The delicate balance between bone formation by osteoblasts and bone resorption by osteoclasts maintains bone homeostasis. Nuclear receptors (NRs) are now understood to be crucial in bone physiology and pathology. However, the function of the Farnesoid X receptor (FXR), a member of the NR family, in regulating bone homeostasis remains incompletely understood. In this study, in vitro and in vivo models revealed delayed bone development and an osteoporosis phenotype in mice lacking FXR in bone marrow mesenchymal stem cells (BMSCs) and osteoblasts due to impaired osteoblast differentiation. Mechanistically, FXR could stabilize RUNX2 by inhibiting Thoc6-mediated ubiquitination, thereby promoting osteogenic activity in BMSCs. Moreover, activated FXR could directly bind to the Thoc6 promoter, suppressing its expression. The interaction between RUNX2 and Thoc6 was mediated by the Runt domain of RUNX2 and the WD repeat of Thoc6. Additionally, Obeticholic acid (OCA), an orally available FXR agonist, could ameliorate bone loss in an ovariectomy (OVX)-induced osteoporotic mouse model. Taken together, our findings suggest that FXR plays pivotal roles in osteoblast differentiation by regulating RUNX2 stability and that targeting FXR may be a promising therapeutic approach for osteoporosis.
Circadian rhythm is ubiquitous in nature. Circadian clock genes such as Bmal1 and Clock form a multi-level transcription-translation feedback network, and regulate a variety of physiological and pathological processes, including bone and cartilage metabolism. Deletion of the core clock gene Bmal1 leads to pathological bone alterations, while the phenotypes are not consistent. Studies have shown that multiple signaling pathways are involved in the process of Bmal1 regulating bone and cartilage metabolism, but the exact regulatory mechanisms remain unclear. This paper reviews the signaling pathways by which Bmal1 regulates bone/cartilage metabolism, the upstream regulatory factors that control Bmal1, and the current Bmal1 knockout mouse models for research. We hope to provide new insights for the prevention and treatment of bone/cartilage diseases related to circadian rhythms.
Tissue clearing combined with high-resolution confocal imaging is a cutting-edge approach for dissecting the three-dimensional (3D) architecture of tissues and deciphering cellular spatial interactions under physiological and pathological conditions. Deciphering the spatial interaction of leptin receptor-expressing (LepR+) stromal cells with other compartments in the bone marrow is crucial for a deeper understanding of the stem cell niche and the skeletal tissue. In this study, we introduce an optimized protocol for the 3D analysis of skeletal tissues, enabling the visualization of hematopoietic and stromal cells, especially LepR+ stromal cells, within optically cleared bone hemisections. Our method preserves the 3D tissue architecture and is extendable to other hematopoietic sites such as calvaria and vertebrae. The protocol entails tissue fixation, decalcification, and cryosectioning to reveal the marrow cavity. Completed within approximately 12 days, this process yields highly transparent tissues that maintain genetically encoded or antibody-stained fluorescent signals. The bone hemisections are compatible with diverse antibody labeling strategies. Confocal microscopy of these transparent samples allows for qualitative and quantitative image analysis using Aivia or Bitplane Imaris software, assessing a spectrum of parameters. With proper storage, the fluorescent signal in the stained and cleared bone hemisections remains intact for at least 2–3 months. This protocol is robust, straightforward to implement, and highly reproducible, offering a valuable tool for tissue architecture and cellular interaction studies.
Osteocytes are the main cells in mineralized bone tissue. Elevated osteocyte apoptosis has been observed in lytic bone lesions of patients with multiple myeloma. However, their precise contribution to bone metastasis remains unclear. Here, we investigated the pathogenic mechanisms driving melanoma-induced osteocyte death. Both in vivo models and in vitro assays were combined with untargeted RNA sequencing approaches to explore the pathways governing melanoma-induced osteocyte death. We could show that ferroptosis is the primary mechanism behind osteocyte death in the context of melanoma bone metastasis. HMOX1 was identified as a crucial regulatory factor in this process, directly involved in inducing ferroptosis and affecting osteocyte viability. We uncover a non-canonical pathway that involves excessive autophagy-mediated ferritin degradation, highlighting the complex relationship between autophagy and ferroptosis in melanoma-induced osteocyte death. In addition, HIF1α pathway was shown as an upstream regulator, providing a potential target for modulating HMOX1 expression and influencing autophagy-dependent ferroptosis. In conclusion, our study provides insight into the pathogenic mechanisms of osteocyte death induced by melanoma bone metastasis, with a specific focus on ferroptosis and its regulation. This would enhance our comprehension of melanoma-induced osteocyte death.
Fibrotic remodeling of nucleus pulposus (NP) leads to structural and mechanical anomalies of intervertebral discs that prone to degeneration, leading to low back pain incidence and disability. Emergence of fibroblastic cells in disc degeneration has been reported, yet their nature and origin remain elusive. In this study, we performed an integrative analysis of multiple single-cell RNA sequencing datasets to interrogate the cellular heterogeneity and fibroblast-like entities in degenerative human NP specimens. We found that disc degeneration severity is associated with an enrichment of fibrocyte phenotype, characterized by CD45 and collagen I dual positivity, and expression of myofibroblast marker α-smooth muscle actin. Refined clustering and classification distinguished the fibrocyte-like populations as subtypes in the NP cells - and immunocytes-clusters, expressing disc degeneration markers HTRA1 and ANGPTL4 and genes related to response to TGF-β. In injury-induced mouse disc degeneration model, fibrocytes were found recruited into the NP undergoing fibrosis and adopted a myofibroblast phenotype. Depleting the fibrocytes in CD11b-DTR mice in which myeloid-derived lineages were ablated by diphtheria toxin could markedly attenuate fibrous modeling and myofibroblast formation in the NP of the degenerative discs, and prevent disc height loss and histomorphological abnormalities. Marker analysis supports that disc degeneration progression is dependent on a function of CD45+COL1A1+ and αSMA+ cells. Our findings reveal that myeloid-derived fibrocytes play a pivotal role in NP fibrosis and may therefore be a target for modifying disc degeneration and promoting its repair.
Mechanical stress modulates bone formation and organization of the extracellular matrix (ECM), the interaction of which affects heterotopic ossification (HO). However, the mechanically sensitive cell populations in HO and the underlying mechanism remain elusive. Here, we show that the mechanical protein Polysyctin-1 (PC1, Pkd1) regulates CTSK lineage tendon-derived mesenchymal stem cell (TDMSC) fate and ECM organization, thus affecting HO progression. First, we revealed that CTSK lineage TDMSCs are the major source of osteoblasts and fibroblasts in HO and are responsive to mechanical cues via single-cell RNA sequencing analysis and experiments with a lineage tracing mouse model. Moreover, we showed that PC1 mediates the mechanosignal transduction of CTSK lineage TDMSCs to regulate osteogenic and fibrogenic differentiation and alters the ECM architecture by facilitating TAZ nuclear translocation. Conditional gene depletion of Pkd1 or Taz in CTSK lineage cells and pharmaceutical intervention in the PC1-TAZ axis disrupt osteogenesis, fibrogenesis and ECM organization, and consequently attenuate HO progression. These findings suggest that mechanically sensitive CTSK-lineage TDMSCs contribute to heterotopic ossification through PC1-TAZ signaling axis mediated cell fate determination and ECM organization.
Gain-of-function mutations in fibroblast growth factor receptor (FGFR) genes lead to chondrodysplasia and craniosynostoses. FGFR signaling has a key role in the formation and repair of the craniofacial skeleton. Here, we analyzed the impact of Fgfr2- and Fgfr3-activating mutations on mandibular bone formation and endochondral bone repair after non-stabilized mandibular fractures in mouse models of Crouzon syndrome (Crz) and hypochondroplasia (Hch). Bone mineralization of the calluses was abnormally high in Crz mice and abnormally low in Hch mice. The latter model presented pseudarthrosis and impaired chondrocyte differentiation. Spatial transcriptomic analyses of the Hch callus revealed abnormally low expression of Col11, Col1a, Dmp1 genes in mature chondrocytes. We found that the expression of genes involved in autophagy and apoptosis (Smad1, Comp, Birc2) was significantly perturbed and that the Dusp3, Dusp9, and Socs3 genes controlling the mitogen-activated protein kinase pathway were overexpressed. Lastly, we found that treatment with a tyrosine kinase inhibitor (BGJ398, infigratinib) or a C-type natriuretic peptide (BMN111, vosoritide) fully rescued the defective endochondral bone repair observed in Hch mice. Taken as a whole, our findings show that FGFR3 is a critical orchestrator of bone repair and provide a rationale for the development of potential treatments for patients with FGFR3-osteochondrodysplasia.
Reproductive hormones associated with the hypothalamic-pituitary-gonadal (HPG) axis are closely linked to bone homeostasis. In this study, we demonstrate that Gonadotropin inhibitory hormone (GnIH, one of the key reproductive hormones upstream of the HPG axis) plays an indispensable role in regulating bone homeostasis and maintaining bone mass. We find that deficiency of GnIH or its receptor Gpr147 leads to a significant reduction in bone mineral density (BMD) in mice primarily by enhancement of osteoclast activation in vivo and in vitro. Mechanistically, GnIH/Gpr147 inhibits osteoclastogenesis by the PI3K/AKT, MAPK, NF-κB and Nfatc1 signaling pathways. Furthermore, GnIH treatment was able to alleviate bone loss in aging, ovariectomy (OVX) or LPS-induced mice. Moreover, the therapy using green light promotes the release of GnIH and rescues OVX-induced bone loss. In humans, serum GnIH increases and bone resorption markers decrease after green light exposure. Therefore, our study elucidates that GnIH plays an important role in maintaining bone homeostasis via modulating osteoclast differentiation and demonstrates the potential of GnIH therapy or green light therapy in preventing osteoporosis.
Craniometaphyseal dysplasia (CMD), a rare craniotubular disorder, occurs in an autosomal dominant (AD) or autosomal recessive (AR) form. CMD is characterized by hyperostosis of craniofacial bones and metaphyseal flaring of long bones. Many patients with CMD suffer from neurological symptoms. The pathogenesis of CMD is not fully understood. Treatment is limited to craniofacial surgery. Here, we report a knock in (KI) mouse model for AR CMD carrying a Cx43R239Q mutation. Cx43 KI/KI mice replicate typical features of AR CMD, including thickening of craniofacial bones, club-shaped femurs, and widened diaphyseal cortical bones. Female Cx43 KI/KI mice display remarkably more bone overgrowth than male Cx43 KI/KI mice as they age. In contrast to Cx43 +/+ littermates, Cx43 KI/KI mice exhibit periosteal bone deposition and increased osteoclast (OC) numbers in the endosteum of long bones. Although formation of resting OCs in Cx43 +/+ and Cx43 KI/KI mice is comparable, the actively resorbing Cx43 KI/KI OCs have reduced resorption on bone chips. Cx43 KI/KI mice display reduced osteocyte dendrites. RNA from Cx43 KI/KI femoral cortical bones show reduced expression levels of Sost, Tnf-α, IL-1β, Esr1, Esr2, and a lower Rankl/Opg ratio. Moreover, the Cx43R239Q mutation results in altered spatial expression of Cx43 protein and mild reduction of gap junction and hemichannel activity. The distinct phenotype seen in Cx43 KI/KI mice but not in Cx43 ablation models suggests that Cx43 loss-of-function is unlikely the main cause of AR CMD. Additional studies are required to investigate new roles of CMD-mutant Cx43.
Intervertebral disc degeneration is a degenerative disease where inflammation and immune responses play significant roles. Macrophages, as key immune cells, critically regulate inflammation through polarization into different phenotypes. In recent years, the role of macrophages in inflammation-related degenerative diseases, such as intervertebral disc degeneration, has been increasingly recognized. Macrophages construct the inflammatory microenvironment of the intervertebral disc and are involved in regulating intervertebral disc cell activities, extracellular matrix metabolism, intervertebral disc vascularization, and innervation, profoundly influencing the progression of disc degeneration. To gain a deeper understanding of the inflammatory microenvironment of intervertebral disc degeneration, this review will summarize the role of macrophages in the pathological process of intervertebral disc degeneration, analyze the regulatory mechanisms involving macrophages, and review therapeutic strategies targeting macrophage modulation for the treatment of intervertebral disc degeneration. These insights will be valuable for the treatment and research directions of intervertebral disc degeneration.
Plp1-lineage Schwann cells (SCs) of peripheral nerve play a critical role in vascular remodeling and osteogenic differentiation during the early stage of bone healing, and the abnormal plasticity of SCs would jeopardize the bone regeneration. However, how Plp1-lineage cells respond to injury and initiate the vascularized osteogenesis remains incompletely understood. Here, by employing single-cell transcriptional profiling combined with lineage-specific tracing models, we uncover that Plp1-lineage cells undergoing injury-induced glia-to-MSCs transition contributed to osteogenesis and revascularization in the initial stage of bone injury. Importantly, our data demonstrated that the Sonic hedgehog (Shh) signaling was responsible for the transition process initiation, which was strongly activated by c-Jun/SIRT6/BAF170 complex-driven Shh enhancers. Collectively, these findings depict an injury-specific niche signal-mediated Plp1-lineage cells transition towards Gli1+ MSCs and may be instructive for approaches to promote bone regeneration during aging or other bone diseases.
Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor whose dysfunction is linked to developmental dysplasia of the hip, osteoporosis and osteoarthritis. Our work addresses the critical question of how these skeletal pathologies emerge. Here, we show the abundant expression of LRP1 in skeletal progenitor cells at mouse embryonic stage E10.5 and onwards, especially in the perichondrium, the stem cell layer surrounding developing limbs essential for bone formation. Lrp1 deficiency in these stem cells causes joint fusion, malformation of cartilage/bone template and markedly delayed or lack of primary ossification. These abnormalities, which resemble phenotypes associated with Wnt signalling pathways, result in severe and persistent skeletal defects including a severe deficit in hip joint and patella, and markedly deformed and low-density long bones leading to dwarfism and impaired mobility. Mechanistically, we show that LRP1 regulates core non-canonical Wnt/planar cell polarity (PCP) components that may explain the malformation of long bones. LRP1 directly binds to Wnt5a, facilitates its cell-association and endocytic degradation and recycling. In the developing limbs, LRP1 partially colocalises with Wnt5a and its deficiency alters abundance and distribution of Wnt5a and Vangl2. Finally, using Xenopus as a model system, we show the regulatory role for LRP1 in Wnt/PCP signalling. We propose that in skeletal progenitors, LRP1 plays a critical role in formation and maturity of multiple bones and joints by regulating Wnt signalling, providing novel insights into the fundamental processes of morphogenesis and the emergence of skeletal pathologies.
The death of osteoblasts induced by glucocorticoid (GC)-mediated oxidative stress plays a crucial role in the development of steroid-induced osteonecrosis of the femoral head (SIONFH). Improving bone formation driven by osteoblasts has shown promising outcomes in the prognosis of SIONFH. Isovitexin has demonstrated antioxidant properties, but its therapeutic effects on GC-induced oxidative stress and SIONFH remain unexplored. In this study, we analyzed clinical samples obtained from SIONFH patients using proteomic and bioinformatic approaches. We found an imbalance in mitochondrial homeostasis and ferroptosis-induced impairment of osteogenic capacity in SIONFH. Subsequently, we investigated the cause-and-effect relationship between mitochondria and ferroptosis, as well as the regulatory role of mitophagy in maintaining mitochondrial homeostasis and controlling ferroptosis. We then identified the critical involvement of SIRT3 in modulating mitochondrial homeostasis and ferroptosis. Furthermore, molecular docking and co-immunoprecipitation confirmed the strong interaction between SIRT3 and BNIP3. Strikingly, restoring SIRT3 expression significantly inhibited pathological mitophagy mediated by the BNIP3/NIX pathway. Additionally, we discovered that Isovitexin, by promoting SIRT3 expression, effectively regulated mitophagy, preserved mitochondrial homeostasis in osteoblasts, suppressed ferroptosis, and restored osteogenic capacity, leading to remarkable improvements in SIONFH. These findings reveal the effects and molecular mechanisms of Isovitexin on SIONFH and highlight the potential of targeting SIRT3 as a promising strategy to suppress mitophagy-mediated ferroptosis in osteoblasts and against SIONFH.
Healthy aging is a common goal for humanity and society, and one key to achieving it is the rejuvenation of senescent resident stem cells and empowerment of aging organ regeneration. However, the mechanistic understandings of stem cell senescence and the potential strategies to counteract it remain elusive. Here, we reveal that the aging bone microenvironment impairs the Golgi apparatus thus diminishing mesenchymal stem cell (MSC) function and regeneration. Interestingly, replenishment of cell aggregates-derived extracellular vesicles (CA-EVs) rescues Golgi dysfunction and empowers senescent MSCs through the Golgi regulatory protein Syntaxin 5. Importantly, in vivo administration of CA-EVs significantly enhanced the bone defect repair rate and improved bone mass in aging mice, suggesting their therapeutic value for treating age-related osteoporosis and promoting bone regeneration. Collectively, our findings provide insights into Golgi regulation in stem cell senescence and bone aging, which further highlight CA-EVs as a potential rejuvenative approach for aging bone regeneration.
Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation and abnormal osteoclast activation, leading to bone destruction. We previously demonstrated that the large extracellular loop (LEL) of Tm4sf19 is important for its function in osteoclast differentiation, and LEL-Fc, a competitive inhibitor of Tm4sf19, effectively suppresses osteoclast multinucleation and prevent bone loss associated with osteoporosis. This study aimed to investigate the role of Tm4sf19 in RA, an inflammatory and abnormal osteoclast disease, using a mouse model of collagen-induced arthritis (CIA). Tm4sf19 expression was observed in macrophages and osteoclasts within the inflamed synovium, and Tm4sf19 expression was increased together with inflammatory genes in the joint bones of CIA-induced mice compared with the sham control group. Inhibition of Tm4sf19 by LEL-Fc demonstrated both preventive and therapeutic effects in a CIA mouse model, reducing the CIA score, swelling, inflammation, cartilage damage, and bone damage. Knockout of Tm4sf19 gene or inhibition of Tm4sf19 activity by LEL-Fc suppressed LPS/IFN-γ-induced TLR4-mediated inflammatory signaling in macrophages. LEL-Fc disrupted not only the interaction between Tm4sf19 and TLR4/MD2, but also the interaction between TLR4 and MD2. μCT analysis showed that LEL-Fc treatment significantly reduced joint bone destruction and bone loss caused by hyperactivated osteoclasts in CIA mice. Taken together, these findings suggest that LEL-Fc may be a potential treatment for RA and RA-induced osteoporosis by simultaneously targeting joint inflammation and bone destruction caused by abnormal osteoclast activation.
Clinical translation of tissue-engineered advanced therapeutic medicinal products is hindered by a lack of patient-dependent and independent in-process biological quality controls that are reflective of in vivo outcomes. Recent insights into the mechanism of native bone repair highlight a robust path dependence. Organoid-based bottom-up developmental engineering mimics this path-dependence to design personalized living implants scaffold-free, with in-build outcome predictability. Yet, adequate (noninvasive) quality metrics of engineered tissues are lacking. Moreover, insufficient insight into the role of donor variability and biological sex as influencing factors for the mechanism toward bone repair hinders the implementation of such protocols for personalized bone implants. Here, male and female bone-forming organoids were compared to non-bone-forming organoids regarding their extracellular matrix composition, transcriptome, and secreted proteome signatures to directly link in vivo outcomes to quality metrics. As a result, donor variability in bone-forming callus organoids pointed towards two distinct pathways to bone, through either a hypertrophic cartilage or a fibrocartilaginous template. The followed pathway was determined early, as a biological sex-dependent activation of distinct progenitor populations. Independent of donor or biological sex, a cartilage-to-bone transition was driven by a common panel of secreted factors that played a role in extracellular matrix remodeling, mineralization, and attraction of vasculature. Hence, the secreted proteome is a source of noninvasive biomarkers that report on biological potency and could be the missing link toward data-driven decision-making in organoid-based bone tissue engineering.
Exosomes have shown good potential in ischemic injury disease treatments. However, evidence about their effect and molecular mechanisms in osteonecrosis of femoral head (ONFH) treatment is still limited. Here, we revealed the cell biology characters of ONFH osteonecrosis area bone tissue in single cell scale and thus identified a novel ONFH treatment approach based on M2 macrophages-derived exosomes (M2-Exos). We further show that M2-Exos are highly effective in the treatment of ONFH by modulating the phenotypes communication between neutrophil and endothelium including neutrophil extracellular traps formation and endothelial phenotype transition. Additionally, we identified that M2-Exos’ therapeutic effect is attributed to the high content of miR-93-5p and constructed miR-93-5p overexpression model in vitro and in vivo based on lentivirus and adeno-associated virus respectively. Then we found miR-93-5p can not only reduce neutrophil extracellular traps formation but also improve angiogenic ability of endothelial cells. These results provided a new theoretical basis for the clinical application of ONFH therapeutic exosomes.
An increasing number of studies have characterized the bone as an endocrine organ, and that bone secreted factors may not only regulate local bone remodeling, but also other tissues and whole-body metabolic functions. The precise nature of these regulatory factors and their roles at bridging the bone, bone marrow adipose tissue, extramedullary body fat and whole-body energy homeostasis are being explored. In this study, we report that KIAA1199, a secreted factor produced from bone and bone marrow, previously described as an inhibitor of bone formation, also plays a role at promoting adipogenesis. KIAA1199-deficient mice exhibit reduced bone marrow adipose tissue, subcutaneous and visceral fat tissue mass, blood cholesterol, triglycerides, free fatty acids and glycerol, as well as improved insulin sensitivity in skeletal muscle, liver and fat. Moreover, these mice are protected from the detrimental effects of high-fat diet feeding, with decreased obesity, lower blood glucose and glucose tolerance, as well as decreased adipose tissue inflammation, insulin resistance and hepatic steatosis. In human studies, plasma levels of KIAA1199 or its expression levels in adipose tissue are positively correlated with insulin resistance and blood levels of cholesterol, triglycerides, free fatty acids, glycerol, fasting glucose and HOMA-IR. Mechanistically, KIAA1199 mediates its effects on adipogenesis through modulating osteopontin-integrin and AKT / ERK signaling. These findings provide evidence for the role of bone secreted factors on coupling bone, fat and whole-body energy homeostasis.
Sclerosteosis, an ultra-rare disorder characterised by high bone mass (HBM) and skeletal overgrowth, leads to facial paralysis, hearing loss and raised intracranial pressure, which is currently managed only through high-risk surgery. Sclerosteosis is caused by SOST mutations and loss of functional sclerostin, a protein that suppresses osteogenesis by antagonising Wnt/β-catenin signalling. Herein, using in vitro and in vivo approaches, we explore whether LGK974, another potent Wnt inhibitor that targets porcupine (PORCN, Wnt-specific acyltransferase), is a promising sclerosteosis therapeutic. In vitro assays showed that 100 nmol/L LGK974 significantly reduced osteoblast alkaline phosphatase (ALP) activity/mineralisation, decreased Wnt/osteoblast marker (Axin2, Runx2 and Ocn) expression, and downregulated ossification and the Wnt signalling pathway, without affecting osteoclast numbers/resorption. To assess in vivo effects, 6-week-old male and female Sost deficient (Sost-/-) mice received LGK974 for 4 weeks and right hindlimbs were subjected to 20 N peak loading to assess mechanoadaptive interactions. µCT revealed significant reductions in vertebral trabecular number and lower cortical bone volume in loaded and non-loaded tibiae in male and female LGK974-treated Sost-/- mice. Interestingly, the target engagement biomarker Axin2 was only significantly reduced in male vertebrae, which may indicate differences in male and female response to LGK974. This study also shows that PORCN inhibition may effectively limit characteristic HBM and skeletal overgrowth in sclerosteosis patients at sites with severe pathology.
Osteogenesis is the process of bone formation mediated by the osteoblasts, participating in various bone-related physiological processes including bone development, bone homeostasis and fracture healing. It exhibits temporal and spatial interconnectivity with angiogenesis, constructed by multiple forms of cell communication occurring between bone and vascular endothelial cells. Molecular regulation among different cell types is crucial for coordinating osteogenesis and angiogenesis to facilitate bone remodeling, fracture healing, and other bone-related processes. The transmission of signaling molecules and the activation of their corresponding signal pathways are indispensable for various forms of cell communication. This communication acts as a “bridge” in coupling osteogenesis to angiogenesis. This article reviews the modes and processes of cell communication in osteogenesis-angiogenesis coupling over the past decade, mainly focusing on interactions among bone-related cells and vascular endothelial cells to provide insights into the mechanism of cell communication of osteogenesis-angiogenesis coupling in different bone-related contexts. Moreover, clinical relevance and applications are also introduced in this review.
Neural EGFL-like 2 (NELL2) is a secreted protein known for its regulatory functions in the nervous and reproductive systems, yet its role in bone biology remains unexplored. In this study, we observed that NELL2 was diminished in the bone of aged and ovariectomized (OVX) mice, as well as in the serum of osteopenia and osteoporosis patients. In vitro loss-of-function and gain-of-function studies revealed that NELL2 facilitated osteoblast differentiation and impeded adipocyte differentiation from stromal progenitor cells. In vivo studies further demonstrated that the deletion of NELL2 in preosteoblasts resulted in decreased cancellous bone mass in mice. Mechanistically, NELL2 interacted with the FNI-type domain located at the C-terminus of Fibronectin 1 (Fn1). Moreover, we found that NELL2 activated the focal adhesion kinase (FAK)/AKT signaling pathway through Fn1/integrin β1 (ITGB1), leading to the promotion of osteogenesis and the inhibition of adipogenesis. Notably, administration of NELL2-AAV was found to ameliorate bone loss in OVX mice. These findings underscore the significant role of NELL2 in osteoblast differentiation and bone homeostasis, suggesting its potential as a therapeutic target for managing osteoporosis.
Chondrocyte senescence is a critical pathological hallmark of osteoarthritis (OA). Aberrant mechanical stress is considered a pivotal determinant in chondrocyte aging; however, the precise underlying mechanism remains elusive. Our findings demonstrate that SPI1 plays a significant role in counteracting chondrocyte senescence and inhibiting OA progression. SPI1 binds to the PERK promoter, thereby promoting its transcriptional activity. Importantly, PERK, rather than GCN2, facilitates eIF2α phosphorylation, activating the mitochondrial unfolded protein response (UPRmt) and impeding chondrocyte senescence. Deficiency of SPI1 in mechanical overload-induced mice leads to diminished UPRmt activation and accelerated OA progression. Intra-articular injection of adenovirus vectors overexpressing SPI1 and PERK effectively mitigates cartilage degeneration. In summary, our study elucidates the crucial regulatory role of SPI1 in the pathogenesis of chondrocyte senescence by activating UPRmt signaling through PERK, which may present a novel therapeutic target for treating OA.
SPI1 alleviates the progression of OA by inhibiting mechanical stress-induced chondrocyte senescence through mitochondrial UPR signaling.
Osteoarthritis (OA) is a degenerative joint disease with significant clinical and societal impact. Traditional diagnostic methods, including subjective clinical assessments and imaging techniques such as X-rays and MRIs, are often limited in their ability to detect early-stage OA or capture subtle joint changes. These limitations result in delayed diagnoses and inconsistent outcomes. Additionally, the analysis of omics data is challenged by the complexity and high dimensionality of biological datasets, making it difficult to identify key molecular mechanisms and biomarkers. Recent advancements in artificial intelligence (AI) offer transformative potential to address these challenges. This review systematically explores the integration of AI into OA research, focusing on applications such as AI-driven early screening and risk prediction from electronic health records (EHR), automated grading and morphological analysis of imaging data, and biomarker discovery through multi-omics integration. By consolidating progress across clinical, imaging, and omics domains, this review provides a comprehensive perspective on how AI is reshaping OA research. The findings have the potential to drive innovations in personalized medicine and targeted interventions, addressing longstanding challenges in OA diagnosis and management.
Bone has long been acknowledged as a fundamental structural entity that provides support and protection to the body’s organs. However, emerging research indicates that bone plays a crucial role in the regulation of systemic metabolism. This is achieved through the secretion of a variety of hormones, cytokines, metal ions, extracellular vesicles, and other proteins/peptides, collectively referred to as bone-derived factors (BDFs). BDFs act as a medium through which bones can exert targeted regulatory functions upon various organs, thereby underscoring the profound and concrete implications of bone in human physiology. Nevertheless, there remains a pressing need for further investigations to elucidate the underlying mechanisms that inform the effects of bone on other body systems. This review aims to summarize the current findings related to the roles of these significant modulators across different organs and metabolic contexts by regulating critical genes and signaling pathways in vivo. It also addresses their involvement in the pathogenesis of various diseases affecting the musculoskeletal system, circulatory system, glucose and lipid metabolism, central nervous system, urinary system, and reproductive system. The insights gained from this review may contribute to the development of innovative therapeutic strategies through a focused approach to bone secretomes. Continued research into BDFs is expected to enhance our understanding of bone as a multifunctional organ with diverse regulatory roles in human health.
With the deepening of epigenetic research, studies have shown that N6-methyladenosine (m6A) is closely related to the development of rheumatoid arthritis (RA), but the mechanism is still unclear. In the study, we collected synovial tissues from normal controls and patients with osteoarthritis (OA) or RA. The levels of m6A and inflammation were analyzed by immunofluorescence staining and western blotting. The roles of IGF2BP3 in cell proliferation and inflammatory activation were explored using transfection and RNA immunoprecipitation assays. IGF2BP3−/− mice were generated and used to establish an arthritis mouse model by transferring serum from adult arthritis K/BxN mice. We found m6A levels were markedly increased in RA patients and mouse models, and the expression of IGF2BP3 was upregulated in individuals with RA and related to the levels of inflammatory markers. IGF2BP3 played an important part in RA-fibroblast-like synoviocytes (FLS) by promoting cell proliferation, migration, invasion, inflammatory cytokine release and inhibiting autophagy. In addition, IGF2BP3 inhibited autophagy to reduce ROS production, thereby decreasing the inflammatory activation of macrophages. More importantly, RASGRF1-mediated mTORC1 activation played a crucial role in the ability of IGF2BP3 to promote cell proliferation and inflammatory activation. In an arthritis model of IGF2BP3−/− mice, IGF2BP3 knockout inhibited RA-FLS proliferation and inflammatory infiltration, and further ameliorated RA joint injury. Our study revealed an important role for IGF2BP3 in RA progression. The targeted inhibition of IGF2BP3 reduced cell proliferation and inflammatory activation and limited RA development, providing a potential strategy for RA therapy.
Osteoarthritis (OA) is a prevalent degenerative joint disorder marked by chronic pain, inflammation, and cartilage loss, with current treatments limited to symptom relief. G protein-coupled receptors (GPCRs) play a pivotal role in OA progression by regulating inflammation, chondrocyte survival, and matrix homeostasis. However, their multifaceted signaling, via G proteins or β-arrestins, poses challenges for precise therapeutic targeting. Biased agonism, where ligands selectively activate specific GPCR pathways, emerges as a promising approach to optimize efficacy and reduce side effects. This review examines biased signaling in OA-associated GPCRs, including cannabinoid receptors (CB1, CB2), chemokine receptors (CCR2, CXCR4), protease-activated receptors (PAR-2), adenosine receptors (A1R, A2AR, A2BR, A3R), melanocortin receptors (MC1R, MC3R), bradykinin receptors (B2R), prostaglandin E2 receptors (EP-2, EP-4), and calcium-sensing receptors (CaSR). We analyze ligands in clinical trials and explore natural products from Traditional Chinese Medicine as potential biased agonists. These compounds, with diverse structures and bioactivities, offer novel therapeutic avenues. By harnessing biased agonism, this review underscores the potential for developing targeted, safer OA therapies that address its complex pathology, bridging molecular insights with clinical translation.
The SH2 domain-containing protein tyrosine phosphatase 2 (SHP2, also known as PTP2C), encoded by PTPN11, is ubiquitously expressed and has context-specific effects. It promotes RAS/MAPK signaling downstream of receptor tyrosine kinases, cytokine receptors, and extracellular matrix proteins, and was shown in various lineages to modulate cell survival, proliferation, differentiation, and migration. Over the past decade, PTPN11 inactivation in chondrocytes was found to cause metachondromatosis, a rare disorder characterized by multiple enchondromas and osteochondroma-like lesions. Moreover, SHP2 inhibition was found to mitigate osteoarthritis pathogenesis in mice, and abundant but incomplete evidence suggests that SHP2 is crucial for cartilage development and adult homeostasis, during which its expression and activity are tightly regulated transcriptionally and posttranslationally, and by varying sets of functional partners. Fully uncovering SHP2 actions and regulation in chondrocytes is thus fundamental to understanding the mechanisms underlying both rare and common cartilage diseases and to designing effective disease treatments. We here review current knowledge, highlight recent discoveries and controversies, and propose new research directions to answer remaining questions.
While bulk RNA sequencing and single-cell RNA sequencing have shed light on cellular heterogeneity and potential molecular mechanisms in the musculoskeletal system in both physiological and various pathological states, the spatial localization of cells and molecules and intercellular interactions within the tissue context require further elucidation. Spatial transcriptomics has revolutionized biological research by simultaneously capturing gene expression profiles and in situ spatial information of tissues, gradually finding applications in musculoskeletal research. This review provides a summary of recent advances in spatial transcriptomics and its application to the musculoskeletal system. The classification and characteristics of data acquisition techniques in spatial transcriptomics are briefly outlined, with an emphasis on widely-adopted representative technologies and the latest technological breakthroughs, accompanied by a concise workflow for incorporating spatial transcriptomics into musculoskeletal system research. The role of spatial transcriptomics in revealing physiological mechanisms of the musculoskeletal system, particularly during developmental processes, is thoroughly summarized. Furthermore, recent discoveries and achievements of this emerging omics tool in addressing inflammatory, traumatic, degenerative, and tumorous diseases of the musculoskeletal system are compiled. Finally, challenges and potential future directions for spatial transcriptomics, both as a field and in its applications in the musculoskeletal system, are discussed.
Age-related osteoporosis poses a significant challenge in musculoskeletal health; a condition characterized by reduced bone density and increased fracture susceptibility in older individuals necessitates a better understanding of underlying molecular and cellular mechanisms. Emerging evidence suggests that osteocytes are the pivotal orchestrators of bone remodeling and represent novel therapeutic targets for age-related bone loss. Our study uses the prematurely aged PolgD257A/D257A (PolgA) mouse model to scrutinize age- and sex-related alterations in musculoskeletal health parameters (frailty, grip strength, gait data), bone and particularly the osteocyte lacuno-canalicular network (LCN). Moreover, a new quantitative in silico image analysis pipeline is used to evaluate the alterations in the osteocyte network with aging. Our findings underscore the pronounced degenerative changes in the musculoskeletal health parameters, bone, and osteocyte LCN in PolgA mice as early as 40 weeks, with more prominent alterations evident in aged males. Our findings suggest that the PolgA mouse model serves as a valuable model for studying the cellular mechanisms underlying age-related bone loss, given the comparable aging signs and age-related degeneration of the bone and the osteocyte network observed in naturally aging mice and elderly humans.
The discontinuation of denosumab [antibody targeting receptor activator of nuclear factor kappa B ligand (RANKL)] therapy may increase the risk of multiple vertebral fractures; however, the underlying pathophysiology is largely unknown. In patients who underwent discontinuation after multiple injections of denosumab, the levels of tartrate-resistant acid phosphatase 5b increased compared to pretreatment levels, indicating a phenomenon known as “overshoot.” The rate of decrease in bone mineral density during the withdrawal period was higher than the rate of decrease associated with aging, suggesting that the physiological bone metabolism had broken down. Overshoot and significant bone loss were also observed in mice receiving continuous administration of anti-RANKL antibody after treatment was interrupted, resembling the original pathology. In mice long out of overshoot, bone resorption recovered, but osteoblast numbers and bone formation remained markedly reduced. The bone marrow exhibited a significant reduction in stem cell (SC) antigen 1- and platelet-derived growth factor receptor alpha-expressing osteoblast progenitors (PαS cells) and alkaline phosphatase-positive early osteoblasts. Just before the overshoot phase, the osteoclast precursor cell population expands and RANKL-bearing extracellular vesicles (EVs) became abundant in the serum, leading to robust osteoclastogenesis after cessation of anti-RANKL treatment. Thus, accelerated bone resorption due to the accumulation of RANKL-bearing EVs and long-term suppression of bone formation uncoupled from bone resorption leads to the severe bone loss characteristic of denosumab discontinuation.
The cranial base synchondroses, comprised of opposite-facing bidirectional chondrocyte layers, drive anteroposterior cranial base growth. In humans, RUNX2 haploinsufficiency causes cleidocranial dysplasia associated with deficient midfacial growth. However, how RUNX2 regulates chondrocytes in the cranial base synchondroses remains unknown. To address this, we inactivated Runx2 in postnatal synchondrosis chondrocytes using a tamoxifen-inducible Fgfr3-creER (Fgfr3-Runx2cKO) mouse model. Fgfr3-Runx2cKO mice displayed skeletal dwarfism and reduced anteroposterior cranial base growth associated with premature synchondrosis ossification due to impaired chondrocyte proliferation, accelerated hypertrophy, apoptosis, and osteoclast-mediated cartilage resorption. Lineage tracing reveals that Runx2-deficient Fgfr3+ cells failed to differentiate into osteoblasts. Notably, Runx2-deficient chondrocytes showed an elevated level of FGFR3 and its downstream signaling components, pERK1/2 and SOX9, suggesting that RUNX2 downregulates FGFR3 in the synchondrosis. This study unveils a new role of Runx2 in cranial base chondrocytes, identifying a possible RUNX2-FGFR3-MAPK-SOX9 signaling axis that may control cranial base growth.
Debate regarding the premature aging of knee joints in acquired immune deficiency syndrome (AIDS) patients has remained contentious, with conjectures pointing towards its correlation with distinct antiviral regimes. Protease inhibitors (PIs) stand as a prominent class of antiviral agents frequently utilized in AIDS management and have been significantly linked to premature senescence. This study aimed to investigate whether PI-containing regimens would accelerate osteoarthritis (OA) development and explore the molecular mechanisms underlying this association. A retrospective cohort of 151 HIV-infected individuals, categorized into PI and non-PI groups, was established. Patients in PI group exhibited lower KOOS and a higher prevalence of radiological knee OA than those in non-PI group. Additionally, 25 anti-HIV drugs were screened and among all antiviral drugs, lopinavir had the most detrimental impact on cartilage anabolism, accelerating cartilage senescence and promoting mouse OA development. Mechanistically, lopinavir accelerated cellular senescence by inhibiting Zmpste24 and interfering nuclear membrane stability, which leads to decreased binding between nuclear membrane-binding protein Usp7 and Mdm2 and activates Usp7/Mdm2/p53 pathway. Zmpste24 overexpression reduces OA severity in mice. These findings suggest that PI-containing regimens accelerate cartilage senescence and OA development through Zmpste24 inhibition, which provides new insights into the selection of HIV regimens.
Musculoskeletal disorders, including osteoarthritis, rheumatoid arthritis, osteoporosis, bone fracture, intervertebral disc degeneration, tendinopathy, and myopathy, are prevalent conditions that profoundly impact quality of life and place substantial economic burdens on healthcare systems. Traditional bulk transcriptomics, genomics, proteomics, and metabolomics have played a pivotal role in uncovering disease-associated alterations at the population level. However, these approaches are inherently limited in their ability to resolve cellular heterogeneity or to capture the spatial organization of cells within tissues, thus hindering a comprehensive understanding of the complex cellular and molecular mechanisms underlying these diseases. To address these limitations, advanced single-cell and spatial omics techniques have emerged in recent years, offering unparalleled resolution for investigating cellular diversity, tissue microenvironments, and biomolecular interactions within musculoskeletal tissues. These cutting-edge techniques enable the detailed mapping of the molecular landscapes in diseased tissues, providing transformative insights into pathophysiological processes at both the single-cell and spatial levels. This review presents a comprehensive overview of the latest omics technologies as applied to musculoskeletal research, with a particular focus on their potential to revolutionize our understanding of disease mechanisms. Additionally, we explore the power of multi-omics integration in identifying novel therapeutic targets and highlight key challenges that must be overcome to successfully translate these advancements into clinical applications.
Itaconate, a macrophage-specific anti-inflammatory metabolite, has recently emerged as a critical regulator in rheumatoid arthritis pathogenesis. We found that itaconate is a TNF-α responsive metabolite significantly elevated in the serum and synovial fluid of rheumatoid arthritis patients and we demonstrated that itaconate is primarily produced by inflammatory macrophages rather than osteoclasts or osteoblasts. In TNF-transgenic and Irg1 −/− hybrid mice, a more severe bone destruction phenotype was observed. Administration of itaconate prevents excessive activation of osteoclasts by inhibiting Tet2 enzyme activity. Furthermore, exogenous administration of itaconate or its derivative, 4-octyl-itaconate, inhibits arthritis progression and mitigates bone destruction, offering a potential therapeutic strategy for rheumatoid arthritis. This study elucidates that TNF-α drives macrophage-derived itaconate production to epigenetically suppress osteoclast hyperactivation through Tet2 inhibition, establishing itaconate and its derivative OI as novel therapeutic agents against rheumatoid arthritis -associated bone destruction.
Rheumatoid arthritis (RA) is a systemic autoimmune disease in which synovial fibroblasts (SFs) maintain chronic inflammation by secreting proinflammatory mediators, leading to joint destruction. While the role of proinflammatory mediators in this process is well-established, the contribution of non-inflammatory regulators in SFs to joint pathology remains poorly understood. In this study, we investigated the non-inflammatory role of SFs in RA using a co-culture model, and found that SFs from RA patients promote apoptosis of human chondrocytes. Mechanistic investigations reveal that SFs can secrete small extracellular vesicles (sEVs), which are taken up by chondrocytes and induce chondrocyte apoptosis in both normal chondrocytes and chondrocytes from patients with RA. sEV-derived miRNA 15-29148 are identified as key signaling molecules mediating the apoptosis effects of chondrocytes. Further studies reveal that SF-derived miRNA 15-29148 targeting CIAPIN1 results in increased chondrocyte apoptosis. We further demonstrate that SF-derived miRNA 15-29148 is transferred to chondrocytes, exacerbating cartilage damage in vivo. Moreover, chondrocyte-specific aptamer-modified polyamidoamine nanoparticles not only ameliorated RA but also prevented its onset. This study suggests that, in RA, the secretion of specific sEV-miRNAs from SFs plays a crucial role in promoting chondrocyte apoptosis, potentially through non-inflammatory regulation, and that sEV-miRNA inhibition in SFs may represent an early preventive treatment strategy for cartilage degradation in RA.
Intervertebral disc degeneration (IDD) is a progressive and dynamic process in which the senescence-associated secretory phenotype (SASP) of nucleus pulposus cells (NPC) plays a significant role. While impaired chaperone-mediated autophagy (CMA) has been associated with inflammation and cellular senescence, its specific involvement in the self-perpetuating feedback loop of NPC senescence remains poorly understood. Through LAMP2A knockout in NPC, we identified a significant upregulation of DYRK1A, a core mediator of premature senescence in Down syndrome. Subsequent validation established DYRK1A as the critical driver of premature senescence in CMA-deficient NPC. Combinatorial transcription factor analysis revealed that under IL1B stimulation or CMA inhibition, elevated DYRK1A promoted FOXC1 phosphorylation and nuclear translocation, initiating transcriptional activation of cell cycle arrest. Intriguingly, CMA impairment concurrently enhanced glutamine metabolic flux in senescent NPC, thereby augmenting their survival fitness. Transcriptomic profiling demonstrated that CMA reactivation in senescent NPC facilitated fate transition from senescence to apoptosis, mediated by decreased glutamine flux via GLUL degradation. Therefore, CMA exerts protective effects against IDD by maintaining equilibrium between premature senescence and senolysis. This study elucidates CMA’s regulatory role in SASP-mediated senescence amplification circuits, providing novel therapeutic insights for IDD and other age-related pathologies.
Rheumatoid arthritis (RA) is a prevalent and debilitating inflammatory disease that significantly impairs functional capacity and quality of life. RA accelerates musculoskeletal aging, leading to complications such as muscle degeneration and sarcopenia. Recent research has identified myopenia as a condition of significant muscle loss associated with illness, distinct from the muscle wasting seen in other chronic diseases like cancer cachexia or heart failure. In RA, myopenia is characterized by muscle depletion without concurrent significant fat loss, and it can affect individuals of all ages. While inflammation plays a central role, it is not the sole factor contributing to the high incidence of muscle wasting in RA. In subsequent discussions, secondary sarcopenia will be considered alongside myopenia, as both involve muscle wasting decline primarily due to disease. This review summarizes recent findings on the impact of RA-related myopenia and secondary sarcopenia on functional capacity, explores its underlying mechanisms, and discusses contemporary strategies to mitigate the process of musculoskeletal aging in RA patients.
Membrane-initiated estrogen receptor α (mERα) signaling has been shown to affect bone mass in murine models. However, it remains unknown which cell types mediate the mERα-dependent effects on bone. In this study, we generated a novel mouse model with a conditional C451A mutation in Esr1, which enables selective knockout of the palmitoylation site essential for the membrane localization of ERα (C451Af/f). First, we used Runx2-Cre mice to generate Runx2-C451Af/f mice with conditional inactivation of mERα signaling in Runx2-expressing osteoblast lineage cells. No significant changes were observed in body weight, weights of estrogen-responsive organs, or serum concentrations of estradiol between female Runx2-C451Af/f and homozygous C451Af/f littermate controls. High-resolution microcomputed tomography analysis showed a consistent decrease in cortical bone mass in the tibia, femur, and vertebra L5 of Runx2-C451Af/f mice and three-point bending analysis of humerus revealed an impaired mechanical bone strength in Runx2-C451Af/f female mice compared to controls. Additionally, primary osteoblast cultures from mice lacking mERα signaling showed impaired differentiation compared to controls. In contrast, conditional inactivation of mERα signaling in hematopoietic cells, by transplantation of bone marrow from mice lacking mERα signaling in all cells to adult wildtype female mice, did not result in any skeletal alterations. In conclusion, this study demonstrates that mERα signaling in osteoblast lineage cells plays a crucial role in the regulation of cortical bone in female mice and shows that mERα inactivation in hematopoietic cells of adult female mice is dispensable for bone regulation.
The effectiveness of cranial suture expansion therapy hinges on the timely and adequate regeneration of bone tissue in response to mechanical stimuli. To optimize clinical outcomes and prevent post-expansion relapse, we delved into the underlying mechanisms governing bone remodeling during the processes of suture expansion and relapse. Our findings revealed that in vitro stretching bolstered mesenchymal stem cells’ antioxidative and osteogenic capacity by orchestrating mitochondrial activities, which governed by force-induced endoplasmic reticulum (ER) stress. Nonetheless, this signal transduction occurred through the activation of protein kinase R-like ER kinase (PERK) at the ER-mitochondria interface, rather than ER-mitochondria calcium flow as previously reported. Subsequently, PERK activation triggered TFEB translocation to the nucleus, thus regulating mitochondrial dynamics transcriptionally. Assessment of the mitochondrial pool during expansion and relapse unveiled a sequential, two-phase regulation governed by the ER stress/p-PERK/TFEB signaling cascade. Initially, PERK activation facilitated TFEB nuclear localization, stimulating mitochondrial biogenesis through PGC1-α, thereby addressing energy demands during the initial phase. Subsequently, TFEB shifted focus towards ensuring adequate mitophagy for mitochondrial quality maintenance during the remodeling process. Premature withdrawal of expanding force disrupted this sequential regulation, leading to compromised mitophagy and the accumulation of dysfunctional mitochondria, culminating in suboptimal bone regeneration and relapse. Notably, pharmacological activation of mitophagy effectively mitigated relapse and attenuated bone loss, while its inhibition impeded anticipated bone growth in remodeling progress. Conclusively, we elucidated the ER stress/p-PERK/TFEB signaling orchestrated sequential mitochondria biogenesis and mitophagy under mechanical stretch, thus ensuring antioxidative capacity and osteogenic potential of cranial suture tissues.
Peripheral neuropathy is a common complication in diabetes, affecting around 50% of the diabetic population. Co-occurrence of diabetic peripheral neuropathy (DPN) and diabetic bone disease has led to the hypothesis that DPN influences bone metabolism, although little experimental evidence has yet supported this premise. To investigate, mice were fed a high-fat diet (HFD) followed by phenotyping of skeletal-innervating neurons and bone architectural parameters. Results showed that HFD feeding resulted in a marked decrease in skeletal innervation (69%–41% reduction in Beta-III-Tubulin-stained nerves, 38% reduction in CGRP-stained nerves in long bone periosteum). These changes in skeletal innervation were associated with significant alterations in bone mass and in cortical and trabecular bone microarchitecture of long bones. Single-cell RNA sequencing (scRNA-Seq) of sensory neurons and bone tissue was next utilized to reconstruct potential nerve-to-bone signaling interactions, including implication of sensory nerve-derived neurotrophins (Bdnf), neuropeptides (Gal, Calca and Calcb), and other morphogens (Vegfa, Pdgfa, and Angpt2). Moreover, scRNA-Seq identified marked shifts in periosteal cell transcriptional changes within HFD-fed conditions, including a reduction in cell proliferation, an increase in adipogenic differentiation markers, and reductions in WNT, TGFβ, and MAPK signaling activity. When isolated, periosteal cells from HFD-fed mice showed deficits in proliferative and osteogenic differentiation potential. Moreover, these cellular changes in proliferation and differentiation capacity were restored by treatment of HFD-exposed periosteal cells to sensory neuron-conditioned medium. In summary, HFD modeling of type 2 diabetes results in skeletal polyneuropathy. Moreover, the combination of multi-tissue scRNA-Seq and isolated in vitro studies strengthen the case for altered nerve-to-bone signaling in diabetic bone disease.