Bone

BackgroundPalliative radiation therapy (RT) for metastatic spine disease significantly increases the risk of vertebral fractures. However, the temporal mechanisms underlying radiation-induced vertebral bone fragility remain poorly understood. ObjectiveTo evaluate the longitudinal effects of a single high-dose irradiation, simulating palliative RT, on vertebral bone mechanical, architectural, and compositional properties in a healthy, skeletally mature rat model. MethodsThirty-one male Sprague Dawley rats received a single 15 Gy lumbar spine irradiation (IR). L4 vertebrae were assessed across all groups (irradiation: 7, 14, and 28 days post-IR, controls: at 0 and 28 days post-IR) for compressive strength and stiffness, micro-CT-derived bone composition and trabecular indices, serum bone turnover markers (NTX and BAP) and advanced glycation endproducts (AGEs). ResultsIrradiation induced progressive deterioration of vertebral bone mechanical properties, with strength decreasing up to 44% and stiffness up to 38% by 28 days post-IR, compared to 0- day controls. Trabecular bone exhibited reduced BMD, BV/TV, and Tb.N with increased Tb.Sp, a shift toward a more rod-like structure. Early post-IR changes suggested disrupted bone remodeling, characterized by elevated NTX and AGEs, but decreased BAP. Multivariable regression demonstrated that Tb.Th and AGEs were independent predictors of stiffness, collectively explaining 61% of its variance. DiscussionHigh-dose irradiation induces sustained temporal degradation of vertebral mechanical properties driven by both trabecular architectural deterioration and alterations in bone matrix quality. Measures of bone composition and non-enzymatic bone turnover suggest this early damage was driven by disruption of bone cellular homeostasis, favoring increased resorption over formation. These findings support that radiation impairs both structural integrity and pre-yield mechanical behavior, providing mechanistic insight into the elevated fracture risk observed clinically after irradiation for metastatic spine disease. Lay summaryThis study used a rat model to mimic palliative radiation therapy for cancer that has spread to the spine and evaluated the changes in bone quality up to 28 days post-therapy. We found that irradiation progressively weakened the structural integrity and composition of the bones in the spine and disrupted the normal balance of bone breakdown and repair, leading to greater bone loss and fragility. Our findings provide insight into the increased risk of fractures observed in patients receiving radiation therapy to the spine and may support efforts to better protect bone health during treatment.

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.

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.

Schwann cells (SCs) provide support for nerves throughout the body. Despite importance for nerve function and repair, the morphology and distribution of SCs in bone remains largely undefined. In this study we used a "Schwann Cell Mapper" mouse (Mpz-Cre+/-;TdT+/+;Ngfr-eGFP+/+) and a "p75 Lineage Tracer" mouse (Ngfr-CreERT2+/-;ZsGreen1+/+) to study SC localization and morphology within the adult mouse calvaria, limb, and vertebrae. We found that all nerves in bone were covered by mature myelinating or non-myelinating SCs, labeled by MPZ and p75-NGFR, respectively. Mature SCs populated the periosteum and entered the bone marrow through transcortical canals. Non-myelinating SCs outnumbered myelinating SCs in bone, with a ratio of [~]2:1 by length density. Non-myelinating SCs in periosteum had more branching and increased size relative to myelinating SCs. In addition, we identified two candidate populations of MPZ lineage+ and p75-NGFR+ immature SCs (iSCs) that were distributed throughout the calvarial periosteum. Similar to mature SCs, p75-NGFR+ candidate iSCs were more prevalent at a ratio of [~]3:1. Overall, neural crest-derived calvarial bone had evidence of increased SC maturity relative to mesoderm-derived sites, identifying niche-level differences in SC maturation. Lastly, lineage tracing revealed that mesenchymal lineages in bone were largely negative for both MPZ and p75-NGFR (<0.1% labeling). These findings provide a framework of SC organization in bone and highlight previously unrecognized diversity across skeletal compartments. By defining distribution and morphology, this work lays the foundation for future studies investigating how SCs contribute to bone biology, including roles in repair, pain, and homeostasis.

Micro-computed tomography ({micro}CT) is widely used to assess trabecular bone microarchitecture, with trabecular separation (Tb.Sp) among the core parameters recommended for reporting. Tb.Sp is typically expressed as a single volume-weighted mean derived from maximal sphere fitting, although the underlying distribution of local separation values is rarely examined. Here, we show that Tb.Sp distributions in metaphyseal trabecular bone are frequently non-Gaussian and bimodal or multimodal. Using {micro}CT datasets from three established models of osteoporosis, spinal cord injury (SCI), ovariectomy (OVX), and ageing, we demonstrate that this behaviour is most evident in metaphyseal trabecular bone and is less apparent in epiphyseal trabecular bone or trabecular thickness distributions. We further show that multimodal metaphyseal Tb.Sp distributions correspond to two spatially distinct contributions within the marrow space: lower-diameter local separation within the residual trabecular network, and higher-diameter regions associated with larger contiguous marrow cavities. Based on this observation, we introduce a simple extension to standard morphometric analysis in which Tb.Sp is decomposed into local trabecular separation (Tb.SpL) and marrow cavity separation (Tb.SpM). Tb.Sp decomposition revealed model-specific patterns of trabecular deterioration. SCI was characterised predominantly by increased Tb.SpM, consistent with expansion of larger marrow cavities, whereas OVX showed a more subtle or distributed alteration. Ageing showed changes in both Tb.SpL and Tb.SpM, with the higher-diameter component becoming most prominent in older animals. Together, these findings demonstrate that mean Tb.Sp can mask structurally distinct forms of metaphyseal marrow-space organisation and support reporting distributional descriptors, and where appropriate Tb.SpL and Tb.SpM, alongside conventional Tb.Sp.

Osteocytes play a central role in bone remodeling, mineral metabolism, and skeletal homeostasis, but direct molecular analysis of human osteocytes remains technically challenging because they are embedded within the mineralized bone matrix. Surgically obtained human bone specimens provide valuable material for studying human bone biology; however, surface-associated cells, marrow-derived cells, and adherent soft tissues can confound downstream transcript analysis. Here, we describe a bone fragment-based protocol for preparing surgically obtained human bone specimens for molecular analysis of osteocyte-associated transcripts. The protocol consists of mechanical trimming, mincing into small bone fragments, repeated washing, and five sequential rounds of collagenase digestion to reduce non-osteocytic cellular components associated with the bone surface and marrow spaces. The remaining mineralized bone fragments are then frozen in liquid nitrogen, cryogenically pulverized, and lysed in TRIzol reagent for total RNA extraction. Histological validation using residual maxillary bone specimens showed that sequential collagenase digestion markedly reduced adherent soft tissue and extra-matrix nuclei while preserving osteocyte lacunar occupancy. This protocol provides a practical workflow for bone fragment-based RNA analysis focused on osteocyte-associated transcripts in human bone specimens. Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@1cec618org.highwire.dtl.DTLVardef@2f746forg.highwire.dtl.DTLVardef@1854247org.highwire.dtl.DTLVardef@1c26c1aorg.highwire.dtl.DTLVardef@1473a88_HPS_FORMAT_FIGEXP M_TBL C_TBL

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.

Fracture nonunion remains a major cause of morbidity, yet patient-specific factors associated with impaired healing remain incompletely characterized. Anemia has been associated with adverse orthopaedic outcomes, but its relationship with fracture nonunion is poorly understood. We examined whether pre-fracture anemia, anemia burden, and clinically relevant anemia subtypes were associated with nonunion following tibial or femoral fractures. Using commercial and Medicare fee-for-service claims from 2016 through 2023, we identified adults aged 19 years or older with a tibial or femoral fracture, continuous enrollment during the preceding year and for at least six months after fracture, and no baseline cancer. Pre-fracture anemia was evaluated as any anemia, the number of distinct anemia diagnoses, and nutritional, hemolytic, aplastic, and other anemia subgroups. Nonunion occurring six to eighteen months after fracture was assessed using incidence rates and multivariable-adjusted hazard models. Among 326,673 adults, 149,704 had pre-fracture anemia and 176,969 did not. The crude incidence of nonunion was 42% higher among individuals with anemia than among those without anemia (incidence rate ratio, 1.42; 95% confidence interval, 1.32 to 1.53) and increased with greater anemia burden. After adjustment for demographic and clinical characteristics, including prior fractures at other anatomical sites, pre-fracture anemia remained associated with nonunion following tibial and femoral fractures, with hazard ratios of 1.83 (95% confidence interval, 1.54 to 2.18) and 1.38 (95% confidence interval, 1.26 to 1.50), respectively. Associations were also observed for nutritional and other anemias, whereas estimates for hemolytic and aplastic anemias were limited by few nonunion events. Within the femur, the association was strongest for distal fractures. These findings demonstrate that pre-fracture anemia is independently associated with nonunion. The increase in risk with greater anemia burden and findings across evaluable subgroups suggest that pre-fracture anemia may help identify patients at increased risk of impaired fracture healing.

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.

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.

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.

Serial in vivo microCT enables quantification of dynamic bone remodeling by capturing formation and resorption over time. This protocol uses Dragonfly to register pre- and post-intervention scans of rodent long bones through an intuitive drag-and-click workflow that requires no coding expertise. Rigid registration enables spatial mapping and quantification of formed, resorbed, and quiescent cortical and trabecular bone at periosteal and endosteal surfaces across multiple tibial regions, providing a non-destructive and complementary alternative to classic, fluorochrome-based dynamic histomorphometry. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=160 HEIGHT=200 SRC="FIGDIR/small/728585v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e20e89org.highwire.dtl.DTLVardef@12872fcorg.highwire.dtl.DTLVardef@951694org.highwire.dtl.DTLVardef@1fd52f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

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.

Low-intensity vibration (LIV) is a non-invasive mechanical stimulus capable of regulating skeletal adaptation and cellular signaling pathways involved in bone remodeling. Despite growing interest in LIV, substantial methodological heterogeneity persists in the selection of experimental vibration parameters such as frequency, expressed in Hertz (Hz) and intensity, defined as earth's gravitational field (g) (9.81 m/s2). Focusing on micro-computed tomography (CT) derived trabecular bone volume fraction (BV/TV) as the main outcome measure, this study sought to synthesize the effects of different LIV frequency and intensity on BV/TV in small rodents (mice and rats) as they remain as the most studied pre-clinical model. To accomplish this, we performed a systematic review searching for publications in English on PubMed, Web of Science, CINAHL, and Embase databases. Two independent investigators followed inclusion criteria to select only peer-reviewed studies with mature mice, using whole-body vibration experiments without other co-variables. We further restricted to include studies that analyzed non-fractured bones and compared pre- and post-intervention or control values. In addition to these core criteria, a detailed hierarchical screening framework was applied during full-text review. The two independent investigators extracted data independently and considered the characteristics of the study, animals' characteristics, intervention characteristics, and results. For this study we considered load-bearing hindlimbs, femur and tibia, separately but did not include vertebrae in the analysis. A Bayesian network meta-analysis and a revised SYRCLE risk of bias (RoB) tool were used to evaluate the risk of bias across included studies. Seven studies met the inclusion criteria. Results showed that an LIV regime applied at 45Hz at 2g presented higher chances to increase trabecular BV/TV of the mouse tibia (estimated effect 3.22 [CrI 1.98, 4.45]), while LIV regimes applied to the femur at 90Hz and 1.4g (estimated effect 3.08 [CrI -1.99, 7.97]) present better chances to increase trabecular BV/TV results compared to other interventions but with no significant differences. Finally, we applied 45Hz at 0.2g LIV to 5 month old male C57BL/6 for 5 weeks (n=10/group) which showed significantly increased Trabecular Thickness (Tb.Th) for both the tibia (10%, p<0.01) and femur (17%, p<0.001), with the femur showing further increases in trabecular BV/TV (32%, p<0.05) compared to non-LIV controls. We conclude that changes in the microarchitectures of the tibia and femur respond differently to the same application of LIV (45Hz, 0.2g) in mice and rats.

Accurate evaluation of extracellular matrix (ECM) mineralization at the nano-scale is essential for establishing relevant in vitro bone models. This is particularly important with the development and increased application of three-dimensional (3D) cell models for biological research. Transmission electron microscopy (TEM) allows to perform ultra-structural analysis of cells and ECM organization, but its application in in vitro bone models remains limited, due to the potential alteration or loss of the mineral phase during sample preparation. In this study, we compared two TEM sample preparation methods - the conventional chemical fixation and the anhydrous methods - to evaluate their ability to preserve the mineralized ECM in MC3T3-E1 cells cultured as monolayers and as alginate-encapsulated bone spheroids. Chemical fixation preserved cellular ultra-structure and collagen organization, allowing for detailed assessment of cells and ECM organization. Although mineral deposits were detected and their needle-like morphology assessed, characterization of more immature deposits was partially limited by the effects of uranyl acetate and the overall sample preparation process, which could lead to alteration or loss of less stable mineral phases. The anhydrous preparation method resulted in limited preservation of cellular and ECM morphology and did not allow reliable identification of mineral deposits. When applied to spheroids, the chemical fixation method preserved the 3D architecture, collagen-rich ECM and inner mineral deposits, confirming spheroids as a relevant model for bone studies. Overall, these results highlight the need for optimized sample preparation strategies that preserve both ultra-structure and mineral components for accurate nano-scale characterization of bone mineralization.

Background Automated pelvic CT segmentation has advanced to reliable coarse bone extraction. Yet the structured anatomical hierarchy required for morphometry, fixation planning, bone quality mapping, and arthroplasty workflows remains unachieved. This study developed and validated a fully automated anatomy-informed pipeline that converts standard pelvic CT into a comprehensive, surgeon-readable subsegmentation of the pelvis and proximal femur. Methods Pelvic CT datasets were retrospectively collected from anonymized archives of hospitals affiliated with the Directorate of Health Affairs, Sharqia, Egypt. After eligibility screening, 757 normal adult cases were processed using a custom one-click 3D Slicer pipeline integrating TotalSegmentator for coarse extraction, followed by deterministic anatomy-based subsegmentation into 81 segments. One hundred randomly selected cases were validated against expert-corrected reference segmentations using Dice similarity coefficient, volume difference, surface distance metrics, and bilateral symmetry analysis. Results Of 1,316 screened cases, 757 met eligibility criteria. Across 8,100 case-segment observations, the pipeline achieved a mean Dice of 0.9926 +/- 0.0465. Complete agreement was observed for the sacrum, ilium, acetabulum, anterior and posterior columns, sciatic buttress, and all landmarks. Relative decreases were confined to boundary-dependent regions. Bilateral symmetry analysis confirmed a median surface agreement of 99.85% within 5 mm. Conclusion The pipeline demonstrated high accuracy and reproducibility across a large normal adult dataset, establishing a structured anatomical foundation for quantitative pelvic analysis and surgical planning workflows. Clinical feasibility across abnormal anatomy and decision-level applications awaits dedicated validation.

Background. Obesity is a modifiable risk factor for osteoarthritis and may contribute to pain, functional impairment, inflammation, and cartilage degradation. Resveratrol has potential anti-inflammatory and chondroprotective effects, but its efficacy as an adjunct to dietary intervention remains unclear. Objective. This study evaluated whether resveratrol supplementation provides additional benefits when combined with a low-calorie diet in postmenopausal women with obesity and knee osteoarthritis. Methods. A total of 97 postmenopausal women with obesity and knee osteoarthritis were included in this randomized controlled clinical study. Participants received either a 10-day low-calorie diet alone or the same diet combined with 150 mg/day trans-resveratrol. Anthropometric parameters, body composition, biochemical markers, pain intensity, functional status, and urinary CTX-II were assessed at baseline and follow-up. Results. Both interventions were associated with reductions in body weight, BMI, waist and hip circumferences, fat mass, glucose, HOMA-IR, lipid parameters, hsCRP, VAS, WOMAC, LAI, and urinary CTX-II. Compared with diet alone, resveratrol supplementation did not provide additional benefits for anthropometric parameters, glucose metabolism, lipid profile, or WOMAC score. However, the resveratrol group showed a greater reduction in hsCRP and urinary CTX-II. The obesity class did not modify the treatment effect. Conclusion. A short-term low-calorie diet improved metabolic, inflammatory, and osteoarthritis-related parameters in postmenopausal women with obesity and knee osteoarthritis. The addition of resveratrol did not enhance weight loss or improve most metabolic outcomes but was associated with greater reductions in hsCRP and urinary CTX-II. These findings suggest a potential anti-inflammatory and cartilage-related effect of resveratrol, which requires confirmation in longer randomized trials.

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.

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.