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Combination treatment of Low-Intensity Vibration (LIV) with zoledronic acid (ZA) was hypothesized to preserve bone mass and muscle strength while reducing adipose tissue accrual associated with complete estrogen (E2)-deprivation in young and skeletally mature mice. Complete E2-deprivation (surgical-ovariectomy (OVX) and daily injection of aromatase inhibitor (AI) letrozole) were performed on 8-week-old C57BL/6 female mice for 4 weeks following commencement of LIV administration or control (no LIV), for 28 weeks. Additionally, 16-week-old C57BL/6 female E2-deprived mice were administered {+/-}LIV twice daily and supplemented with {+/-}ZA (2.5 ng/kg/week). By week 28, lean tissue mass quantified by dual-energy X-ray absorptiometry was increased in younger OVX/AI+LIV(y) mice, with increased myofiber cross-sectional area of quadratus femorii. Grip strength was greater in OVX/AI+LIV(y) mice than OVX/AI(y) mice. Fat mass remained lower in OVX/AI+LIV(y) mice throughout the experiment compared with OVX/AI(y) mice. OVX/AI+LIV(y) mice exhibited increased glucose tolerance and reduced leptin and free fatty acids than OVX/AI(y) mice. Trabecular bone volume fraction and connectivity density increased in the vertebrae of OVX/AI+LIV(y) mice compared to OVX/AI(y) mice; however, this effect was attenuated in the older cohort of E2-deprived mice, specifically in OVX/AI+ZA mice, requiring combined LIV with ZA to increase trabecular bone volume and strength. Similar improvements in cortical bone thickness and cross-sectional area of the femoral mid-diaphysis were observed in OVX/AI+LIV+ZA mice, resulting in greater fracture resistance. Our findings demonstrate that the combination of mechanical signals in the form of LIV and anti-resorptive therapy via ZA improve vertebral trabecular bone and femoral cortical bone, increase lean mass, and reduce adiposity in mice undergoing complete E2-deprivation. One Sentence Summary: Low-magnitude mechanical signals with zoledronic acid suppressed bone and muscle loss and adiposity in mice undergoing complete estrogen deprivation. Translational RelevancePostmenopausal patients with estrogen receptor-positive breast cancer treated with aromatase inhibitors to reduce tumor progression experience deleterious effects to bone and muscle subsequently develop muscle weakness, bone fragility, and adipose tissue accrual. Bisphosphonates (i.e., zoledronic acid) prescribed to inhibit osteoclast-mediated bone resorption are effective in preventing bone loss but may not address the non-skeletal effects of muscle weakness and fat accumulation that contribute to patient morbidity. Mechanical signals, typically delivered to the musculoskeletal system during exercise/physical activity, are integral for maintaining bone and muscle health; however, patients undergoing treatments for breast cancer often experience decreased physical activity which further accelerates musculoskeletal degeneration. Low-magnitude mechanical signals, in the form of low-intensity vibrations, generate dynamic loading forces similar to those derived from skeletal muscle contractility. As an adjuvant to existing treatment strategies, low-intensity vibrations may preserve or rescue diminished bone and muscle degraded by breast cancer treatment.

SUMMARYZoledronic acid (ZA) prevents muscle weakness in mice with bone metastases; however, its role in muscle weakness in non-tumor-associated metabolic bone diseases and as an effective treatment modality for the prevention of muscle weakness associated with bone disorders, is unknown. We demonstrate the role of ZA-treatment on bone and muscle using a mouse model of accelerated bone remodeling, which represents the clinical manifestation of non-tumor associated metabolic bone disease. ZA increased bone mass and strength and rescued osteocyte lacunocanalicular organization. Short-term ZA treatment increased muscle mass, whereas prolonged, preventive treatment improved muscle mass and function. In these mice, muscle fiber-type shifted from oxidative to glycolytic and ZA restored normal muscle fiber distribution. By blocking TGF{beta} release from bone, ZA improved muscle function, promoted myoblast differentiation and stabilized Ryanodine Receptor-1 calcium channel. These data demonstrate the beneficial effects of ZA in maintaining bone health and preserving muscle mass and function in a model of metabolic bone disease. Context and significanceTGF{beta} is a bone regulatory molecule which is stored in bone matrix, released during bone remodeling, and must be maintained at an optimal level for the good health of the bone. Excess TGF{beta} causes several bone disorders and skeletal muscle weakness. Reducing excess TGF{beta} release from bone using zoledronic acid in mice not only improved bone volume and strength but also increased muscle mass, and muscle function. Progressive muscle weakness coexists with bone disorders, decreasing quality of life and increasing morbidity and mortality. Currently, there is a critical need for treatments improving muscle mass and function in patients with debilitating weakness. Zoledronic acids benefit extends beyond bone and could also be useful in treating muscle weakness associated with bone disorders.

BackgroundOsteoporosis is prevalent in elderly women, which causes fragility fracture and hence increased mortality and morbidity. Predicting osteoporotic fracture risk is both clinically-beneficial and cost-effective. However, traditional tools using clinical factors and bone mineral density (BMD) fail to reflect bone microstructure. Here we aim to use high-resolution peripheral quantitative CT (HR-pQCT) images to construct deep-learning models which predict fragility fracture history in elderly Chinese women. MethodsWe used ChiVOS, a community-based national cohort of 2,664 Chinese elderly women. Demographic data, BMD, and HR-pQCT from 216 patients were used to construct three groups of models: BMD, pQCT-index, and DeepQCT. For DeepQCT, we used ResNet34 as classifier, and logistic regression for late fusion. Models were developed using 6-fold cross-validation in development set (90%, N=195), and tested in internal test set (10%, N=21). We applied unsupervised clustering on HR-pQCT indices to derive patient subgroups. FindingsDeepQCT (best model AUC 0.86-0.94) was superior or similar to pQCT-index (best model AUC 0.8-0.93), which both outperformed BMD (best model AUC 0.54-0.78). Surprisingly, DeepQCT built from non-weight-bearing bones performed similarly to weight-bearing bones. Furthermore, two distinct patient groups were classified using HR-pQCT indices. The one with higher DeepQCT risk score showed lower volumetric BMD, bone more microarchitectural abnormalities, and had higher probability of osteoporosis and fragility fracture history. InterpretationDeepQCT scores and HR-pQCT-index permit early recognition of patients with high risk of fragility fracture. This established framework can be easily adapted for other diagnostic tasks using HR-pQCT scans, which promotes bone health management via digital medicine. FundingThis research was supported by the National Natural Science Foundation of China (LC, 82100946; WX, 82270938), CAMS Innovation Fund for Medical Sciences (WX, 2021-I2M-1-002), National Key R&D Program of China (WX, 2021YFC2501700), National High Level Hospital Clinical Research Funding (WX, 2022-PUMCH-D-006), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (LC, 2023-PT320-10), and Young Elite Scientists Sponsorship Program by BAST (LC, No.BYESS2023171). Part of the study was supported by Merck Sharp & Dohme China, Hangzhou, China. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSBone mineral density (BMD) from dual X-ray absorptiometry was firstly used to predict fragility fracture, but had low sensitivity. Tools like FRAX, QFracture, and Garvan, which also incorporated clinical factors into prediction models, showed improved performance. Models containing standard HR-pQCT indices (FRAC) further surpassed most clinical tools. Nevertheless, direct learning from original HR-pQCT images is always desired to reduce labor and bias. Deep learning being the most common method for image-based learning, we searched PubMed for articles published up to Mar 25, 2024, using keywords "( fragility fracture OR osteoporotic fracture) and ( prediction model) and ( HR-pQCT or High-resolution peripheral quantitative CT) and ( deep-learning OR deep learning)". Results showed that no study has built deep learning models from HR-pQCT for fragility fracture prediction. Added value of this studyWe developed DeepQCT from HR-pQCT of 216 elderly Chinese women from a national cohort (ChiVOS), which calculated risk scores using individual bone images and clinical features. BMD and pQCT-index models were compared to DeepQCT. We found both DeepQCT (best model AUC 0.86-0.94) and pQCT-index (best model AUC 0.8-0.93) outperformed BMD (best model AUC 0.54-0.78). DeepQCT using non-weight-bearing bones (ulna, fibula) performed similarly to weight-bearing bones (tibia, radius). Specifically, HR-pQCT revealed one patient subgroup with higher DeepQCT risk scores, which showed lower BMD and multiple bone microarchitectural abnormalities, associated with osteoporosis and fragility fracture history. Implications of all the available evidenceDeepQCT is the first method which uses deep-learning to predict fragility fracture directly from HR-pQCT images. It is also the first to use single bones individually in prediction models, including non-weight-bearing bones, which are excluded in HR-pQCT-index computation. Of note, DeepQCT risk score is highly clinically relevant, as showed in bone density or microarchitectural features differences between patient subgroups. The non-inferior performance of DeepQCT compared to the manual annotation-dependent pQCT-index, supported its application to reduce labor and enhance efficiency. Performance of non-weight-bearing bones also challenges traditional perception of using load-bearing bones only in predicting osteoporotic conditions. Most importantly, the DeepQCT framework can be easily adapted for other tasks using HR-pQCT scans, which greatly expands application of digital medicine in bone mineral disease diagnosis or management.

BackgroundOsteoporosis progresses through stages characterized by declining bone mineral density, vertebral deterioration, and muscle atrophy, with bone-muscle interactions driving synergistic degeneration. MethodsThis study retrospectively collected data from 444 patients aged 50 and older, who underwent DXA, CT, and MRI scans at the First Affiliated Hospital of Soochow University. CT values were measured for 6 vertebrae (L1-S1) and 30 adjacent muscle groups (psoas major, erector spinae, quadratus lumborum) to assess vertebral and muscle density. After analyzing changes in CT values across osteoporosis stages development to capture vertebrae and muscles degeneration pattern, we use multiple interpretable machine learning models to construct classification model and construct bone-muscle interaction network. ResultsThis study found that osteoporosis progresses with age, with faster degeneration in females. Early stages show significant bone degradation, especially in L5 and S1 vertebrae, while later stages highlight muscle atrophy. Machine learning models, enhanced by Recursive Feature Elimination (RFE), effectively predicted disease progression (with Normal vs. Osteopenia 0.788, Normal vs. Osteoporosis 0.909, Normal vs. Osteoporotic fracture 0.942, Osteopenia vs. Osteoporosis 0.708, Osteopenia vs. Osteoporotic fracture 0.820 and Osteoporosis vs. Osteoporotic fracture 0.770). The Combined bone muscle interaction network reveals that vertebrae dominate early interactions, shifting to the muscle-clustered module in advanced stages, reflecting the complex degeneration of both bone and muscle. ConclusionThis study develops classification models and analyze bone-muscle interactions in osteoporosis, uncovering synergistic degradation patterns across disease stages. The innovative BMINet toolkit offers an efficient, interpretable framework for personalized analysis, advancing precision medicine and integrated care for osteoporosis patients.

Genome-wide association studies (GWASs) have revolutionized our understanding of the genetics of complex diseases, such as osteoporosis; however, the challenge has been converting associations to causal genes. Studies have demonstrated the utility of transcriptomics data in linking disease-associated variants to genes; though for osteoporosis, few population transcriptomics datasets have been generated on bone or bone cells, and an even smaller number have profiled individual cell-types. To begin to evaluate approaches to address this challenge, we profiled the transcriptomes of bone marrow-derived stromal cells (BMSCs) cultured under osteogenic conditions, a popular model of osteoblast differentiation and activity, from five Diversity Outbred (DO) mice using single-cell RNA-seq (scRNA-seq). The goal of the study was to determine if BMSCs could serve as a model for the generation of cell-type specific transcriptomic profiles of mesenchymal lineage cells derived from large populations of mice to inform genetic studies. We demonstrate that dissociation of BMSCs from a heavily mineralized matrix had little effect on viability or their transcriptomic signatures. Furthermore, we show that BMSCs cultured under osteogenic conditions are diverse and consist of cells with characteristics of mesenchymal progenitors, marrow adipogenic lineage precursors (MALPs), osteoblasts, osteocyte-like cells, and immune cells. Importantly, all cells were nearly identical from a transcriptomic perspective to cells isolated directly from bone. We also demonstrated the ability to multiplex single cells and subsequently assign cells to their "mouse-of-origin" using demultiplexing approaches based on genotypes inferred from coding SNPs. We employed scRNA-seq analytical tools to confirm the biological identity of profiled cell-types. SCENIC was used to reconstruct gene regulatory networks (GRNs) and we showed that identified cell-types show GRNs expected of osteogenic and pre-adipogenic lineage cells. Further, CELLECT analysis showed that osteoblasts, osteocyte-like cells, and MALPs captured a significant component of BMD heritability. Together, these data suggest that BMSCs cultured under osteogenic conditions coupled with scRNA-seq can be used as a scalable and biologically informative model to generate cell-type specific transcriptomic profiles of mesenchymal lineage cells in large mouse, and potentially human, populations.

PurposeOsteoporosis and associated hip fractures are a major public health concern. Dualenergy X-ray Absorptiometry (DXA) remains the diagnostic gold standard, but its areal (a) bone mineral density (BMD) measurements have limited sensitivity, as many fractures occur at T-scores above -2.5. Three-dimensional (3D) DXA provides compartment-specific volumetric parameters of the hip, potentially improving osteoporosis management. This study aimed to establish reference data for 3D-DXA parameters to improve osteoporosis management and investigate potential compartmental imbalances at the hip. MethodThis multicenter, cross-sectional, population-based observational study (SEIOMM-3D-DXA project), supported by the Spanish Society for Bone and Mineral Metabolism (SEIOMM), analyzed hip DXA scans from 1366 Spanish men and women across six centers. 3D-DXA analyses were conducted using the 3D-Shaper software (3D-Shaper Medical, Barcelona, Spain), producing estimates of trabecular volumetric (v) BMD and cortical surface (s) BMD. Age- and sex-specific reference curves were generated using the LMS method, and thresholds were established based on regression with T-score values. Moreover, trabecular vBMD and cortical sBMD Z-scores were calculated to evaluate potential compartmental imbalances. ResultsThe derived aBMD curves closely aligned with the NHANES III Caucasian reference. Sex-specific thresholds for trabecular vBMD and cortical sBMD were established for patient stratification. Z-score comparisons revealed significant discrepancies between trabecular and cortical compartments in 52.0% of females and 48.7% of males, underlining the importance of compartment-specific bone assessment. ConclusionsThis study establishes reference curves for clinical interpretation of 3D-DXA parameters and demonstrates the potential of 3D-DXA to capture compartmental imbalances at the hip. Mini AbstractIn this study, hip scans from over a thousand men and women in Spain were analyzed to create normative reference values for 3D-DXA parameters. These results can help doctors better stratify people based on the status of each part of the bone and improve the management of osteoporosis.

BackgroundBone formation during development and repair is divergently modulated by osteoblast (OB)-derived vascular endothelial growth factor (VEGF) which drives the skeletal sexual dimorphism of the bone vasculature. While the extracellular matrix (ECM) provides both structural and instructive cues to developing vasculature, the contributions of the bone matrix to this skeletal vascular dimorphism in bone remains undefined at the cellular level. MethodsPrimary OBs were isolated from neonatal female and male C57BL/6 long bones and cultured under basal or osteogenic conditions. ECM composition was quantified by Raman spectroscopy. Primary murine bone marrow endothelial cells (BMECs) were seeded directly onto established OB layers and maintained in heterotypic cocultures to assess contact-mediated effects of OB ECM on BMEC survival and expansion. OB-conditioned media (CM) was used to evaluate soluble-factor contributions, with VEGF-A concentration quantified by ELISA. ResultsRaman spectroscopy, on individual OBs from monotypic cultures, revealed sexually dimorphic ECM signatures that were independent of cellular growth profiles. Female OB matrices were enriched with type I collagen-specific proline and hydroxyproline and octacalcium phosphate with enhanced collagen intra-strand stability consistent with a matrix-dominant signature. Male OB matrices exhibited relatively lower type I collagen content and higher carbonated apatite resulting in an elevated mineral-to-matrix ratio indicative of advanced mineral maturation. After 24-hours of heterotypic culture of BMECs with OBs, BMEC numbers were 1.39-fold higher when in contact with male OBs. CM treatment of BMECs did not recapitulate these effects despite higher VEGF-A release from male OBs. ConclusionsSex differences in OB ECM are linked to divergent, contact-dependent regulation of BMEC behaviour. These findings suggest that differences in matrix maturation stat contribute to the sex-specific regulation of the skeletal vascular niche. Elucidating the mechanisms that regulate sex-specific OB-ECM production may reveal new therapeutic targets for selectively modulating pathological skeletal angiogenesis in men and women. SummaryBone is a sexually dimorphic organ, with men and women differing in bone size, strength and risk of fracture. The skeletal vasculature is essential for bone growth and repair, with bone forming osteoblast (OB) cells influencing blood vessel development through the skeletal extracellular matrix (ECM). Although the interactions between OB and vascular cells are crucial for lifelong skeletal health, it is not yet known whether sex differences in bone structure between men and women arise from differences in OB activity, or whether this divergence is driven by sex differences in blood vessel growth. Here, we show that male and female mouse OBs deposit distinct ECMs that differentially influence vascular endothelial cell behaviour. Female OBs produce a collagen-rich matrix with low mineral content. In contrast, male OB matrices contain less collagen and more mineral while releasing elevated levels of blood vessel promoting VEGF-A than females. When placed directly onto these OBs, vascular cell growth was greater when in contact with male than female OBs. Notably, this sex-dependent effect requires direct contact between both cell types and was not reproduced by exposure to OB-derived substances alone. These findings identify a cellular mechanism by which sex differences in OB matrix composition influences vascular cell behaviour in bone. Understanding how OB-vascular interactions differ by sex may help explain variation in bone health, healing capacity and disease risk between men and women. Further, our approach may support the discovery of new therapeutic targets that support bone growth and repair while targeting abnormal blood vessel growth in a sex-specific manner. HighlightsO_LIPrimary OBs from male and female C57BL/6 mouse long bones synthesise compositionally distinct ECMs. C_LIO_LIFemale OB matrices are type I collagen-rich and enriched with octacalcium phosphate, whereas male OB matrices contain less type I collagen and higher levels of carbonated apatite. C_LIO_LIBone marrow-derived endothelial cell (BMEC) growth is enhanced in heterotypic cocultures with male, but not female, OBs after 24 hours. C_LIO_LIMale OBs release higher levels of the pro-angiogenic factor VEGF-A than female OBs. C_LIO_LIThe sex-specific effects of the OB ECM on BMECs is contact-dependent and are not reproduced by treatment with OB-derived conditioned media. C_LI

microRNAs (miRNAs) are non-coding RNAs which modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. miR-324 is a miRNA previously implicated in bone and cartilage maintenance, defects of which result in common age-related diseases, such as osteoporosis or osteoarthritis (OA). In global miR-324-null mice cartilage damage was increased in both surgically and ageing-induced OA, despite minimal changes to the cartilage transcriptome, with few predicted miR-324 targets dysregulated. However, micro-computed tomography and histology demonstrated that global miR- 324-null the mice had an increase in bone mineral density, trabecular thickness and cortical thickness, with many parameters increasing with age. The bone marrow of miR-324-null mice also had reduced lipid content while and in vivo TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation in miR-324-null mice. Ex vivo assays revealed that the high bone mass phenotype of the miR-324-null mice resulted from increased osteoblast activity and decreased osteoclastogenesis. RNA-seq and qRT-PCR followed by miR-324 target prediction and validation in osteoblasts, osteoclasts and bone marrow macrophages identified the osteoclast fusion regulator Pin1 as a miR-324 target in the osteoclast lineage and the master osteogenic regulator Runx2 as a target of miR-324-5p in osteoblasts, the in vitro overexpression of which recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo. These data point to important roles of miR-324 in skeletal biology with altered bone homeostasis in miR-324-null mice potentially causal for the increased cartilage damage observed during OA and ageing. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis.

Osteoblasts generate bone by secreting collagen and mineralizing it in response to various signaling cues. We have previously shown that the majority of ATP generated by differentiated osteoblasts in response to glucose is through glycolysis in contrast to undifferentiated cells that are more dependent on oxidative phosphorylation. To confirm our previous findings, metabolomics was performed for unlabeled polar metabolites, revealing elevated glycolytic metabolites at the later stages of differentiation. Krebs cycle (TCA cycle) metabolites were also changed confirming metabolic rerouting with differentiation. We hypothesized that an increase in mitophagy shifts ATP generation towards glycolysis resulting in the observed bioenergetic and metabolic changes. Utilizing calvarial osteoblasts isolated from a mitophagy reporter mouse model (MitoQC), an increase in mitophagy and the mitophagy receptor, Bnip3, was observed with osteoblast differentiation. KD of Bnip3 in osteoblasts inhibited differentiation and mineralization arising from impaired mitochondrial function. In vivo, male Bnip3 null mice exhibited a significant decrease in osteoblast numbers resulting in lower bone mass. Mechanistically we identified decreased fusion and increased fission factors, impaired stress signaling and increased proapoptotic factors in the absence of Bnip3. These data demonstrate for the first time that BNIP3 expression and mitophagy during osteoblast differentiation are necessary for relieving mitochondrial stress to maintain optimal bone mass.

The scavenger receptor class B member 1 (SCARB1), encoded by Scarb1, is a cell surface receptor for high density lipoproteins, low density lipoproteins (LDL), oxidized LDL (OxLDL), and phosphocholine-containing oxidized phospholipids (PC-OxPLs). Scarb1 is expressed in multiple cell types, including osteoblasts and macrophages. PC-OxPLs, present on OxLDL and apoptotic cells, adversely affect bone metabolism. Overexpression of E06 IgM - a natural antibody that recognizes PC-OxPLs- increases cancellous and cortical bone at 6 months of age in both sexes and protects against age- and high fat diet- induced bone loss, by increasing bone formation. We have reported that SCARB1 is the most abundant scavenger receptor for OxPLs in osteoblastic cells, and osteoblasts derived from Scarb1 knockout mice (Scarb1 KO) are protected from the pro-apoptotic and anti-differentiating effects of OxLDL. Skeletal analysis of Scarb1 KO mice produced contradictory results, with some studies reporting elevated bone mass and others reporting low bone mass. To clarify if Scarb1 mediates the negative effects of PC-OxPLs in bone, we deleted it in osteoblast lineage cells using Osx1-Cre transgenic mice. Bone mineral density (BMD) measurements and micro-CT analysis of cancellous and cortical bone at 6 months of age did not reveal any differences between Scarb1{Delta}OSX-l mice and their wild-type (WT), Osx1-Cre, or Scarb1fl/fl littermate controls. We then investigated whether PC-OxPLs could exert their anti-osteogenic effects via activation of SCARB1 in myeloid cells by deleting Scarb1 in LysM-Cre expressing cells. BMD measurements and micro-CT analysis at 6 months of age did not show any differences between Scarb1{Delta}LysM mice and their WT, LysM-Cre, or Scarb1fl/fl controls. Based on this evidence, we conclude that Based on this evidence, we conclude that the adverse skeletal effects of PC-OxPLs in adult mice are not mediated by Scarb1 expressed in osteoblast lineage cells or myeloid cells.

Recent research has identified metabolic pathways which play key roles in the differentiation and function of osteoblasts and osteoclasts. However, the mechanisms by which osteocytes, the most numerous cells in bone, meet their energetic demands are still unknown. To address this, we used the IDG-SW3 osteocyte cell line to examine changes in metabolism during differentiation from late osteoblasts to mature osteocytes. There was a significant increase in the expression of glycolysis genes (including Pkm and Ldha), glucose consumption and lactate production during late differentiation of these cells. This was concurrent with the onset of the expression of mature osteocyte markers. Inhibition of glycolysis using the glucose analogue 2-deoxy-d-glucose (2-DG) inhibited IDG-SW3 cell mineralization and differentiation into osteocytes. To examine the effect of glycolysis inhibition on mature osteocytes, we treated differentiated IDG-SW3 cells and long bone osteocytes with 2-DG. Glycolysis inhibition resulted in decreased expression of the bone formation inhibitor Sost and mineralization inhibitor Fgf23. Concurrently, there was an increase in genes associated with lipolysis (Lpl) fatty acid {beta}-oxidation (Ppar{delta} and Cpt1a). Treatment of differentiated IDG-SW3 cells with the unsaturated fatty acid oleic acid increased Cpt1a expression and downregulated Sost and Fgf23. Application of mechanical stress to IDG-SW3 cells resulted in upregulation of oxidative metabolism, Ppar{delta} and Cpt1a expression. Long and short chain acylcarnitines were increased in the cortical bone of axially loaded tibiae compared to non-loaded controls, indicative of increased {beta}-oxidation. Overall, our data suggests that while glycolysis is essential for osteocyte differentiation, mature osteocytes are metabolically flexible. Furthermore, {beta}-oxidation may play an important role in the osteocyte response to mechanical stress.

ObjectivesStudies documented the association of melanoregulin (MREG), a cargo-sorting protein, with its binding partner, the autophagic protein, microtubule-associated protein 1 light chain 3B (LC3B) in macrophages which could affect bone physiology due to the importance of autophagy in osteoclast function. Herein we propose to test the hypothesis that MREG modulates bone remodeling. Therefore, we analyzed the Mregdsu/dsu mutant mice for bone mass, growth plate microarchitecture, and bone marrow-derived osteoclast function to understand how lack of MREG affects bone and mass at two different time points. MethodsMice femurs from wild type and MREG-/- male mice (on C57BL6)/J background) were harvested at 4 and 10 months and imaged by microcomputed tomography to assess bone mass parameters. Femurs were processed for histology by H&E and TRAP staining for assessment of osteoclast numbers. Primary bone marrow-derived macrophages from 3-week-old mice were harvested to assess osteoclast differentiation and function via TRAP, resorptive assay and Western Blot for osteoclast differentiation markers. In addition, a separate cohort of mice were analyzed via EchoMRI to characterize total lean vs. fat whole body mass. ResultsThere was a statistically significant difference in bone volume of 10-month old mice in wild type vs. MREG-/- with MREG mutation suggesting a preservative effect on phenotypical bone parameters as the mice age. A reduction in adipose tissue but an increase in osteoclast numbers was found histologically in MREG mutant femurs. Bone marrow-derived cells, however, showed reduced osteoclastic function in MREG-/-. The mutant mice presented a total lean mass significantly increased compared to wild type per EchoMRI. ConclusionsMREG deficiency seems to impact osteoclast numbers in vivo but not in vitro, although in vitro function was reduced. MREG deficiency favors lean mass preservation over fat accumulation in bones and body composition as mice age. This study provides the foundation for a more in-depth investigation of MREGs role in bone and systemic metabolism. It is possible that MREG can be a future target for new therapeutic modalities in inflammatory and metabolic bone diseases.

Increased physical loading of the skeleton activates new bone formation ensuring its ability to meet mechanical demands over time; however, the capacity of bone to respond to mechanical stimulation diminishes with age. Osteocytes, the cells embedded and dispersed throughout mineralized bone matrix, are master regulators of mechanoadaptation through recruitment of new bone-forming cells, the osteoblasts, via signaling to osteoprogenitors located on bone surfaces. We previously demonstrated that in vivo and in vitro mechanical stimulation significantly upregulated the chemokine C-X-C Motif Chemokine Ligand 12 (CXCL12) and its receptor, CXCR4, in osteocytes and bone lining cells, and that CXCR4 antagonism with AMD3100 attenuated in vivo load-induced bone formation. Here, we extended this work by showing that ablation of CXCL12+ cells and deletion of cxcl12 in late-stage osteoblasts and osteocytes significantly attenuated in vivo load-induced bone formation in the mouse tibia. This bone loading phenotype was rescued by treatment with recombinant CXCL12. To address mechanism, we showed that in vitro deletion of cxcl12 and cxcr4, separately, in bone marrow stromal cells resulted in significantly reduced osteogenic differentiation. Furthermore, CXCL12 treatment enhanced GSK-3b phosphorylation and {beta}-catenin translocation to the nucleus, the former of which was partially blocked by AMD3100. Finally, CXCL12 synergized Wnt signaling leading to significantly increased total {beta}-catenin protein and Axin2 expression, a Wnt signaling target gene. These findings together demonstrate that CXCL12 expression in late-stage osteoblasts and osteocytes is essential for load-induced bone formation, in part, by regulating osteogenic differentiation through activation of the Wnt signaling pathway. SignificanceSkeletal adaptation to mechanical loading is contingent on the recruitment of new osteoblasts to bone surfaces. CXCL12, a chemokine expressed by osteolineage cells, targets effector cells expressing its receptor CXCR4, including osteoprogenitors. Exogenous mechanical loading of mouse hind limbs upregulates CXCL12 in osteocytes, bone lining cells and marrow cells, while antagonizing CXCR4 led to significantly attenuated load-induced bone formation. Here, we show that CXCL12 expression in late-stage osteoblasts and osteocytes is required for load-induced bone formation. Treatment with recombinant CXCL12 rescued the bone loading phenotype suggesting that the CXCL12/CXCR4 signaling pathway may be a feasible drug target for promoting load-induced bone formation when exercise alone is insufficient to counteract low bone mass and osteoporosis.

Type 2 diabetes mellitus (T2DM) is associated with an increased fracture risk independent of bone mass. The exact origin of this increased fracture risk is still not well understood. Using a polygenic diabetic rat model, synchrotron radiation micro-computed tomography (SRCT), and in situ scanning electron microscope (SEM) fracture toughness, we related the changes at the microscale to toughness and material properties of diabetic rat femurs. The diabetic rat model (ZDSD) displayed overnight fasting hyperglycemia and an increased AGEs content. Additionally, we measured the impairment of post-yield properties and toughness in diabetic rats. The cortical geometry and porosity were also affected in this ZDSD model. We measured a decrease in osteocyte lacunar density associated with a decreased lacunar volume. Moreover, we found decreased canal density while maintaining a similar canal diameter. These results indicate that diabetes impairs bone remodeling, affecting bone microstructure. Because canals and lacunae are also linked with extrinsic toughening mechanisms, we attribute the decreased toughness largely to these microstructural changes. In conclusion, we showed that changes in lacunae and canal density, combined with AGEs accumulation, decreased toughness in T2DM rat bone.

Bones ability to adapt to mechanical demands is governed by mechanoregulation, the process by which cells sense and respond to mechanical stimuli to maintain skeletal integrity. In osteoporosis, increased bone resorption activity leads to structural deterioration and elevated fracture risk. While existing pharmacological therapies aim to restore bone mass to reduce fracture risk, it is unclear how they modulate mechanoregulation, especially when combined with physical interventions. Here, we investigate the joint effects of load-bearing physical and pharmacological treatment in a female mouse model of osteoporosis using longitudinal in vivo micro-computed tomography and computational mechanics. We demonstrate that mechanical loading additively and synergistically enhanced predicted strength, bone volume, and mechanoregulation parameters when combined with anabolic therapies (parathyroid hormone and sclerostin antibody) but not with anti-catabolic treatments (bisphosphonates). Increases in predicted strength are associated with reductions in bone resorption rates, shifts in the (re)modeling thresholds as anticipated by Frost in the mechanostat theory, and the modeling capacity of anabolic pharmacological treatments. These findings underscore the therapeutic potential of combining anabolic pharmacological therapies with load-bearing physical activity, particularly in early treatment phases, to optimize bone adaptation and fracture prevention in osteoporosis management.

Although body mass index is positively associated with bone mineral density, suggesting obesity is protective against fracture, elderly obese individuals experience greater fracture risk at certain sites than non-obese peers, suggesting bone structural or material changes contribute to fragility. Diet-induced obesity rodent studies have reported detrimental changes to bone microstructure and some apparent-level material properties, but tissue-level material changes are not well understood. Because adipose tissue is highly vascularized, and bone remodeling depends critically on functional vascular supply, concurrent effects on osteovascular perfusion and structure may provide insight about obesity-related bone fragility. This study aimed to determine the effects of obesity on both tissue-level bone properties and osteovascular properties that could negatively impact bone strength. Five-week-old male C57Bl/6J mice were fed either high fat diet (HFD) or control fat diet (CFD) for 17 weeks and received daily treadmill exercise or remained sedentary for eight weeks at ages 14-22 weeks. HFD negatively affected femur bending strength, with 18% lower yield load than CFD. Although HFD negatively altered cancellous microstructure in the distal femur, with 32% lower bone volume fraction than CFD, it did not affect cortical bone geometry in the femoral metaphysis or diaphysis. HFD caused increased carbonate substitution but had no effect on other composition metrics or apparent- or tissue-level material properties in the femoral diaphysis. Exercise did not affect bone strength or microstructure but increased endosteal mineralizing surface in the tibial diaphysis, mineral crystallinity and mineral-to-matrix ratio in the femur, and blood supply to the proximal tibial metaphysis. HFD did not affect blood supply in the tibia or 2D osteovascular structure in the distal femoral metaphysis, indicating that HFD negatively affects cancellous bone without affecting osteovasculature. This study reveals that HFD negatively affected cancellous microstructure without affecting osteovascular structure, and whole-bone strength without altering cortical geometry or material properties.

Impaired callus remodeling significantly contributes to the delayed healing of osteoporotic fractures; however, the underlying mechanisms remain unclear. Sensory neuronal signaling plays a crucial role in bone repair. In this study, we demonstrate that in ovariectomized (OVX) mice, the loss of CGRP+TrkA+ sensory neuronal signaling during callus remodeling correlates with increased Cx3cr1+iOCs expression within the bone callus. Conditional knockout of Cx3cr1+iOCs restored CGRP+TrkA+ sensory neuronal, enabling normal callus remodeling progression. Mechanistically, we further demonstrate that Cx3cr1+iOCs secrete seme3A in the osteoporotic fracture repair microenvironment, inhibiting CGRP+TrkA+ sensory neurons axonal regeneration and suppressing nerve-bone signaling exchange, thus hindering bone remodeling. Lastly, in human samples, we observed an association between the loss of CGRP+TrkA+ sensory neuronal signaling and increased expression of Cx3cr1+iOCs. In conclusion, enhancing CGRP+TrkA+ sensory nerve signaling by inhibiting Cx3cr1+iOCs activity presents a potential strategy for treating delayed healing in osteoporotic fractures.

BackgroundFZD8 could be a promising therapeutic target in osteoporosis (OP), although the signal transduction mechanism in OP regarding FZD8 has not been completely elucidated. AimsWe used the CRISPR/Cas9 technique to develop an Fzd8-knockout mouse model to study whether Fzd8 inactivation results in genetic changes with potential correlations to OP. Materials and MethodsGenotypes of distinguished classified knockout mice, i.e., heterozygous, homozygous, and wild-type were identified through PCR. Applying the murine model, third generation mice were used for the downstream experiments. We investigated the potential relevance of differentially expressed genes (DEGs) in OP. ResultsWe found that osteoclasts significantly increased in Fzd8-knockout homozygous mice, compared to wild-type mice, while osteoblasts reduced significantly. Before transcription, heterozygous and homozygous mice possessed DEGs related to exons SNP, which are associated with exons CNV. After transcription, DEGs related to exons SNP in heterozygous and homozygous mice were observed, some of which are potentially associated with OP based on pathway and gene set enrichment analyses. ConclusionsOur Fzd8-knockout murine model showed that there were significant alternations in Fzd10 and Lta gene expressions and Itgb3 and RANK protein expressions among the wild-type and homozygous mice, which are significantly associated with bone remodeling. Our results revealed that FZD8 could be a therapeutic target in OP. This study elucidates the molecular mechanisms in OP, providing evidence-based data for OP drug development and treatment.

PurposeTo evaluate the interrater reliability and clinical applicability of a novel MRI-based radiological staging system for osteochondritis dissecans (OCD) that incorporates a short echo time GRE sequence to assess progressive ossification and healing status. Methods and MaterialsThis retrospective HIPAA-compliant study was approved by the institutional IRB. MRI exams from April 2017 to December 2023 were reviewed for patients undergoing diagnostic OCD evaluation. Inclusion required the first MRI with a short echo time GRE (Gradient-Recalled Echo) and TSE (Turbo Spin Echo) sequences. Fifty-two MRIs (mean patient age: 13.4 {+/-} 3.8 years; 28 male, 24 female knees) were randomly selected to ensure balanced stage distribution. Five musculoskeletal radiologists and fellows independently applied the proposed staging system based on progressive ossification, bridging, and lesion stability. Interrater reliability was measured using Fleiss Kappa. Healing outcomes were stratified as: (i) early surgery, (ii) successful non-operative therapy, or (iii) delayed surgery after failed non-operative management. Mean healing times were compared across groups using ANOVA. ResultsSubstantial interrater reliability (Fleiss Kappa = 0.71, 95% CI: 0.65-0.77; p < 0.01) indicates strong agreement across five readers. Among 43 cases with clinical data, 19% (n=8) underwent immediate surgery, while 81% (n=35) received initial non-operative care; 29% (n=10) later required surgery. Healing times differed significantly (p = 0.002, ANOVA): 0.75 {+/-} 0.38 years for early surgery, 0.86 {+/-} 0.62 years for successful non-operative treatment, and 2.4 {+/-} 1.5 years for failed non-operative management with delayed surgery. Findings support the reproducibility of the staging system and its potential to identify lesions at risk of failed non-operative healing. ConclusionThis novel MRI-based radiological staging system demonstrates substantial interrater reliability and enables tracking of progressive ossification, improving assessment of OCD healing. Its integration of short echo time GRE sequences supports broader application in musculoskeletal imaging, including monitoring of fracture healing. Key ResultsO_ST_ABSHigh Interrater ReliabilityC_ST_ABSThe novel MRI-based Osteochondritis Dissecans (OCD) staging system demonstrated substantial interrater reliability (Fleiss Kappa = 0.71), supporting its reproducibility for clinical and research use. Healing Time DifferentiationHealing timelines differed significantly across treatment groups--patients with failed non-operative therapy required nearly three times longer to heal than those who underwent early surgery or successfully completed conservative treatment. MRI-based Ossification Tracking with broader ApplicabilityThe staging system effectively visualized progressive ossification using short echo time GRE sequences (TE < 2.6 ms), enabling assessment of healing stages not captured by conventional MRI sequences. Beyond OCD, this framework may be applicable to other conditions involving endochondral ossification, such as fracture healing. Summary StatementA novel MRI-based radiological staging system for osteochondritis dissecans demonstrates substantial interrater reliability and enables assessment of healing through improved visualization of progressive ossification using short echo time GRE.

Bone formation is critical to maintain bone integrity. Here, we studied the importance of intact energy metabolism for bone formation in humans. The skeletal impact of impaired oxidative phosphorylation (OXPHOS) was investigated in adult individuals with genetically defective mitochondrial DNA translation (m.3243A>G). Although impaired mitochondrial ATP production in m.3243A>G human bone marrow stromal cells (hBMSC) was compensated by increased glycolytic ATP production (unchanged net ATP production), both in vitro osteoblast differentiation and in vivo ectopic bone formation were decreased. The impaired OXPHOS was associated with mitochondrial stress and disruption of the pro-osteogenic transcriptional program characteristic of hBMSC. Supporting OXPHOS pharmacologically in hBMSC restored mitochondrial ATP production, their transcriptional program and metabolism, leading to upregulation of osteogenic genes and restoration of bone formation capacity. These findings demonstrate a mitochondrial regulation mechanism of the osteogenic capacity of hBMSCs and identify OXPHOS as a potential target for increasing bone formation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/629993v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@fca817org.highwire.dtl.DTLVardef@17fb2d3org.highwire.dtl.DTLVardef@b5667forg.highwire.dtl.DTLVardef@15c35db_HPS_FORMAT_FIGEXP M_FIG C_FIG