Journal of Bone and Mineral Research

Introduction Low bone mineral density (BMD) is associated with altered bone remodeling and osteoimmune regulation. We aimed to prioritize transcript-level signals supported across human expression cohorts and evaluate their attribution within local cis-regulatory architecture. Materials and Methods Nominal differential-expression signals from GSE56815 were restricted with a predefined 25-term MSigDB thematic library. Features measurable in GSE56815 and GSE2208 underwent linear SVM ranking, LASSO, and XGBoost selection, followed by cross-cohort transcriptomic assessment. OCEL1 was examined in a four-exposure locus-aware CisMRBEEX model with NR2F6, MRPL34, BABAM1, eQTLGen cis-eQTLs, heel eBMD GWAS statistics, and UKBB337K LD. Results The expression screen identified 2,568 genes at nominal P<0.05; thematic restriction retained 190 candidates and 107 common features entered machine learning. Linear SVM, LASSO, and XGBoost retained 24, 18, and 9 genes and converged on CPNE1, EZR, and OCEL1. Only OCEL1 showed concordant expression direction with P<0.05 in both cohorts. In the primary 268-variant four-exposure model, OCEL1 was positively associated with heel eBMD ({beta}=0.010293, SE=0.004107, 95% CI 0.002244-0.018342, P=0.01220, PIP=0.4001; conditional coefficient on the standardized analysis scale); BABAM1 retained an oppositely directed component. The no-palindromic reconstruction remained positive with wider uncertainty ({beta}=0.007652, P=0.06739). Conclusion Convergent transcriptomic evidence prioritized OCEL1, whose conditional genetic-expression component was positively associated with heel eBMD after local shared cis regulation was modeled. Keywords: low bone mineral density; OCEL1; transcriptomics; machine learning; cis-Mendelian randomization

Objective: To examine associations between volume and intensity of leisure time physical activity (LTPA) and fracture risk during midlife and determine whether adherence to World Health Organisation (WHO) physical activity recommendations was associated with fracture risk. Methods: Cross-sectional analysis of UK Biobank participants aged 40 to 65 years using self-reported physical activity and fracture data. LTPA volume (LTPAV) was derived from walking, moderate and vigorous activity. Participants were categorised into LTPAV quintiles and by adherence to WHO physical activity recommendations. Multivariable logistic regression examined associations with self-reported fracture within the previous five years, with sex stratified analyses. Results: Among 322,749 participants, 53% were female with a mean age of 47 years. Fracture risk increased with higher LTPAV, although the association was non-linear and varied by age and sex. The strongest associations were in the highest quintile among men and women aged 40 to 49 years (OR 1.81, 95% CI 1.64 to 1.99 and OR 1.42, 95% CI 1.28 to 1.58 respectively). In mutually adjusted models, vigorous activity demonstrated the strongest independent association with fracture risk. Meeting WHO physical activity recommendations, without exceeding them, was not associated with increased fracture risk. Conclusions: Higher fracture risk was observed in individuals undertaking higher volumes of leisure-time physical activity, with the association largely driven by vigorous-intensity activity. The association was strongest in adults aged 40 to 49 years and in men. Physical activity consistent with current WHO recommendations was not associated with increased fracture risk, supporting continued promotion of physical activity during midlife.

Purpose: Psychosocial factors are underexplored in relation to osteoporosis. This study examined whether depressive symptoms are associated with incident osteoporosis in middle aged and older adults, and whether this association is modified by age or sex. Methods: Prospective cohort study of 8,577 adults aged [&ge;]50 years from the English Longitudinal Study of Ageing. The sample were free of osteoporosis at baseline (2002-2003) and were followed up for incident osteoporosis until wave 11 (2023-2024). Depressive symptoms were measured at baseline (2002-03) using the 8-item Center for Epidemiologic Studies-Depression Scale (CES-D). Cox proportional hazards regressions were used to estimate associations, adjusting for age, sex and wealth. Results: Over 20 years of follow-up, 948 (11.1%) participants reported incident osteoporosis. Each one-point increase in baseline CES-D was associated with a higher hazard of incident osteoporosis adjusted for age, sex, and wealth (hazard ratio (HR) 1.07, 95% CI 1.04-1.11, p<0.001). The association was modified by age (interaction p=0.008) and concentrated in participants aged 50-64 years at baseline (HR 1.10, 95% CI 1.06-1.14), with no significant association in older age strata. Osteoporosis incidence was higher in women, but this was not modified by depressive symptoms. Findings were robust to sequential adjustment (informed by a directed acyclic graph) for education, wealth, inflammatory conditions and health behaviours (HR 1.04, 95% CI 1.01-1.08) Conclusion: Depressive symptoms are associated with incident osteoporosis over two decades of follow-up, particularly among adults aged 50-64. Mid-life may represent a target for psychosocial intervention to support bone health.

Leisure time physical activity (LTPA) in mid-life may have beneficial effects on risk factors for hip fracture in later life. Conversely, physical activity may increase fracture risk through risk of injury, and the optimal amount of physical activity for reducing hip fracture risk remains unclear. We explored the associations between volume and intensity of LTPA in mid-life, and subsequent risk of hip fracture ascertained from linked health data. This prospective observational study used middle aged adults aged 40- 65 years from UK Biobank. LTPA volume (LTPAv) was derived from self-reported walking, moderate and vigorous activity at baseline (2006- 2010). Cox proportional hazard and Fine-Gray sub-distribution models were used to estimate the association between activity and hip fracture, which was sex-stratified. From 324,284 participants with complete data 53% were female and mean age at baseline was 54.5 (SD 7.3). Across 16 years of follow-up there were 2,674 hip fractures. In terms of LTPA volume, the lowest risk of hip fracture occurred in quintile 3 for women (HR 0.78, 95% CI 0.67-0.91), and men (HR 0.74, 95% CI 0.60- 0.90), relating to approximately one hour/day of leisure time physical activity. When activity intensities were examined separately, the lowest estimated hazards were observed for vigorous activity, at quintile 4 in women (HR 0.65, 95% CI 0.55 - 0.76) and quintile 3 in men (HR 0.64, 95% CI 0.52- 0.79), corresponding to approximately 60- 90 minutes of vigorous activity per week. In conclusion, moderately high volumes of mid-life physical activity, particularly vigorous activity, were related to lower risk of subsequent hip fracture, but these relationships appear to attenuate at higher volumes.

Implant-associated bone infection involves a complex interplay between pathogenic stimuli and host cell responses, yet analysis in preclinical models has typically relied on qualitative or semi-quantitative measures. We aimed to establish a quantified evaluation framework to define host-pathogen relationships in a preclinical implant infection model. Staphylococcus aureus-coated stainless-steel implants were inserted trans-cortically in mouse tibiae and bone changes recorded longitudinally by in vivo micro-CT. An automated segmentation task list was developed to independently isolate and quantify cortical, periosteal-reactive, and trabecular bone compartments. RGB trichrome histomorphometry was used to quantify bone matrix integrity, osteocyte lacunar geometry, and osteoclastic activity. Droplet digital PCR was used to determine absolute bacterial and host genome copy number. Infected implants produced marked reductions in trabecular bone volume fraction, number, and bone mineral density (BMD), together with decreased cortical bone volume fraction and increased cortical porosity, accompanied by significant elevations in periosteal bone volume fraction. Histologically, infected bone exhibited increased eroded surface indicative of osteoclastic resorption, extensive degraded bone matrix and pathological remodelling of osteocyte lacunae towards circularity, consistent with an osteocytic osteolysis response. Infection-induced changes to cortical bone structure correlated mostly with host cell rather than bacterial load; however, cortical BMD negatively correlated with the bacterial:host genome ratio. This multifaceted, quantified framework reveals distinct pathobiological effects of implant-associated infection on trabecular, cortical, and periosteal bone compartments, bone matrix and osteocyte and osteoclast populations, consistent with reports in human patients, suggesting that major pathological changes are driven by the host bone cell response to infection.

Abstract Calcific deposits in the arterial media have been associated with a number of metabolic and genetic disorders including diabetes, chronic kidney disease and generalized arterial calcification of infancy. While medial calcification and physiologic hard tissue mineralization in the skeleton are both regulated by several common determinants, emerging data suggest that there might be fundamental differences in the mechanisms underlying these two processes. Objective: We previously demonstrated that elastin haploinsufficiency delays medial calcification in MGP-deficient mice. Here, using mice in which a human ELN transgene rescues mouse elastin deficiency, we investigated whether the origin and abundance of arterial elastin differentially affect the initiation and progression of medial calcification. Approach and Results: We pursued a transgenic approach to alter the arterial elastin scaffold in MGP-deficient mice. Our analyses of a humanized MGP-deficient model with 40% reduction of medial elastin content showed a complete absence of the early-stage vascular calcification. Additionally, we showed that mouse and human elastin orthologues affect vascular calcification in a comparable manner. Conclusion: Arterial elastin abundance, rather than orthologue origin, modulates the initiation and progression of medial calcification in MGP-deficient mice. A further reduction in arterial elastin beyond that achieved by elastin haploinsufficiency profoundly delays mineral deposition and maturation, whereas restoration of elastin abundance through transgenic human ELN expression restores arterial calcification.

Postmenopausal osteoporosis is a highly prevalent disease characterized by low bone mass and structural deterioration of bone tissue leading to increased bone fragility and fracture risk. Osteocytes orchestrate local bone (re)modeling and therefore, characterizing their 3D morphological and spatial distribution across distinct (re)modeling microenvironments is essential for understanding the cellular mechanisms underlying divergent tissue-level responses. To address this, we used a 3D-3D multiscale registration framework mapping endpoint osteocyte lacunae onto dynamic, time-lapsed in vivo (re)modeling sites. Ovariectomized 15-week-old mice were subjected to 4 weeks of anabolic parathyroid hormone (PTH) or anti-catabolic bisphosphonate (BIS) treatment as monotreatment or combined with cyclic mechanical loading (ML). Biweekly in vivo micro-computed tomography (micro-CT) to identify (re)modeling sites and endpoint ultra-high resolution (ultra-HR) micro-CT to characterize osteocyte lacunae was performed. Osteocyte lacunae were characterized by lacunar density, volume and sphericity. Our findings showed that physical and pharmacological therapies differentially regulate the osteocyte lacunar network through distinct perilacunar adaptations across functional microenvironments. While BIS stabilizes local lacunar architecture, PTH drives asymmetrical lacunar expansion (increased volume with reduced sphericity) through perilacunar remodeling that supersedes any added ML effects. Crucially, estrogen loss disrupts the natural spatial maturation gradient by inducing rounding of lacunae in newly formed bone. Therapies counteract this disruption, restoring the healthy gradients in lacunar morphometry between spatially distinct microenvironments. Additionally, despite restoring tissue-level bone volume, physical and pharmacological osteoporosis therapies do not fully recover persistent lacunar density deficits. The overall lacunar morphological spatial signatures confirm active osteocytic tuning to endocrine, physical and matrix cues.

Many hormones and substances acting through G-protein coupled receptors and protein kinase A (PKA) activation inhibit the salt-inducible kinases (SIKs) by phosphorylation. SIKs tonically phosphorylate CREB-regulated transcriptional coactivators (CRTC1, 2 and 3), sequestering them in the cytoplasm and, thus, preventing their translocation into the nucleus. Once in the nucleus, CRTCs bind CREB family member transcription factors and enhance their activity. We and others have shown that parathyroid hormone (PTH) activation of PKA and resultant SIK2/3 inhibition allows CRTC2/3 nuclear translocation. One of the major actions of CRTC2/3 in the osteoblast lineage is the regulation of transcription of Rankl, as well as other PTH-controlled genes. However, little is known about the role of these co-activators in the osteoblast lineage in vivo. Here, we have investigated whether there are basal effects in vivo on bone examined at 2 different ages of conditional deletion of these two co-activators in the osteoblast lineage using Col2.3-Cre. We found significant increases in body weight, length, bone mineral density, bone volume/total volume, trabecular thickness and number with decreased trabecular separation in young (2 months old) male mice, all of which dissipated by 6 months of age. Female mice showed minimal changes in the bone phenotype at either age. Nevertheless, there were gene expression changes in bones of both sexes at both ages, and in particular decreases in Rankl, Runx2 and Sost, and accompanying changes in Wnt pathway genes. These effects may explain the changes in the bone phenotype in the young male mice, but it is notable that there is a sexual dimorphism in the action of CRTC2 and CRTC3. Overall, the work supports the data from research in vitro and forms a basis for investigation of the role of these co-activators in PTH action in vivo.

Mechanical stability critically influences bone fracture healing, yet how the early mechanical environment directs the transition from inflammation to regeneration remains unclear. Using a murine femoral osteotomy model, we compared rigid, semirigid, and dynamically adjusted semirigid-to-rigid fixation. Semirigid fixation delayed healing on day 21, whereas a strategy of early compliance followed by increased stiffness after 7 days restored bridging and improved bone regeneration beyond constant rigid fixation, pointing to a critical early mechanosensitive window after fracture. Single-cell RNA sequencing across the first week, comparing semirigid with rigid fixation, showed that fixation stiffness altered intercellular signaling within one day of injury. Signaling among myeloid populations was more broadly increased under semirigid fixation, with monocytes the single exception, and signaling associated with resolution of inflammation was reduced. From day 5, periosteal and skeletal progenitor populations increasingly directed cartilage-associated matrix and growth factor signaling toward macrophages and chondrocytes, accompanied by increased Sox9 regulon activity and increased COL10+ hypertrophic cartilage matrix by day 7. Reduced fixation stiffness therefore promotes a chondrogenic trajectory of repair, but increasing stiffness is required to redirect this response toward bone formation. Piezo1 was expressed across the responding populations and shifted between compartments as healing progressed. PIEZO1 activation with Yoda1 under rigid fixation enhanced bone formation but provided no additional benefit under semirigid or dynamically adjusted fixation, whereas inhibition of mechanosensitive signaling with GsMTx4 impaired repair across all conditions. Together, these findings establish the early mechanical environment as a determinant of fracture-healing trajectory, shaping the transition from inflammation to regeneration, and show that successful repair depends not on maximal stability but on when stiffness is applied. Increasing either early mechanical stimulation or cellular mechanosensitivity enhanced regeneration, supporting mechanosensitive signaling as a key component of the early healing response.

At physiological levels glucocorticoid (GC) signalling within the osteoblast lineage has been proposed to be required for skeletal homeostasis, whereas supraphysiological activation supresses bone formation and causes bone loss. While these observations suggest that GC exert dose- and context-dependent effects, the molecular mechanisms underlying these opposing actions remain unresolved. Here we systematically dissected the receptor-, lineage-, and concentration-dependent effects of GC signalling on human bone marrow stromal cell (BMSC) differentiation using integrated transcriptional, epigenomic, and functional analyses. Our findings suggest that GC opposing effects are not simply due to a GR dose-response but instead reflect distinct context- and concentration-dependent modes of action. During early BMSC differentiation, the cellular context induced GR-dependent licensing of lineage-specific gene and enhancer programs. The adverse effects of high-dose GC developed over time and could not be predicted from the early transcriptional response. We found that high-dose GC caused osteoblast maturation arrest, accompanied with a cellular stress response sustained by myeloid zinc finger 1 (MZF1) also identified as an endogenous inhibitor of osteoblast differentiation.

Neutrophils are among the first immune cells recruited to fractures, yet their contribution to bone repair remains poorly understood. Using live imaging and neutrophil perturbation in adult zebrafish, we show that neutrophils establish the early fracture microenvironment. Neutrophils rapidly accumulated at fractures, underwent NETosis, and exhibited transcriptional programmes associated with matrix remodelling. Compromising neutrophil recruitment, via ibuprofen treatment or orthogonal perturbations, increased fracture non-union, delayed osteoblast differentiation, impaired mineralisation, and altered callus architecture. Mechanistically, neutrophils actively interacted with extracellular matrix proteins, including laminin, fibronectin, and collagen I, through uptake, trafficking and secretion-associated pathways. Together, our findings reveal that neutrophils establish a provisional emergency fracture matrix that templates subsequent bone repair, providing a mechanistic link between early non-steroidal anti-inflammatory drug (NSAID) exposure and impaired skeletal regeneration.

Background: Viable pediatric human growth plate (GP) tissue is rarely available for translational research, limiting direct investigation of human longitudinal bone growth and pediatric growth disorders. In this proof-of-concept study, we aimed to determine whether it is feasible to establish a clinically integrated ex vivo human GP model using tissue obtained during routine percutaneous epiphysiodesis (PE) procedures in adolescents treated for extreme tall stature or leg length difference due to trauma. Methods: GP tissue and cells were collected during PE and processed using protocols adapted from established methods of human osteoarthritic cartilage processing within the RAAK study. Feasibility was assessed by evaluating tissue collection, cell isolation, contamination rate, monolayer expansion, and generation of three-dimensional cartilage pellets. Proliferation of GP-derived chondrocytes was compared with osteoarthritis-derived articular chondrocytes, and histological assessment was performed to evaluate cartilage-like matrix formation. Results: Across consecutive surgical procedures, viable GP tissue could be obtained reproducibly, with only few samples failing to yield cells and no relevant contamination issues. Isolated GP chondrocytes expanded successfully in two-dimensional culture and showed a strong early proliferative response compared with RAAK-derived chondrocytes. In addition, GP-derived cells formed three-dimensional organoids and histology confirmed cartilage-like matrix deposition supporting their capacity to generate neo-cartilage tissue in vitro. Conclusion: This study demonstrates feasibility to obtain, culture, and functionally assess viable human GP tissue from routine PE surgery. As such, the Leiden ex vivo human GP model provides a unique platform to study local mechanisms of endochondral bone growth, link genetic determinants of height to functional GP biology, and support future therapeutic research in pediatric growth disorders.

The pace of fracture repair declines with age, increasing the risk of delayed union and nonunion. Delayed union and nonunion are associated with significant morbidity and even mortality with few effective non-surgical interventions. Here, we identify cathelicidin antimicrobial peptide (CAMP) as an age-dependent regulator of bone repair. Single-cell RNA sequencing and immunohistochemistry demonstrated higher CAMP expression during fracture healing in young compared with aged mice. Global or myeloid lineage-depletion of Camp delayed fracture repair in young mice, whereas Camp depletion in aged mice increased callus bone formation. Heterochronic bone marrow transplantation using the conditional genetic knockout further implicated hematopoietic-lineage derived cells in these effects and demonstrated that myeloid-specific Camp expression is important in the rejuvenation of the pace of fracture healing. CAMP promotes osteogenic differentiation through P2X7 receptor (P2X7R)-dependent signaling, influencing the downstream regulation of NLRP3 expression. NLRP3 expression is also correlated with osteogenesis following Camp modulation in both young and old mice, supporting CAMP-P2X7R-NLRP3 signaling as a regulator of osteogenesis. Together, these findings identify CAMP as a hematopoietic-derived regulator of bone repair whose function is age-dependent, promoting fracture healing in young animals while constraining it in aged animals.

Objectives. Observational studies report large, conflicting associations between common drugs and hip or knee replacement. We tested them in the serious adverse event (SAE) tables of randomized trials on ClinicalTrials.gov. Methods. We included randomized drug trials with at least 500 participants. A language model classified arms from summaries without outcome counts (masked by instruction only). We pooled osteoarthritis-related SAEs by drug class as Mantel-Haenszel risk ratios (RRs). Because the plan followed a preliminary analysis of 72 trials, we report estimates rather than tests, checked by positive and negative controls and calibration. Results. The 881 placebo- or usual-care-controlled trials included 1.75 million participants and 9,408 osteoarthritis-related SAEs. The tables reproduced CANTOS's own canakinumab effect (RR 0.57, 95% CI 0.39 to 0.82), the anti-nerve growth factor excess (2.31, 1.82 to 2.92) and two of three modest effects specified before extraction: gallbladder disease with glucagon-like peptide-1 receptor agonists (GLP-1RAs; 1.36, 1.22 to 1.51) and ketoacidosis with sodium-glucose cotransporter-2 (SGLT2) inhibitors (1.96, 1.48 to 2.60). In the same trials, osteoarthritis-related SAEs did not differ detectably with GLP-1RAs (0.97, 0.86 to 1.10; calibrated 0.87, 0.54 to 1.41) or SGLT2 inhibitors (0.96, 0.82 to 1.12; calibrated 0.86, 0.53 to 1.40). Other proposed drugs gave imprecise estimates. Post hoc, 8 of 21 published observational estimates differed from randomized ones, in both directions (3 to 6 after calibration). Conclusions. Over about two years, GLP-1RAs and SGLT2 inhibitors had no detectable effect on osteoarthritis-related SAEs in trials that detected their known effects. Slow or modest structural effects remain possible.

Abstract Purpose: Haemochromatosis due to HFE-C282Y homozygosity can lead to excess iron absorption and is typically associated with liver malignancy, plus widespread arthritis. Recent evidence suggests that limb fractures are more common, but little is known about vertebral effects. This study investigated the association of vertebral compression fractures, assessed with intelligent dual-energy X-ray absorptiometry (iDXA), and HFE genotype in a large community cohort. Methods: UK Biobank data from 227 European genetic ancestry C282Y homozygotes (mean 64.6 years) and 234 age, sex, and BMI-matched controls without common HFE haemochromatosis variants were included. Lateral vertebral assessment scans (iDXA, GE-Lunar) were acquired at imaging reassessment (2014-2020) and reviewed, blind to genotype, for radiological evidence of vertebral fracture. Matched logistic regression models assessed associations between C282Y homozygosity and vertebral fractures. Results: 78 vertebral fractures (16.9%) were identified within 461 participants. Male C282Y homozygotes had increased odds of vertebral fracture (n=22/89, 24.7%) compared to participants without HFE alleles (n=9/90, 10.0%); Odds Ratio [OR]: 2.95, 95%CI: 1.28-6.85, p=0.01. The association persisted after excluding individuals with a diagnosis of haemochromatosis (OR: 3.37, 95% CI: 1.41-8.10, p=0.007). No excess fracture risk was observed in female C282Y homozygotes (n=23/138, 16.7%) vs those without HFE alleles (n=24/144, 16.7%); OR: 0.99, 95%CI: 0.53-1.87, p=1.00. Conclusion: In this community-based imaging study, male HFE C282Y homozygotes had a markedly higher likelihood of vertebral fractures than those without HFE variants. These findings support further evaluation of vertebral fracture assessment in C282Y homozygous men to ensure prompt treatment to prevent future fracture if appropriate.

Abstract Fractures in older adults cause high morbidity and mortality. This study aims to identify fracture incidence and risk factors, and to develop the Fracture Risk Model-Abu Dhabi (FRM-AD). Method This retrospective cohort study of 1757 males and females aged 40+ from Abu Dhabi, United Arab Emirates, who participated in a screening program from 2016 to 2020, followed until 2024 for 4.7 years, SD =1.8. It utilized Electronic Medical Records (EMRs) data and telephone interviews. The outcome assessed was the occurrence of fractures. Results There were 58 out of the 1149 females (5.0%) and 26 out of the 608 males (4.3%) who had at least one incident fracture. Hip and vertebral fractures accounted for 12.1% and 11.5%, respectively. There was an annual incidence of 10.5 fractures per 1,000 person-years, 10.7 cases among females and 9.9 among males, rising after age 80 to 39.1 among females and 25.3 among males. Fracture Predictors identified using Poisson regression analysis were older age, smoking, history of previous fracture(s), interaction of history of fracture in a parent with a family history of osteoporosis, positive history of hip fracture in a parent after the age of 40, lack of dyslipidemia diagnosis and the interaction of diabetes mellitus diagnosis and family history of osteoporosis. A higher Body Mass Index (BMI) increased the risk of fractures in this cohort, especially in obese males. Logistic regression of variables at the end of follow-up showed that lower latest vitamin D levels were associated with increased fracture risk. FRM-AD, derived using Poisson regression, performed well in predicting fractures, with Receiver Operating Characteristic (ROC) curves of 0.736 (0.677-0.794) and 0.742 (0.684-0.800) without and with BMD, respectively. The FRAX AUC to predict MOF and hip fracture ranged between 0.624 and 0.683. Conclusion In this Emirati cohort, the locally derived FRM-AD showed moderate discrimination for incident fracture and outperformed FRAX-AD, suggesting that a locally derived model may improve fracture risk stratification. Several risk factors and predictors were identified that can be targeted for prevention.

Background: Bone mechanical competence depends not only on bone mass but also on the spatial organization of mineral and organic matrix properties. Whereas quantitative backscattered electron imaging (qBEI) characterizes mineral heterogeneity using Bone Mineral Density Distributions (BMDDs), no equivalent framework currently exists for describing the biochemical heterogeneity of the bone matrix by Raman microspectroscopy. Methods: We developed a quantitative Raman analysis (qRA) workflow to generate Bone Material Properties Distributions (BMPDs). Raman spectra were acquired along full-thickness line scans crossing successive bone structural units in human trabecular and cortical bone. Pixel-wise Raman-derived biochemical parameters describing collagen organization, advanced glycation end-products, glycosaminoglycans, mineral-to-matrix ratios, carbonate substitution, and mineral crystallinity were converted into normalized frequency distributions and modeled using gaussian functions. BMPDs were generated from undecalcified pMMA-embedded iliac biopsies (n=38) and routinely processed decalcified paraffin-embedded osteomedullary biopsies (n=21). The analytical framework was validated against conventional two-dimensional Raman mapping, and reproducibility, interindividual variability, and demographic influences were evaluated. Results: BMPDs were consistently well described by gaussian functions (typically R2 > 0.95), allowing extraction of three descriptors for each biochemical parameters: Mean, Peak, and Width. One-dimensional line scans showed excellent agreement with conventional two-dimensional Raman mapping while reducing acquisition time approximately 100-fold. Technical variability remained substantially lower than biological variability for all parameters, and a single trabecular line scan provided reliable estimates of BMPD descriptors for nearly all Raman-derived indices. Cortical bone exhibited narrower BMPDs than trabecular bone, indicating lower biochemical heterogeneity. Comparable BMPDs were obtained from undecalcified and routinely decalcified specimens, demonstrating robustness to tissue processing. No significant associations with age or sex were observed after correction for multiple testing. Reference BMPDs further enabled visualization and standardized quantification of deviations in representative pathological bone biopsies. Conclusions: Quantitative Raman analysis introduces BMPDs as a novel framework for assessing the spatial heterogeneity of bone extracellular matrix composition. By extending Raman microspectroscopy beyond conventional mean measurements, BMPDs provide robust, reproducible descriptors of bone material organization and establish a methodological foundation for investigating alterations of bone quality in metabolic bone diseases.

Osteocytes are the most abundant cells in bone and are increasingly recognised not only for their role in skeletal remodelling and inflammatory signalling but also for their potential involvement in immune responses. In this study, we searched available gene expression datasets of human primary osteocyte-like cells exposed acutely to Staphylococcus aureus and identified significantly induced expression of key genes related to antigen processing and presentation. We then confirmed that human bone explant-derived osteoblastic cells, representative of a mature osteoblast-pre-osteocyte stage, expressed, as expected, high cell surface levels of major histocompatibility complex (MHC) Class I but also, low basal levels of the MHC Class II family member, HLA-DR. However, confocal imaging revealed high expression of MHC Class II molecules and the peptide-loading chaperone HLA-DM within the lysosomal compartments, consistent with canonical antigen-processing machinery. Differentiation towards a mature osteocyte phenotype increased MHC Class II protein levels and maintained expression of intracellular HLA-DM. Exposure of mature osteocyte-like cells to S. aureus further up-regulated both intracellular and cell surface MHC Class II expression. Demonstrative of antigen presenting cell functionality, S. aureus-exposed osteocytes induced autologous CD4+ T cell proliferation. Furthermore, MHC Class II expression in osteocytes was detected in bone sampled from patients with periprosthetic joint infections, providing evidence that these mechanisms operate in vivo. Together, our findings reveal that human osteocytes are capable of inducible MHC Class II-associated antigen presentation in response to bacterial challenge, pointing to a novel role for osteocytes in adaptive immune surveillance within bone.

Background Lean mass is positively associated with bone mineral density (BMD), and this relationship may vary according to age and sex. Sedentary behavior has also been associated with BMD, although findings differ across populations and skeletal sites. However, it remains unclear whether sedentary time modifies the relationship between regional lean mass and BMD and whether such modification varies across sex and age. These relationships were explored using nationally representative U.S. data. Methods Adults aged 18-59 years from the 2017-2018 National Health and Nutrition Examination Survey (NHANES) were analyzed. Mean bilateral leg lean mass and mean bilateral leg BMD were derived from dual-energy X-ray absorptiometry measurements, and sedentary time was obtained from self-report. Survey-weighted regression models incorporated NHANES examination weights, strata, and primary sampling units. The primary model examined the four-way interaction among leg lean mass, sedentary time, sex, and continuous age, with adjustment for race/ethnicity and height. Quadratic lean-mass terms were examined in sensitivity analyses. Results The primary complete-case analysis included 2,543 adults (1,191 men and 1,352 women). Women showed a more apparent decline in both leg lean mass and leg BMD at ages 50-59 years, whereas sedentary time did not increase in parallel with these age-related changes. Leg lean mass was positively associated with leg BMD in both sexes, with modest nonlinearity, particularly among women. In the primary survey-weighted model, the four-way interaction among leg lean mass, sedentary time, sex, and age was positive (beta = 0.000111 per year, 95% CI 0.000039-0.000183; P = 0.0051), indicating increasing sex divergence in sedentary-time modification of the lean mass-BMD association with age. At younger ages, conditional interaction estimates were similar between sexes; at older ages, estimates became increasingly positive in women while remaining negative but imprecise in men. Quadratic sensitivity analyses attenuated the four-way interaction estimate by approximately 9-12% and reduced its statistical precision, while broadly preserving its direction and magnitude. Conclusions In this exploratory cross-sectional analysis, sedentary-time modification of the association between DXA-derived leg lean mass and leg BMD varied according to sex and age. The sex difference was small at younger ages and became progressively more apparent with increasing age. These findings suggest that behavioral and demographic context may contribute to variation in the muscle-bone relationship itself, although confirmation in independent and longitudinal datasets is needed.

Individuals with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder caused by mutations in the bone morphogenetic protein receptor ACVR1 (also known as ALK2), experience progressive and severely debilitating endochondral heterotopic ossification (HO). Previous studies have identified fibro-adipogenic progenitors (FAPs) as a major cellular source of HO in FOP mouse models. Here, we used mRNA sequencing of FAPs isolated from injured muscle of FOP mice to capture the earliest gene expression changes associated with the transformation of muscle tissues into bone. Whereas the transcriptomes of mutant and wild-type FAPs were indistinguishable prior to injury and at 1 day post injury, dramatic dysregulation of gene expression was observed at 3 and 5 days after injury. Surprisingly, gene expression changes suggested early specification of osteochondral lineages by 3 days post injury, approximately 3 to 4 days before the appearance of histologically identifiable cartilage. We identified dysregulation of genes encoding components of major signaling pathways, cell cycle regulators, extracellular matrix components, and hypoxia response mediators, and showed that these gene expression changes are temporally associated with abnormal population growth of mutant FAPs. We further demonstrated that treatment of FOP mice with either an anti-activin A monoclonal antibody or the ACVR1 kinase inhibitor BLU-782 (IPN60130; fidrisertib) normalized the expression of the majority of dysregulated FAP genes. However, the relative efficacy of the drugs differed, and distinct sets of overlapping genes were normalized by the treatments. The substantial transcriptional changes caused by these drugs in FAPs from normally regenerating muscle were also enumerated and described. These findings define early transcriptional events associated with pathogenic FAP reprogramming and provide a molecular framework for understanding the shared and distinct effects of activin A and ACVR1 inhibition.