascorbic acid), without the addition of any stimulator, gave rise to “osteoclast colonies” (i.e. ALP-positive osteoblastic colonies containing TRAP-positive osteoclasts) that exceeded 30% of total CFU-F, whereas those from younger mice produced very few osteoclast colonies. Adipogenic cultures of bone marrow cells from the aged mice yielded even more osteoclasts than did osteogenic cultures, and RT-PCR analysis revealed RANKL was up-regulated in adipogenic cultures of bone marrow stromal cells. These results suggest that bone marrow cells in aged animals have a potent capacity for generating osteoclast colonies under adipogenic conditions without the need of any osteotrophic hormones, and that adipogenic cells in bone marrow may contribute to accelerated osteoclastogenesis in senile or steroid-induced osteoporosis by producing RANKL as an adipokine.

Disclosures: S. Takeshita, None.

W080

The Matrix PRoline/arginine-rich End Leucine-rich Repeat Protein (PRELP) Inhibits Osteoclastogenesis Inactivating the NF-kappaB Signal. M. Alamanou*¹, N. Rucci¹, A. Rufo*¹, P. Heinegård*², A. Teti¹. ¹Experimental Medicine, University of L'Aquila, L'Aquila, Italy, ²Experimental Medical Science, Lund University, Lund, Sweden.

PRELP originally found in cartilage is also found at certain basement membranes where it may serve as an anchor by binding to heparan sulfate side chains of perlecan and concomitantly to collagens type I/II collagens. Loss of function mutations of PRELP are involved in the pathogenesis of Hutchinson-Gilford progeria, characterized, among other symptoms, by severe osteoporosis. PRELP is highly expressed in cartilage and developing bone. Herein we demonstrate that a peptide corresponding to the entire heparin-binding domain of PRELP inhibits mouse osteoclast formation. Treatment with the PRELP-peptide decreased osteoclastogenesis by 80% in non-fractionated mouse bone marrow cultures exposed to 1,25(OH)2VitD3, and in purified bone marrow macrophages treated with M-CSF and RANKL (IC50 = 7.3 microM). Pre-treatment of mature osteoclasts with the PRELP-peptide decreased adhesion to fetal calf serm protein substrate by 60%. In contrast, the peptide showed no activity with osteoclast precursors and neither their number, nor TRAcP activity and adhesion were affected by the treatment. Consistently, the PRELP-peptide inhibited osteoclastogenesis when added at the 4^th day of culture, suggesting a late effect on pre-fusion osteoclast precursors. Although a terminally-tagged PRELP-peptide co-localized with heparan sulphate proteoglycans (HSPGs), they did not appear to physically interact. Consistently, treatment with heparinase to remove the heparan sulfate chains, failed to prevent PRELP from inhibiting osteoclastogenesis. In contrast, the peptide activity was shown to depend on binding to the chondroitin sulphate chains of cell surface proteoglycans, and the effect was abolished by treatment with chondroitinase. Tagged PRELP was internalised by osteoclast precursors by an annexin II-dependent mechanism. It translocated to the nucleus where it bound NF-kappaB reducing its transcriptional activity by 50%. Finally, the PRELP-peptide was found to affect osteoclastogenesis and bone resorption only by a direct mechanism as real-time RT-PCR failed to detect any change in expression of cytokines promoting osteoclastogenesis in mouse calvarial osteoblasts. Accordingly, the PRELP-peptide showed no effect on their alkaline phosphatase activity, nodule mineralization, gene expression nor intracellular signalling protein phosphorylations. We conclude that PRELP is a direct negative regulator of osteoclast formation, which blocks NF-kappa B signaling in late stage pre-fusion committed osteoclast precursors, with no apparent effect on the osteoblast lineage.

Disclosures: A. Teti, None.

This study received funding from: EU project OSTEOGENE LSHM-CT-2003-502941.

W081

Comparative Analysis of Osteoclast Bone Resorption Activity and Apoptosis in Young and Old Mice. S. J. Wimalawansa, X. Wang, J. Ghosh*, F. Azeez*., Medicine, Robert Wood Johnson Medical School, New Brunswick, NJ, USA.

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with consequent increase in bone fragilely and susceptibility to fracture. Besides the effects of hormone, environmental factors and other secondary causes on bone, aging itself is believed to be a major cause of bone loss in the elderly.

In the present study, we compared the osteoclasts bone-resorbing activity and life span of old and young mice. Five, 4–6 week old (young) female mice and five, 20–24 months old (aged) female mice were utilized to obtain osteoclast progenitors and osteoclasts. The osteoclast progenitor cells were obtained from mice bone marrow of long bones in simultaneously, and cultured in 10% FBS α-MEN with 5ng/ml MCSF for two days. The non-adherent cells were further cultured for 11 days in vitro with OC medium contained 30 ng/ml MCSF and 60 ng/ml RANK. 200,000 cells were seeded on each calcium phosphate coated disc (BD Biosciences Discovery Labware, Canada) for pit resorption assay; and 200,000 cells were seeded on each pre-treated cover slip for TRAP stain and apoptosis assay. The pit area is measured using the Metavue Software version 6.0 on the Nikon 2000U confocal microscope. TRAP stain was used to quantify matured osteoclasts (>3 nuclears). The Image-iT LIVE Green Poly Caspases Detection Kit, Molecular Probes Invitrogen Technologies was used for apoptosis assay. The protocol of molecular Probes was adopted and the number of mature osteoclasts with three or more nuclei were counted using ACT-1 Software, Nikon 2000U confocal microscope. The fluorescence intensity indicated the apoptotic status of the cells.

The osteoclasts from aged mice showed more bone resorbing activity than those from young mice; the resorption area of age osteoclasts are 11.6 ± 1.2%, while the resorption area from young osteoclast are 5.1 ± 2.5% (p = 0.02). Trap stain results indicated that the matured osteoclasts from aged mice are almost twice as much as those from young mice (122 vs. 69). Furthermore, the apoptotic mature osteoclasts in young were more than those in old mice (90% vs. 81%).

The etiology of age-related bone loss is complicated, and although some mechanisms involving osteoblast and osteoclast dysfunction have been reported, the process remains elusive. Our data indicate that matured osteoclast from old mice A). Survived longer, B). Less apoptotic, and thus, C). Absorbed more bone than osteoclast from young mice. Osteoclastic apoptotic-induction agents should help to combat osteoporosis in the elderly.

Disclosures: S.J. Wimalawansa, None.

W082

High D(+)glucose Concentration Inhibits RANKL-induced Osteoclastogenesis. Y. Wittrant*¹, Y. Gorin*², K. Woodruff*¹, D. Horn*¹, S. Mohan*¹, H. E. Abboud*², S. Werner¹. ¹Pathology, University of Texas Health Science Center San Antonio and Audie Murphy VA Hospital, San Antonio, TX, USA, ²Nephrology, University of Texas Health Science Center San Antonio, San Antonio, TX, USA.

Diabetes is associated with altered bone metabolism that may lead to increased risk of osteoporosis and fracture. Hyperglycemia has been implicated in the pathogenesis of diabetic bone disease; however, the biologic effect of glucose on osteoclastogenesis is unclear. Although glucose is a source of energy for osteoclast resorption, effect of sustained high glucose levels on osteoclast (OC) differentiation and function have not been explored. We examined the effect of high D(+)glucose and L(-)glucose (osmotic control) on RANKL-induced osteoclastogenesis using RAW264.7 cells and Bone Marrow Macrophages as models. OC formation was analyzed using TRAP assay, expression of CTR and cathepsin K mRNAs. Since reactive oxygen species (ROS), caspase-3 and NF-kB are crucial for RANKL-induced osteoclastogenesis, the effect of glucose on these factors was examined. Intracellular ROS generation was determined by measuring fluorescence intensity in cultures incubated with 2′7′-dichlorofluorescein diacetate (DCF-DA). Caspase-3 activity was quantified in cell lysates and NF-kB-dependent luciferase activity was analyzed following transient transfection with a vector containing NF-kB response elements linked to the luciferase reporter gene. The effect of glucose on cellular function was assessed using a migration assay. Results showed that high D-Glc, but not L-Glc, caused a profound decreased in RANKL-induced TRAP-positive OC and led to a predominance of mononuclear cells; this effect correlated with reduced expression of CTR and Cathepsin K. Compared to cultures incubated with RANKL alone, high D-Glc markedly reduced ROS levels by 90%, decreased caspase-3 activity and inhibited NF-kB transcriptional activity, whereas high L-Glc had little or no effect. In separate experiments, incubation of cells with H₂O₂ in the absence of RANKL induced NF-kB luciferase activity, indicating the ability of ROS to directly affect NF-kB activity. In migration assays, fewer cells migrated into and covered the wounded site in high D-Glc-treated cultures compared to control cultures. These findings indicate, for the first time, that high D-Glc inhibits OC formation, ROS production, caspase-3 activity and migration in response to RANKL through a metabolic pathway. High D-Glc may alter RANKL-induced OC formation by inhibiting NF-kB activity via a ROS-dependent mechanism. Our data suggest that high glucose may prevent excessive osteoclast-mediated bone loss in diabetes and provide new insight into the biologic effects of glucose on osteoclastogenesis.

Disclosures: Y. Wittrant, None.

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

W083

The Role of Bcl2 in Osteoclastogenesis Ex Vivo. D. Yang, F. Liu, L. K. McCauley, J. Yamashita.. University of Michigan School of Dentistry, Ann Arbor, MI, USA.

Activation of B-cell leukemia/lymphoma 2 (Bcl2) suppresses programmed cell death in many cell types. It has been reported that forced expression of Bcl2 in M-CSF deficient monocytes partially rescued osteopetrosis of op/op mice. We recently reported that Bcl2 homozygous mutant mice had increased bone mass and that such skeletal phenotype was at least partially attributed to osteoclast defects. These findings suggest an important role of Bcl2 in osteoclastogenesis. However, the function of Bcl2 in osteoclast differentiation and survival has not been well investigated. In this study the impact of Bcl2 on osteoclastogenesis was studied using bone marrow cells. Mononuclear cells were obtained from freshly isolated bone marrow of Bcl2 wild type (+/+) and knockout (-/-) mice and cultured. Osteoclastogenesis was induced using M-CSF and RANKL. Cell enumeration and morphometric analysis of TRAP-stained osteoclasts revealed substantial differences in a course of osteoclastogenesis between Bcl2 +/+ and -/- cultures. At day-5 a more vigorous osteoclastogenesis was observed in Bcl2-/- cultures compared to those in +/+ (p<0.01). Not only were numbers increased in Bcl2-/- cultures at day-5, but the average size of Bcl2-/- osteoclasts was approximately twice that of +/ + (p<0.001). However, at day-7 and −9, Bcl2-/- osteoclasts were no longer large and no difference was detected between genotypes. To determine the mechanism of vigorous Bcl2-/-osteoclastogenesis, M-CSF dependent bone marrow macrophages were stimulated with RANKL and M-CSF and the expression of NFATcl, TRAP, and DC-STAMP at the RNA level was quantitatively analyzed with real-time PCR. It was revealed that the expression of DC-STAMP and TRAP was more rapid and considerably stronger in Bcl2 -/- than that in +/+ preosteoclasts. Higher expression of NFATcl was also noted in Bcl2 -/- than +/+ preosteoclasts. The early expression of osteoclast markers fits the results of cell enumeration and morphometric analysis. These data suggest that Bcl2 plays a role in regulation of RANK signaling in myeloid cells. In summary, Bcl2-/- osteoclastogenesis was rapid and robust, and vanished quickly thereafter, while +/+ osteoclastogenesis occurred more gently. Such vigorous Bcl2 -/- osteoclastogenesis was associated with early and elevated expression of DC-STAMP and TRAP.

Disclosures: J. Yamashita, None.

W084

Lysine-Specific Gingipain (Kgp) and Lipopolysaccharide (LPS) Synergistically Induces Osteoclast Differentiation. R. Yasuhara*¹, Y. Miyamoto*¹, M. Takami¹, T. Imamura*², R. Kamijo¹. ¹Department of Biochemistry, Showa University School of Dentistry, Tokyo, Japan, ²Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.

Porphyromonas gingivalis is known as a major pathogen of periodontitis associated with alveolar bone loss due to excessive bone resorption by osteoclasts. P. gingivalis produces cysteine proteases called gingipain divided into two types, Lys-gingipain (Kgp) and Arg-gingipains (Rgps), according to their cleavage-sites. In this study we examined the effects of gingipains on osteoclast differentiation. In cocultures of mouse bone marrow cells and osteoblasts, the addition of Kgp but not Rgp induced the formation of multinucleated osteoclasts (Kgp: 23 +/- 3.5 cells/well, Rgp: 0 cells/well). On the other hand, LPS, a constituent of gram-negative bacteria including P. gingivalis, induced mononuclear osteoclasts and small number of multinucleated osteoclasts (0–1 cells/well). Interestingly, simultaneous addition of Kgp and LPS to the cocultures induced significantly larger number of multinucleated osteoclasts (97 +/- 4.5 cells/well) than that induced by Kgp or LPS alone. Numerous resorption pits were developed on the surface of dentin slices on which cells were cocultured in the presence of Kgp and LPS together. However, LPS alone did not induce any resorption pits. Not only LPS but also the other bacterial constituents such as CpG ODN, poly(I:C) RNA and peptidoglycan, synergistically induced osteoclast differentiation in the combination with Kgp. We further examined the mechanisms of osteoclast differentiation by Kgp. Inactivated Kgp by Z-FK-cmk, a specific inhibitor for Kgp, completely inhibited osteoclast differentiation induced by Kgp in the cocultures, indicating that the specific enzymatic activity of Kgp was essential for the induction of osteoclasts. Kgp did not induce the expression level of mRNAs for the receptor activator of NF-kB ligand (RANKL) in osteoblasts, whereas Kgp significantly reduced the protein level of OPG in coculture media. In an in-vitro study, Kgp cleaved recombinant OPG protein suggesting that degradation of OPG by Kgp caused the enhancement of osteoclast differentiation in cocultures. These results suggest that Kgp exhibits synergistic virulence with LPS resulting in the inflammatory bone resorption characteristically seen in periodontitis. Therefore, we propose here that Kgp is a new target for the prevention and treatment of periodontitis.

Disclosures: R. Yasuhara, None.

W085

Analysis of Matrix Vesicle Poteins from Long Bone Growth Plates. J. Jeong*¹, H. Kim*¹, H. Kim*¹, E. Park¹, J. Cho¹, R. Garimella², N. N. Nahar², H. C. Anderson², J. Lim*¹, S. Kim¹, J. Choi¹. ¹Biochemistry & Cell Biology, Kyungpook Natl. Univ. School of Medicine, Skeletal Diseases Genome Research Center, Daegu, Republic of Korea, ²Pathology, Kansas University Medical Center, Kansas, KS, USA.

Initiation of bone mineralization is a key step to make a proper bone formation. Matrix vesicles (MVs) have been known as an initiation site for mineralization. Thus, identification of proteins from MV will provide fundamental clues to access the mechanism of the initiation step in bone mineralization. Although many components of MV have been identified by tedious single protein confirmation approach, comprehensive protein component analysis from MV has not been studied. In this study, we isolated MVs from growth plates of rachitic rat bones. Proteins from MVs were separated by SDS-PAGE and the gels were cut into 15 pieces from top to bottom. After in-gel digestion with trypsin, eluted peptides were extracted for LC-MS/MS analysis. Identified proteins from MVs were various including previously known proteins such as annexins and peptidases, along with other variety of enzymes, extracellular matrix, growth factors, and signal molecules. The systematic proteomic analysis of MVs gives an important opportunity to understand the molecular mechanism of initiation step of bone mineralization.

Disclosures: J. Choi, None.

W086

Comparison of In Vivo Model of Functional Bone Adaptation to the Calculated Mechanical Environment. C. J. MacKay*¹, G. C. Goulet*², D. Coombe*³, R. F. Zernicke*¹. ¹Faculty of Medicine, University of Calgary, Calgary, AB, Canada, ²Faculty of Engineering, University of Calgary, Calgary, AB, Canada, ³Computer Modeling Group, Ltd., Calgary, AB, Canada.

Interest in functional adaptation of bone has led to the development of numerous experimental models [1,2,3,4]. Animal models provide insights into the molecular signaling pathways by which bone senses and adapts to changes in functional demand, but ambiguity persists about the mechanical components contributing to the principal determinants of skeletal morphology. Here, experimental results from an in vivo model of functional bone adaptation, using 12 adult female Sprague-Dawley rats, were compared to a finite element model (FEM). Animals were subjected to 28 day, daily lateral-medial cantilever tibial loading regime, consisting of 1 s pulses of a 1 Hz trapezoidal wave followed by 9 s of rest to a maximal strain magnitude of 1000 μϵ, for a total duration of 120 s. Bone-targeted flourochromes, calcein AM (days 6 and 18) and xylenol orange (days 13 and 27) were administered to estimate relative bone formation rates and localize osteogenic mineralization in response to exogenous loading. Histology and comparison of tibial cross-sections, obtained from experimental and contralateral limbs, indicated increases in bone mineralization rates on several ossification fronts across the tibial cross-section. These results were compared to the FEM model. The model calculated areas of maximal compression and tension across the experimental region of interest during loading, and estimates fluid flow vectors and pressure gradients, thereby quantifying the mechanical milieu during experimental loading. The comparison of the resultant osteogenic responses in this model with previously calculated results provided insight into the specific mechanical signals that may influence dynamic skeletal morphology.

[1] Judex et al. (1997). J. Bone Min. Res. 12, 1737–1745. [2] Judex & Zernicke (2000). J. Appl. Physiol. 88, 2183–2191. [3] Gross et al. (1992). J. Biomech. 25, 1081–1087. [4] Rubin & Lanyon (1987). J. Orthop. Res. 5, 300–310.

Disclosures: C.J. MacKay, None.

W087

Temporal Expression of PHOSPHO1 During Chick Limb Bud Mesenchymal Cell Differentiation and Mineralization. V. E. MacRae*¹, M. G. Davey*¹, J. L. Milan², C. Farquharson¹. ¹Gene Function and Development, Roslin Institute, Midlothian, United Kingdom, ²Burnham Institute for Medical Research, La Jolla, CA, USA.

PHOSPHO1 is a phosphatase with up-regulated expression in growth plate chondrocytes and osteoblasts, and is implicated in the initiation of inorganic phosphate generation for matrix mineralization. This study has established the temporal expression of PHOSPHO1 during chondrogenesis and endochondral ossification within the developing chick limb bud. This model permits the monitoring of PHOSPHO1 expression during mesenchymal cell differentiation into mineral producing chondrocytes. Micromasses were prepared from stage 24 limb bud mesenchymal cells which were cultured for 0, 3, 7 and 10d. Alcian blue (proteoglycan synthesis) and alizarin red (mineral formation) staining was negligible at 0d, with a marked increase in staining intensity at 7d and 10d. This confirmed that the mesenchymal cultures had differentiated into chondrocytes with subsequent mineralization. mRNA expression of phosphol, tissue non-specific alkaline phosphatase (Akp2) and collagen II was also determined. Phosphol expression was observed at all time points, however a notable increase in expression was seen by 7d. Interestingly, phosphol expression decreased between 7d and 10d, which is in agreement with previous studies that have suggested that PHOSPHO1 is involved in the initial events of mineral formation. Akp2 was expressed at 0d and 3d, with a marked decrease at 7d and 10d. A comparable pattern has previously been reported for alkaline phosphatase activity in chick limb bud cultures. Collagen II was absent at 0d and was upregulated at 3d onwards. Further studies using chick limb tissue at E3.5 (stage 24), E5.5 (stage 28), E6.5 (stage 30) and E10.5 (stage 36) were undertaken. A marked increase in PHOSPHO1 protein expression was seen at E6.5 and E10.5. These data were complemented by increased phosphol mRNA expression in E6.5 and E10.5 compared to E3.5 and E5.5. TNAP protein expression was noted at all developmental stages, with no obvious differences in expression levels. Collagen II gene expression was present from E5.5 onwards. Using whole-mount in-situ hybridisation, Phosphol mRNA expression was observed in chick metatarsi (E6.5) around the mid-shaft of the bone. Immunohistochemical staining of tibia sections (E6.5) revealed that PHOSPHO1 staining was localized to the osteoid (bone collar) and associated periosteal osteoblasts within the mid-diaphyseal region. Some chondrocytes within the rudiment also stained positively for PHOSPHO1. These studies concur with our hypothesis that PHOSPHO1 has a pivotal role in the first phase of the mineralization process.

Disclosures: V.E. MacRae, None.

W088

Mineralization of Murine Mesenchymal C3H10T1/2 Cells in Micromass Culture. R. Roy¹, I. Binderman*², S. Doty*¹, V. Kudryashov*¹, A. Boskey¹. ¹Hospital for Special Surgery, New York, NY, USA, ²Ichilov Hospital, Tel Aviv, Israel.

Chick limb bud mesenchymal cells have been shown to undergo mineralization in micromass culture with the addition of inorganic phosphate (iP) (J Cell Biochem. 84(3):509–19). The murine multipotential mesenchymal cell line C3H10T1/2 udergoes chondrogenic differentiation in micromass cultures when stimulated by bone morphogenic protein-2 (Differentiation. Jan;64(2):67–76). These murine cells were used in micromass culture to determine whether they mineralize similar to the chick system. Cells were expanded in monolayer culture with DMEM supplemented with 10% FBS and 1% antibiotics/antimycotics (AB/AM). For micromass cultures cells were trysinized, counted, and resuspended in Ca free DMEM supplemented with 10% FBS and 1% AB/AM. Cells were plated at a density of 100,000 cells per 10μl spot at the center of 35mm poly-I-lysine coated cell culture dishes to aid in attachment. Cells were allowed to attach at 37°C for 2 hours. Dishes were then flooded with 2mL of Ca free DMEM with 10% FBS and 1% AB/AM along with 100ng/mL of BMP-2. Media was changed 3X/wk and on day 2 of micromass cultures L-glutamine, 25μg/ml ascorbic acid, and 1mM Ca were added to all cultures, while 4mM iP was added to some cultures to promote mineralization. Alizarin red (AR), and von Kossa (VK) stains were performed on whole mount cultures and sections to visualize mineralization. Type I and type II collagen immunohistochemistry was used to determine whether these cultures form calcified cartilage or mineralized bone. VK and AR stains showed mineralization on day 28 in 4mM (iP) cultures, while control cultures showed no mineralization (Fig). In all cultures both type I and type II collagen was present at day 16, 21, and 28. At early time points there was more type II collagen in mineralizing cultures than in control cultures. These results indicate that this murine cell line does mineralize in the presence of exogenous phosphate which has been supported by previous ⁴⁵Ca uptake data, although mineralization may be delayed relative to the chick cultures. This murine culture system is comparable to the chick system and may be more useful for study of gene expression and signaling.

Disclosures: R. Roy, None.

This study received funding from: NIH AR037661.

W089

Alterations in Collagen Structure and Mineral Composition in Calcified Cartilage and Subchondral Bone in a Monkey Model of OA. M. E. Ruppel¹, C. S. Carlson², L. M. Miller³. ¹Biomedical Engineering, SUNY Stony Brook, Stony Brook, NY, USA, ²Dept. of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA, ³National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY, USA.

Osteoarthritis (OA) can be a debilitating disease which is characterized by disorganization and calcification of the collagen matrix in the cartilage and increased turnover and thickening of the subchondral bone. In the present study, collagen structure and chemical composition were studied in the calcified cartilage and subchondral bone in plastic-embedded mid-coronal sections of proximal tibia from cynomolgus monkeys (n = 26 animals; ages 6–30 yrs). These sites contained a range of histological OA lesions, from normal morphology to severe OA. Synchrotron-assisted Fourier transform infrared microscopy (FTIRM) was used to determine the level of mineralization (mineral/protein ratio), carbonate accumulation (carbonate/phosphate ratio), and collagen structure (protein amide band ratio) as a function of tissue compartment and OA severity. Preliminary results showed that the protein amide band ratios were significantly different between subchondral bone and calcified cartilage, suggesting differences in collagen organization between the two tissue compartments; however, the differences were not affected by OA severity. Both the level of mineralization (p = 0.0431) and carbonate accumulation (p<0.0001) were significantly higher in subchondral bone when compared to calcified cartilage. Although the mineralization level of the calcified cartilage increased (p = 0.0037) with increasing OA severity, the subchondral bone remained unchanged. Carbonate accumulation increased significantly in both tissue compartments as a function of OA severity. These compositional changes that are observed as a function of OA severity are consistent with the progression of a “mineralization front” where endochondral ossification occurs and calcified cartilage is replaced by subchondral bone. These findings indicate that calcified cartilage and subchondral bone have distinctly different collagen structure and mineral compositions that are affected by OA severity and could increase stress on the afflicted joints.

Disclosures: M.E. Ruppel, None.

This study received funding from: The Department of Energy (contract DE-AC02-98CH10886) and National Institutes of Health Grant RR14099.

W090

Distribution and Localization of Aluminum in Bone Tissues of Aluminum Osteopathy. Y. Teraki¹, H. Sasakabe*². ¹Internal Medicine, Shinjo Tokushukai Hospital, Mitaka, Japan, ²Internal Medicine, Shinjo Tokushukai Hospital, Shinjo, Japan.

The pathophysiology of the so-called aluminum (Al) osteopathy is yet to be fully clarified. To investigate the dynamic behavior of Al in bone tissues, we have examined aluminum in bone tissue sections by laser scanning confocal microscopy using the staining with lumogalion, a new chelating reagent we have developed. On account of the reported finding that Al is not excreted in urine in the presence of impaired renal function, this study was performed in 20 5/6-nephrectomized rats which were dosed orally or parenterally with an Al preparation for 3–6 months, followed by sacrifice and preparation of bone tissue slides. Thanks to the high detection sensitivity of Al-lumogalion complex, the technique allowed Al identification with a lower detection limit of 19.0 μg/g. Results: The study showed diffuse or dense granular Al deposits in osteoids rather than in the generally believed calcification front. No Al was detected in calcified bone tissues. Furthermore, Z-scans of the stained bone tissues enabled observation of a three-dimensional Al deposition status. The results indicate potential usefulness of the lumogalion stain technique in the diagnosis of Al osteopathy.

Disclosures: Y. Teraki, None.

W091

Role of Cell Death and Osteogenic Activity in Injury-induced Calcification from TNF-alpha Receptor p55-/- p75-/- Mice. Y. Zhao¹, A. L. Urganus*¹, Y. Chen*², P. H. Stern³, A. L. Boskey⁴, L. M. Pachman*⁵, ¹Childrens Memorial Research Center, Northwestern University, Chicago, IL, USA, ²Research Center for Genetic Medicine, Children's National Medical Center, Washington D.C., DC, USA, ³Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA, ⁴Hospital for Special Surgery, Cornell university-Weill Medical College, New York, NY, USA, ⁵Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Background: TNF-α receptor (p55-/-, p75-/-) null mice develop calcifications along with increased inflammation after an injection with cardiotoxin (CTX), a snake venom component. While TNF- α inhibits osteoblast differentiation in vitro and promotes osteoclast formation the mechanism of the pathological mineralization in this mouse model remains unknown.

Objective: To elucidate the potential roles of cell death and formation of osteoblasts and osteoclasts in CTX-induced calcifications.

Materials and Methods: Four-week-old wild type C57B6 mice and TNF-α receptor null mice were injected with CTX into the skeletal muscle (Tibialis anterior) and sacrificed after 3, 7, 14 and 28 days. The muscles were stained for calcifications by von Kossa or calcein pre-labeling. The osteoblast markers, core binding factor alpha 1 (Cbfal), osteocalcin and the osteoclast marker, tartrate-resistant acid phosphatase (TRAP) were stained in the injured tissues after 3, 7 and 14 day treatment. Total RNA was extracted from the muscle for microarray analysis and realtime PCR analysis of Cbfal and Receptor Activator for Nuclear Factor k B Ligand (RANKL) from the 3 day group.

Results: Calcifications were apparent in all of the TNF-α receptor null mice at day 3 and day 7 after CTX administration whereas they were observed in only 29% of wild type mice at day 3 and 7. By day 14, no calcifications were observed in either strain. Microarray result indicated that the anti-apoptotic genes (BCL2A1(+1.51), BCL2L1(+1.8)), pro-apoptotic genes (CASP7(+1.22), BID(+1.8)), Cbfal(+1.7) and TRAP(+1.3) were marginally up-regulated in TNF-α receptor null mice compared to wild type mice. Realtime PCR failed to show difference of Cbfal and RANKL in the injured muscles between the mutant and control mice. Osteocalcin was expressed in mineralized muscle in both strains. TRAP staining was positive in TNF-α receptor null mice at day 7 but was much less at day 14. We speculate that the formation of osteoclasts contribute to the resorption of the minerals.

Conclusion: Cell death and osteoblastic differentiation may lead to muscle calcifications following CTX administration to TNF-α receptor null mice. Osteoclastic resorption may account for the transient nature of the calcifications.

Disclosures: Y. Zhao, None.

This study received funding from: Cure JM.

W092

Msx2 Promotes Late Stages of Chondrocyte Differentiation by Up-regulating Ihh Expression. K. Amano*¹, F. Ichida*², A. Sugita*², K. Hata*², M. Kogo*¹, R. Nishimura², T. Yoneda². ¹Oral and Maxillofac, Osaka Univ Grad Dent, Suita City, Osaka, Japan, ²Biochem, Osaka Univ Grad Dent, Suita City, Osaka, Japan.

A homeobox gene, Msx2 (muscle segment homeobox 2), plays important roles in the development, growth and differentiation of various types of cells and tissues including ectodermal organs, teeth, vascular cells and cancer. Accumulating data also suggest that Msx2 regulates the skeletal development by controlling endochondral ossification and/or membranous ossification. Mice deficient in Msx2 gene exhibit disturbed chondrogenesis with reduced bone formation. However, little is known about the molecular basis by which Msx2 modulates chondrogenesis. In the present study, we investigated the role of Msx2 using murine primary rib chondrocytes. Treatment of chondrocytes with BMP2 up-regulated the expression of Msx2 mRNA along with the differentiation. Overexpression of Msx2 stimulated calcification of primary chondrocytes in the presence of BMP2, whereas Msx2 alone had no effects. We also found that constitutively-active Msx2 (caMsx2; Msx2P148H) more strongly enhanced BMP2-stimulated calcification than wild-type Msx2. Consistent with this, caMsx2 overexpression up-regulated the expression of alkaline phosphatase and type × collagen that were stimulated by BMP2. However, caMsx2 had no effects on type II collagen expression. Furthermore, organ culture experiments using mouse embryonic metatarsals showed that caMsx2 markedly stimulated the differentiation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. The stimulatory effects of Msx2 on chondrocyte differentiation were enhanced by co-overexpression of Smad1 and Smad4, but inhibited by Smad6, an inhibitory Smad of BMP2 signaling. Of note, caMsx2 overexpression enhanced BMP2-induced indian hedgehog (Ihh) expression in mouse primary chondrocytes and Ihh overexpression promoted calcification of primary chondrocytes. Importantly, cyclopamine, a specific inhibitor of hedgehogs, blocked Msx2- or Ihh-induced calcification of primary chondrocytes. In conclusion, our results suggest that Msx2 plays important roles in the regulation of late, but not early, stages of chondrocyte differentiation by up-regulating Ihh expression in collaboration with BMP2/Smad signaling.

Disclosures: K. Amano, None.

W093

Effects of Pro-inflammatory Cytokines on Supressor of Cytokine Signalling-2 (SOCS-2) Expression in the Growth Plate. V. E. MacRae*¹, S. Pells*¹, S. F. Ahmed², C. Farquharson¹. ¹Gene Function and Development, Roslin Institute, Midlothian, United Kingdom, ²Child Health, Royal Hospital for Sick Children, Glasgow, United Kingdom.

Abnormal growth patterns are commonly observed in children suffering from chronic inflammatory diseases, which are associated with the increased production of pro-inflammatory cytokines. Supressor of Cytokine Signalling-2 (SOCS-2) negatively regulates the signal transduction of several cytokines and this study examined the role of SOCS2 in regulating bone growth, and the response of growth plate chondrocytes to proinflammatory cytokines. Initial studies indicated that SOCS2 was expressed by both murine primary chondrocytes and the ATDC5 chondrogenic cell line. Foetal (E14–E18) and neonatal (1 and 9-day-old) primary murine chondrocyte expressed SOCS-2 mRNA at similar levels whereas SOCS-2 protein expression was greater (78%) during chondrogenesis than terminal differentiation. IL-1beta and TNFalpha (both 10ng/ml) exposure for 48h increased SOCS-2 protein expression in primary 1-day-old murine chondrocytes (79% and 65% increase respectively). 7-week-old female mice lacking SOCS-2 (SOCS2-/-) expression were significantly heavier (26%; P<0.0001) and longer (6%; P<0.001) compared to wild-type (WT) mice. The tibiae of the null mice were longer (8%; P<0.001), wider (18%; P<0.001) and had wider growth plates (24%; P<0.001) than WT mice. Proliferative and hypertrophic zones were significantly wider in SOCS2-/- mice compared to WT mice (10% and 14% respectively; P<0.001). The growth of metatarasals from 1-day-old female SOCS2-/- and WT mice over 8-days was inhibited by TNFalpha (47% and 33% respectively; both p<0.001) and IL-1beta (71% and 86% respectively; both p<0.001) by comparable amounts. No effect of genotype was observed. In contrast, matrix gene expression by primary chondrocytes exposed to pro-inflammatory cytokines over 48h was dependent upon the genotype of the host animal. RT-PCR studies revealed that aggrecan expression was notably reduced only in chondrocytes from SOCS2-/- exposed to IL-1beta and TNFalpha. Collagen Type × expression was markedly reduced in chondrocytes from both SOCS2-/- and WT mice whereas collagen Type II expression was not altered by cytokine exposure. This is the first study to show the expression of SOCS-2 in growth plate chondrocytes and its dramatic effect on suppressing skeletal growth. IL-1beta and TNFalpha may mediate their effects through SOCS-2, influencing the growth of children with inflammatory diseases through a local effect at the growth plate.

Disclosures: V.E. MacRae, None.

W094

Hormonal Regulation of Hypertrophic Chondrocyte Apoptosis. S. U. Miedlich*, M. B. Demay.. Endocrine Unit, MGH, Boston, MA, USA.

In vivo studies in genetically modified (Npt2a knockout, VDR knockout and hyp) and dietary manipulated mouse models have demonstrated that hypophosphatemia leads to impaired apoptosis of hypertrophic chondrocytes in the growth plate and plays a critical role in the development of rickets. However, studies in the Npt2a knockout mice demonstrate that the expansion of the growth plate and impaired apoptosis observed at 16 days of age, has reverted to normal by 35 days of age, despite the presence of persistent hypophosphatemia. The transient nature of these findings in the Npt2a null mice strongly suggests the presence of additional factors that modulate the response of hypertrophic chondrocytes to circulating phosphate levels. Thus, we are undertaking investigations in an in vitro model of chondrocyte maturation in order to investigate whether 1,25-dihydroxyvitamin D or parathyroid hormone modulate the effects of phosphate on hypertrophic chondrocyte apoptosis.

The clonal chondrocytic cell line (RCJ3.1C5.18) has been shown to recapitulate the program of chondrocytic differentiation in culture, including acquistion of markers of hypertrophic differentiation and formation of mineralized matrix nodules. To address whether these cells were susceptible to phosphate-mediated hypertrophic chondrocyte apoptosis, they were subjected to 7mM sodium phosphate or 7mM sodium chloride (control) for 18 to 72 hours. The Guava Nexin assay was employed to measure plasma membrane changes associated with apoptosis (Annexin V-PE) and to identify dead cells (7-AAD). An MTT based assay was also used to evaluate mitochondrial activity, a surrogate for cellular viability.

Western analyses were performed to insure that collagen X, a marker of hypertrophic chondrocyte differentiation, was observed in RCJ3.1C5.18 cells after 7–10 days of culture in the presence of ascorbic acid. Treatment of hypertrophic chondrocytes with phosphate (7 mM) resulted in a significant increase in apoptotic as well as dead cells after > 24 hours of treatment. Studies are underway to evaluate whether 1,25-dihydroxyvitamin D enhances the susceptibility of hypertrophic chondrocytes to phosphate-mediated apoptosis in this model. Investigations will also examine the effects of parathyroid hormone on phosphate-induced apoptosis in hypertrophic RCJ3.1C5.18 cells.

These investigations demonstrate that the RCJ3.1C5.18 cell model is an appropriate system in which to investigate the effect of hormones on phosphate-induced apoptosis. Studies of these potential regulatory factors will not only further our understanding of the pathophysiological mechanisms underlying rickets but may also provide insights into the universal regulation of cellular apoptosis.

Disclosures: S.U. Miedlich, None.

This study received funding from: NIH.

W095

A-Raf and B-Raf Are Dispensable for Endochondral Bone Development and PTHrP Suppresses ERK Activation in Hypertrophic Chondrocytes. S. Provot¹, G. Nachtrab*², J. Paruch*², A. P. Chen*³, A. Silva*³, H. M. Kronenberg². ¹Anatomy, UCSF, San francisco, CA, USA, ²Endocrine Unit, MGH - Harvard Medical School, Boston, MA, USA, ³Neurobiology, UCLA, Los Angeles, CA, USA.

Parathyroid hormone-related peptide (PTHrP) and its PTH/PTHrP receptor (PPR) increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development, at least partly through cAMP-dependent signaling pathways. Because some data suggest that the ability of cAMP to stimulate cell proliferation involves the MAPKKK B-Raf, we hypothesized that PTHrP's proliferative action in chondrocytes might be mediated by B-Raf. While B-raf gene expression is detected by in situ hybridization (ISH) in proliferative chondrocytes, its conditional removal in cartilage using a type II collagen promoter-driven cre (col2-cre) and a floxed B-raf allele does not affect chondrocyte proliferation and maturation, as indicated by histologic analyses, BrdU assays, and ISH with various chondrogenic markers on embryonic and postnatal samples. Lack of B-raf does not alter either PTHrP-induced increased chondrocyte proliferation and delayed maturation, both in vivo in mice expressing a constitutively active PPR in cartilage (col2-caPPR), and in vitro in metatarsal explants treated acutely with PTHrP. Similar results were obtained by conditionally removing B-Raf from osteoblasts using Osterix- and coll-cre mice. Because A-raf expression detected by ISH is identical to that of B-raf in cartilage, whereas that of C-raf is restricted to hypertrophic chondrocytes, we speculated that A-Raf might play a redundant function with B-Raf in this tissue. Surprisingly, mice deficient for both A-Raf (universal A-raf knockout) and B-Raf in chondrocytes exhibit normal endochondral bone development, suggesting that the Raf-ERK pathway is not critical for chondrocyte proliferation. Consistent with this idea, the presence of activated ERK detected by immunohistochemistry is essentially restricted to hypertrophic chondrocytes. Notably, acute treatment of bone explants with PTHrP or prolong activation of PPR in col2-caPPR mice suppress ERK activation in these cells, while ERK activation is increased in PTHrP KO mice. Thus, PTHrP signals suppress ERK activation in normal conditions. Taken together, these results demonstrate that B-raf and A-Raf are dispensable for endochondral bone development, and they suggest that ERK might regulate hypertrophic differentiation rather than proliferation in cartilage, possibly through its activation by C-Raf in hypertrophic chondrocytes.

Disclosures: S. Provot, None.

This study received funding from: NIH.

W096

Cell Death and Cell Proliferation of Cartilage Layer in Human Anterior Cruciate Ligament Tibial Insertion after Rupture. H. Mutsuzaki¹, M. Sakane², K. Honda*³, K. Ikeda*⁴, S. Hattori*⁵, N. Ochiai*². ¹Department of Orthopaedic Surgery, Tsukuba Central Hospital, Ushiku, Japan, ²Department of Orthopaedic Surgery, Institute of Clinical Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan, ³Department of Legal Medicine, Doctoral Program in Social and Environmental Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan, ⁴Department of Orthopaedic Surgery, Ichihara Hospital, Tsukuba, Japan, ⁵Department of Agriculture, Ibaraki University, Ami, Japan.

The purpose of this study is to investigate cellular responses of cartilaginous layers in human anterior cruciate ligament (ACL) tibial insertions compared with normal ones. Sixteen tibial insertions of ruptured ACLs were obtained during primary ACL reconstructions. We also obtained sixteen normal ACL tibial insertions from cadavers. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining assay to detect apoptosis, proliferating cell nuclear antigen (PCNA) staining and a histological examination was performed. The percentage of TUNEL-positive chondrocytes in ruptured ACL insertions (43.6 ± 14.8%) was higher than that of normal ones (9.6 ± 5.8%). The percentage of PCNA-positive chondrocytes was not significant deference between ruptured ACL insertions (23.1 ± 26.3%) and normal ones (12.3 ± 7.3%). The average thickness of the cartilage layer, the glycosaminoglycan-stained area and the number of chondrocytes per millimeter in ruptured ACL insertions were lower than that of normal ones. The decrease of chondrocytes because of imbalance of the cell death and the cell proliferation in the ACL insertions after rupture compared with those in normal could lead to histological changes of the cartilage layer in the insertions. The results may help elucidate the etiology of the histological changes, the function and the significant of existence of the ACL insertion.

Disclosures: M. Sakane, None.

W097

Intra-articular Osteoclastogenesis Inhibitory Factor/Osteoprotegerin Prevents Cartilage Degeneration in a Murine Model of Osteoarthritis. S. Shimizu*¹, Y. Asou², S. Itoh*³, U. Chung⁴, H. Kawaguchi⁴, K. Shinomiya*², T. Muneta*¹. ¹Section of Orthopaedic Surgery, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan, ²Department of Orthopaedic Surgery, Tokyo Medical and Dental University, Tokyo, Japan, ³Human Gene and Science Center, Tokyo Medical and Dental University, Tokyo, Japan, ⁴Department of Orthopaedic Surgery, University of Tokyo, Tokyo, Japan.

Osteoarthritis (OA) is a chronic degenerative joint disorder characterized by articular cartilage destruction and osteophyte formation. RANK, RANKL, and Osteoclastogenesis Inhibitory Factor (OCIF)/Osteoprotegerin (OPG) mRNAs and proteins are expressed in normal cartilage. Cartilage from patients with OA contains increased levels of OPG mRNA. However, the functions of OPG expressed during OA pathogenesis are poorly understood. The purpose of this study was to investigate the effect of endogenous and exogenous OPG on chondrocytes during OA pathogenesis in vivo. First, OA was surgically induced in young adult OPG+/- mice and wild-type (WT) littermates to investigate the effect of chondrocyte-derived OPG deficiency. Four weeks after surgically induction of OA, histological evaluation of tibial cartilage demonstrated typical features of cartilage degeneration both in WT and OPG+/- mice. The degenerative changes in the articular cartilage, however, were significantly enhanced in OPG+/- mice compared to WT littermates. Cartilage thickness was significantly reduced in OA-induced OPG^+/− mice. The morphology of subchondral bone structures was not affected by OPG haploinsufficiency. Next, we investigated the effect of exogenously administered rhOPG by daily intraarticular injections following surgical induction of OA. Histological evaluation indicated that OA-induced mice in rhOPG treated group were protected from cartilage degeneration compared to vehicle group four weeks after the operation. Cartilage thickness was significantly preserved in rhOPG administred mice. Subchondral bone volume was not affected by rhOPG treatment, indicating that chondro-protective effect of rhOPG was independent of subchondral bone metabolism. TUNEL assay indicated that rhOPG administration prevented chondrocyte apoptosis in an experimental model of OA. Pro-apoptotic ligand TRAIL, one of the ligand for OPG, was co-expressed with OPG in articular chondrocytes irrespective of OPG administration. These data indicated that endogenous OPG had a protective effect against the mechanical stress-induced cartilage degeneration. Furthermore, direct administration of rhOPG to articular chondrocytes prevented cartilage degeneration via prevention of chodrocyte apoptosis.

Disclosures: S. Shimizu, None.

W098

Mechanisms of Osterix Control of Endochondral Ossification. D. Y. Soung, L. A. Kaback*, A. Naik*, N. Smith*, E. M. Schwarz, R. J. O'Keefe, H. Drissi.. Orthopaedics, University of Rochester, Rochester, NY, USA.

We investigated the expression and regulation of the zinc finger protein Osterix (Osx) during endochondral ossification in the developing embryo. Our in situ hybridization and immunohistochemical analyses of Osx expression showed a restricted expression of Osx transcript and protein to the immature chondro/osteoprogenitor cells and mature osteoblasts but excluded from hypertrophic chondrocytes. Using a fracture injury model we show consistent expression of Osx protein in mesenchymal progenitor cells in the periosteum and immature chondrocytes and osteoblasts embedded in the fracture callus while hypertrophic chondrocytes, vessels and fibrous tissue remain devoid of Osx expression. Additionally, using RNAs isolated from fracture calluses throughout the healing process and achievement of bony unions, we observe that Osx transcripts parallel that of Runx2 and differentially overlap both cartilage and bone phenotypic markers. Furthermore, using the limb bud-derived MLB13MYC Clone 17 cells, we show that PTHrP inhibited chondrocyte maturation while it enhanced mRNA levels of Osx in these chondro/osteoprogenitor cells. Our viral over-expression studies, complemented with siRNA mediated loss of Osx function experiments indicate that Osx serves as an inhibitor of chondrogenesis and chondrocyte terminal differentiation while it promotes osteoblast maturation. Together, our findings provide novel evidence for the molecular mechanisms underlying Osx inhibition of chondrocyte differentiation, and further suggest a role for this transcription factor in promoting endochondral ossification during bone repair.

Disclosures: D.Y. Soung, None.

This study received funding from: HIAMS/NIH.

W099

Epigenetic Modulatory Agents Suppress the Chondrocytic Phenotype. M. Stewart¹, E. Caporali*¹, A. Stewart*¹, P. Jones*². ¹Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA, ²Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Epigenetic processes impact gene transcription by directly altering DNA/chromatin structure, resulting in transcriptional repression. There is increasing recognition that genes active in chondrocyte differentiation, proliferation and arthritis are subject to epigenetic control, however epigenetic regulatory processes can have global impacts on cellular transcriptional activity. This study was carried out to assess the effects of epigenetic-modifying agents on the articular chondrocytic (AC) phenotype.

Cartilage was collected from equine articular joints and murine epiphyses. Chondrocytes were cultured as explants or isolated by collagenase digestion and cultured as non-adherent aggregates. 5-azacytidine (5AZA) and zebularine (ZEB) were administered to inhibit DNA methylation, while valproic acid (VA) and Trichostatin A (TSA) was used to block histone deacetylation. The phenotypic effects on these epigenetic modifying reagents on the chondrocytic phenotype were assessed by Northern blot analyses of Coll II and aggrecan mRNAs, DMMB measurement of sulfated GAG secretion and Col II ELISA assays.

Treatment of chondrocyte aggregate cultures with epigenetic modulating reagents resulted in profound suppression of Coll II and aggrecan mRNA levels, reduced sulfated GAG secretion and Col II deposition. Similar effects were noted in cartilage explants. Consequent experiments showed these profound effects were reversible, with return to control levels within 3 days of 5AZA or VA removal. In contrast, chondrocytes treated continuously for 10 days of the experiments showed persistent transcriptional suppression of chondrocyte-specific genes. In contrast to previous reports, no Coll × mRNA was detected in articular chondrocytes treated with 5AZA or co-treated BMP-2.

These data show that epigenetic-modifying agents have profound but reversible effects on the chondrocytic phenotype. The conventional effect of these reagents is to [de-repress' genes normally silenced epigenetically. Inhibition of AC-specific genes could follow unmasking of repressive elements in the gene promoters or by activation of genes that repress the AC phenotype. The effects might also result from non-epigenetic pharmacological actions; however multiple epigenetic reagents induce similar effects on two distinct AC-specific genes, arguing against this possibility.

Disclosures: M. Stewart, None.

This study received funding from: Arthritis Foundation.

W100

Runx2 Haploinsufficiency Ameliorates the Ectopic Calcification in a Mouse Model of Ossification of the Posterior Longitudinal Ligament (OPLL). S. Takeda¹, M. Iwasaki*², S. Sato*¹, A. Kimura*¹, H. Inose*¹, K. Shinomiya*¹. ¹Department of Orthopedics and 21COE, Tokyo Medical and Dental University, Tokyo, Japan, ²Department of Orthopedics, Tokyo Medical and Dental University, Tokyo, Japan.

Ossification of the Posterior Longitudinal Ligament (OPLL) is a disease characterized by the ectopic calcification of the ligament, but its pathogenesis remains to be addressed. Histologically, calcification in OPLL is characterized by the existence of chondrocytes and bone marrow, suggesting that it is caused by endochondral bone formation-like mechanism. In an effort to elucidate the molecular pathogenesis of OPLL, we focused on Runx2. Runx2 is an essential transcription factor for osteoblastogenesis, whose deficiency causes the complete lack of endochondral bone formation. Induction of Runx2 expression was demonstrated in the ectopically-calcified ligaments of ttw mice, a mice model of OPLL, by monitoring the lacZ activity inserted into the Runx2 genomic locus. To address the functional role of Runx2 in the promotion of OPLL, we crossed Runx2 heterozygote-deficient mice with ttw mice. Analyzing the area of calcification in the posterior longitudinal ligament by micro-computed tomography revealed that ttw mice with Runx2 haploinsufficiency carry less calcification than ttw mice without Runx2 haploinsufficiency. Histologically, reduction of mineralized bony structures and bone marrow stroma was observed in ttw mice with Runx2 haploinsufficiency; Runx2 haploinsufficiency ameliorated the ectopic calcification in ttw mice. Taken together, our results demonstrated that Runx2 is intimately involved in the cause of OPLL in vivo.

Disclosures: S. Takeda, None.

W101

Transcriptional Induction of SOX9 by NF-kB Subunit p65 During Chondrogenesis. M. Ushita, T. Ikeda, T. Saito, A. Kan, K. Nakamura, U. Chung, H. Kawaguchi.. Sensory and Motor System Medicine, University of Tokyo, Tokyo, Japan.

Although SOX9 is an essential molecule for chondrogenic differentiation from mesenchymal cells, little is known about the upstream signaling. The present study attempted to identify transcription factors to induce SOX9 expression and examined the mechanism. By exhaustive comparison of the genomic sequences between human and mouse SOX9 proximal promoters, we identified several highly conserved regions within which NF-kB, NFAT, and CREB/ATF motifs were found as putative transcription factor binding sites. We then created expression vectors of transcription factors and co-factors for these sites: 11 for NF-kB, 5 for NFAT, and 11 for CREB/ATF, and transfected them in mouse chondrogenic ATDC5 cells and non-chondrogenic HeLa cells with a luciferase-reporter construct containing a human 1,000 bp SOX9 promoter. Among them, an NF-kB subunit p65 most strongly activated the luciferase activity. Deletion analysis using a series of 5′-deletion constructs of the SOX9 promoter confirmed that the core responsive element to p65 included the estimated NF-kB motifs located around −250 bp relative to the transcriptional start site. The transcriptional activity of p65 was decreased by a site-directed mutagenesis in the NF-kB motif, and the tandem-repeat constructs responded to p65 depending on its repeat number. Electrophoretic mobility shift assay revealed specific binding of the in vitro-translated p65 protein with the wild-type NF-kB motif oligonucleotide probe, but not with the mutated probe. Cold competition with an excess of the unlabelled probe, but not with the unlabelled mutated probe, cancelled the complex formation. In addition, the complex underwent supershift by the antibody to p65, confirming the specific binding of p65 and the NF-kB motif. In vivo expression of p65 was shown by immunohistochemistry to be co-localized with SOX9 in the resting and proliferative chondrocytes of the mouse growth plate. To further investigate the functional relevance of p65 to chondrogenic differentiation, we established HeLa cells or mouse primary costal chondrocytes overexpressing p65. Expressions of type II collagen and SOX9 were markedly increased in both cells by the overexpression as compared with respective GFP-transfected control cells. In conclusion, we identified NF-kB/p65 as a potent transcriptional signaling for SOX9 promoter activation and chondrogenic differentiation. Elucidation of the molecular network related to the SOX9 - NF-kB/p65 axis will greatly benefit development of the regenerative medicine of cartilage.

Disclosures: M. Ushita, None.

W102

Tissue Specific Expression of Murine Aggrecan Is Regulated Through Both Regulatory Enhancer and Repressor Elements. N. A. Wigner, T. A. Einhorn, L. C. Gerstenfeld.. Orthopaedics, Boston Univeristy School of Medicine, Boston, MA, USA.

Aggrecan deposition is a hallmark of the mature chondrocyte. Thus the analysis of aggrecan's unique temporal and spatial expression regulation can extend current understanding of cartilage specific transcriptional control. Previous studies, using collagen type II as a model gene, have identified a complex transcription network during skeletal tissue development. As a result, transcription factors such as Sox9, Runx2, and Snail1 have been shown to regulate collagen type II expression and have been implicated in chondrogensis. However, few studies have explored the cis-acting elements and the interacting transcriptional regulators of other cartilage extracellular matrix proteins such as aggrecan. The goal of these experiments was to characterize the cis-transcriptional regulatory motifs that mediate aggrecan expression during chondrogenic differentiation and to identify the temporal interaction of transcription factors that impart cartilage specific expression. The 5′ proximal region of the aggrecan gene and various permutations of it's promoter and 5′UTR were cloned into luciferase reporter vectors and assessed by transient transfection into both chondrogenic and non chondrogenic cell lines and C3H10T1/2 stems cells induced to undergo chondrogenic differentiation. The temporal and functional nature of specific cis-elements was further assessed by electromobility shift analysis. During the sequential pattern of chondrogenic differentiation, an increased expression of Sox9 was accompanied with a decrease in Runx2 and Snail1. Concomitant with this, was an increase in aggrecan mRNA. We have identified regulatory regions within the aggrecan promoter and 5′UTR that mediate it's tissue specific regulation. This study has shown aggrecan's 5′UTR to be a robust tissue-specific regulator of expression. In undifferentiated chondrocytes, placing this 5′UTR immediately downstream of both it's own proximal promoter and the promoter of collagen type II, enhanced reporter activity by 5.6-fold and 3.4-fold relative to their respective basal promoters alone. In addition, the presence of the 5′UTR was necessary to maintain observable reporter activity in differentiated chondrocytes. Co-transfection of the aggrecan reporters with Sox9 resulted in an enhanced reporter activity by 1.6-fold relative to controls, although the presence of the 5′UTR did not mediate this effect. Thus, the potential identification of both cis and trans acting factors involved in the regulation of aggrecan can provide insight into the regulation of cartilage as a whole and identify key molecular switches mediating cell differentiation.

Disclosures: N.A. Wigner, None.

W103

Cartilage Extracellular Matrix Homeostasis Is Regulated by RARy Repression Function. J. A. Williams*¹, N. Kondo*¹, N. Takeshita*², T. Okabe*¹, M. Enomoto-Iwamoto*¹, M. Pacifici¹, M. Iwamoto¹. ¹Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA, USA, ²Orthopaedic Surgery, Tohoku University, Sendai, Japan.

Cartilage extracellular matrix is maintained at homeostasis in the upper regions of growth plate and is rapidly degraded in hypertrophic zone, but the mechanisms underlying such critical metabolic shift are not fully understood. We previously showed that transcription factor RARγ is strongly expressed in the upper growth plate zones and mice lacking RARγ and another family member exhibit dwarfism and low cartilage matrix content. RARs are known to serve as transcriptional repressors and activators depending on absence or presence of ligands, respectively. Given that the upper growth plate zones contain scanty endogenous retinoid levels if any, we asked whether RARγ exerts ligand-independent repressor function and contributes to matrix homeostasis. Primary rib chondrocytes in monolayer culture were treated with a retinoid pan-agonist or pan-antagonist. As expected, agonist treatment markedly increased matrix catabolism and MMP gene expression compared to untreated control cultures. However, treatment with antagonist inhibited endogenous matrix catabolism and MMP expression to levels below control. The same was seen when RARE reporter activity was measured; the agonist increased it above control levels, whereas the antagonist reduced it to below control levels. To determine whether these responses were attributable to RAR function, we compared double-floxed and double-null RARβ/RARγ chondrocytes. The former behaved just as wild type cells, whereas the double-null chondrocytes failed to respond to either agonist or antagonist. The zinc finger transcription factor Zacl modulates nuclear hormone receptor function and is co-expressed with RARγ in cartilage. We found that Zacl over-expression enhanced RARγ repressor activity in RARα/RARβ double-null chondrocytes that express RARγ only. Mammalian two hybrid tests showed that Zacl physically associates with RARγ and such association is lost when the LxxLL motif of Zacl is mutated. The results of our study provide the first evidence that RARγ acts as a ligand-independent repressor in cartilage matrix homeostasis and may do so in strict cooperation with Zacl.

Disclosures: J.A. Williams, None.

W104

Phosphate Regulates Chondrocyte Differentiation in a model of Embryonic Endochondral Bone Formation. A. A. Zalutskaya, M. Cox*, M. B. Demay.. Endocrine Unit, MGH, Boston, MA, USA.

Phosphate is required for terminal differentiation of hypertrophic chondrocytes during postnatal growth plate maturation. To investigate whether extracellular phosphate plays a role during embryonic endochondral bone formation, we performed investigations in the mouse metatarsal culture model. This model recapitulates in vivo bone development in culture and circumvents the homeostatic changes that occur in the intact animal.

Metatarsals were isolated from day 15.5 C57BL6/J embryos and cultured in phosphate-free high glucose DMEM, 1% Pen/Strep, 0.05mg/ml ascorbic acid and 0.25% heat-inactivated FBS. The estimated phosphate content of this basal medium is 0.3 μM. Three culture conditions were used to address the effect of extracellular phosphate on chondrocyte differentiation: 0 mM added phosphate, 1.25 mM added phosphate (control) and 7 mM added phosphate. Metatarsals were cultured for 4, 8 and 12 days at 37°C, 5% CO₂. The length of each metatarsal was measured at the time of isolation, as well as at the end of the 12 day culture period. At the end of the culture period, frozen sections were obtained to permit in situ hybridization analyses with digoxigenin-UTP labeled probes. Metatarsals cultured in 1.25mM phosphate demonstrated a 120 ± 6.66% increase in length over 12 days. While the absence of supplemental phosphate impaired growth, as did 7mM added phosphate, there was still a significant increase in the length of the metatarsals cultured under these conditions over a 12 day period (90 ± 10.18% and 70± 2.59% increase respectively). In situ hybridization analyses did not reveal a significant change in the expression of Sox 5 or Sox 6, early markers of chondrocyte differentiation, among the three phosphate concentrations. However, analyses of later markers of chondrocyte differentiation, including Col II, Col × and osteopontin demonstrated an attenuation of chondrocyte differentiation in metatarsal elements cultured under hyper- or hypophosphatemic conditions, relative to that observed in those cultured under normophosphatemic conditions. While, von Kossa staining demonstrated significant mineralized matrix formation in the metatarsals cultured under control phosphate conditions, there was marked impairment in mineral deposition in the elements cultured without added phosphate and with 7mM phosphate. Thus, extracellular phosphate has a significant effect on the differentiation of chondrocytes in the metatarsal culture system. The effects of phosphate are biphasic: both high and low phosphate attenuate growth and impair chondrocyte differentiation. Studies are underway to determine the molecular basis for these effects.

Disclosures: A.A. Zalutskaya, None.

W105

The Role of Nkx3.2 in Muscle/Cartilage Cell Fate Determination. D. Cairns*, L. Zeng*., Anatomy and Cell Biology, Tufts University School of Medicine, Boston, MA, USA.

Significance. Bone and muscle are distinct tissues that are situated adjacent to each other in vertebrates. Failure to establish and maintain their identities may lead to serious genetic diseases such as fibtodysplasia ossificans progressiva (FAP), where a mutation in the BMP receptor causes the muscle tissue to be transformed into cartilage and bone. The mechanism of how BMP signaling leads to this transformation is not fully understood.

Results. In the embryo, BMP pathway is crucial for inhibiting muscle cell fate and promotes cartilage cell fate determination. Nkx3.2 is a BMP-induced transcription factor that is expressed exclusively in cartilage progenitors and is absent in the muscle progenitors. Prior work has demonstrated that Nkx3.2 promotes Sox9 expression and chondrocyte differentiation and survival. Here we show that in vivo Nkx3.2 ectopic expression in the progenitors of the back muscle (via electroporation of the somite) prevents the expression of Pax3, Pax7 and terminal muscle marker myosin. In addition, virally-expressed Nkx3.2 prevents muscle marker expression in the chicken limb micromass culture. Furthermore, C2C12 cells that are transfected with Nkx3.2 fail to differentiate into myotubes upon serum deprivation. Conclusion. Our work demonstrates that BMP-induced factor Nkx3.2 plays a critical role in muscle/cartilage cell fate decision in the embryo.

Disclosures: L. Zeng, None.

This study received funding from: Arthritis National Research Foundation.

W106

Localization of the Cis-enhancer Element for Mouse Col10a1 Expression in Hypertrophic Chondrocytes In Vivo. O. Zheng¹, B. Keller*¹, G. Zhou*², D. Napierala*¹, Y. Chen*¹, B. Zabel*³, A. Parker*⁴, B. Lee⁵. ¹Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, ²Orthopedics, Case Western Reserve University, Cleveland, OH, USA, ³Center of Pediatrics and Adolescence Medicine, University Hospital of Freiburg, D-79106, Germany, ⁴Respiratory and Inflammation Research Area, Astrazeneca, Cheshire, United Kingdom, ⁵Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX, USA.

We and others had previously shown that 4kb or 4.6kb Col10a1 promoter containing Runx2 or AP-1 (Activator Protein-1) elements contribute to its hypertrophic chondrocyte-specific expression in vivo (Zheng et al., 2003; Gebhard et al., 2004). These data suggest that the Col10a1 distal promoter (-4.4 to −3.8 kb) harbors a critical enhancer that mediates its tissue specificity. To further localize the tissue-specific enhancer element, we have generated series of transgenic reporter constructs containing 600, 300 or 150 bp of fragments derived from this region upstream of the Col10a1 basal promoter driving LacZ gene. The results demonstrated that 150bp Col10a1 promoter element (-4.3 to −4.15 kb) is sufficient to direct its tissue-specific expression in vivo. In silico analysis of this region identified several transcription factor binding sites including two potential AP-1 sites within 5′- and 3′- end respectively. Interestingly, transgenic mice using reporter construct without the two putative AP-1 elements showed tissue-specific reporter activity. We have also performed EMSA studies and specific DNA/Protein complexes were observed when oligos derived from this region were incubated with the MCT cell nuclear extracts. In vitro transfection studies using reporter constructs derived from these oligos showed up-regulated reporter activity. These data together with previous observations suggest that there are transacting factors in addition to Runx2 or AP-1 that may bind the cis-elements within this Col10a1 distal promoter and mediate its tissue-specific expression in vivo.

Disclosures: Q. Zheng, None.

This study received funding from: NIH, Arthritis Foundation.

W107

The Diverge Effects of Noggin on BMP-2-induced Osteogenesis. T. Engstrand*¹, C. Aulin*², J. Hilborn*². ¹Department of Reconstructive Plastic Surgery, Karolinska Institute, Stockholm, Sweden, ²Department of Materials Chemistry, Uppsala University, Uppsala, Sweden.

The purpose of the study was to evaluate the in vitro-effects of noggin on BMP-2 in mesenchymal cells and the in vivo-effects of transplanted mesenchymal cells co-expressing noggin and BMP-2.

The W20-17 murine mesenchymal celline was retrovirally infected with either BMP-2 cDNA or co-infected with BMP-2 and noggin cDNAs. The expression and production of BMP-2 and noggin was determined by mRNA dot blot and western blot, respectively. The in vitro effect was measured by alkaline phosphatase activity. Ectopic bone formation induced by transplanted BMP-2- or bmp-2/noggin- overexpressing cells was visualised by radiography and histology.

The amounts of secreted BMP-2 in culture medium was dramatically enhanced in BMP-2/ noggin co-expressing cells as compared to cells expressing BMP-2 alone (control). The co-expressing cells induced a 5-fold increase in ALP activity as compared to the control in vitro. The enhanced effects in co-expressing cells was verified in vivo where transplanted BMP-2/noggin cells induced aboundant bone formation. However, the enhanced osteoinductive effect of BMP-2/noggin co-expressing cells was found to be concentration-dependent since noggin exerted a inhibitory effect on BMP-2 when expressed at higher rate. Using added recombinant BMP-2 and noggin we demonstrate that noggin had a stabilizing effect on BMP-2 as determined by ALP-activity in long-term cell cultures.

Noggin exerts an inhibitory effect by direct binding to BMP-2. Unexpectedly, we have found that noggin enhances the secreted amounts of BMP-2 in osteoprogenitor cells co-expressing noggin and BMP-2. These cells also demonstrate an increased osteoinduction in vitro and in vivo as compared to cells expressing BMP-2 alone. The positive effects of noggin on BMP-2 osteoinduction was dependent on the relative concentrations of BMP-2 and noggin since higher noggin expression completely inhibited BMP-2 action. Our results suggest that noggin stabilizes BMP-2 and functions as a BMP-carrier. The mechanisms of the dissociation of BMP-2 from noggin still remain to be solved.

Figure legend:. Transplanted noggin/BMP-2-overexpressing cells induced aboundant bone formation when implanted ectopically in rats (III) as compared to cells expressing BMP-2 alone (II). Cells transduced with vector alone had no activity in this assay (I).

Disclosures: T. Engstrand, None.

This study received funding from: Vinnova/SSF.

W108

In vitro Characterization of Bidirectional Tet-inducible Expression Systems for Bone Tissue Engineering. G. Feichtinger*, B. Klösch*, M. van Griensven*, H. Redl*., Austrian Cluster of Tissue Regeneration, Ludwig Boltzmann Institute for exp. and clin. Traumatology, Vienna, Austria.

Gene medicine approaches for osteoinduction could provide potent alternatives to current growth factor or stem-cell based approaches being cheaper and safer. The additional employment of inducible promoters could maximize safety of these approaches. Furthermore inducible systems that express more than 1 therapeutic gene can be useful regarding differentiation processes in which more than 1 gene is required to generate a sufficient impulse. Therefore, the aim of this study was to characterize a Tet-inducible system for bidirectional expression of hBMP2 and 7, hBMP4 and 7 or Runx2 and Osterix. The TetON-system (TREtight-BI) was modified to express the reverse tet-transactivator (rtTA) on the response vector making it a single vector system. This new system mediates inducible expression of 2 different genes of interest and constitutive expression of the rtTA in only 1 plasmid making it more convenient for practical use. In vitro characterization was carried out in C2C12 cells transfected with the described TetON systems at different doxycycline (doxy) concentrations (0; 50–1000ng/ml). Osteogenic differentiation was detected by alkaline phosphatase, RT-PCR and von Kossa staining (day 10). Time and dose dependent kinetics were studied for 14 days using EYFP and dsRED. Reportergene expression levels were detected by fluorescence microscopy and RT-PCR compared to constitutive promoters (CMV, human ubiquitin C promoter).

Osteogenic differentiation was confirmed for all therapeutic expression systems. These new single vector systems did not exhibit lower expression capacity as the original double vector system. The maximal level of expression was comparable to constitutive promoters. No expression was detectable at 0ng/ml doxy. There was initial expression activation 12 hours after doxy application in a dose dependent manner decreasing within 14 days.

Our in vitro data strongly supports the potential of expression systems to induce differentiation in targeted cells. We conclude from our in vitro results that the constructed single vector, bidirectional inducible expression systems provide a promising alternative to currently employed growth factor therapies in bone tissue engineering. Furthermore expression of two therapeutic genes generates more potent responses compared to single gene approaches. The TRE element together with the rtTA is a reliable and stringent inducible expression system. The major concerns for future in vivo application of the described systems are efficient non-viral in vivo transfection methods and nephrotoxic features at certain doxy concentrations.

Disclosures: G. Feichtinger, None.

W109

Identification of Novel Factors Associated with Osteoblast Differentiation. M. S. Friedman¹, W. Luo*², S. M. Oyserman*¹, H. Shitaye*³, P. J. Woolf*², K. D. Hankenson¹. ¹Animal Biology, University of Pennsylvania, Philadelphia, PA, USA, ²Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA, ³University of Michigan, Ann Arbor, MI, USA.

A variety of signaling molecules, transcription factors, and extracellular matrix (ECM) proteins are known to be involved in osteoblast differentiation. How these factors interact in human mesenchymal stem cells (hMSC) to yield functional osteoblasts is poorly understood. We hypothesized that by using high-throughput expression analysis of hMSC we could identify individual genes, gene expression patterns, and transcriptional networks associated with osteoblast differentiation and function. We examined how BMP treatment duration correlated with osterix and Dlx-5 expression. hMSC treated with BMP6 (20 nM) for one hour and cultured for 4 days without BMP6 show increased DLX5 and osterix expression (6 and 100 fold, respectively). However, only MSC treated with BMP for a minimum of 24 hours and cultured for 14 days without BMP, demonstrate terminal osteoblast differentiation and function (mineralization). Using this preliminary expression and function data, we designed a novel growth factor induction strategy coupled with microarray analysis. RNA was harvested following treatment, or cells were further cultured without BMP for 24h, 96h, or 10d and RNA harvested (13 Affymetrix U133+ microarrays). Genes were sorted into 20 clusters based on BMP treatment duration, gene expression levels, and gene expression patterns. The transcription factors DLX2, 3, 5, ID1, 3, and 4, and SMAD5, and 6 clustered together and were highly elevated with only 8 hours of BMP treatment and remained elevated for 10 days without additional BMP. However, expression of these genes did not correlate with osteoblast function (increased expression with increased BMP treatment). In contrast, expression of Osterix and the Notch associated transcription factors HEY1 and HEY2 correlated with osteoblast function. Osteopontin, BSP, aggrecan 1, syndecan 1, osteomodulin, and GPM6B, clustered with these transcription factors, showing expression patterns associated with osteoblast function. Results for select genes were validated by Q-PCR. To evaluate if genes in these clusters were involved in osteoblast function, we performed overexpression and loss of function studies. Importantly, antagonism of Notch signaling by gamma secretase inhibitors (L685,458) greatly inhibits BMP induced mineralization, suggesting a role for Notch signaling in BMP induced osteoblast differentiation. In contrast, neither osterix nor osteomodulin are sufficient for osteoblast induction. In conclusion, by using a novel functional microarray analysis, we identified novel factors that may be critical to osteoblast differentiation and function.

Disclosures: M.S. Friedman, None.

W110

Bone Morphogenetic Protein-4 and −6 Are Over-Expressed in the Multiple Myeloma Bone Marrow Samples. D. Grcevic*¹, R. Kusec*², A. Luki*¹, I. K. Lukic³, N. Kovacic³, D. Nemet*⁴, A. Marusic*³. ¹Department of Physiology and Immunology, Zagreb University School of Medicine, Zagreb, Croatia, ²Department of Hematology, Clinical Hospital “Dubrava”, Zagreb University School of Medicine, Zagreb, Croatia, ³Department of Anatomy, Zagreb University School of Medicine, Zagreb, Croatia, ⁴Department of Hematology, Clinical Hospital “Zagreb”, Zagreb University School of Medicine, Zagreb, Croatia.

Multiple myeloma (MM) is a B-lymphocyte neoplasia characterized by the slow proliferation of plasma-cells in bone marrow (BM) and bone destruction. Although several bone morphogenetic proteins (BMP) have been shown to elicit apoptosis of myeloma cells in vitro, we proposed that BMPs may have other non-apoptotic and pro-survival effects.

BM was obtained from MM patients (n = 25) and control subjects (n = 14) after the informed consent and approval of the Ethical Board. Selected BM MM samples were magnetically separated for CD138 positive cells. Primary MM samples and myeloma lines (NCI H-929 and Thiell) were cultured for 48 hours and treated by recombinant human (rh)BMP-2 or −6 (500 ng/mL for both) with or without BMP inhibitor rhNoggin. cDNA was extracted from mononuclear BM cells or cell lines, amplified by quantitative PCR using TaqMan assays for BMPs, corresponding receptors and downstream regulatory molecules, and normalized to GPDH. For detection of apoptotic and dead cells, we used flow cytometric annexin V/propidium iodide staining. ELISA assays for p53, Bax and Bcl-2 were performed on cultured cell lysates.

Expression of BMP-4, −6 and Alk-2 was significantly higher in MM BM samples compared with control BM (around 10-fold for BMP-4, 35-fold for BMP-6, 2-fold for Alk-2), with significant positive correlation of BMP-6 expression and plasma-cell percentage in MM samples (p<0.01). Upon in vitro BMP-2 or −6 treatment, insignificant changes in the percentage of death/apoptotic cells were observed paralleled by down-regulation of pro-apoptotic Bax and p21 (up to 2-fold and 5-fold respectively), and up-regulation of inhibitors of differentiation ID-1 and −2 (up to 200-fold and 5-fold respectively). Changes induced by BMPs were blocked by the addition of Noggin. ELISA confirmed the involvement of p53 pathway and decrease in the expression of Bax. Selected CD138 positive myeloma cells showed relatively higher endogenous expression of BMPs/receptors compared with corresponding full BM samples and CD138 negative population, providing a basis for autocrine action.

We found that BMP-4 and −6 are over-expressed in MM BM samples and selected CD138 positive myeloma cells, indicating the role of BMPs as proliferative and survival autocrine factors in MM. Our study opened the possibility to further test clinical application of BMP inhibitors in MM treatment.

Disclosures: D. Grcevic, None.

W111

Similar Effects of Proteasome Inhibition on Osteoblast Differentiation and Anagen Induction. G. E. Gutierrez¹, I. R. Garrett², W. Gallwitz*², G. Rossini*², C. Christiansen³, G. R. Mundy¹. ¹Bone Center and Dept of Medicine/Clinical Pharmacology, Vanderbilt University and Medical Center, Nashville, TN, USA, ²OsteoScreen, LTD, San Antonio, TX, USA, ³Center for Clinical and Basic Research, Ballerup, Denmark.

We have noticed that the mammalian bone remodeling process shares many features in common with the hair cycle. In both, there are phases of growth, resorption or regression, and rest. Moreover, the regulatory controls of both involve many of the same extracellular and intracellular factors. For example, both anagen (growth phase of the hair cycle) and the bone formation phase of the bone remodeling cycle are controlled by the BMP-2 ligand-signal transduction pathway and downstream molecules such as Wnt and beta-catenin. Therefore, we hypothesized that manipulation of specific molecules in this pathway should increase both bone formation and anagen induction. Accordingly, we examined the effects of compounds that stimulated transcription of the BMP-2 gene for their effects on osteoblasts and the hair follicle. We have shown that inhibitors of the proteasome stimulate osteoblast differentiation and bone formation via this mechanism. During studies to determine the effects of proteasome inhibition on bone growth in vivo, we injected proteasome inhibitors (PI) into the subcutaneous tissue over murine calvaria. There was increase in bone formation and marked morphologic changes in hair follicles, with an increase in size, in the inner and outer root sheath diameters and extension into the dermal adipose tissue, all characteristic features of the anagen phase of the hair cycle. All PI tested had similar affects, independent of their chemical structure. Other non-proteasome anti-proteolytic agents had no effect. To confirm that the PI were stimulating hair growth, we examined the effects of the PI PSI on hair on the right dorsal trunk after depilation. There was a rapid increase in hair growth in the treated mice. We next examined PI on hair follicles in explants of murine skin, assessed by changes in hair follicle diameter and hair elongation. rhBMP-2 (100ng/ml) increased follicle area in these cultures. We found that increase in follicle area stimulated by both PSI and epoxomicin was inhibited by noggin, indicating that these effects are BMP-dependent. We then determined the effects of proteasome inhibition on anagen induction in subjects with male pattern baldness. Topical administration of the proteasome inhibitor PSI led to a 40% increase in anagen induction in 8 subjects over a two week treatment period.

These results point to the similarities between the bone remodeling process and the cycling of the hair follicle and suggest both processes are responsive to proteasome inhibition and are dependent on the BMP-2 ligand-signal transduction pathway.

Disclosures: G.E. Gutierrez, Neosil 1.

W112

Increased Expression of BMP Antagonists During Distraction Osteogenesis in a Mouse Model. T. Hague*¹, F. Hamadeh*¹, N. Alam*¹, M. Kotsiopriftis*², D. Lauzier*¹, R. St-Arnaud*¹, V. Rosen*³, R. C. Hamdy¹. ¹Orthopaedics, Shriners Hospital for Children, Montreal, PQ, Canada, ²Orthopaedics, Montreal Childrens Hospital, Montreal, PQ, Canada, ³Oral and Developmental Biology, Harvard School of Dental Medicine, Boston, MA, USA.

Distraction osteogenesis (DO) is a widely used technique for limb lengthening and replacement of bone loss due to trauma, infection or malignancies. The procedure involves gradually applying controlled distraction of two bony segments post osteotomy resulting in the generation of new bone within the distracted gap. Although the technique is widely used, one of its limitations is the long period of time required for the newly formed bone to consolidate. It has previously been shown that the application of bone morphogenetic proteins (BMPs) can increase bone formation during DO, however, exogenous BMPs have many drawbacks. An alternative method for accelerating the rate of bone formation may be to modulate the intrinsic BMP signaling pathway. The aim of this study was to identify BMP antagonists involved in distraction osteogenesis in order to determine if targeting BMP antagonists can be used for increasing BMP signaling and hence the rate of bone consolidation during DO.

DO was applied to the right tibia of 60 adult wild type mice. The surgery involved applying a mini ilizarov fixator and performing an osteotomy in the centre of the tibia. Distraction began after a latency period of 5 days at a rate of 0.2mm/12hrs for 2 weeks. Mice were sacrificed in groups of 12 during the following times post surgery: day 5 (latency), days 11 and 17 (distraction) and days 34 and 51 (consolidation). Specimens were examined using x-rays, μCT, histology, real-time PCR and immunohistochemistry. The expression of the extracellular BMP inhibitors: BMP3, Noggin, Gremlin and Chordin; intracellular BMP inhibitors: Smad 6, 7, Smurf 1, 2; as well as the receptor level inhibitor: BAMBI was analyzed. In addition, the expression of the BMP ligands 2, 4, 6 and 7 and their receptors were also monitored.

Results revealed an increase in BMP 2, 4 and 6 during the distraction phase. Most interestingly, a significant increase in the expression of BMP3, noggin, chordin, BAMBI, Smad 7 and Smurf 1 were observed. Activin receptor expression was also upregulated in the distraction and consolidation phases. Altogether, our results suggests that regulation of BMP signaling through noggin as well as BMP3 and Activin receptors may be important in the distraction system.

The current study provides a clearer understanding of the genes involved in DO. According to the results, future studies should be targeted at blocking specific intrinsic BMP inhibitors as a means for accelerating the rate of bone formation during DO.

Disclosures: T. Haque, None.

W113

Regulation of Mouse BAMBI Gene Expression. T. Kondo, R. Kitazawa, K. Mori, S. Kitazawa.. Division of Pathology, Kobe University Graduate School of Medicine, Kobe, Japan.

BMP and activin membrane-bound inhibitor (BAMBI) is a transmembrane glycoprotein structurally similar to the transforming growth factor (TGF)-β family type I receptor, except that it lacks an intracellular kinase domain. As a pseudoreceptor, BAMBI interacts with many of the type I and II TGF-β receptors and functions as a negative regulator of TGF-β signaling. To elucidate the role of BAMBI during skeletogenesis and osteoblastic differentiation, we first immunohistochemically analyzed the localization of mouse BAMBI expression in mouse bone tissue. BAMBI expression was seen in some of periosteal osteoblasts, in chondrocytes in articular cartilage and in hypertrophic chondrocytes in the growth plate. Most of the osteocytes embedded in trabecular bone were, however, negative for BAMBI. In vitro studies revealed that BAMBI mRNA expression increased 95-fold in recombinant human bone morphogenetic protein 2 (rhBMP-2)-treated C2C12 myoblastic cells induced into the osteoblastic lineage. Furthermore, BAMBI knockdown by siRNA increased alkaline phosphatase and Runx2 expression in ST2, suggesting that BAMBI expression plays an important role as part of the negative feedback loop system in TGF-β/BMP signaling during osteoblastic differentiation. Additionally, transient transfection studies with the use of the mouse BAMBI promoter reporter construct showed that BAMBI promoter activity in ST2 increased 1.8-fold by TGF-β treatment, 1.2-fold by rhBMP-2 treatment, and 1.9-fold by treatment with lithium chloride, an agonist of the β-catenin signaling pathway. Since both BMP responsive elements and TCF-1 binding sites are conserved in mouse and human BAMBI promoters, BAMBI gene expression is regulated by both TGF-β/BMP and Wnt/β-catenin signaling pathways. We then assessed the methylation status of the BAMBI promoter region by Southern blot analysis and found tissue-specific distribution of promoter hypermethylation. Sodium bisulfite mapping, however, revealed no CpG locus methylation around the transcription start site in primary cultured osteoblasts or osteocytes. We therefore speculate that the epigenetic regulation of BAMBI gene expression by CpG hypermethylation is physiologically involved in tissue- and development-specific BAMBI expression, and that decreased BAMBI expression in terminally-differentiated osteocytes is due not to epigenetic gene silencing but passively to decreased β-catenin and/or BMP signaling in osteocytes. Thus, BAMBI is a target gene of both BMP and Wnt/β-catenin signaling and may be involved in preventing some of the osteoblast precursors and chondrocytes from terminal differentiation.

Disclosures: T. Kondo, None.

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Type III TGFβ Receptor Regulates BMP Signaling in Differentiating Osteoblasts In Vitro and In Vivo. M. Ni*¹, G. R. Mundy², R. L. Caldwell³. ¹Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA, ²Department of Medicine/Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA, ³Department of Orthopaedics and Rehabilitation, Vanderbilt University School of Medicine, Nashville, TN, USA.

Several cell surface receptors for TGF-beta are now well established. Type I and II receptors (TRI and TRII) express intracellular kinase domains responsible for receptor activation and signaling. The type III TGF-beta receptor (TRIII) is an 849-amino acid transmembrane proteoglycan that exists as a co-receptor for TGF-beta, yet maintains biological functions distinct from TRI and TRII. TRIII has a short, highly conserved 43-amino acid cytoplasmic domain (CD). TRIII regulates TGF-beta signaling by recruiting TGF-beta ligand to TRII, driving endocytosis of the signaling complex, and guiding regiospecificity of cellular differentiation. BMPs are necessary for osteogenesis from early embryonic tissue patterning to postnatal skeletal development. BMPs promote bone development by stimulating proliferation and differentiation of osteoblasts, and recent work has suggested that non-union of bone may be the result of decreased levels of BMP activity. Due to signaling homology shared between TGF-beta and BMP, we hypothesized that BMP-induced bone formation is regulated by TRIII from early embryogenesis through development of mature skeleton, and that the TRIII CD is central to these functions. Our hypothesis was tested by phenotypic analysis of TRIII knockout mice, immunohistochemical (IHC) localization of TRIII in developing and mature bone, and transfection assays using 2T3 osteoblasts. We observed TRIII null mice exhibit a shorter skeletal phenotype (<10%) than WT mice. IHC analysis of femoral bone from WT 2-month old mice revealed enhanced TRIII expression in bone marrow as well as the growth plate, while IHC analyses for TRIII in adult mice reveal little TRIII expression in mature bone. In committed bone cells, TRIII attenuates BMP signaling. We found that forced TRIII expression down-regulates SMAD 1, 5 and 8 phosphorylation after BMP2 stimulation, and deletion of the TRIII CD further decreases SMAD phosphorylation. Bone cells demonstrate decreased alk phos activity upon TRIII forced expression. TRIII up-regulates SMAD6 after BMP2 stimulation, and represses BMPRII promoter activity via the TRIII CD. We conclude that TRIII plays a dual role in regulating BMP signaling throughout bone development. Furthering our understanding of TRIII in bone biology will give insights into the control of normal bone formation and provide better understanding of disorders of bone formation.

Disclosures: M. Ni, None.

This study received funding from: Developmental Funds from the Dept. of Orthopaedics and Rehabilitation and the Vanderbilt University Tumor Microenvironment Network.

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Protein Related to DAN and Cerberus (PRDC) Is Expressed in Skeletogenesis and Prevents Osteoblastic Differentiation. A. Nifuji¹, H. Ideno*², R. Takanabe*², A. Shimada*¹, R. Araki*², M. Abe*². ¹Department of Pharmacology, Tsurumi University of Dental Medicine, Yokoha