Journal of Bone and Mineral Research

Bone remodeling is a tightly regulated physiological process that maintains bone health through coordinated action of bone-resorbing osteoclasts and bone-forming osteoblasts. Disruption of this balance, such as the one induced by estrogen decline after menopause, results in bone loss and osteoporosis. Genetic factors play an important role in determining bone mineral density (BMD) loss over time. However, translating genetic associations into individualized risk prediction remains challenging due to small effect size of individuals variants and non-linear interactions within the bone remodeling unit. Here, we present a bone cell population dynamics model that includes major regulatory pathways, such as the RANK/RANKL/OPG axis, Wnt signaling, and hormonal regulation by estrogen, parathyroid hormone, and TGF-{beta}. We calibrate the model on clinical data from healthy postmenopausal women, and women with reduced BMD undergoing anti-osteoporotic therapy. The calibrated model captures healthy BMD decline in postmenopausal women and therapeutic response to anti-osteoporotic medications. We mechanistically incorporate the effect of 22 variants across 8 genes involved in bone remodeling and simulate BMD trajectories in 1,000 virtual subjects differing by ancestry and genetic makeup. The median predicted 5-year BMD loss was 3.57% (95% prediction interval: 1.31-5.24), consistent with the values reported in the literature. The virtual individuals with African ancestry were predicted to experience the highest average 5-year BMD loss. The strongest genetic risk factors for bone loss were predicted to be CYP19A1 rs727479 and OPG rs3102735, while LRP5 rs11228240 emerged as a protective factor that could partially counteract the detrimental effects of other variants. Several epistatic effects were observed in the genetic interaction analysis. Mechanistically, our model suggested that estrogen exerts its effect on bone remodeling primarily by modulating osteoclast apoptosis. Overall, this framework demonstrates a proof-of-concept for integration of genetic risk factors into mechanistic models of disease and can be extended to other conditions with polygenic inheritance.

Teriparatide (PTH 1-34) is an anabolic agent used to treat osteoporosis, yet clinical response varies widely among patients. To investigate genetic and sex-specific determinants of skeletal response, we administered intermittent PTH to male and female mice from eight genetically diverse inbred strains. Mice were treated for four weeks, and bone phenotypes were assessed via DXA, microCT, and mechanical testing. Response to PTH was highly strain- and sex-dependent, with some strains responding at the femur but not the spine, and vice versa. Heritability estimates for PTH-induced changes in bone mineral density (BMD), cortical area, breaking strength, and trabecular bone volume fraction (BV/TV) ranged from moderate to high, with BV/TV showing the strongest genetic influence. Cortical bone response mechanisms differed by sex: males exhibited periosteal expansion, while females showed endosteal remodeling. These findings mirror clinical observations where hip non-response is more prevalent than spine non-response and suggest that genetic background and sex significantly influence therapeutic outcomes. Our data support the use of genetically diverse mouse models to elucidate the genetic architecture of PTH response and highlight the potential for personalized approaches in osteoporosis treatment. Future genome-wide association studies in outbred mice may identify specific loci mediating skeletal responsiveness to PTH, advancing precision medicine strategies for bone anabolic therapies. LAY SUMMARYTeriparatide, a drug used to treat osteoporosis, consists of the active portion of parathyroid hormone (PTH). Information from clinical studies suggests that not all patients will respond to this medication. We used eight strains of inbred mice to study the impact of genetic background and sex on the response to PTH. We learned that response to PTH is driven by both genetics and sex. Some strains responded at the femur, but not the spine and vice versa. These results may explain why a failure to respond at the hip in humans is more common than at the spine.

Vitamin D receptor (VDR) signaling is essential for osteoblast maturation and skeletal mineralization, yet the intracellular mechanisms linking VDR activity to matrix production remain poorly defined. Here, we show that vdr-/- calvarial osteoblasts initiate differentiation but fail to complete the transition to a mature, mineralizing state, exhibiting suppressed late-stage markers such as Dmp1, Phex, and Col1a1 and defective nodule formation both in vivo and in vitro. Transcriptomic profiling revealed a SMAD network imbalance, with elevated inhibitory SMADs and attenuated phosphorylation of both SMAD1/5/9 and SMAD2/3. Whereas SMAD1/5/9 activation was preserved in vivo and restored by exogenous BMP2 in vitro, consistent with in vivo paracrine BMP availability, reduced SMAD2/3 phosphorylation persisted in both contexts, indicating a cell-autonomous defect. Mechanistically, vdr-/- osteoblasts displayed impaired intracellular Ca{superscript 2} dynamics and diminished CaMKII activation, with VDR/RXR occupancy detected near the Camk2g locus. Pharmacological CaMKII inhibition (KN-93) recapitulated the phenotype, reducing SMAD2/3 phosphorylation and mineralization and establishing CaMKII as an upstream regulator of SMAD2/3. A milk-based diet rescued skeletal defects in vdr-/- mice in a calcium-independent manner and selectively restored CaMKII-SMAD2/3 signaling. Metabolomic profiling identified the omega-6 fatty acid {gamma}-linolenic acid (GLA) as an elevated circulating mediator; exogenous GLA restored Ca{superscript 2} flux, CaMKII activation, SMAD2/3 phosphorylation, matrix production, and mineralization, without reactivating Runx2, Sp7, or BMP-SMAD1/5/9 signaling, and dependent on CaMKII activity. These findings reveal a VDR-independent, GLA-inducible CaMKII-SMAD2/3 mineralization program, positioning metabolic modulation of calcium signaling as a strategy to restore osteoblast function under impaired vitamin D signaling.

Erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. Recent evidence demonstrates that ERFE is also expressed in osteoblasts and osteoclasts and Erfe-/- mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. To mechanistically dissect how bone-derived ERFE exerts an osteoprotective effect, we first created Erfefl/fl mice, which were then crossed with Col2.3-Cre mice to generate osteoblast-selective Erfe mutants (or Erfefl/fl;Col2.3-Cre mice). We now demonstrate that ERFE derived from osteoblasts is not responsible for the decreased BMD noted in Erfe-/- mice, revealing enhanced BMD during anabolic stress in Erfefl/fl;Col2.3-Cre mice. Consistently, in contrast to global ERFE loss, osteoblast-selective ERFE loss does not increase osteoclasts in vivo. Furthermore, our results demonstrate that ERFE loss in osteoblasts induces osteoclast Erfe expression in co-culture experiments in vitro. Finally, the osteoclastogenesis gene program is induced in co-culture with osteoblasts only when ERFE is lost in osteoclasts. Taken together, our finding provide strong evidence of osteoclast-derived ERFE as a central osteoprotective regulator of bone mass, its loss resulting in net bone loss in Erfe-/- mice. BRIEF SUMMARYLoss of erythroferrone derived from osteoclasts enhances osteoclastogenesis resulting in accelerated bone loss. SIGNIFICANCE STATEMENTCanonical erythroferrone (ERFE) function includes hepcidin suppression through bone morphogenic proteins (BMPs) sequestration, establishing the rationale for ERFE-mediated regulation of bone homeostasis. We previously showed that global ERFE loss controls bone mass. Here, we report that osteoclast-derived ERFE is a major regulator of osteoclastogenesis. For this, we crossed Erfefl/fl with Col2.3-Cre mice to generate osteoblast-selective Erfe mutants, demonstrating that osteoblast-derived ERFE does not recapitulate bone loss found in global ERFE knockout mice. In contrast, bone mineral density is enhanced during anabolic stress in Erfefl/fl;Col2.3-Cre mice. Finally, we document that osteoclast ERFE loss enhances osteoclastogenesis in co-culture with osteoblasts. Together, the data provide compelling evidence that osteoclast-derived ERFE modulates communication between osteoblasts and osteoclasts.

The terminal differentiation of osteoblasts into osteocytes, the most abundant cell type in cortical bone, is critical for skeletal homeostasis. Osteocyte loss is a hallmark of bone aging and fragility, yet the mechanisms regulating osteocyte formation and survival are poorly understood. We show that inactivation of fibroblast growth factor receptor 1 (Fgfr1) in the mature osteoblast lineage results in extensive osteocyte death, identifying FGFR1 signaling as essential for osteocyte viability and bone integrity. Lineage tracing and analysis of endogenous and induced appositional bone formation revealed that newly embedded osteocytes fail to survive without FGFR1. These osteocytes exhibited ectopic expression of osteocalcin and podoplanin within sclerostin-positive, TUNEL-reactive lacunae, along with defective dendrite formation and disruption of the local lacunocanalicular network. RNA sequencing of cortical bone demonstrated reduced expression of extracellular matrix (ECM) genes and neuronal regulatory genes, while histological and ultrastructural analyses showed disorganized collagen fibrils, diminished osteoid, and abnormal mineralization. In vitro, FGF signaling in Ocy454 cells regulated gene programs involved in development, axon guidance, and bone ECM organization, highlighting a dual function for FGF signaling in which it controls both matrix-dependent and intrinsic cell differentiation mechanisms during the osteoblast-to-osteocyte transition. We propose that FGFR1 deficiency causes ECM disorganization and impaired dendrite formation, disrupting osteocyte communication with neighboring bone and vascular cells, ultimately leading to cell death. These findings establish FGFR signaling as a critical regulator of osteocyte differentiation, viability of bone-embedded osteocytes, and bone homeostasis. Summary StatementFGFR signaling has a profound effect on adult bone extracellular matrix that is vital to maintaining the viability and morphology of newly formed osteocytes, their lacunocanalicular network and the maintenance of bone homeostasis.

Osteoporosis is a prevalent skeletal disorder characterized by deterioration of bone microarchitecture and loss of bone mineral density, leading to increased fracture risk and substantial health and economic burdens, particularly among older adults. Bone remodeling is orchestrated by a complex interplay of systemic and local regulators, among which vitamin D plays a central role in maintaining skeletal homeostasis. Although numerous studies have examined the effects of vitamin D on bone metabolism, outcomes have been inconsistent across populations, dosing regimens, and experimental models. To clarify the net skeletal impact of vitamin D, we investigated its effects in postmenopausal osteoporosis (PMO). Vitamin D (1,25-dihydroxyvitamin D3- active form of vitamin D) supplementation effectively prevented bone loss in ovariectomized mice, at both lower and higher concentrations. Mechanistically, vitamin D promoted osteoclast differentiation in vitro, consistent with its RANKL-dependent pro-osteoclastogenic activity, yet paradoxically conferred bone protection in vivo. This discrepancy was explained by vitamin Ds profound immunomodulatory effects, which reshaped both innate and adaptive immune responses to suppress osteoclast formation and function. Concurrently, vitamin D improved intestinal barrier integrity and restored gut microbial composition, thereby stabilizing the gut-immune-bone axis and reducing pro-resorptive inflammatory signaling. Together, these findings demonstrate that vitamin D prevents bone loss through the coordinated regulation of immune and gut homeostasis, reconciling its apparent pro-resorptive effects in vitro with its overall anti-resorptive outcomes in vivo. This integrated mechanism highlights immune-gut microbial modulation as a key mediator of vitamin D-induced bone preservation and supports the development of vitamin D as an immunotherapeutic adjunct for the prevention and management of PMO. Altogether, our findings for the first time dissect the paradox surrounding the osteoprotective property of vitamin D supplementation.

Rett syndrome is a severe neurodevelopmental disorder caused predominantly by loss-of-function mutations in the X-linked gene MECP2. Besides a vast array of neurological and physiological impairments, patients also frequently develop severe osteopenia with increased fracture risk, however, the mechanisms underlying these skeletal defects are not completely understood. Previous work in Mecp2-null mouse models has suggested that osteopenia is mainly due to impaired osteoblast function and reduced bone formation. Here, we examined bone mass, microarchitecture, and remodeling parameters in a Mecp2-null mouse model during postnatal development, with a particular focus on osteoclast involvement. Micro-computed tomography and histomorphometric analyses showed reduced bone mineral density and trabecular bone volume, associated with increased trabecular separation and cortical thinning. These structural alterations were accompanied by increased osteoclast number per bone surface, elevated urinary deoxypyridinoline, and higher expression of osteoclast-associated genes, including Cathepsin K. Furthermore, gene expression analysis revealed an age-dependent shift in bone remodeling. At postnatal day 35, mutant mice showed reduced expression of Dlx5 and Dlx6, consistent with low bone turnover. By postnatal day 55, Rankl and Cathepsin K were markedly upregulated, suggesting an increase in osteoclast resorptive activity, while key osteoblast markers and the RANKL/OPG ratio did not change significantly. A potential cell-autonomous contribution of Mecp2 to osteoclast maturation is also suggested by the analysis of public transcriptomic datasets on human osteoclast differentiation. Together, our findings identify increased osteoclast activity as a significant contributor to Rett-associated osteopenia and suggest that skeletal pathology in Mecp2 deficiency progresses from an early low-turnover state to a later phase of increased osteoclast resorption. HIGHLIGHTSO_LIWhat are the main findings. O_LIMecp2-null mice display reduced bone mass and altered bone microarchitecture during postnatal development, associated not only with reduced osteoblast activity, but also with increased osteoclast number, elevated urinary deoxypyridinoline, and increased expression of osteoclast-associated genes. C_LIO_LIBone remodelling shows an age-dependent shift in Mecp2 deficiency, from an early low-turnover state at postnatal day 35 to increased osteoclast resorptive activity at postnatal day 55. C_LI C_LIO_LIWhat are the implications of the main findings? O_LIRett-associated osteopenia is not explained solely by impaired osteoblast function, but also involves a significant osteoclast contribution to skeletal deterioration. C_LIO_LIThese findings refine the pathophysiological model of bone involvement in Rett syndrome and support the idea that skeletal alterations evolve dynamically during disease progression. C_LI C_LI

Social isolation is a known modifiable risk factor for many chronic diseases including cardiovascular, metabolic, and brain disorders. Recent research has demonstrated that social isolation is similarly detrimental to skeletal health, but these effects may be sexually dimorphic. In rodents, isolation negatively affects bone in adult male mice, but not in females. However, these sex differences have not been systematically investigated, and it is unknown if they persist with long-term social isolation. The goal of our study was to investigate if isolation-induced bone loss may occur on different timescales between female and male mice, as well as investigate the potential roles of estrogen and testosterone. We examined bone changes in grouped (4 mice/cage) or isolated (1 mouse/cage) female and male 16-week-old C57BL/6J mice after 2, 4, or 8 weeks of treatment. We found that social isolation through single housing significantly reduced bone parameters across treatment lengths in male mice (20% reduction in Tb.BV/TV; 8% reduction in Ct.Th.) but not in females even with prolonged isolation. Isolation also decreased biomechanical properties in the femur of male but not female mice. While the females overall bone phenotype was unaffected, isolated females did show an increase in bone turnover markers with as little as 2 weeks of isolation. Isolation also altered estrogen-related gene expression in male mice isolated for 4 or 8 weeks. Overall, our results demonstrate that short- and long-term social isolation has sexually dimorphic effects on murine bone. These findings have important clinical implications for individuals at risk for social isolation, as well as for pre-clinical rodent models utilizing single housing.

Introduction Denosumab increases bone mineral density and reduces fracture risk in patients with osteoporosis. However, whether BMD response to denosumab differs by age, particularly during longer term treatment, remains unclear. This study investigated the association between baseline age and BMD gain during 3 years of denosumab treatment in patients with osteoporosis. Methods This retrospective study included patients with osteoporosis who were treated with denosumab. DXA-based BMD and bone turnover markers were followed for up to 3 years. Percent BMD gain from baseline, defined as %BMD gain, was evaluated. The longitudinal association between baseline age and %BMD gain was assessed using multivariable linear mixed-effects models for the lumbar spine and total hip. Analyses were performed in the treatment naive cohort and the overall cohort according to prior osteoporosis treatment status. Results A total of 255 patients were included in the analysis, of whom 110 had not received prior osteoporosis treatment. In multivariable linear mixed-effects models, older baseline age was associated with smaller lumbar spine %BMD gain in the treatment naive cohort at both 1 and 3 years. Each 1-year increase in age was associated with a 0.187 percentage-point lower lumbar spine %BMD gain at 1 year and a 0.293 percentage-point lower gain at 3 years (1 year: {beta} = -0.187, p = 0.006, 3 years: {beta} = -0.293, p = 0.031). In contrast, baseline age was not significantly associated with total hip %BMD gain in the treatment naive cohort (1 year: {beta} = -0.011, p = 0.826; 3 years: {beta} = 0.028, p = 0.727). In the overall cohort, baseline age was not significantly associated with %BMD gain at either the lumbar spine or total hip at 1 or 3 years (all p > 0.05). Conclusion Older baseline age was associated with a modestly smaller lumbar spine BMD gain in treatment naive patients, whereas no significant age-related association was observed at the total hip. In the overall cohort, age was not significantly associated with BMD gain at either site. These findings suggest that age may have a limited, site specific influence on BMD response to denosumab, particularly in treatment naive patients, and may support more individualized treatment planning in patients with osteoporosis.

BackgroundPregnancy and lactation impose substantial demands on maternal calcium homeostasis, leading to pronounced skeletal remodeling during lactation followed by recovery after weaning. Although bone mass is largely returned at the tissue level after weaning, it remains unclear whether osteocyte-level remodeling exhibits a similarly reversible pattern. Osteocytes regulate mineral mobilization through perilacunar/canalicular remodeling (PLR), which is elevated during lactation. However, its spatial and temporal regulation in response to reproduction remains poorly defined. Objective/HypothesisThis study aims to determine whether PLR-regulated lacunar remodeling during reproduction varies with osteocyte location and relative age. We hypothesized that osteocyte PLR-mediated lacunar change is spatially heterogeneous during lactation, varies with osteocyte location and relative age, and may persist after weaning. MethodsFemale wild-type (WT) and osteocyte-specific PTH/PTHrP receptor knockout (cKO) mice were studied across reproductive stages. Longitudinal in vivo DXA and CT were used to assess skeletal changes. Sequential fluorochrome labeling tracked mineral dynamics and defined osteocyte relative age. Osteocyte PLR activity was evaluated by MMP13 immunohistochemistry. Lacunar-canalicular structure (LCS) was assessed using Ploton silver nitrate staining, and spatially resolved lacunar morphology was quantified using high-resolution backscattered scanning electron microscopy (bSEM). ResultsAt the tissue level, reproduction induced distinct skeletal responses, characterized by reversible cortical bone loss and persistent trabecular deterioration. Cortical bone loss during lactation was spatially asymmetric and confined to the posterior cortex. Fluorochrome labeling further resolved surface-specific remodeling patterns during reproduction, including endocortical resorption at the posterior cortex and sustained deposition at anterior endocortical and posterior periosteal surfaces. At the cellular level, osteocyte PLR activity increased during lactation in WT mice and returned after weaning, whereas no changes were observed in cKO mice. Consistently, lacunar size increased during lactation and returned toward baseline after weaning in WT mice but remained unchanged in cKO mice. Spatially resolved analysis demonstrated that lacunar remodeling was heterogeneous across cortical thickness. At the anterior cortex, lacunar enlargement occurred near the endocortical surface during lactation and was reversible after weaning. In contrast, at the posterior cortex, lacunar enlargement occurred near the periosteal surface and persisted after weaning. These spatial patterns corresponded to cortical regions enriched with newly formed osteocytes, whereas pre-existing osteocytes exhibited minimal changes. This spatial heterogeneity was absent in cKO mice. ConclusionOsteocyte PLR-mediated lacunar remodeling during reproduction is spatially heterogeneous and varies with osteocyte location and relative age. These findings demonstrate that recovery at the tissue level does not necessarily extend to the osteocyte microenvironment and identify osteocyte PLR-mediated lacunar remodeling as a spatially structured and potentially persistent component of reproductive skeletal adaptation. Together, these results highlight a previously unrecognized layer of maternal skeletal health.

Geroprotectors extend lifespan and improve several aspects of healthspan, yet their effects on skeletal ageing remain poorly understood. They hold potential advantages over current bone-targeted osteoporosis therapies, as they may simultaneously improve bone, neuromuscular function, and vision, thereby reducing the risk of falls, the major cause of fractures. Here we examined, for the first time, the long-term effects of rapamycin, acarbose, and 17-estradiol, administered at lifespan-extending doses on trabecular and cortical bone architecture in male and female UM-HET3 mice measured with micro-computed tomography at 12 and 22 months of age. Bayesian modelling analysis reveals that all interventions produced responses in trabecular bone in females at 22 months. These effects were driven mainly by increases in trabecular number, with little evidence for changes in trabecular thickness. In contrast, treatment effects in males were generally negligible. Cortical responses were modest. Moderate increases in cortical area fraction were observed in females treated with rapamycin or 17-estradiol at 22 months, whereas cortical thickness remained largely unchanged, suggesting a geometrical rather than anabolic effect. Interestingly, geroprotectors strongest skeletal responses in females contrasts with the predominantly male-biased lifespan extension reported for acarbose and 17-estradiol, suggesting differential mechanisms mediating lifespan extension and bone structure preservation.

Purpose: The Fracture Risk Assessment Tool (FRAX) excludes objective skeletal muscle health and genetic variables. We evaluated the prognostic associations of handgrip-defined probable/possible sarcopenia and genome-wide polygenic scores (GPS) with 10-year fracture risk, and their incremental predictive value beyond FRAX across racial/ethnic groups and GPS strata. Methods: We analyzed 2,051 postmenopausal women from the Women's Health Initiative. Race-specific analyses focused on Black, Hispanic, and White participants (n=2,009), excluding American Indian/Alaska Native and Asian/Pacific Islander individuals due to sparse fracture events. Sarcopenia status was operationalized by low handgrip strength alone via EWGSOP2 (<16.0 kg) and AWGS 2025 (<18.0-20.0 kg) criteria. Fine-Gray models estimated subdistribution hazard ratios (sHR), treating death as a competing risk. Predictive performance at 10 years was assessed using time-dependent AUC, Brier scores, and decision curve analysis (DCA). Results: Handgrip-defined probable or possible sarcopenia prevalence was 4.4% (EWGSOP2) and 6.4% (AWGS 2025). Black women demonstrated lower risk for major osteoporotic fractures (MOF) (adjusted sHR=0.19, 95% CI: 0.08-0.48) and hip fractures (adjusted sHR=0.07, 95% CI: 0.01-0.52) compared to White women. Neither sarcopenia status nor high GPS showed statistically significant independent associations with fractures after FRAX adjustment. Adding sarcopenia status to baseline FRAX (AUC: 0.71 for MOF; 0.69 for hip) yielded near-identical AUCs, Brier scores, and within-sample net benefit. Conclusion: Handgrip-defined probable/possible sarcopenia and current GPS do not provide independent or incremental predictive value beyond the clinical FRAX framework within this genomic sub-sample of older women.

In the context of muscle loss, bone repair is impaired, suggesting that muscle derived signals contribute to bone regeneration. However, how muscle surrounding the injury site communicates with the bone repair niche remains unclear. Here we found that CX3CL1 expression was induced in endothelial cells in muscle surrounding a femoral bone injury site. Deletion of Cx3cl1 impaired bone healing, demonstrating a functional role for CX3CL1 in bone repair. A CX3CL1 receptor, CX3CR1, was expressed by PDGFR stromal progenitors and lineage tracing showed that CX3CR1 expressing osteoprogenitor lineage cells accumulated at the injury site during repair. PDGFR stromal progenitors showed enhanced osteoblastogenesis in response to recombinant CX3CL1. In older mice, local CX3CL1 delivery increased PDGFRCX3CR1 osteoprogenitor accumulation and improved bone repair. These findings identify a muscle bone signaling pathway in which endothelial CX3CL1 promotes bone repair through CX3CR1 expressing osteoprogenitors.

Osteoclasts originate from bone marrow-derived macrophages, and their maturation relies on fusion of pre-osteoclasts into mature osteoclasts that maintain bone homeostasis. Previous studies established that mechanical signals regulate osteoblast activity in bone homeostasis, although whether such signals regulate bone homeostasis by acting on osteoclasts remains unclear. Herein, membrane tension decreased during pre-osteoclast fusion, accompanied by reduced Piezo1. This suggests that mechanical stimulation inhibits fusion of monocytes into osteoclasts through Piezo1. Piezo1-knockout monocytes (CTSKCre; Piezo1fl/fl) attenuated the anabolic effect of exercise on bone mass in vivo, whereas shear stress inhibited osteoclast fusion in vitro. Piezo1 activation elevated E-cadherin, which anchors Merlin. This led to hyperactivation of the Yes-associated protein (YAP) signaling pathway, subsequently activating TAX1BP1 and suppressing NF-{kappa}B signaling as well as osteoclast differentiation and maturation. Thus, physical exercise activates a Piezo1-E-cadherin-Merlin-YAP axis that prevents osteoclast fusion, presenting a druggable mechanotransduction pathway for osteoporosis.

Sex hormones are essential regulators of skeletal maintenance, but the cell-specific mechanisms by which osteocytes mediate the skeletal consequences of sex hormone deficiency remain incompletely understood. Osteocyte estrogen receptor {beta} (Ot-ER{beta}) has been implicated in sex-specific regulation of bone mass, particularly in male mice, yet its role in coordinating bone morphology and mechanical competence following sex hormone withdrawal is unclear. In this study, male and female mice with osteocyte-targeted ER{beta} deletion (ER{beta}-dOT) and littermate controls were subjected to orchiectomy (ORX), ovariectomy (OVX), or sham surgery at 20 weeks of age. Four weeks later, vertebral and tibial bone morphology were assessed by micro-computed tomography, and tibial mechanical behavior was evaluated using strain gauge-calibrated, microCT-based finite element modeling. ORX induced substantial cancellous bone loss in the lumbar vertebra and proximal tibia of male mice regardless of genotype. In cortical bone, however, ORX reduced tibial cortical area and minimum moment of inertia in male littermate controls, whereas these cortical deficits were attenuated in male ER{beta}-dOT mice. Consistent with these morphological changes, ORX increased finite element-predicted peak tensile and compressive strains in tibial cortical and cancellous compartments and reduced whole-bone stiffness in male controls, but these mechanical deteriorations were largely prevented by Ot-ER{beta} deletion. In contrast, OVX produced modest changes in female tibial cortical geometry and increased cancellous bone strains, but these responses were not strongly dependent on Ot-ER{beta}. Together, these findings reveal that Ot-ER{beta} mediates the skeletal response to sex hormone withdrawal in a sex- and compartment-dependent manner. Specifically, Ot-ER{beta} contributes to ORX-induced deterioration of tibial cortical morphology and mechanical competence in male mice, whereas it is largely dispensable for OVX-induced skeletal changes in female mice. This work highlights osteocyte ER{beta} as a sex-specific regulator linking hormonal status, bone morphology, and load-induced strain environments.

PurposeEstrogen deficiency drives bone loss through interacting endocrine, oxidative, inflammatory and bone-remodeling disturbances. Ergothioneine (EGT) is a diet-derived thiol/thione antioxidant whose effects on the estrogen-deficient skeleton are unknown. We evaluated whether EGT, alone or combined with vitamin K2, vitamin D3 and magnesium L-threonate, attenuates the skeletal and systemic consequences of ovariectomy (OVX) in mice. MethodsForty-eight female C57BL/6J mice underwent sham surgery or OVX and received daily oral gavage for 12 weeks of vehicle, alendronate (1.53 mg/kg), EGT (30 mg/kg/day), EGT with vitamin K2 (40 {micro}g/kg/day) and vitamin D3 (500 IU/kg/day), or EGT with vitamin K2, magnesium L-threonate (350 mg/kg/day) and vitamin D3 (n = 5-6 analysed per group). Outcomes included the uterine index, tibial micro-computed tomography, distal-femoral histology, and serum bone turnover markers (CTX-I, PINP, osteocalcin), sex hormones, TNF-, IL-6, SOD and MDA. OVX lowered the uterine index and induced tibial trabecular deterioration, with increased CTX-I, decreased PINP and osteocalcin, elevated TNF- and IL-6, reduced SOD and increased MDA (all P < 0.01 vs sham). Alendronate restored tibial micro-CT bone-volume fraction (BV/TV) and trabecular number (P < 0.01 vs OVX). The EGT-based regimens did not significantly restore tibial micro-CT BV/TV, trabecular thickness or trabecular number (all P > 0.05 vs OVX), but significantly increased trabecular area on distal-femoral histology (OVX 7.6% vs 14.2-15.0% across regimens; P < 0.05 vs OVX) and lowered CTX-I, TNF-, IL-6 and MDA while raising SOD and partially restoring PINP and osteocalcin (P < 0.05-0.01 vs OVX). Because the histological and micro-CT endpoints were assessed at different skeletal sites, structural interpretation is cautious. Apparent increases in serum estradiol were assay-dependent and are regarded as exploratory. Ergothioneine-based nutritional regimens improved the systemic oxidative, inflammatory and bone-turnover environment of estrogen-deficient bone loss and preserved distal-femoral trabecular area on histology, although tibial three-dimensional microarchitecture by micro-CT was not restored. Because the histological and micro-CT endpoints were assessed at different skeletal sites, the structural interpretation is necessarily cautious. These findings support further evaluation of EGT as a dietary adjunct, with mechanistic and dose-optimization studies warranted.

The clinical utility of monitoring longitudinal changes in musculoskeletal trajectories, including bone mineral density (BMD), muscle strength, height, and weight for fracture prediction, remains underutilized, as current gold-standard tools such as the Fracture Risk Assessment Tool (FRAX) rely solely on cross-sectional baseline data. This study aimed to determine whether a deep learning model integrating individualized musculoskeletal trajectories improves fracture prediction accuracy compared to established static benchmarks. We developed the Hybrid Trajectory-Based model (HyTrax), a Transformer-based deep learning model that encodes sequential measurements of hip and spine BMD, grip strength, height, and weight as temporal tokens, incorporating subject-specific slopes derived from linear mixed-effects models. The model was trained and internally validated in 27,512 postmenopausal women from the Women's Health Initiative (WHI) and externally evaluated in 1,193 participants from the Framingham Heart Study (FHS). In the WHI validation set, the HyTrax + FRAX (BMD) ensemble model achieved a time-dependent Area Under the Curve (AUC) of 0.85 for Major Osteoporotic Fracture, outperforming both the longitudinal Transformer alone (AUC = 0.80) and the standard FRAX-BMD model (AUC = 0.82). The HyTrax + FRAX (BMD) ensemble model demonstrated favorable discrimination and improved risk stratification (Net Reclassification Improvement +26.5%) in WHI. Evaluation in the FHS cohort demonstrated the transportability of the longitudinal embeddings, with the HyTrax + Baseline 2 ensemble model (integrating longitudinal embeddings with clinical risk factors, BMD, and grip strength) achieving an AUC of 0.74. Explainability analyses identified early longitudinal weight fluctuations and overall height loss trajectories as important predictors of future fracture risk, alongside static factors such as age and genetic predisposition. By leveraging individualized trajectories through deep sequential modeling with baseline FRAX probability, the HyTrax + FRAX (BMD) ensemble model improved fracture discrimination over static assessments, offering a framework for incorporating repeated clinical measures into fracture prediction.

Osteoarthritis is increasingly recognised as a disease of failed integration across the whole joint unit; however, the mechanisms that co-ordinate tissue integrity from development through to adult homeostasis remain largely unresolved. The LIM-homeodomain transcription factor LMX1B is a key determinant of embryonic skeletal patterning, but how it functions to regulate skeletal integrity in the mature skeleton is unknown. Recently, LMX1B was identified as a key driver of osteoarthritis. Here we show that loss of lmx1ba in zebrafish causes premature and progressive severe osteoarthritic pathology in adult spines and jaws, despite largely normal early skeletal patterning, revealing a conserved and continuous requirement for lmx1ba in joint maintenance beyond development. At a cellular level, loss of lmx1ba decouples osteoblast and osteoclast-mediated remodelling leading to bone overgrowth, heterogeneity of bone properties causing increased incidence of spontaneous fractures, and progressive abnormalities in spine morphology. In parallel, we observe degeneration of the intervertebral disc and dysregulation of the proteome and glycosaminoglycans indicative of disrupted extracellular matrix and a breakdown of the coordinated regulation of hard and soft tissue interfaces, which at the organismal level leads to altered joint performance. Notably, degeneration is restricted to mobile joints, and is not observed in cranial sutures, demonstrating a selective requirement for lmx1ba in mechanically active tissues. These changes are consistent with a model of spatially disrupted matrix properties that, under cyclic loading, promote progressive tissue damage. Our findings support a model in which continued expression of LMX1B in adulthood is required to maintain joint structures throughout life.

Osteoblastic bone metastasis, in which disseminated tumor cells drive net bone formation, is a clinically distinct and mechanistically underexplored form of skeletal disease that is enriched in hormone receptor-positive breast cancers. Preclinical models of bone metastasis from breast cancer predominantly rely on immunodeficient hosts inoculated with osteolytic human breast cancer cell lines, limiting the study of immune-dependent mechanisms of bone remodeling. Here we describe the development and characterization of an immunocompetent, syngeneic osteoblastic bone metastasis model using intratibial injection of PyMT-CK(OB), a luciferase-expressing derivative of the MMTV-PyMT mammary carcinoma cell line, in FVB/N mice. PyMT-CK(OB) cells produced detectable bioluminescent signal after intratibial injection, enabling longitudinal monitoring of tumor progression. Micro-computed tomography (microCT) revealed significant increases in trabecular bone volume fraction and trabecular number at three and four weeks post-injection, consistent with osteoblastic remodeling. Histological analysis confirmed dense bone lesion formation in tumor-bearing bones. Critically, this osteoblastic phenotype was entirely absent in immunodeficient NOD SCID hosts, despite robust tumor growth, supporting a role for immune competence in tumor-induced bone formation. Loss of bioluminescent signal in immunocompetent mice reflected either immune pressure on reporter gene expression or limited space for cancer cell expansion in the bone, rather than tumor regression or hypoxia, as confirmed by hypoxia imaging and histological endpoint analysis. In contrast, a second PyMT cell subline, PyMT-CF, maintained sustained bioluminescent signal and produced predominantly osteolytic lesions, providing a complementary syngeneic model of osteolytic disease from the same parental background. In vitro hydrogel coculture experiments and protein array analysis of conditioned media revealed that the PyMT sublines have differing impact on MC3T3 osteoblast mineralization, identifying candidate mediators of divergent bone remodeling phenotypes. R7 mammary carcinoma cells derived from MMTV-RON transgenic mouse mammary tumors did not induce measurable bone remodeling under equivalent experimental conditions. Together, these models provide a validated, immunologically intact framework for studying the mechanistic basis of osteoblastic bone metastasis and evaluating therapeutic interventions targeting the tumor-bone microenvironment.

Osteolytic bone diseases are driven by excessive osteoclast formation and bone resorption. While cGAS-STING signaling is known to regulate bone homeostasis via macrophage-intrinsic mechanisms, its role in osteoblast-mediated control of osteoclastogenesis remains poorly defined. Here, we show that cGAS-STING activation of macrophages suppresses their osteoclastogenic potential while promoting immune activation. In osteoblasts, cGAS-STING triggers IRF3-mediated IFN-{beta} production and, notably, shifts the OPG-RANKL axis toward increased osteoprotegerin. In transwell co-culture, pre-activated osteoblasts reduce osteoclast differentiation of strain-matched macrophages. Mechanistically, osteoblast-derived IFN-{beta} is sufficient to inhibit osteoclastogenesis in a paracrine manner. Furthermore, autocrine IFN-{beta} signaling appears to modulate the OPG-RANKL axis, although additional regulatory factors may contribute. Together, these findings identify cGAS-STING-IFN-{beta} signaling as a dual regulator of osteoclastogenesis, acting directly on macrophages and indirectly via osteoblast-derived anti-osteoclastogenic mediators. This highlights osteoblasts as cGAS-STING-responsive bystander cells within the bone microenvironment that can be targeted as an alternative strategy to limit pathological bone resorption. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/724040v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@167dfcorg.highwire.dtl.DTLVardef@a95477org.highwire.dtl.DTLVardef@e88c77org.highwire.dtl.DTLVardef@15de567_HPS_FORMAT_FIGEXP M_FIG C_FIG