Adiponectin is the most abundant circulating adipokine and is primarily involved in glucose metabolism and insulin resistance. Within the bone, osteoblasts and osteoclasts express the adiponectin receptors, however, there are conflicting reports on the effects of adiponectin on bone formation and turnover. Many studies have shown a pro-osteogenic role for adiponectin in in vivo murine models and in vitro: with increased osteoblast differentiation and activity, alongside lower levels of osteoclastogenesis. However, human studies often demonstrate an inverse relationship between adiponectin concentration and bone activity. Moreover, the presence of multiple isoforms of adiponectin and multiple receptor subtypes has the potential to lead to more complex signalling and functional consequences. As such, we still do not fully understand the importance of the adiponectin signalling pathway in regulating bone homeostasis and repair in health, with age and in disease. In this review, we explore our current understanding of adiponectin bioactivity in the bone; the significance of its different isoforms; and how adiponectin biology is altered in disease. Ultimately, furthering our understanding of adiponectin regulation of bone biology is key to developing pharmacological and non-pharmacological (lifestyle) interventions that target adiponectin signalling to boost bone growth and repair in healthy ageing, following injury or in disease.

Osteoporosis is a metabolic bone disease with dysregulated coupling between bone resorption and bone formation, which results in decreased bone mineral density. The MEF2C locus, which encodes the transcription factor MADS box transcription enhancer factor 2, polypeptide C (MEF2C), is strongly associated with adult osteoporosis and osteoporotic fractures. Although the role of MEF2C in bone and cartilage formation by osteoblasts, osteocytes, and chondrocytes has been studied, the role of MEF2C in osteoclasts, which mediate bone resorption, remains unclear. In this study, we identified MEF2C as a positive regulator of human and mouse osteoclast differentiation. While decreased MEF2C expression resulted in diminished osteoclastogenesis, ectopic expression of MEF2C enhanced osteoclast generation. Using transcriptomic and bioinformatic approaches, we found that MEF2C promotes the RANKL-mediated induction of the transcription factors c-FOS and NFATc1, which play a key role in osteoclastogenesis. Mechanistically, MEF2C binds to FOS regulatory regions to induce c-FOS expression, leading to the activation of NFATC1 and downstream osteoclastogenesis. Inducible deletion of Mef2c in mice resulted in increased bone mass under physiological conditions and protected mice from bone erosion by diminishing osteoclast formation in K/BxN serum induced arthritis, a murine model of inflammatory arthritis. Our findings reveal direct regulation of osteoclasts by MEF2C, thus adding osteoclasts as a cell type in which altered MEF2C expression or function can contribute to pathological bone remodeling.

Bone remodeling is precisely coordinated by bone resorption and formation. Apoptotic osteoclasts generate large amounts of apoptotic bodies (ABs) marking the end of the bone resorption phase, whereas the functions of osteoclast-derived ABs remain largely unknown. Here, we identified the molecular profile of ABs derived from osteoclasts at distinct differentiation stages and investigated their corresponding functions. ABs were isolated from apoptotic bone marrow macrophages, preosteoclasts, and mature osteoclasts induced by staurosporine. Proteomic signature analysis with liquid chromatography-tandem mass spectrometry suggested marked protein cargo differences among the different ABs. Further bioinformatic analysis showed that the proteomic signatures of the ABs were highly similar to those of their parental cells. Functionally, pOC-ABs induced endothelial progenitor cell differentiation and increased CD31^hiEmcn^hi endothelial cell formation in a murine bone defect model via their PDGF-BB cargo. mOC-ABs induced osteogenic differentiation of mesenchymal stem cells and facilitated osteogenesis via RANKL reverse signaling. In summary, we mapped the detailed proteomic landscapes of ABs derived from osteoclasts and showed that their potential biological roles are important in coupling bone formation with resorption during bone remodeling.

The maturation and function of osteoblasts (OBs) rely heavily on the reversible phosphorylation of signaling proteins. To date, most of the work in OBs has focused on phosphorylation by tyrosyl kinases, but little has been revealed about dephosphorylation by protein tyrosine phosphatases (PTPases). SHP2 (encoded by PTPN11) is a ubiquitously expressed PTPase. PTPN11 mutations are associated with both bone and cartilage manifestations in patients with Noonan syndrome (NS) and metachondromatosis (MC), although the underlying mechanisms remain elusive. Here, we report that SHP2 deletion in bone gamma-carboxyglutamate protein-expressing (Bglap ⁺) bone cells leads to massive osteopenia in both trabecular and cortical bones due to the failure of bone cell maturation and enhanced osteoclast activity, and its deletion in Bglap ⁺ chondrocytes results in the onset of enchondroma and osteochondroma in aged mice with increased tubular bone length. Mechanistically, SHP2 was found to be required for osteoblastic differentiation by promoting RUNX2/OSTERIX signaling and for the suppression of osteoclastogenesis by inhibiting STAT3-mediated RANKL production by osteoblasts and osteocytes. These findings are likely to explain the compromised skeletal system in NS and MC patients and to inform the development of novel therapeutics to combat skeletal disorders.

Low back pain (LBP), as a leading cause of disability, is a common musculoskeletal disorder that results in major social and economic burdens. Recent research has identified inflammation and related signaling pathways as important factors in the onset and progression of disc degeneration, a significant contributor to LBP. Inflammatory mediators also play an indispensable role in discogenic LBP. The suppression of LBP is a primary goal of clinical practice but has not received enough attention in disc research studies. Here, an overview of the advances in inflammation-related pain in disc degeneration is provided, with a discussion on the role of inflammation in IVD degeneration and pain induction. Puncture models, mechanical models, and spontaneous models as the main animal models to study painful disc degeneration are discussed, and the underlying signaling pathways are summarized. Furthermore, potential drug candidates, either under laboratory investigation or undergoing clinical trials, to suppress discogenic LBP by eliminating inflammation are explored. We hope to attract more research interest to address inflammation and pain in IDD and contribute to promoting more translational research.

Mechanical loading opens connexin 43 (Cx43) hemichannels (HCs), leading to the release of bone anabolic molecules, such as prostaglandins, from mechanosensitive osteocytes, which is essential for bone formation and remodeling. However, the mechanotransduction mechanism that activates HCs remains elusive. Here, we report a unique pathway by which mechanical signals are effectively transferred between integrin molecules located in different regions of the cell, resulting in HC activation. Both integrin α5 and αV were activated upon mechanical stimulation via either fluid dropping or flow shear stress (FSS). Inhibition of integrin αV activation or ablation of integrin α5 prevented HC opening on the cell body when dendrites were mechanically stimulated, suggesting mechanical transmission from the dendritic integrin αV to α5 in the cell body during HC activation. In addition, HC function was compromised in vivo, as determined by utilizing an antibody blocking αV activation and α5-deficient osteocyte-specific knockout mice. Furthermore, inhibition of integrin αV activation, but not that of α5, attenuated activation of the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway upon mechanical loading, and the inhibition of PI3K/AKT activation blocked integrin α5 activation and HC opening. Moreover, HC opening was blocked only by an anti-integrin αV antibody at low but not high FSS levels, suggesting that dendritic αV is a more sensitive mechanosensor than α5 for activating HCs. Together, these results reveal a new molecular mechanism of mechanotransduction involving the coordinated actions of integrins and PI3K/AKT in osteocytic dendritic processes and cell bodies that leads to HC opening and the release of key bone anabolic factors.

The vast majority of patients with advanced breast cancer present skeletal complications that severely compromise their quality of life. Breast cancer cells are characterized by a strong tropism to the bone niche. After engraftment and colonization of bone, breast cancer cells interact with native bone cells to hinder the normal bone remodeling process and establish an osteolytic “metastatic vicious cycle”. The sympathetic nervous system has emerged in recent years as an important modulator of breast cancer progression and metastasis, potentiating and accelerating the onset of the vicious cycle and leading to extensive bone degradation. Furthermore, sympathetic neurotransmitters and their cognate receptors have been shown to promote several hallmarks of breast cancer, such as proliferation, angiogenesis, immune escape, and invasion of the extracellular matrix. In this review, we assembled the current knowledge concerning the complex interactions that take place in the tumor microenvironment, with a special emphasis on sympathetic modulation of breast cancer cells and stromal cells. Notably, the differential action of epinephrine and norepinephrine, through either α- or β-adrenergic receptors, on breast cancer progression prompts careful consideration when designing new therapeutic options. In addition, the contribution of sympathetic innervation to the formation of bone metastatic foci is highlighted. In particular, we address the remarkable ability of adrenergic signaling to condition the native bone remodeling process and modulate the bone vasculature, driving breast cancer cell engraftment in the bone niche. Finally, clinical perspectives and developments on the use of β-adrenergic receptor inhibitors for breast cancer management and treatment are discussed.

Overnutrition causes hyperactivation of mTORC1-dependent negative feedback loops leading to the downregulation of insulin signaling and development of insulin resistance. In osteoblasts (OBs), insulin signaling plays a crucial role in the control of systemic glucose homeostasis. We utilized mice with conditional deletion of Rptor to investigate how the loss of mTORC1 function in OB affects glucose metabolism under normal and overnutrition dietary states. Compared to the controls, chow-fed Rptor _ob ^−/− mice had substantially less fat mass and exhibited adipocyte hyperplasia. Remarkably, upon feeding with high-fat diet, mice with pre- and post-natal deletion of Rptor in OBs were protected from diet-induced obesity and exhibited improved glucose metabolism with lower fasting glucose and insulin levels, increased glucose tolerance and insulin sensitivity. This leanness and resistance to weight gain was not attributable to changes in food intake, physical activity or lipid absorption but instead was due to increased energy expenditure and greater whole-body substrate flexibility. RNA-seq revealed an increase in glycolysis and skeletal insulin signaling pathways, which correlated with the potentiation of insulin signaling and increased insulin-dependent glucose uptake in Rptor-knockout osteoblasts. Collectively, these findings point to a critical role for the mTORC1 complex in the skeletal regulation of whole-body glucose metabolism and the skeletal development of insulin resistance.

Tissue-resident macrophages are highly specialized to their tissue-specific microenvironments, activated by various inflammatory signals and modulated by genetic and environmental factors. Osteoclasts and microglia are distinct tissue-resident cells of the macrophage lineage in bone and brain that are responsible for pathological changes in osteoporosis and Alzheimer’s disease (AD), respectively. Osteoporosis is more frequently observed in individuals with AD compared to the prevalence in general population. Diagnosis of AD is often delayed until underlying pathophysiological changes progress and cause irreversible damages in structure and function of brain. As such earlier diagnosis and intervention of individuals at higher risk would be indispensable to modify clinical courses. Pleiotropy is the phenomenon that a genetic variant affects multiple traits and the genetic correlation between two traits could suggest a shared molecular mechanism. In this review, we discuss that the Pyk2-mediated actin polymerization pathway in osteoclasts and microglia in bone and brain, respectively, is the horizontal pleiotropic mediator of shared risk factors for osteoporosis and AD.

The demand for biomaterials that promote the repair, replacement, or restoration of hard and soft tissues continues to grow as the population ages. Traditionally, smart biomaterials have been thought as those that respond to stimuli. However, the continuous evolution of the field warrants a fresh look at the concept of smartness of biomaterials. This review presents a redefinition of the term “Smart Biomaterial” and discusses recent advances in and applications of smart biomaterials for hard tissue restoration and regeneration. To clarify the use of the term “smart biomaterials”, we propose four degrees of smartness according to the level of interaction of the biomaterials with the bio-environment and the biological/cellular responses they elicit, defining these materials as inert, active, responsive, and autonomous. Then, we present an up-to-date survey of applications of smart biomaterials for hard tissues, based on the materials’ responses (external and internal stimuli) and their use as immune-modulatory biomaterials. Finally, we discuss the limitations and obstacles to the translation from basic research (bench) to clinical utilization that is required for the development of clinically relevant applications of these technologies.

Silk fibroin (SF) can be used to construct various stiff material interfaces to support bone formation. An essential preparatory step is to partially transform SF molecules from random coils to β-sheets to render the material water insoluble. However, the influence of the SF conformation on osteogenic cell behavior at the material interface remains unknown. Herein, three stiff SF substrates were prepared by varying the β-sheet content (high, medium, and low). The substrates had a comparable chemical composition, surface topography, and wettability. When adsorbed fibronectin was used as a model cellular adhesive protein, the stability of the adsorbed protein-material interface, in terms of the surface stability of the SF substrates and the accompanying fibronectin detachment resistance, increased with the increasing β-sheet content of the SF substrates. Furthermore, (i) larger areas of cytoskeleton-associated focal adhesions, (ii) higher orders of cytoskeletal organization and (iii) more elongated cell spreading were observed for bone marrow-derived mesenchymal stromal cells (BMSCs) cultured on SF substrates with high vs. low β-sheet contents, along with enhanced nuclear translocation and activation of YAP/TAZ and RUNX2. Consequently, osteogenic differentiation of BMSCs was stimulated on high β-sheet substrates. These results indicated that the β-sheet content influences osteogenic differentiation of BMSCs on SF materials in vitro by modulating the stability of the adsorbed protein-material interface, which proceeds via protein-focal adhesion-cytoskeleton links and subsequent intracellular mechanotransduction. Our findings emphasize the role of the stability of the adsorbed protein-material interface in cellular mechanotransduction and the perception of stiff SF substrates with different β-sheet contents, which should not be overlooked when engineering stiff biomaterials.

Ultra-processed foods have known negative implications for health; however, their effect on skeletal development has never been explored. Here, we show that young rats fed ultra-processed food rich in fat and sugar suffer from growth retardation due to lesions in their tibial growth plates. The bone mineral density decreases significantly, and the structural parameters of the bone deteriorate, presenting a sieve-like appearance in the cortices and poor trabecular parameters in long bones and vertebrae. This results in inferior mechanical performance of the entire bone with a high fracture risk. RNA sequence analysis of the growth plates demonstrated an imbalance in extracellular matrix formation and degradation and impairment of proliferation, differentiation and mineralization processes. Our findings highlight, for the first time, the severe impact of consuming ultra-processed foods on the growing skeleton. This pathology extends far beyond that explained by the known metabolic effects, highlighting bone as a new target for studies of modern diets.

Osteoporosis (OP) is a common skeletal disease involving low bone mineral density (BMD) that often leads to fragility fracture, and its development is affected by multiple cellular pathologies and associated with marked epigenetic alterations of osteogenic genes. Proper physical exercise is beneficial for bone health and OP and reportedly possesses epigenetic modulating capacities; however, whether the protective effects of exercise on OP involve epigenetic mechanisms is unclear. Here, we report that epigenetic derepression of nuclear factor erythroid derived 2-related factor-2 (Nrf2), a master regulator of oxidative stress critically involved in the pathogenesis of OP, mediates the significant osteoprotective effects of running exercise (RE) in a mouse model of OP induced by ovariectomy. We showed that Nrf2 gene knockout (Nfe2l2^−/−) ovariectomized mice displayed a worse BMD reduction than the controls, identifying Nrf2 as a critical antiosteoporotic factor. Further, femoral Nrf2 was markedly repressed with concomitant DNA methyltransferase (Dnmt) 1/Dnmt3a/Dnmt3b elevations and Nrf2 promoter hypermethylation in both patients with OP and ovariectomized mice. However, daily 1-h treadmill RE significantly corrected epigenetic alterations, recovered Nrf2 loss and improved the femur bone mass and trabecular microstructure. Consistently, RE also normalized the adverse expression of major osteogenic factors, including osteoblast/osteoclast markers, Nrf2 downstream antioxidant enzymes and proinflammatory cytokines. More importantly, the RE-conferred osteoprotective effects observed in the wild-type control mice were largely abolished in the Nfe2l2^{−/ −} mice. Thus, Nrf2 repression due to aberrant Dnmt elevation and subsequent Nrf2 promoter hypermethylation is likely an important epigenetic feature of the pathogenesis of OP, and Nrf2 derepression is essential for the antiosteoporotic effects of RE.

Mechanobiological stimuli, such as low-intensity pulsed ultrasound (LIPUS), have been shown to promote bone regeneration and fresh fracture repair, but the fundamental biophysical mechanisms involved remain elusive. Here, we propose that a mechanosensitive ion channel of Piezo1 plays a pivotal role in the noninvasive ultrasound-induced mechanical transduction pathway to trigger downstream cellular signal processes. This study aims to investigate the expression and role of Piezo1 in MC3T3-E1 cells after LIPUS treatment. Immunofluorescence analysis shows that Piezo1 was present on MC3T3-E1 cells and could be ablated by shRNA transfection. MC3T3-E1 cell migration and proliferation were significantly increased by LIPUS stimulation, and knockdown of Piezo1 restricted the increase in cell migration and proliferation. After labeling with Fluo-8, MC3T3-E1 cells exhibited fluorescence intensity traces with several high peaks compared with the baseline during LIPUS stimulation. No obvious change in the fluorescence intensity tendency was observed after LIPUS stimulation in shRNA-Piezo1 cells, which was similar to the results in the GsMTx4-treated group. The phosphorylation ratio of ERK1/2 in MC3T3-E1 cells was significantly increased (P < 0.01) after LIPUS stimulation. In addition, Phalloidin-iFluor-labeled F-actin filaments immediately accumulated in the perinuclear region after LIPUS stimulation, continued for 5 min, and then returned to their initial levels at 30 min. These results suggest that Piezo1 can transduce LIPUS-induced mechanical signals into intracellular calcium. The influx of Ca²⁺ serves as a second messenger to activate ERK1/2 phosphorylation and perinuclear F-actin filament polymerization, which regulate the proliferation of MC3T3-E1 cells.

Alveolar bone is the thickened ridge of jaw bone that supports teeth. It is subject to constant occlusal force and pathogens invasion, and is therefore under active bone remodeling and immunomodulation. Alveolar bone holds a distinct niche from long bone considering their different developmental origin and postnatal remodeling pattern. However, a systematic explanation of alveolar bone at single-cell level is still lacking. Here, we construct a single-cell atlas of mouse mandibular alveolar bone through single-cell RNA sequencing (scRNA-seq). A more active immune microenvironment is identified in alveolar bone, with a higher proportion of mature immune cells than in long bone. Among all immune cell populations, the monocyte/macrophage subpopulation most actively interacts with mesenchymal stem cells (MSCs) subpopulation. Alveolar bone monocytes/macrophages express a higher level of Oncostatin M (Osm) compared to long bone, which promotes osteogenic differentiation and inhibits adipogenic differentiation of MSCs. In summary, our study reveals a unique immune microenvironment of alveolar bone, which may provide a more precise immune-modulatory target for therapeutic treatment of oral diseases.

Bone tumors, especially those in osteosarcoma, usually occur in adolescents. The standard clinical treatment includes chemotherapy, surgical therapy, and radiation therapy. Unfortunately, surgical resection often fails to completely remove the tumor, which is the main cause of postoperative recurrence and metastasis, resulting in a high mortality rate. Moreover, bone tumors often invade large areas of bone, which cannot repair itself, and causes a serious effect on the quality of life of patients. Thus, bone tumor therapy and bone regeneration are challenging in the clinic. Herein, this review presents the recent developments in bifunctional biomaterials to achieve a new strategy for bone tumor therapy. The selected bifunctional materials include 3D-printed scaffolds, nano/microparticle-containing scaffolds, hydrogels, and bone-targeting nanomaterials. Numerous related studies on bifunctional biomaterials combining tumor photothermal therapy with enhanced bone regeneration were reviewed. Finally, a perspective on the future development of biomaterials for tumor therapy and bone tissue engineering is discussed. This review will provide a useful reference for bone tumor-related disease and the field of complex diseases to combine tumor therapy and tissue engineering.

Ankylosing spondylitis (AS) is chronic inflammatory arthritis with a progressive fusion of axial joints. Anti-inflammatory treatments such as anti-TNF-α antibody therapy suppress inflammation but do not effectively halt the progression of spine fusion in AS patients. Here we report that the autoimmune inflammation of AS generates a microenvironment that promotes chondrogenesis in spine ligaments as the process of spine fusion. Chondrocyte differentiation was observed in the ligaments of patients with early-stage AS, and cartilage formation was followed by calcification. Moreover, a large number of giant osteoclasts were found in the inflammatory environment of ligaments and on bony surfaces of calcified cartilage. Resorption activity by these giant osteoclasts generated marrow with high levels of active TGF-β, which induced new bone formation in the ligaments. Notably, no Osterix⁺ osteoprogenitors were found in osteoclast resorption areas, indicating uncoupled bone resorption and formation. Even at the late and maturation stages, the uncoupled osteoclast resorption in bony interspinous ligament activates TGF-β to induce the progression of ossification in AS patients. Osteoclast resorption of calcified cartilage-initiated ossification in the progression of AS is a similar pathologic process of acquired heterotopic ossification (HO). Our finding of cartilage formation in the ligaments of AS patients revealed that the pathogenesis of spinal fusion is a process of HO and explained why anti-inflammatory treatments do not slow ankylosing once there is new bone formation in spinal soft tissues. Thus, inhibition of HO formation, such as osteoclast activity, cartilage formation, or TGF-β activity could be a potential therapy for AS.

Osteoarthritis comprises several joint disorders characterized by articular cartilage degeneration and persistent pain, causing disability and economic burden. The incidence of osteoarthritis is rapidly increasing worldwide due to aging and obesity trends. Basic and clinical research on osteoarthritis has been carried out for decades, but many questions remain unanswered. The exact role of subchondral bone during the initiation and progression osteoarthritis remains unclear. Accumulating evidence shows that subchondral bone lesions, including bone marrow edema and angiogenesis, develop earlier than cartilage degeneration. Clinical interventions targeting subchondral bone have shown therapeutic potential, while others targeting cartilage have yielded disappointing results. Abnormal subchondral bone remodeling, angiogenesis and sensory nerve innervation contribute directly or indirectly to cartilage destruction and pain. This review is about bone-cartilage crosstalk, the subchondral microenvironment and the critical role of both in osteoarthritis progression. It also provides an update on the pathogenesis of and interventions for osteoarthritis and future research targeting subchondral bone.

Angiogenesis and osteogenesis are coupled. However, the cellular and molecular regulation of these processes remains to be further investigated. Both tissues have recently been recognized as endocrine organs, which has stimulated research interest in the screening and functional identification of novel paracrine factors from both tissues. This review aims to elaborate on the novelty and significance of endocrine regulatory loops between bone and the vasculature. In addition, research progress related to the bone vasculature, vessel-related skeletal diseases, pathological conditions, and angiogenesis-targeted therapeutic strategies are also summarized. With respect to future perspectives, new techniques such as single-cell sequencing, which can be used to show the cellular diversity and plasticity of both tissues, are facilitating progress in this field. Moreover, extracellular vesicle-mediated nuclear acid communication deserves further investigation. In conclusion, a deeper understanding of the cellular and molecular regulation of angiogenesis and osteogenesis coupling may offer an opportunity to identify new therapeutic targets.

L-plastin (LPL) was identified as a potential regulator of the actin-bundling process involved in forming nascent sealing zones (NSZs), which are precursor zones for mature sealing zones. TAT-fused cell-penetrating small molecular weight LPL peptide (TAT- MARGSVSDEE, denoted as an inhibitory LPL peptide) attenuated the formation of NSZs and impaired bone resorption in vitro in osteoclasts. Also, the genetic deletion of LPL in mice demonstrated decreased eroded perimeters and increased trabecular bone density. In the present study, we hypothesized that targeting LPL with the inhibitory LPL peptide in vivo could reduce osteoclast function and increase bone density in a mice model of low bone mass. We injected aging C57BL/6 female mice (36 weeks old) subcutaneously with the inhibitory and scrambled peptides of LPL for 14 weeks. Micro-CT and histomorphometry analyses demonstrated an increase in trabecular bone density of femoral and tibial bones with no change in cortical thickness in mice injected with the inhibitory LPL peptide. A reduction in the serum levels of CTX-1 peptide suggests that the increase in bone density is associated with a decrease in osteoclast function. No changes in bone formation rate and mineral apposition rate, and the serum levels of P1NP indicate that the inhibitory LPL peptide does not affect osteoblast function. Our study shows that the inhibitory LPL peptide can block osteoclast function without impairing the function of osteoblasts. LPL peptide could be developed as a prospective therapeutic agent to treat osteoporosis.

Osteoporosis is a common skeletal disease, affecting ~200 million people around the world. As a complex disease, osteoporosis is influenced by many factors, including diet (e.g. calcium and protein intake), physical activity, endocrine status, coexisting diseases and genetic factors. In this review, we first summarize the discovery from genome-wide association studies (GWASs) in the bone field in the last 12 years. To date, GWASs and meta-analyses have discovered hundreds of loci that are associated with bone mineral density (BMD), osteoporosis, and osteoporotic fractures. However, the GWAS approach has sometimes been criticized because of the small effect size of the discovered variants and the mystery of missing heritability, these two questions could be partially explained by the newly raised conceptual models, such as omnigenic model and natural selection. Finally, we introduce the clinical use of GWAS findings in the bone field, such as the identification of causal clinical risk factors, the development of drug targets and disease prediction. Despite the fruitful GWAS discoveries in the bone field, most of these GWAS participants were of European descent, and more genetic studies should be carried out in other ethnic populations to benefit disease prediction in the corresponding population.

Understanding the role of neuropilin 2 (NRP2) in prostate cancer cells as well as in the bone microenvironment is pivotal in the development of an effective targeted therapy for the treatment of prostate cancer bone metastasis. We observed a significant upregulation of NRP2 in prostate cancer cells metastasized to bone. Here, we report that targeting NRP2 in cancer cells can enhance taxane-based chemotherapy with a better therapeutic outcome in bone metastasis, implicating NRP2 as a promising therapeutic target. Since, osteoclasts present in the tumor microenvironment express NRP2, we have investigated the potential effect of targeting NRP2 in osteoclasts. Our results revealed NRP2 negatively regulates osteoclast differentiation and function in the presence of prostate cancer cells that promotes mixed bone lesions. Our study further delineated the molecular mechanisms by which NRP2 regulates osteoclast function. Interestingly, depletion of NRP2 in osteoclasts in vivo showed a decrease in the overall prostate tumor burden in the bone. These results therefore indicate that targeting NRP2 in prostate cancer cells as well as in the osteoclastic compartment can be beneficial in the treatment of prostate cancer bone metastasis.

Bone mainly functions as a supportive framework for the whole body and is the major regulator of calcium homeostasis and hematopoietic function. Recently, an increasing number of studies have characterized the significance of bone as an endocrine organ, suggesting that bone-derived factors regulate local bone metabolism and metabolic functions. In addition, these factors can regulate global energy homeostasis by altering insulin sensitivity, feeding behavior, and adipocyte commitment. These findings may provide a new pathological mechanism for related metabolic diseases or be used in the diagnosis, treatment, and prevention of metabolic diseases such as osteoporosis, obesity, and diabetes mellitus. In this review, we summarize the regulatory effect of bone and bone-derived factors on energy metabolism and discuss directions for future research.

Mechanical loading to the bone is known to be beneficial for bone homeostasis and for suppressing tumor-induced osteolysis in the loaded bone. However, whether loading to a weight-bearing hind limb can inhibit distant tumor growth in the brain is unknown. We examined the possibility of bone-to-brain mechanotransduction using a mouse model of a brain tumor by focusing on the response to Lrp5-mediated Wnt signaling and dopamine in tumor cells. The results revealed that loading the tibia with elevated levels of tyrosine hydroxylase, a rate-limiting enzyme in dopamine synthesis, markedly reduced the progression of the brain tumors. The simultaneous application of fluphenazine (FP), an antipsychotic dopamine modulator, enhanced tumor suppression. Dopamine and FP exerted antitumor effects through the dopamine receptors DRD1 and DRD2, respectively. Notably, dopamine downregulated Lrp5 via DRD1 in tumor cells. A cytokine array analysis revealed that the reduction in CCN4 was critical for loading-driven, dopamine-mediated tumor suppression. The silencing of Lrp5 reduced CCN4, and the administration of CCN4 elevated oncogenic genes such as MMP9, Runx2, and Snail. In summary, this study demonstrates that mechanical loading regulates dopaminergic signaling and remotely suppresses brain tumors by inhibiting the Lrp5-CCN4 axis via DRD1, indicating the possibility of developing an adjuvant bone-mediated loading therapy.

Bone undergoes a constant and continuous remodeling process that is tightly regulated by the coordinated and sequential actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Recent studies have shown that histone demethylases are implicated in osteoblastogenesis; however, little is known about the role of histone demethylases in osteoclast formation. Here, we identified KDM4B as an epigenetic regulator of osteoclast differentiation. Knockdown of KDM4B significantly blocked the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. Mice with myeloid-specific conditional knockout of KDM4B showed an osteopetrotic phenotype due to osteoclast deficiency. Biochemical analysis revealed that KDM4B physically and functionally associates with CCAR1 and MED1 in a complex. Using genome-wide chromatin immunoprecipitation (ChIP)-sequencing, we revealed that the KDM4B–CCAR1–MED1 complex is localized to the promoters of several osteoclast-related genes upon receptor activator of NF-κB ligand stimulation. We demonstrated that the KDM4B–CCAR1–MED1 signaling axis induces changes in chromatin structure (euchromatinization) near the promoters of osteoclast-related genes through H3K9 demethylation, leading to NF-κB p65 recruitment via a direct interaction between KDM4B and p65. Finally, small molecule inhibition of KDM4B activity impeded bone loss in an ovariectomized mouse model. Taken together, our findings establish KDM4B as a critical regulator of osteoclastogenesis, providing a potential therapeutic target for osteoporosis.

The regulation of whole-body homeostasis by the skeleton is mediated by its capacity to secrete endocrine signaling molecules. Although bone-derived hormones confer several adaptive benefits, their physiological functions also involve trade-offs, thus eventually contributing to disease. In this manuscript, we discuss the origins and functions of two of the best-studied skeletal mediators, fibroblast growth factor 23 and osteocalcin, in an evolutionary context. Moreover, we provide a theoretical framework seeking to explain the broad involvement of these two hormones in amniote physiology as well as their potential to fuel the development and progression of diseases. Vice versa, we outline which perturbations might be amenable to manipulation of these systems and discuss limitations and ongoing challenges in skeletal endocrine research. Finally, we summarize unresolved questions and potential future studies in this thriving field.

Atrophic fracture nonunion poses a significant clinical problem with limited therapeutic interventions. In this study, we developed a unique nonunion model with high clinical relevance using serum transfer-induced rheumatoid arthritis (RA). Arthritic mice displayed fracture nonunion with the absence of fracture callus, diminished angiogenesis and fibrotic scar tissue formation leading to the failure of biomechanical properties, representing the major manifestations of atrophic nonunion in the clinic. Mechanistically, we demonstrated that the angiogenesis defect observed in RA mice was due to the downregulation of SPP1 and CXCL12 in chondrocytes, as evidenced by the restoration of angiogenesis upon SPP1 and CXCL12 treatment in vitro. In this regard, we developed a biodegradable scaffold loaded with SPP1 and CXCL12, which displayed a beneficial effect on angiogenesis and fracture repair in mice despite the presence of inflammation. Hence, these findings strongly suggest that the sustained release of SPP1 and CXCL12 represents an effective therapeutic approach to treat impaired angiogenesis and fracture nonunion under inflammatory conditions.

Chondrocyte differentiation is a critical process for endochondral ossification, which is responsible for long bone development and fracture repair. Considerable progress has been made in understanding the transcriptional control of chondrocyte differentiation; however, epigenetic regulation of chondrocyte differentiation remains to be further studied. NSD1 is a H3K36 (histone H3 at lysine 36) methyltransferase. Here, we showed that mice with Nsd1 deficiency in Prx1⁺ mesenchymal progenitors but not in Col2⁺ chondrocytes showed impaired skeletal growth and fracture healing accompanied by decreased chondrogenic differentiation. Via combined RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we identified sex determining region Y box 9 (Sox9), the key transcription factor of chondrogenic differentiation, as a functional target gene of NSD1. Mechanistically, NSD1 regulates Sox9 expression by modulating H3K36me1 and H3K36me2 levels in the Sox9 promoter region, constituting a novel epigenetic regulatory mechanism of chondrogenesis. Moreover, we found that NSD1 can directly activate the expression of hypoxia-inducible factor 1α (HIF1α), which plays a vital role in chondrogenic differentiation through its regulation of Sox9 expression. Collectively, the results of our study reveal crucial roles of NSD1 in regulating chondrogenic differentiation, skeletal growth, and fracture repair and expand our understanding of the function of epigenetic regulation in chondrogenesis and skeletal biology.

Patients with Alzheimer’s disease (AD) often have lower bone mass than healthy individuals. However, the mechanisms underlying this change remain elusive. Previously, we found that Tg2576 mice, an AD animal model that ubiquitously expresses Swedish mutant amyloid precursor protein (APP_swe), shows osteoporotic changes, reduced bone formation, and increased bone resorption. To understand how bone deficits develop in Tg2576 mice, we used a multiplex antibody array to screen for serum proteins that are altered in Tg2576 mice and identified hepcidin, a master regulator of iron homeostasis. We further investigated hepcidin’s function in bone homeostasis and found that hepcidin levels were increased not only in the serum but also in the liver, muscle, and osteoblast (OB) lineage cells in Tg2576 mice at both the mRNA and protein levels. We then generated mice selectively expressing hepcidin in hepatocytes or OB lineage cells, which showed trabecular bone loss and increased osteoclast (OC)-mediated bone resorption. Further cell studies suggested that hepcidin increased OC precursor proliferation and differentiation by downregulating ferroportin (FPN) expression and increasing intracellular iron levels. In OB lineage cells, APP_swe enhanced hepcidin expression by inducing ER stress and increasing OC formation, in part through hepcidin. Together, these results suggest that increased hepcidin expression in hepatocytes and OB lineage cells in Tg2576 mice contributes to enhanced osteoclastogenesis and trabecular bone loss, identifying the hepcidin-FPN-iron axis as a potential therapeutic target to prevent AD-associated bone loss.

Osteocytes are the most abundant cells in bone, which is a frequent site of breast cancer metastasis. Here, we focused on Wnt signaling and evaluated tumor-osteocyte interactions. In animal experiments, mammary tumor cells were inoculated into the mammary fat pad and tibia. The role of Lrp5-mediated Wnt signaling was examined by overexpressing and silencing Lrp5 in osteocytes and establishing a conditional knockout mouse model. The results revealed that administration of osteocytes or their conditioned medium (CM) inhibited tumor progression and osteolysis. Osteocytes overexpressing Lrp5 or β-catenin displayed strikingly elevated tumor-suppressive activity, accompanied by downregulation of tumor-promoting chemokines and upregulation of apoptosis-inducing and tumor-suppressing proteins such as p53. The antitumor effect was also observed with osteocyte-derived CM that was pretreated with a Wnt-activating compound. Notably, silencing Lrp5 in tumors inhibited tumor progression, while silencing Lrp5 in osteocytes in conditional knockout mice promoted tumor progression. Osteocytes exhibited elevated Lrp5 expression in response to tumor cells, implying that osteocytes protect bone through canonical Wnt signaling. Thus, our results suggest that the Lrp5/β-catenin axis activates tumor-promoting signaling in tumor cells but tumor-suppressive signaling in osteocytes. We envision that osteocytes with Wnt activation potentially offer a novel cell-based therapy for breast cancer and osteolytic bone metastasis.

Osteoporosis is an osteolytic disorder commonly associated with excessive osteoclast formation. Transcriptional coactivator with PDZ-binding motif (TAZ) is a key downstream effector of the Hippo signaling pathway; it was suggested to be involved in the regulation of bone homeostasis. However, the exact role of TAZ in osteoclasts has not yet been established. In this study, we demonstrated that global knockout and osteoclast-specific knockout of TAZ led to a low-bone mass phenotype due to elevated osteoclast formation, which was further evidenced by in vitro osteoclast formation assays. Moreover, the overexpression of TAZ inhibited RANKL-induced osteoclast formation, whereas silencing of TAZ reduced it. Mechanistically, TAZ bound to TGF-activated kinase 1 (TAK1) and reciprocally inhibited NF-κB signaling, suppressing osteoclast differentiation. Collectively, our findings highlight an essential role of TAZ in the regulation of osteoclastogenesis in osteoporosis and its underlying mechanism.

Bone-produced fibroblast growth factor 23 (FGF23) increases in response to inflammation and iron deficiency and contributes to cardiovascular mortality in chronic kidney disease (CKD). Neutrophil gelatinase-associated lipocalin (NGAL or lipocalin 2; LCN2 the murine homolog) is a pro-inflammatory and iron-shuttling molecule that is secreted in response to kidney injury and may promote CKD progression. We investigated bone FGF23 regulation by circulating LCN2. At 23 weeks, Col4a3^KO mice showed impaired kidney function, increased levels of kidney and serum LCN2, increased bone and serum FGF23, anemia, and left ventricular hypertrophy (LVH). Deletion of Lcn2 in CKD mice did not improve kidney function or anemia but prevented the development of LVH and improved survival in association with marked reductions in serum FGF23. Lcn2 deletion specifically prevented FGF23 elevations in response to inflammation, but not iron deficiency or phosphate, and administration of LCN2 increased serum FGF23 in healthy and CKD mice by stimulating Fgf23 transcription via activation of cAMP-mediated signaling in bone cells. These results show that kidney-produced LCN2 is an important mediator of increased FGF23 production by bone in response to inflammation and in CKD. LCN2 inhibition might represent a potential therapeutic approach to lower FGF23 and improve outcomes in CKD.

Skeletal interoception regulates bone homeostasis through the prostaglandin E2 (PGE2) concentration in bone. Vertebral endplates undergo ossification and become highly porous during intervertebral disc degeneration and aging. We found that the PGE2 concentration was elevated in porous endplates to generate spinal pain. Importantly, treatment with a high-dose cyclooxygenase 2 inhibitor (celecoxib, 80 mg·kg⁻¹ per day) decreased the prostaglandin E2 concentration and attenuated spinal pain in mice with lumbar spine instability. However, this treatment impaired bone formation in porous endplates, and spinal pain recurred after discontinuing the treatment. Interestingly, low-dose celecoxib (20 mg·kg⁻¹ per day, which is equivalent to one-quarter of the clinical maximum dosage) induced a latent inhibition of spinal pain at 3 weeks post-treatment, which persisted even after discontinuing treatment. Furthermore, when the prostaglandin E2 concentration was maintained at the physiological level with low-dose celecoxib, endplate porosity was reduced significantly, which was associated with decreased sensory nerve innervation and spinal pain. These findings suggest that low-dose celecoxib may help to maintain skeletal interoception and decrease vertebral endplate porosity, thereby reducing sensory innervation and spinal pain in mice.

A comprehensive understanding of the cellular heterogeneity and molecular mechanisms underlying the development, homeostasis, and disease of human intervertebral disks (IVDs) remains challenging. Here, the transcriptomic landscape of 108 108 IVD cells was mapped using single-cell RNA sequencing of three main compartments from young and adult healthy IVDs, including the nucleus pulposus (NP), annulus fibrosus, and cartilage endplate (CEP). The chondrocyte subclusters were classified based on their potential regulatory, homeostatic, and effector functions in extracellular matrix (ECM) homeostasis. Notably, in the NP, a PROCR⁺ resident progenitor population showed enriched colony-forming unit-fibroblast (CFU-F) activity and trilineage differentiation capacity. Finally, intercellular crosstalk based on signaling network analysis uncovered that the PDGF and TGF-β cascades are important cues in the NP microenvironment. In conclusion, a single-cell transcriptomic atlas that resolves spatially regulated cellular heterogeneity together with the critical signaling that underlies homeostasis will help to establish new therapeutic strategies for IVD degeneration in the clinic.

Glucose metabolism is fundamental for the functions of all tissues, including cartilage. Despite the emerging evidence related to glucose metabolism in the regulation of prenatal cartilage development, little is known about the role of glucose metabolism and its biochemical basis in postnatal cartilage growth and homeostasis. We show here that genetic deletion of the glucose transporter Glut1 in postnatal cartilage impairs cell proliferation and matrix production in growth plate (GPs) but paradoxically increases cartilage remnants in the metaphysis, resulting in shortening of long bones. On the other hand, articular cartilage (AC) with Glut1 deficiency presents diminished cellularity and loss of proteoglycans, which ultimately progress to cartilage fibrosis. Moreover, predisposition to Glut1 deficiency severely exacerbates injury-induced osteoarthritis. Regardless of the disparities in glucose metabolism between GP and AC chondrocytes under normal conditions, both types of chondrocytes demonstrate metabolic plasticity to enhance glutamine utilization and oxidation in the absence of glucose availability. However, uncontrolled glutamine flux causes collagen overmodification, thus affecting extracellular matrix remodeling in both cartilage compartments. These results uncover the pivotal and distinct roles of Glut1-mediated glucose metabolism in two of the postnatal cartilage compartments and link some cartilage abnormalities to altered glucose/glutamine metabolism.

Back pain is a common condition with a high social impact and represents a global health burden. Intervertebral disc disease (IVDD) is one of the major causes of back pain; no therapeutics are currently available to reverse this disease. The impact of bone mineral density (BMD) on IVDD has been controversial, with some studies suggesting osteoporosis as causative for IVDD and others suggesting it as protective for IVDD. Functional studies to evaluate the influence of genetic components of BMD in IVDD could highlight opportunities for drug development and repurposing. By taking a holistic 3D approach, we established an aging zebrafish model for spontaneous IVDD. Increased BMD in aging, detected by automated computational analysis, is caused by bone deformities at the endplates. However, aged zebrafish spines showed changes in bone morphology, microstructure, mineral heterogeneity, and increased fragility that resembled osteoporosis. Elements of the discs recapitulated IVDD symptoms found in humans: the intervertebral ligament (equivalent to the annulus fibrosus) showed disorganized collagen fibers and herniation, while the disc center (nucleus pulposus equivalent) showed dehydration and cellular abnormalities. We manipulated BMD in young zebrafish by mutating sp7 and cathepsin K, leading to low and high BMD, respectively. Remarkably, we detected IVDD in both groups, demonstrating that low BMD does not protect against IVDD, and we found a strong correlation between high BMD and IVDD. Deep learning was applied to high-resolution synchrotron µCT image data to analyze osteocyte 3D lacunar distribution and morphology, revealing a role of sp7 in controlling the osteocyte lacunar 3D profile. Our findings suggest potential avenues through which bone quality can be targeted to identify beneficial therapeutics for IVDD.

Chronic high-fat diet (HFD) consumption not only promotes obesity and insulin resistance, but also causes bone loss through mechanisms that are not well understood. Here, we fed wild-type CD-1 mice either chow or a HFD (43% of energy from fat) for 18 weeks; HFD-fed mice exhibited decreased trabecular volume (−28%) and cortical thickness (−14%) compared to chow-fed mice. In HFD-fed mice, bone loss was due to reduced bone formation and mineral apposition, without obvious effects on bone resorption. HFD feeding also increased skeletal expression of sclerostin and caused deterioration of the osteocyte lacunocanalicular network (LCN). In mice fed HFD, skeletal glucocorticoid signaling was activated relative to chow-fed mice, independent of serum corticosterone concentrations. We therefore examined whether skeletal glucocorticoid signaling was necessary for HFD-induced bone loss, using transgenic mice lacking glucocorticoid signaling in osteoblasts and osteocytes (HSD2^OB/OCY-tg mice). In HSD2^OB/OCY-tg mice, bone formation and mineral apposition rates were not suppressed by HFD, and bone loss was significantly attenuated. Interestingly, in HSD2^OB/OCY-tg mice fed HFD, both Wnt signaling (less sclerostin induction, increased β-catenin expression) and glucose uptake were significantly increased, relative to diet- and genotype-matched controls. The osteocyte LCN remained intact in HFD-fed HSD2^OB/OCY-tg mice. When fed a HFD, HSD2^OB/OCY-tg mice also increased their energy expenditure and were protected against obesity, insulin resistance, and dyslipidemia. Therefore, glucocorticoid signaling in osteoblasts and osteocytes contributes to the suppression of bone formation in HFD-fed mice. Skeletal glucocorticoid signaling is also an important determinant of glucose uptake in bone, which influences the whole-body metabolic response to HFD.

Emerging insights into cellular senescence highlight the relevance of senescence in musculoskeletal disorders, which represent the leading global cause of disability. Cellular senescence was initially described by Hayflick et al. in 1961 as an irreversible nondividing state in in vitro cell culture studies. We now know that cellular senescence can occur in vivo in response to various stressors as a heterogeneous and tissue-specific cell state with a secretome phenotype acquired after the initial growth arrest. In the past two decades, compelling evidence from preclinical models and human data show an accumulation of senescent cells in many components of the musculoskeletal system. Cellular senescence is therefore a defining feature of age-related musculoskeletal disorders, and targeted elimination of these cells has emerged recently as a promising therapeutic approach to ameliorate tissue damage and promote repair and regeneration of the skeleton and skeletal muscles. In this review, we summarize evidence of the role of senescent cells in the maintenance of bone homeostasis during childhood and their contribution to the pathogenesis of chronic musculoskeletal disorders, including osteoporosis, osteoarthritis, and sarcopenia. We highlight the diversity of the senescent cells in the microenvironment of bone, joint, and skeletal muscle tissue, as well as the mechanisms by which these senescent cells are involved in musculoskeletal diseases. In addition, we discuss how identifying and targeting senescent cells might positively affect pathologic progression and musculoskeletal system regeneration.

High erythropoietin (Epo) levels are detrimental to bone health in adult organisms. Adult mice receiving high doses of Epo lose bone mass due to suppressed bone formation and increased bone resorption. In humans, high serum Epo levels are linked to fractures in elderly men. Our earlier studies indicated that Epo modulates osteoblast activity; however, direct evidence that Epo acts via its receptor (EpoR) on osteoblasts in vivo is still missing. Here, we created mice lacking EpoR in osteoprogenitor cells to specifically address this gap. Deletion of EpoR in osteoprogenitors (EpoR:Osx-cre, cKO) starting at 5 weeks of age did not alter red blood cell parameters but increased vertebral bone volume by 25% in 12-week-old female mice. This was associated with low bone turnover. Histological (osteoblast number, bone formation rate) and serum (P1NP, osteocalcin) bone formation parameters were all reduced, as were the number of osteoclasts and TRAP serum level. Differentiation of osteoblast precursors isolated from cKO versus control mice resulted in lower expression of osteoblast marker genes including Runx2, Alp, and Col1a1 on day 21, whereas the mineralization capacity was similar. Moreover, the RANKL/OPG ratio, which determines the osteoclast-supporting potential of osteoblasts, was substantially decreased by 50%. Similarly, coculturing cKO osteoblasts with control or cKO osteoclast precursors produced significantly fewer osteoclasts than coculture with control osteoblasts. Finally, exposing female mice to Epo pumps (10 U·d⁻¹) for 4 weeks resulted in trabecular bone loss (−25%) and increased osteoclast numbers (1.7-fold) in control mice only, not in cKO mice. Our data show that EpoR in osteoprogenitors is essential in regulating osteoblast function and osteoblast-mediated osteoclastogenesis via the RANKL/OPG axis. Thus, osteogenic Epo/EpoR signaling controls bone mass maintenance and contributes to Epo-induced bone loss.

Disruption of bone homeostasis caused by metastatic osteolytic breast cancer cells increases inflammatory osteolysis and decreases bone formation, thereby predisposing patients to pathological fracture and cancer growth. Alteration of osteoblast function induces skeletal diseases due to the disruption of bone homeostasis. We observed increased activation of pERK1/2 in osteolytic breast cancer cells and osteoblasts in human pathological specimens with aggressive osteolytic breast cancer metastases. We confirmed that osteolytic breast cancers with high expression of pERK1/2 disrupt bone homeostasis via osteoblastic ERK1/2 activation at the bone-breast cancer interface. The process of inflammatory osteolysis modulates ERK1/2 activation in osteoblasts and breast cancer cells through dominant-negative MEK1 expression and constitutively active MEK1 expression to promote cancer growth within bone. Trametinib, an FDA-approved MEK inhibitor, not only reduced breast cancer-induced bone destruction but also dramatically reduced cancer growth in bone by inhibiting the inflammatory skeletal microenvironment. Taken together, these findings suggest that ERK1/2 activation in both breast cancer cells and osteoblasts is required for osteolytic breast cancer-induced inflammatory osteolysis and that ERK1/2 pathway inhibitors may represent a promising adjuvant therapy for patients with aggressive osteolytic breast cancers by altering the shared cancer and bone microenvironment.

Mechanotransduction is a fundamental ability that allows living organisms to receive and respond to physical signals from both the external and internal environments. The mechanotransduction process requires a range of special proteins termed mechanotransducers to convert mechanical forces into biochemical signals in cells. The Piezo proteins are mechanically activated nonselective cation channels and the largest plasma membrane ion channels reported thus far. The regulation of two family members, Piezo1 and Piezo2, has been reported to have essential functions in mechanosensation and transduction in different organs and tissues. Recently, the predominant contributions of the Piezo family were reported to occur in the skeletal system, especially in bone development and mechano-stimulated bone homeostasis. Here we review current studies focused on the tissue-specific functions of Piezo1 and Piezo2 in various backgrounds with special highlights on their importance in regulating skeletal cell mechanotransduction. In this review, we emphasize the diverse functions of Piezo1 and Piezo2 and related signaling pathways in osteoblast lineage cells and chondrocytes. We also summarize our current understanding of Piezo channel structures and the key findings about PIEZO gene mutations in human diseases.

Osteoclasts are bone-resorbing cells that play an essential role in homeostatic bone remodeling and pathological bone erosion. Macrophage colony stimulating factor (M-CSF) is abundant in rheumatoid arthritis (RA). However, the role of M-CSF in arthritic bone erosion is not completely understood. Here, we show that M-CSF can promote osteoclastogenesis by triggering the proteolysis of c-FMS, a receptor for M-CSF, leading to the generation of FMS intracellular domain (FICD) fragments. Increased levels of FICD fragments positively regulated osteoclastogenesis but had no effect on inflammatory responses. Moreover, myeloid cell-specific FICD expression in mice resulted in significantly increased osteoclast-mediated bone resorption in an inflammatory arthritis model. The FICD formed a complex with DAP5, and the FICD/DAP5 axis promoted osteoclast differentiation by activating the MNK1/2/EIF4E pathway and enhancing NFATc1 protein expression. Moreover, targeting the MNK1/2 pathway diminished arthritic bone erosion. These results identified a novel role of c-FMS proteolysis in osteoclastogenesis and the pathogenesis of arthritic bone erosion.

Tissue engineering is rapidly progressing toward clinical application. In the musculoskeletal field, there has been an increasing necessity for bone and cartilage replacement. Despite the promising translational potential of tissue engineering approaches, careful attention should be given to the quality of developed constructs to increase the real applicability to patients. After a general introduction to musculoskeletal tissue engineering, this narrative review aims to offer an overview of methods, starting from classical techniques, such as gene expression analysis and histology, to less common methods, such as Raman spectroscopy, microcomputed tomography, and biosensors, that can be employed to assess the quality of constructs in terms of viability, morphology, or matrix deposition. A particular emphasis is given to standards and good practices (GXP), which can be applicable in different sectors. Moreover, a classification of the methods into destructive, noninvasive, or conservative based on the possible further development of a preimplant quality monitoring system is proposed. Biosensors in musculoskeletal tissue engineering have not yet been used but have been proposed as a novel technology that can be exploited with numerous advantages, including minimal invasiveness, making them suitable for the development of preimplant quality control systems.

Osteoporosis (OP) is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures. Pharmaceutical therapies that promote anabolic bone formation in OP patients and OP-induced fracture are needed. We investigated whether a neutralizing antibody against Siglec-15 can simultaneously inhibit bone resorption and stimulate bone formation. We found that the multinucleation of osteoclasts was inhibited in SIGLEC-15 conditional knockout mice and mice undergoing Siglec-15 neutralizing antibody treatment. The secretion of platelet-derived growth factor-BB (PDGF-BB), the number of tartrate-resistant acid phosphatase-positive (TRAP⁺) mononuclear cells, and bone formation were significantly increased in the SIGLEC-15 conditional knockout mice and antibody-treated mice. The anabolic effect of the Siglec-15 neutralizing antibody on bone formation was blunted in mice with Pdgfb deleted in TRAP⁺ cells. These findings showed that the anabolic effect of the Siglec-15 neutralizing antibody was mediated by elevating PDGF-BB production of TRAP⁺ mononuclear cells. To test the therapeutic potential of the Siglec-15 neutralizing antibody, we injected the antibody in an ovariectomy-induced osteoporotic mouse model, which mimics postmenopausal osteoporosis in women, and in two fracture healing models because fracture is the most serious health consequence of osteoporosis. The Siglec-15 neutralizing antibody effectively reduced bone resorption and stimulated bone formation in estrogen deficiency-induced osteoporosis. Of note, the Siglec-15 neutralizing antibody promoted intramembranous and endochondral ossification at the damaged area of cortical bone in fracture healing mouse models. Thus, the Siglec-15 neutralizing antibody shows significant translational potential as a novel therapy for OP and bone fracture.

The recent identification of homozygous WNT1 mutations in individuals with osteogenesis imperfecta type XV (OI-XV) has suggested that WNT1 is a key ligand promoting the differentiation and function of bone-forming osteoblasts. Although such an influence was supported by subsequent studies, a mouse model of OI-XV remained to be established. Therefore, we introduced a previously identified disease-causing mutation (G177C) into the murine Wnt1 gene. Homozygous Wnt1 ^G177C/G177C mice were viable and did not display defects in brain development, but the majority of 24-week-old Wnt1 ^G177C/G177C mice had skeletal fractures. This increased bone fragility was not fully explained by reduced bone mass but also by impaired bone matrix quality. Importantly, the homozygous presence of the G177C mutation did not interfere with the osteoanabolic influence of either parathyroid hormone injection or activating mutation of LRP5, the latter mimicking the effect of sclerostin neutralization. Finally, transcriptomic analyses revealed that short-term administration of WNT1 to osteogenic cells induced not only the expression of canonical WNT signaling targets but also the expression of genes encoding extracellular matrix modifiers. Taken together, our data demonstrate that regulating bone matrix quality is a primary function of WNT1. They further suggest that individuals with WNT1 mutations should profit from existing osteoanabolic therapies.

In a previous transcriptomic study of human bone marrow stromal cells (BMSCs, also known as bone marrow-derived “mesenchymal stem cells”), SFRP2 was highly over-represented in a subset of multipotent BMSCs (skeletal stem cells, SSCs), which recreate a bone/marrow organ in an in vivo ectopic bone formation assay. SFRPs modulate WNT signaling, which is essential to maintain skeletal homeostasis, but the specific role of SFRP2 in BMSCs/SSCs is unclear. Here, we evaluated Sfrp2 deficiency on BMSC/SSC function in models of skeletal organogenesis and regeneration. The skeleton of Sfrp2-deficient (KO) mice is overtly normal; but their BMSCs/SSCs exhibit reduced colony-forming efficiency, reflecting low SSC self-renewal/abundancy. Sfrp2 KO BMSCs/SSCs formed less trabecular bone than those from WT littermates in the ectopic bone formation assay. Moreover, regeneration of a cortical drilled hole defect was dramatically impaired in Sfrp2 KO mice. Sfrp2-deficient BMSCs/SSCs exhibited poor in vitro osteogenic differentiation as measured by Runx2 and Osterix expression and calcium accumulation. Interestingly, activation of the Wnt co-receptor, Lrp6, and expression of Wnt target genes, Axin2, C-myc and Cyclin D1, were reduced in Sfrp2-deficient BMSCs/SSCs. Addition of recombinant Sfrp2 restored most of these activities, suggesting that Sfrp2 acts as a Wnt agonist. We demonstrate that Sfrp2 plays a role in self-renewal of SSCs and in the recruitment and differentiation of adult SSCs during bone healing. SFRP2 is also a useful marker of BMSC/SSC multipotency, and a factor to potentially improve the quality of ex vivo expanded BMSC/SSC products.

Runt-related transcription factor-1 (Runx1) is required for chondrocyte-to-osteoblast lineage commitment by enhancing both chondrogenesis and osteogenesis during vertebrate development. However, the potential role of Runx1 in joint diseases is not well known. In the current study, we aimed to explore the role of Runx1 in osteoarthritis induced by anterior cruciate ligament transaction (ACLT) surgery. We showed that chondrocyte-specific Runx1 knockout (Runx1 ^f/f Col2a1-Cre) aggravated cartilage destruction by accelerating the loss of proteoglycan and collagen II in early osteoarthritis. Moreover, we observed thinning and ossification of the growth plate, a decrease in chondrocyte proliferative capacity and the loss of bone matrix around the growth plate in late osteoarthritis. We overexpressed Runx1 by adeno-associated virus (AAV) in articular cartilage and identified its protective effect by slowing the destruction of osteoarthritis in cartilage in early osteoarthritis and alleviating the pathological progression of growth plate cartilage in late osteoarthritis. ChIP-seq analysis identified new targets that interacted with Runx1 in cartilage pathology, and we confirmed the direct interactions of these factors with Runx1 by ChIP-qPCR. This study helps us to understand the function of Runx1 in osteoarthritis and provides new clues for targeted osteoarthritis therapy.