Journal of Bone and Mineral Research

BackgroundRandomized controlled trials (RCT) of vitamin D supplementation to reduce fracture risk in children are lacking. MethodsWe conducted a Phase 3 RCT of weekly oral supplementation with 14,000 IU vitamin D3 for 3 years in Mongolian schoolchildren aged 6-13 years. Serum 25-hydroxyvitamin D (25[OH]D) concentrations and the proportion of participants reporting [&ge;]1 fracture were secondary outcomes for the main trial. Radial bone mineral density (BMD) was assessed in a nested sub-study, with serum concentrations of parathyroid hormone (PTH) and bone-specific alkaline phosphatase (BALP) determined in a subset of participants. Findings8851 children were enrolled in the main trial, of whom 1465 also participated in the sub-study. Vitamin D deficiency was prevalent at baseline (25[OH]D <20 ng/mL in 90.1%). The intervention elevated 25(OH)D concentrations (adjusted inter-arm mean difference [aMD] 20.3 ng/mL, 95% CI 19.9 to 20.6) and suppressed PTH concentrations (aMD -13.6 pmol/L, 95% CI -23.5 to -3.7), but it did not influence fracture risk (adjusted risk ratio 1.10, 95% CI 0.93 to 1.29, P=0.27) or radial BMD z-score (aMD -0.06, 95% CI -0.18 to 0.07, P=0.36). Vitamin D suppressed serum BALP concentrations more among participants with baseline 25(OH)D concentrations <10 vs. [&ge;]10 ng/mL (Pinteraction=0.04). However, effects of the intervention on fracture risk and radial BMD were not modified by baseline vitamin D status (Pinteraction[&ge;]0.67). InterpretationWeekly oral vitamin D supplementation elevated serum 25(OH)D concentrations and suppressed PTH concentrations in vitamin D-deficient schoolchildren in Mongolia. However, this was not associated with reduced fracture risk or increased radial BMD. FundingNational Institutes of Health RESEARCH IN CONTEXTO_ST_ABSEVIDENCE BEFORE THIS STUDYC_ST_ABSWe searched PubMed from inception to 31st December 2022 for randomized controlled trials (RCT) evaluating effects of vitamin D supplementation on bone mineral content (BMC), bone mineral density (BMD) and fracture risk in HIV-uninfected schoolchildren. A meta-analysis of data from 884 participants in six RCT reported no statistically significant effects of vitamin D on total body BMC, hip BMD, or forearm BMD, but a trend towards a small positive effect on lumbar spine BMD. RCT investigating fracture outcomes were lacking, as were RCT investigating effects of vitamin D on bone outcomes in children with baseline serum 25-hydroxyvitamin D (25[OH]D) concentrations <20 ng/mL. ADDED VALUE OF THIS STUDYThis is the first RCT to investigate effects of vitamin D supplementation on fracture risk and BMD in Mongolian schoolchildren. Vitamin D deficiency was prevalent among the study population at baseline, and weekly oral supplementation with 14,000 IU vitamin D3 for 3 years elevated serum 25(OH)D concentrations into the physiologic range and suppressed serum PTH concentrations. However, the intervention did not influence fracture risk or radial BMD, either in the study population as a whole or in the large sub-group of participants with baseline serum 25(OH)D concentrations <10 ng/mL. IMPLICATIONS OF ALL THE AVAILABLE EVIDENCETaken together with null findings from another recenty-completed phase 3 RCT of weekly oral vitamin D supplementation conducted in South African schoolchildren, our findings do not support a role for vitamin D supplementation to reduce fracture risk or increase BMD in primary schoolchildren.

Bone fracture is accompanied by mechanical stresses and inflammation - conditions that impair mitochondria via the phenomenon of permeability transition. This phenomenon occurs due to opening of the mitochondrial permeability transition pore (MPTP) promoted by cyclophilin D (CypD). MPTP opening exacerbates inflammation and cell death and, thus can disrupt fracture repair. Here we tested a hypothesis that protecting mitochondria from MPTP opening via inhibition of CypD improves fracture repair. Our data indicate that osteoblast activity, bone formation, and biomechanical properties of repaired bones were significantly increased in CypD knock-out mice when compared to controls during fracture repair. These effects were observed in male but not female mice, thus showing sexual dimorphism. Pharmacological inhibition of CypD with NIM811 in male mice also stimulated fracture repair. In addition, CypD knock-out or pharmacological inhibition produced pro-osteogenic effect in isolated bone marrow osteoprogenitors. This in vitro effect was associated with higher mitochondrial respiration and increased {beta}-catenin activity regulated by mitochondria-dependent acetylation. Our findings implicate a sex-specific role of MPTP in bone fracture and suggest CypD inhibition as a modality to promote fracture repair.

ObjectivesHip shape is thought to be an important causal risk factor for hip osteoarthritis and fracture. We aimed to identify genetic determinants of hip shape and use these to assess causal relationships with hip osteoarthritis. MethodsStatistical hip shape modelling was used to derive 10 hip shape modes (HSMs) from DXA images in UK Biobank and Shanghai Changfeng cohorts (ntotal=43,485). Genome-wide association study meta-analyses were conducted for each HSM. Two-sample Mendelian randomisation (MR) was used to estimate causal effects between HSM and hip osteoarthritis using hip fracture as a positive control. ResultsAnalysis of the first 10 HSMs identified 290 independent association signals (P<5x10-8). Hip shape SNPs were also associated (P<1.7x10-4) with hip osteoarthritis (n=29) and hip fracture (n=4). Fine mapping implicated SMAD3 and PLEC as candidate genes that may be involved in the development of hip shape and hip osteoarthritis. MR analyses suggested there was no causal effect between any HSM and hip osteoarthritis, however there was evidence that HSM2 (higher neck-shaft angle) and HSM4 (wider femoral neck) have a causal effect on hip fracture (ORIVW 1.27 [95% CI 1.12-1.44], P=1.79x10-4 and OR 0.74 [0.65-0.84], P=7.60x10-6 respectively) ConclusionsWe report the largest hip shape GWAS meta-analysis that identifies hundreds of novel loci, some of which are also associated with hip osteoarthritis and hip fracture. MR analyses suggest hip shape may not cause hip osteoarthritis but is implicated in hip fractures. Consequently, interventions aimed at modifying hip shape in older adults to prevent hip osteoarthritis may prove ineffective. Key messagesO_ST_ABSWHAT IS ALREADY KNOWN ON THIS TOPICC_ST_ABSHip shape in many forms has been linked with an increased risk of hip osteoarthritis and hip fracture. These observational associations have led to the inference of causality, prompting the development of surgical treatments aimed at modifying hip shape to potentially prevent hip osteoarthritis. Unfortunately, observational studies are susceptible to confounding and reverse causation. WHAT THIS STUDY ADDSThis study provides a comprehensive catalogue of genetic associations related to variations in hip shape, in the form of 10 orthogonal hip shape modes. Substantial genetic overlap was observed between hip shape and both hip osteoarthritis and fracture. However, MR analyses suggested there was no causal effect between hip shape and hip osteoarthritis. Conversely, there was strong evidence that hip shape variation, including greater neck-shaft angle, is causal for hip fractures. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYThis study suggests that, at a population level, moderate hip shape variation does not cause hip osteoarthritis, meaning previously seen observational associations are likely confounded or due to reverse causality. Therefore, targeting these variations of hip shape through surgery, especially in older populations, may prove ineffective in preventing hip osteoarthritis.

ImportanceFragility fractures are a public health problem. Over 70% of women having fractures have osteopenia or normal BMD, but they remain unidentified and untreated because the definition of osteoporosis, a bone mineral density (BMD) T-Score [&le;] -2.5SD, is often used to signal bone fragility. ObjectiveAs deep learning facilitates investigation of bones multi-level hierarchical structure and soft tissue, we tested whether this approach might better identify women at risk of fracture before fracture. DesignWe pooled data from three French and Swiss prospective population-based cohorts (OFELY, QUALYOR, GERICO) that collected clinical risk factors for fracture, areal BMD and distal radius measurements with high resolution peripheral quantitative tomography (HRpQCT). Using only three-dimensional images of the distal radius, ulna and soft tissue acquired by HRpQCT, an algorithm, a Structural Fragility Score-Artificial Intelligence (SFS-AI), was trained to distinguish 277 women having fractures from 1401 remaining fracture-free during 5 years and then was tested in a validation cohort of 422 women. SettingEuropean postmenopausal women ParticipantsWe have studied postmenopausal women considered as representative of the general population, who were followed for a median 9.4 years in OFELY, 5.4 years in QUALYOR and 5.7 years in GERICO. Main outcome and measureAll types of incident fragility fractures ResultsWe used data from 2666 postmenopausal women, with age range of 42-94. In women [&ge;] 65 years having All Fragility Fractures or Major Fragility Fractures, SFS-AI generated an AUC of 66-70%, sensitivities of 60-68% and specificity of 71%. Sensitivities were greater than achieved by the fracture risk assessment (FRAX) with BMD or BMD (6.7-26.7%) with lower specificities than these diagnostics ([~]95%). Conclusion and relevanceThe SFS-AI is a holistic surrogate of fracture risk that pre-emptively identifies most women needing prompt treatment to avert a first fracture. Key PointsO_ST_ABSQuestionC_ST_ABSCan a deep learning model (DL){degrees} based on high resolution images of the distal forearm predict fragility fractures? FindingsIn the setting of 3 pooled population-based cohorts, the DL model predicted fractures substantially better than areal bone mineral density and FRAX, especially in women [&ge;]65 years. MeaningOur DL model may become an easy to use way to identify postmenopausal women at risk for fracture to improve fracture prevention.

Glucagon-like peptide-1 (GLP-1) has previously been shown to be indispensable for optimal bone strength by acting at the bone material level. However, it was not fully clear whether the effects of GLP-1 were mediated by direct or indirect actions on bone cells. In the present study, we were unable to demonstrate the expression of the GLP-1 receptor (GLP-1r) in bone tissue at the gene expression level using qPCR and in situ hybridization, or at the protein level. Furthermore, the peripheral administration of exendin-4, a specific GLP-1r agonist, in ovariectomized (OVX) BALB/c mice enhanced post-yield displacement (18%) and energy-to-fracture (24%), as well as bone volume/total volume (BV/TV) (11%), trabecular number (Tb.N) (6%), and collagen maturity (18%). These bone effects were still observed when exendin-4 was centrally administered into the lateral cerebral ventricle. On the other hand, the peripheral administration of exendin-4 coupled to bovine serum albumin, a GLP-1r agonist that cannot penetrate the brain, failed to replicate the positive effects on bone despite increased calcitonin secretion. Altogether, these data confirm that GLP-1r agonists represent an interesting approach for managing bone fragility due to ovariectomy, but also suggest that GLP-1r agonists require a central relay yet to be identified to exert positive effects on bone physiology. Further studies are needed to decipher the mechanisms of action of GLP-1 and GLP-1r agonists on bone physiology.

Biomineralisation is essential for skeletal integrity, yet the synergistic roles of tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 in postnatal bone mineralisation remain poorly defined. To decipher this, we generated a novel murine model in which Alpl was deleted in Prx1- expressing cells (AlplPrx1/Prx1) in mice with a global Phospho1-/- deficiency to overcome the perinatal lethality that arises upon dual global deletion. Using a multi-modal approach to spatially phenotype the limbs of these animals, we reveal mice lacking both TNAP and PHOSPHO1 exhibit a distinct lack of mineralisation and altered anatomical structure at postnatal day 1 (PN1) and 3-weeks of age. Although viable, these mice did not thrive due to their reduced size, thus further investigations were conducted on mice with a heterozygous deletion of TNAP (Alplwt/Prx1;Phospho1-/-). Although smaller than wild-types at PN1 and 3 weeks old, these mice did not display the gross limb deformations observed in the homozygous animals and the single, functioning Alpl allele rescued the loss of biomineralisation observed following dual phosphatase deletion. At 6-weeks of age, compromised epiphyses and metaphyses were only seen in AlplPrx1/Prx1 animals. Further, we found that tibial geometry and porosity was significantly altered by Phospho1 deletion (Phospho1-/-), which was compounded in the Alplwt/Prx1;Phospho1-/- mice and linked to alterations in collagen configuration, matrix mineralisation and growth plate deformities. Together, our findings establish the mechanistic framework for TNAP and PHOSPHO1 in permissive biomineralisation, providing critical insights into this fundamental process. Significance StatementBiomineralisation is essential for skeletal development and is critically dependent on phosphatases that release inorganic phosphate for hydroxyapatite formation. Our study investigates the dual role of PHOSPHO1 and TNAP in this process, using a novel murine knockout model. Deletion of both enzymes results in complete loss of bone mineralisation, demonstrating their critical synergistic function. Further, we show that PHOSPHO1 and TNAP exhibit distinct, spatially-restricted functions in the tibia and thus enhances our understanding of the fundamentals processes underpinning biomineralisation. These findings also have clinical relevance as they have the potential to inform on treatment strategies for hypo- and hyper-mineralised pathologies.

BackgroundTransposable elements (TEs) such as LINE-1 and HERV-K are mobile retroviral elements that have biological functions in several tissues. In a prior study, we demonstrated the positive contribution of LINE-1 activation to bone homeostasis and microfracture repair. People living with HIV experience a high prevalence of low bone mineral density (BMD) and fragility fractures, particularly when treated with nucleoside reverse transcriptase inhibitor (NRTI)-based antiretroviral therapy (ART). The mechanisms underlying ART-associated bone loss remain unclear. Several NRTIs inhibit not only viral but also endogenous reverse transcriptases (RTs) encoded by LINE-1 and HERV-K. Therefore, we hypothesized that since endogenous RT activity supports bone function, its inhibition by NRTIs may contribute to bone loss and secondary osteoporosis, as frequently observed in patients receiving ART. MethodsWe quantified L1-Ta and HERV-K DNA content, used as a proxy for endogenous RT activity, in trans-iliac bone biopsies, skeletal muscle, and peripheral blood mononuclear cells (PBMCs) in a clinically well-characterized cohort of postmenopausal women stratified into healthy, osteopenic or osteoporotic. Bone status was assessed through BMD measurements and bone remodeling was evaluated by extensive serum analyses combined with expression of osteoblastic and osteocytic biomarkers. The ex vivo results were corroborated in vitro, where the functional impact of RT inhibition was tested in IDG-SW3 cells differentiating from osteoblasts into osteocytes using three NRTIs (tenofovir, lamivudine, abacavir). The cell cultures were analyzed for osteocyte maturation, mineralization, and responsiveness to 1,25-dihydroxyvitamin D and parathyroid hormone (PTH). FindingsL1-Ta and HERV-K DNA content was significantly higher in bone than in muscle or PBMCs and declined with age, specifically in bone. Both elements were markedly reduced in osteoporotic bone, but unchanged in matched non-skeletal control tissues. L1-Ta and HERV-K DNA content correlated positively with BMD at three skeletal sites and with the expression of osteocyte-specific genes, particularly SOST and MEPE. Treatment of IDG-SW3 cells with NRTIs selectively impaired osteocyte endocrine function. Induction of FGF23 by 1,25-dihydroxyvitamin D and PTH-mediated suppression of SOST were inhibited, and gene expression in osteocytes and mineralization were slightly affected. Abacavir had the strongest impact, almost completely abolishing vitamin D3 induction of FGF23 and PTH responsiveness. InterpretationThese findings identify endogenous TE-derived RT activity as a previously unrecognized regulator of osteocyte function and bone homeostasis. Reduced L1-Ta and HERV-K DNA contents is associated with impaired osteocyte function and low BMD, while pharmacological RT inhibition disrupts key endocrine pathways in osteocytes. NRTI-mediated inhibition of endogenous osteocyte RT activity may therefore contribute to ART-associated osteoporosis. Assessment of TE-derived RT activity may improve the identification of individuals at risk for skeletal deterioration. Preservation of endogenous osteocyte RT activity represents a potential therapeutic strategy for mitigating bone loss in HIV-infected patients undergoing long-term ART. FundingThis research was supported by KAUST BAS/1/1037-01-01, the South East Norway Health Authority and Oslo University Hospital, Ullevaal (52009/8029); The 6th EU Framework Program (LSHM-CT-2003-502941); Legat til Forskning, Lovisenberg Diaconal Hospital.

ObjectiveTo identify genetic factors associated with hip morphology in Chinese populations. MethodsAn 85-point Statistical Shape Model (SSM) was applied to extract hip shape modes (HSMs). Diameter of the femoral head (DFH), femoral neck width (FNW) and hip axis length (HAL) were obtained from SSM points using Python scripts. Genome-wide association study (GWAS) was conducted in the Shanghai Changfeng (SC) cohort (N=5,310) for each phenotype of DXA-derived hip morphology. Replication of GWAS was conducted in the Core cohort (N=917). ResultsGWAS identified a total of 331 SNPs in 14 loci that were associated with features of hip morphology in the SC cohort. 4 of 14 loci were replicated in the Core cohort: rs143383 (GDF5) associated with HAL (P = 9.4x10-10), rs11614913 (MIR196A2) associated with HSM9 (P = 2.8 x10-10), rs35049516 (SUPT3H) associated with HSM4 (P = 4.3 x10-10) and rs7761119 (UST) associated with HSM8 (P = 1.7x10-8). Of these, two loci were known to affect hip morphology, including rs143383 (GDF5) and rs35049516 (SUPT3H), whereas rs11614913 (MIR196A2) and rs7761119 (UST) were novel. There was also overlap with previous GWAS of HSM and other hip-based metrics. ConclusionsIn the largest East Asian ancestry hip shape GWAS to date we identified and replicated four loci associated with different aspects of hip morphology (GDF5, MIR196A2, SUPT3H, UST). Strong SNP-to-gene evidence was found. All four loci have previously been implicated in musculoskeletal development, however this is the first report that rs11614913 (MIR196A2) and rs7761119 (UST) are associated with hip morphology. Despite the small sample size, this study paves the way for trans-ancestry meta-analyses.

Protein kinase A (PKA) is involved in bone biology and is a key mediator of parathyroid hormone signaling in the osteoblast. However, the consequences of sustained PKA activation in bone are unclear. In this study, we inducibly activated PKA in osteoblasts by deleting its major regulatory subunit, Prkar1a, using a Col11-driven Cre system. Prkar1aob-/-mice demonstrated rapid and profound bone pathologies in their femurs, lumbar and caudal vertebrae with cortical bone breakdown and cortical trabecularization. This phenotype was characterized by increased bone turnover and elevated osteoblastic and osteoclastic activities. Transcriptomic and qPCR analyses showed an impairment of osteoblast differentiation with a defect in ossification, expansion of stromal cells, and numbers of both osteoblastic and osteoclastic precursors. Moreover, there were alterations in gene expression of chemokines and Wnt members with enhanced osteoclastogenesis. Altogether, activation of PKA in osteoblasts by inducible deletion of Prkar1a causes a profound high bone turnover phenotype resembling several human bone diseases.

PurposeThis study aimed to compare the effects of sodium glucose cotransporter 2 inhibitors (SGLT2I) and dipeptidyl peptidase-4 inhibitors (DPP4I) on new-onset hip fractures. MethodsThis was a retrospective population-based cohort study including type-2 diabetes mellitus patients treated with either SGLT2I or DPP4I between January 1st 2015 and December 31st 2020 in Hong Kong. The primary outcome was new-onset hip fracture and the secondary outcome was all-cause mortality. Propensity score matching (1:1 ratio) using the nearest neighbour search was performed. Univariable and multivariable Cox regression were applied to identify significant predictors. Competing risks models and multiple approaches using the propensity score were performed. ResultsThis cohort included 56393 patients with type-2 diabetes mellitus (median age: 62.1 years old [interquantile range, IQR]: 54.2-71.1; 57.45% males), of which 20432 patients ([incidence rate, IR]: 36.23%) used SGLT2I and 35961 patients (IR: 63.77%) used DPP4I. After the 1:1 propensity score matching, 449 (IR: 1.09%) patients had hip fractures, and 2012 patients (IR: 4.92%) died. SGLT2I was associated with significantly lower risks of hip fractures after adjusting for the demographics, past comorbidities, non-SGLT2I/DPP4I medications and laboratory results (hazard ratio: 0.55; 95% confidence interval: 0.42-0.89; P=0.0036). The results were consistent in the competing risk models and the different propensity matching approaches. ConclusionsSGLT2I was associated with lower risks of new-onset hip fractures after propensity score matching and adjustments. SummaryThis study compared the risks of hip fractures in between users of sodium glucose cotransporter 2 inhibitors (SGLT2I) and dipeptidyl peptidase-4 inhibitors in type-2 diabetes mellitus. After propensity score matching, SGLT2I was associated with lower risks of hip fractures adjusting for confounders (hazard ratio: 0.55; 95% confidence interval: 0.42-0.89; P=0.0036).

Osteoporosis is a prevalent cause of fractures in older adults and remains a source of morbidity that requires efforts to develop therapeutics. Circulating proteins play a critical role in the pathophysiology of osteoporosis and offer opportunities to identify new causal determinants of bone health. We therefore performed a large-scale proteome-wide Mendelian randomization (MR) analysis to estimate the effects of genetically determined circulating proteins levels on bone mineral density (BMD) and fracture risk. Genetic instruments were derived from cis-protein quantitative trait loci (cis-pQTLs) for 2,110 plasma proteins across four European ancestry cohorts and applied to genome-wide association studies (GWAS) of heel estimated BMD, femoral neck BMD, lumbar spine BMD, any fracture, and forearm fracture in up to 426,824 individuals of European ancestry. Across proteins and outcomes, 192 protein-skeletal outcome associations showed MR evidence of association, without evidence for heterogeneity or horizontal pleiotropy, and 128 of these further showed strong colocalization with osteoporosis-related loci. We then prioritized proteins that replicated across cohorts, exhibited concordant effect directions, and were likely to be active in circulation, yielding 18 high-confidence causal proteins for BMD and fracture risk. These included established skeletal regulators such as sclerostin (SOST) and R-spondin-3 (RSPO3), which showed opposing effects consistent with their known biology, along with less well-characterized proteins. Higher genetically predicted tissue inhibitor of metalloproteinases 2 (TIMP2) levels was associated with lower BMD and increased forearm fracture risk. Gene-level and variant-level phenome-wide association analyses converged on skeletal traits, and rare predicted damaging or loss-of-function variants in TIMP2 were associated with higher BMD at the heel, spine and hip. Our findings implicate several circulating proteins as putatively causal factors for osteoporosis and, among them, provide multiple layers of evidence supporting TIMP2 as a genetically supported candidate for further functional and translational evaluation. Lay summaryOsteoporosis is characterized by decreased bone density, and despite available medications, it remains a key risk factor for fractures, requiring continued effort for development of new therapeutics. We used genetic data to estimate the effect of genetically predicted levels of 2,110 blood proteins on strength and fracture risk. We found 18 proteins with effects on bone mineral density and fractures. One protein, tissue inhibitor of metalloproteinases 2 (TIMP2), showed robust evidence linking its higher levels to decreased bone density and increased risk of forearm fracture, highlighting TIMP2 as a promising new treatment target for osteoporosis.

Osteoglophonic Dysplasia (OGD) is an autosomal dominant skeletal dysplasia characterized by impaired bone growth resulting in short stature, severe craniofacial abnormalities, and in some patients FGF23-mediated hypophosphatemia. It is caused by gain-of-function variants in FGFR1, particularly in or near the transmembrane domain of the receptor. We used CRISPR in mice to knock-in the FGFR1 p.N330I variant, chosen based on its association with FGF23 excess. Skeletal phenotyping of this Fgfr1+/N330I model demonstrated markedly reduced body weight and naso-anal length, shortened long bones, and craniosynostosis, all hallmarks of the human disease. Mutant mice exhibited profound microarchitectural changes in cortical bone and severe disorganization of the growth plate and articular cartilage, driven by decreased cell proliferation and increased apoptosis in skeletal tissues. In addition to osteochondrodysplasia, we noted dramatic increases in plasma FGF23 and hypophosphatemia, driven by upregulated Fgf23 expression and protein levels in bone, with consequent undermineralization. An in vivo ossicle assay allowed longitudinal evaluation of mineral metabolism. We modulated the signaling pathway by repurposing an inhibitor of the overactive receptor, infigratinib, resulting in partial restoration of naso-anal length in treated mutant mice. This first model of OGD offers insights into the disease pathogenesis and open avenues for targeted therapeutic strategies.

In the United States, there are over ten million adults diagnosed with osteoporosis and many more are at risk of developing the condition. Osteoporosis affects both males and females, mostly post-menopausal. Bisphosphonates and denosumab have been widely used globally to treat the condition. The use of bisphosphonates and denosumab had been associated with rare adverse effects including osteonecrosis of the jaw, ONJ, and atypical femur fracture, AFF. However, it remained unclear whether those side effects were class-wide or drug-specific. By analyzing over 230,000 osteoporosis patient reports from the FDA adverse event reporting system, FAERS, we confirmed the association of bisphosphonates and denosumab use with AFF and ONJ. Additionally, comparing each of the four frequently used bisphosphonates with denosumab-treated patients used as a control, we identified: (i) varying significance of association with ONJ and AFF for alendronate, risedronate, ibandronate and zoledronic acid, (ii) over two fold increase in risk of both side effects in alendronate patients, particularly in females, (iii) over a six fold increase in AFF risk in both males and females taking risedronate, and (iv) lower risk of both AFF and ONJ, for zoledronic acid patients compared to denosumab. Key pointsO_LIWe performed a disproportionality analysis of over 230,000 post-marketing reports of patients treated for osteoporosis to measure the risk of developing atypical femur fracture (AFF) and osteonecrosis of the jaw (ONJ). C_LIO_LIAlendronate, ibandronate, risedronate, zoledronic acid, and denosumab were all significantly associated with AFF and ONJ when compared to teriparatide. C_LIO_LIWhen compared to denosumab, patients taking alendronate, ibandronate, risedronate, or zoledronic acid had a variable risk of ONJ and AFF, which correlated with the frequency of drug administration. The trend in variable risk was observed in both females and males. C_LI

Fractures heal by rapid formation of mineralized callus. Essential to this process is proliferation of periosteal cells to supply bone-forming osteoblasts. To better understand the role of cell proliferation in fracture healing, we asked: How is callus composition altered when osteolineage cells proliferation is blocked? Do mature osteoblasts proliferate to contribute to callus formation? First, mice expressing herpes simplex virus-thymidine kinase (HSV-TK) driven by the 3.6Col1a1 promoter were treated with ganciclovir (GCV) to ablate proliferating osteolineage cells for 5 or 10 days. Analysis of callus cells using single-cell RNA-seq revealed that GCV-treated Col1-TK mice had fewer osteoblasts and chondrocytes than control mice, with more myofibroblasts and immune cells, consistent with fibrous nonunion. In controls, 15-30% of callus cells that expressed the early osteoblast markers osterix (OSX) and the late marker osteocalcin (OCN) were in the cell cycle. Next, we targeted proliferating osteoblasts at different stages of differentiation by crossing Osx-CreERT2, Ocn-Cre and Dmp1-CreERT2 mice with novel ROSA-TK mice. Following fracture, each Cre;ROSA-TK mouse line exhibited poorer radiographic healing, decreased callus bone volume and a shift from callus bone to fibrous tissue. We conclude that osteoblasts, often considered post-mitotic, proliferate after fracture to contribute to formation of mineralized callus essential to healing.

BACKGROUNDFibrous dysplasia (FD) is a rare, disabling disease with no established treatments. Growing evidence supports inhibiting the pro-osteoclastic factor receptor activator of nuclear Kappa-B ligand (RANKL) as a potential treatment strategy. We conducted a phase 2 trial evaluating the anti-RANKL drug denosumab in adults with FD, with an emphasis on investigating post-discontinuation bone turnover rebound, and cellular mechanisms underlying anti-RANKL effects on FD osteoprogenitors. METHODSEight subjects received denosumab for 6-months and were observed for 8-months post-discontinuation. Efficacy and safety were evaluated using bone turnover markers, 18F-NaF PET/CT, and lesion biopsies. RANKL neutralization effects were assessed by histology, RNASeq, and an FD mouse model. Interplay between osteoclasts and FD osteoprogenitors was assessed in an ex vivo lesion model. RESULTSDenosumab markedly reduced bone turnover and radiographic lesional activity in all subjects. Denosumab was well-tolerated during the treatment period, however post-discontinuation turnover reached or exceeded pre-treatment in six subjects, associated with severe hypercalcemia in one. Histology and whole-exome RNA sequencing showed reduced FD cell proliferation and increased osteogenic maturation, with increased lesional bone formation. The ex vivo model supported the dependence of FD cell proliferation on osteoclast activation. CONCLUSIONSOsteoclast inhibition by anti-RANKL decreased FD cell proliferation and lesional activity, enabling osteogenic maturation and bone formation. These findings provide new understanding of FD pathogenesis as driven by crosstalk between osteoclasts and pre-osteoblast/osteoblasts, and support denosumab as a mechanistically-driven treatment strategy. Marked bone turnover rebound with post-discontinuation hypercalcemia occurs in a subset of patients, particularly younger individuals with high disease burden. TRIAL REGISTRATIONClinicalTrials.gov NCT03571191 FUNDINGThis work was supported by the Intramural Research Program of the NIDCR, NICHD, and Clinical Center, National Institutes of Health. Clinical trial NCT03571191 was conducted as an investigator-sponsored study supported by Amgen, Inc. This research was supported in part by the NIDCR Genomics and Computational Biology Core: ZIC DC000086 and Veterinary Resources Core: ZIC DE000740-05. Work in MTC lab and LVC labs were supported by the of Research on Womens Health (ORWH) through the Bench to Bedside Program award #884515.

The protective effect of estrogens against cortical bone loss is mediated via direct actions on mesenchymal lineage cells, but functional evidence for the precise molecular mechanism(s) and the mediators of these effects has only recently began to emerge. We report that the matrix metalloproteinase 13 (MMP-13) is the highest up-regulated gene in calvaria or bone marrow cells from mice lacking the estrogen receptor (ER) alpha in osteoprogenitors. We, therefore, generated mice with conditional Mmp-13 deletion in Prrx1 expressing cells (Mmp-13{Delta}Prrx1) and compared the effect of estrogen deficiency on their bone phenotype to that of control littermates (Mmp-13f/f). Femur and tibia length was decreased in sham-operated Mmp-13{Delta}Prrx1 mice as compared to Mmp-13f/f. Cortical thickness and trabecular bone volume in the femur and tibia were increased and osteoclast number at the endocortical surfaces was decreased in the sham-operated female Mmp-13{Delta}Prrx1 mice; whereas bone formation rate was unaffected. Ovariectomy (OVX) caused a decrease of cortical thickness in the femur and tibia of Mmp-13f/f control mice. This effect was attenuated in the Mmp-13{Delta}Prrx1 mice; but the decrease of trabecular bone caused by OVX was not affected. These results reveal that mesenchymal cell-derived MMP-13 regulates osteoclast number, bone resorption, and bone mass. We have recently reported that the loss of cortical, but not trabecular bone, caused by OVX is also attenuated in Cxcl12{Delta}Prrx1 mice. Together with the present report, this functional genetic evidence provides proof of principle that increased production of mesenchymal cell-derived factors, such as CXCL12 and MMP-13, are important mediators of the adverse effect of estrogen deficiency on cortical, but not trabecular, bone. Therefore, the mechanisms responsible for the protective effect of estrogens on these two major bone compartments are different.

Aging is associated with decreased bone formation and bone mass and increased fracture risk. Wnt pathway activation by mechanical loading is a potent strategy to improve bone mass, however, load-induced bone formation is diminished with aging. Neutralizing antibody (Ab) therapies targeting Wnt pathway inhibitors Sclerostin (Scl) and Dickkopf-related protein 1 (Dkk1) have proven successful in preclinical and clinical osteoporotic conditions. We asked whether treatment combining Scl-Ab and Dkk1-Ab can increase load-induced bone formation in a preclinical model of osteoporosis. Aged (22-month) C57BL/6N female mice underwent combination Scl-Ab plus Dkk1-Ab therapy (15 mg/kg each; subcutaneously; 2x/wk; saline control) for 2 weeks, concomitant with a mechanical loading regimen previously shown to induce modest bone formation in tibias of aged mice (-2200 {varepsilon}, 1200 cycles/day, 5 day/wk). Changes in bone morphology and formation were assessed by longitudinal microCT and dynamic histomorphometry, respectively. Molecular indices of bone formation and Wnt pathway activation were assessed by qPCR of cortical bone. Treatment with Scl-Ab plus Dkk1-Ab induced significant improvements in cancellous (BV/TV +50%) and cortical morphology (Ct.Th +25%) in non-loaded limbs of antibody-treated mice vs. vehicle control mice. Importantly, periosteal bone formation rate was 10-fold higher in loaded limbs of antibody versus vehicle treated mice, indicating a synergistic effect. Gene expression analysis showed that antibody treatment and loading synergistically upregulated Wnt1 expression, which may have contributed to the observed synergistic effect on bone formation. These results confirm the potent anabolic effect of combination Scl plus Dkk1 antibody treatment. Moreover, they show that antibody treatment and skeletal loading are more effective at increasing periosteal bone formation in aged mice than either treatment alone. These findings support the concept that combinatorial therapy using dual Scl and Dkk1 antibodies plus weight-bearing exercise may be an effective treatment for age-related osteoporosis.

Chondrocytes within the fracture callus transform into osteoblasts during bone regeneration, but the molecular mechanisms regulating this process are unknown. Wnt ligands are expressed within the fracture callus, and hypertrophic chondrocytes undergoing transformation to osteoblasts exhibit nuclear localization of {beta}-catenin, indicating active Wnt signaling in these cells. Here, we show that conditional knock out (cKO) of {beta}-catenin in chondrocytes inhibits the transformation of chondrocytes to osteoblasts, while stabilization of {beta}-catenin in chondrocytes accelerates this process. After cKO, chondrocyte-derived cells were located in the bone marrow cavity and upon re-fracture formed cartilage. Lineage tracing in wild type mice revealed that in addition to osteoblasts, chondrocytes give rise to stem cells that contribute to repair of subsequent fractures. These data indicate that Wnt signaling directs cell fate choices of chondrocytes during fracture healing by stimulating transformation of chondrocytes to osteoblasts, and provide a framework for developing Wnt-therapies to stimulate repair.

ObjectiveMinimum joint space width (mJSW) from 2-dimensional images provides a proxy for cartilage thickness. This study aimed to conduct a genome-wide association study (GWAS) of mJSW to (i) identify new genetic determinants of mJSW and use them to (ii) examine causal effects of mJSW on hip osteoarthritis (HOA) risk. MethodsGWAS meta-analysis of hip mJSW derived from plain X-rays (four cohorts) or DXA (one cohort) was performed, stratified by sex and adjusted for age and ancestry principal components. Mendelian randomisation (MR) and cluster analyses were used to examine causal effect of mJSW on HOA. Results50,745 individuals were included in the meta-analysis. 42 SNPs, which mapped to 39 loci (35 novel), were identified. Mendelian randomisation (MR) revealed little evidence of a causal effect of mJSW on HOA ({beta}IVW -0.01 [95% CI -0.19, 0.17]). However, MR-Clust analysis suggested the null MR estimates reflected the net effect of two distinct causal mechanisms cancelling each other out, one of which was protective, whereas the other increased HOA susceptibility. For the latter mechanism, all loci were positively associated with height, suggesting mechanisms leading to greater height and mJSW increase the risk of HOA in later life. ConclusionsGWAS and MR analyses suggested one group of mJSW loci reduces HOA risk via increased mJSW, suggesting possible utility as targets for chondroprotective therapies. The second group of mJSW loci increased HOA risk, despite increasing mJSW, but were also positively related to height, suggesting they contribute to mJSW and HOA risk via a growth-related mechanism.

The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-indian hedgehog (Ihh) feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we utilized a tamoxifen-inducible PTHrP-creER line with Patched-1 (Ptch1) floxed and tdTomato reporter alleles to specifically activate Hedgehog signaling in PTHrP+ resting chondrocytes and trace the fate of their descendants. Hedgehog-activated PTHrP+ chondrocytes formed large concentric clonally expanded cell populations within the resting zone ( patched roses) and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell-descendants migrated away from the growth plate and eventually transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.