The periosteum, a highly vascularized thin tissue, has excellent osteogenic and bone regenerative abilities. The generation of periosteum-mimicking tissue has become a novel strategy for bone defect repair and regeneration, especially in critical-sized bone defects caused by trauma and bone tumor resection. Here, we utilized a bone morphogenetic protein-2 (BMP-2)-loaded scaffold to create periosteum-like tissue (PT) in vivo, mimicking the mesenchymal condensation during native long bone development. We found that BMP-2-induced endochondral ossification plays an indispensable role in the construction of PTs. Moreover, we confirmed that BMP-2-induced PTs exhibit a similar architecture to the periosteum and harbor abundant functional periosteum-like tissue-derived cells (PTDCs), blood vessels, and osteochondral progenitor cells. Interestingly, we found that the addition of chondroitin sulfate (CS), an essential component of the extracellular matrix (ECM), could further increase the abundance and enhance the function of recruited PTDCs from the PTs and finally increase the regenerative capacity of the PTs in autologous transplantation assays, even in old mice. This novel biomimetic strategy for generating PT through in vivo endochondral ossification deserves further clinical translation.

The intervertebral disc (IVD) is the largest avascular tissue. Hypoxia-inducible factors (HIFs) play essential roles in regulating cellular adaptation in the IVD under physiological conditions. Disc degeneration disease (DDD) is one of the leading causes of disability, and current therapies are ineffective. This study sought to explore the role of HIFs in DDD pathogenesis in mice. The findings of this study showed that among HIF family members, Hif1α was significantly upregulated in cartilaginous endplate (EP) and annulus fibrosus (AF) tissues from human DDD patients and two mouse models of DDD compared with controls. Conditional deletion of the E3 ubiquitin ligase Vhl in EP and AF tissues of adult mice resulted in upregulated Hif1α expression and age-dependent IVD degeneration. Aberrant Hif1α activation enhanced glycolytic metabolism and suppressed mitochondrial function. On the other hand, genetic ablation of the Hif1α gene delayed DDD pathogenesis in Vhl-deficient mice. Administration of 2-methoxyestradiol (2ME2), a selective Hif1α inhibitor, attenuated experimental IVD degeneration in mice. The findings of this study show that aberrant Hif1α activation in EP and AF tissues induces pathological changes in DDD, implying that inhibition of aberrant Hif1α activity is a potential therapeutic strategy for DDD.

Osteoporosis is a highly prevalent public health burden associated with an increased risk of bone fracture, particularly in aging women. Estrogen, an important medicinal component for the preventative and therapeutic treatment of postmenopausal osteoporosis, induces osteogenesis by activating the estrogen receptor signaling pathway and upregulating the expression of osteogenic genes, such as bone morphogenetic proteins (BMPs). The epigenetic regulation of estrogen-mediated osteogenesis, however, is still unclear. In this report, we found that estrogen significantly induced the expression of lysine-specific demethylase 6B (KDM6B) and that KDM6B depletion by shRNAs led to a significant reduction in the osteogenic potential of DMSCs. Mechanistically, upon estrogen stimulation, estrogen receptor-α (ERα) was recruited to the KDM6B promoter, directly enhancing KDM6B expression. Subsequently, KDM6B was recruited to the BMP2 and HOXC6 promoters, resulting in the removal of H3K27me3 marks and activating the transcription of BMP2 and HOXC6, the master genes of osteogenic differentiation. Furthermore, we found that estrogen enhanced DMSC osteogenesis during calvarial bone regeneration and that estrogen’s pro-osteogenic effect was dependent on KDM6B in vivo. Taken together, our results demonstrate the vital role of the ERα/KDM6B regulatory axis in the epigenetic regulation of the estrogen-dependent osteogenic response.

Tendon heterotopic ossification (HO) is characterized by bone formation inside tendon tissue, which severely debilitates people in their daily life. Current therapies fail to promote functional tissue repair largely due to our limited understanding of HO pathogenesis. Here, we investigate the pathological mechanism and propose a potential treatment method for HO. Immunofluorescence assays showed that the Mohawk (MKX) expression level was decreased in human tendon HO tissue, coinciding with spontaneous HO and the upregulated expression of osteochondrogenic and angiogenic genes in the tendons of Mkx ^−/− mice. Single-cell RNA sequencing analyses of wild-type and Mkx ^−/− tendons identified three cell types and revealed the excessive activation of osteochondrogenic genes during the tenogenesis of Mkx ^−/− tendon cells. Single-cell analysis revealed that the gene expression program of angiogenesis, which is strongly associated with bone formation, was activated in all cell types during HO. Moreover, inhibition of angiogenesis by the small-molecule inhibitor BIBF1120 attenuated bone formation and angiogenesis in the Achilles tendons of both Mkx mutant mice and a rat traumatic model of HO. These findings provide new insights into the cellular mechanisms of tendon HO and highlight the inhibition of angiogenesis with BIBF1120 as a potential treatment strategy for HO.

Intervertebral disc (IVD) degeneration (IVDD) is the main cause of low back pain with major social and economic burdens; however, its underlying molecular mechanisms remain poorly defined. Here we show that the focal adhesion protein Kindlin-2 is highly expressed in the nucleus pulposus (NP), but not in the anulus fibrosus and the cartilaginous endplates, in the IVD tissues. Expression of Kindlin-2 is drastically decreased in NP cells in aged mice and severe IVDD patients. Inducible deletion of Kindlin-2 in NP cells in adult mice causes spontaneous and striking IVDD-like phenotypes in lumbar IVDs and largely accelerates progression of coccygeal IVDD in the presence of abnormal mechanical stress. Kindlin-2 loss activates Nlrp3 inflammasome and stimulates expression of IL-1β in NP cells, which in turn downregulates Kindlin-2. This vicious cycle promotes extracellular matrix (ECM) catabolism and NP cell apoptosis. Furthermore, abnormal mechanical stress reduces expression of Kindlin-2, which exacerbates Nlrp3 inflammasome activation, cell apoptosis, and ECM catabolism in NP cells caused by Kindlin-2 deficiency. In vivo blocking Nlrp3 inflammasome activation prevents IVDD progression induced by Kindlin-2 loss and abnormal mechanical stress. Of translational significance, adeno-associated virus-mediated overexpression of Kindlin-2 inhibits ECM catabolism and cell apoptosis in primary human NP cells in vitro and alleviates coccygeal IVDD progression caused by mechanical stress in rat. Collectively, we establish critical roles of Kindlin-2 in inhibiting Nlrp3 inflammasome activation and maintaining integrity of the IVD homeostasis and define a novel target for the prevention and treatment of IVDD.

Bone metastases occur in patients with advanced-stage prostate cancer (PCa). The cell-cell interaction between PCa and the bone microenvironment forms a vicious cycle that modulates the bone microenvironment, increases bone deformities, and drives tumor growth in the bone. However, the molecular mechanisms of PCa-mediated modulation of the bone microenvironment are complex and remain poorly defined. Here, we evaluated growth differentiation factor-15 (GDF15) function using in vivo preclinical PCa-bone metastasis mouse models and an in vitro bone cell coculture system. Our results suggest that PCa-secreted GDF15 promotes bone metastases and induces bone microarchitectural alterations in a preclinical xenograft model. Mechanistic studies revealed that GDF15 increases osteoblast function and facilitates the growth of PCa in bone by activating osteoclastogenesis through osteoblastic production of CCL2 and RANKL and recruitment of osteomacs. Altogether, our findings demonstrate the critical role of GDF15 in the modulation of the bone microenvironment and subsequent development of PCa bone metastasis.

The outer coverings of the skeleton, which is also known as the periosteum, are arranged in concentric layers and act as a reservoir for tissue-specific bone progenitors. The cellular heterogeneity within this tissue depot is being increasingly recognized. Here, inducible PDGFRα reporter animals were found to mark a population of cells within the periosteum that act as a stem cell reservoir for periosteal appositional bone formation and fracture repair. During these processes, PDGFRα reporter⁺ progenitors give rise to Nestin⁺ periosteal cells before becoming osteoblasts and osteocytes. The diphtheria toxin-mediated ablation of PDGFRα reporter⁺ cells led to deficits in cortical bone formation during homeostasis and a diminutive hard callus during fracture repair. After ossicle transplantation, both mouse PDGFRα reporter⁺ periosteal cells and human Pdgfrα⁺ periosteal progenitors expand, ossify, and recruit marrow to a greater extent than their counterpart periosteal cells, whereas PDGFRα reporter⁻ periosteal cells exhibit a predisposition to chondrogenesis in vitro. Total RNA sequencing identified enrichment of the secreted factors Fermt3 and Ptpn6 within PDGFRα reporter⁺ periosteal cells, which partly underlie the osteoblastogenic features of this cell population.

A gain-of-function mutation in the fibroblast growth factor receptor 3 gene (FGFR3) results in achondroplasia (ACH), the most frequent form of dwarfism. Constitutive activation of FGFR3 impairs bone formation and elongation and many signal transduction pathways. Identification of new and relevant compounds targeting the FGFR3 signaling pathway is of broad importance for the treatment of ACH, and natural plant compounds are prime drug candidate sources. Here, we found that the phenolic compound (-)-epicatechin, isolated from Theobroma cacao, effectively inhibited FGFR3’s downstream signaling pathways. Transcriptomic analysis in an Fgfr3 mouse model showed that ciliary mRNA expression was modified and influenced significantly by the Indian hedgehog and PKA pathways. (-)-Epicatechin is able to rescue mRNA expression impairments that control both the structural organization of the primary cilium and ciliogenesis-related genes. In femurs isolated from a mouse model (Fgfr3 ^Y367C/+) of ACH, we showed that (-)-epicatechin eliminated bone growth impairment during 6 days of ex vivo culture. In vivo, we confirmed that daily subcutaneous injections of (-)-epicatechin to Fgfr3 ^Y367C/+ mice increased bone elongation and rescued the primary cilium defects observed in chondrocytes. This modification to the primary cilia promoted the typical columnar arrangement of flat proliferative chondrocytes and thus enhanced bone elongation. The results of the present proof-of-principle study support (-)-epicatechin as a potential drug for the treatment of ACH.

Intermittent injections of parathyroid hormone (iPTH) are applied clinically to stimulate bone formation by osteoblasts, although continuous elevation of parathyroid hormone (PTH) primarily results in increased bone resorption. Here, we identified Calca, encoding the sepsis biomarker procalcitonin (ProCT), as a novel target gene of PTH in murine osteoblasts that inhibits osteoclast formation. During iPTH treatment, mice lacking ProCT develop increased bone resorption with excessive osteoclast formation in both the long bones and axial skeleton. Mechanistically, ProCT inhibits the expression of key mediators involved in the recruitment of macrophages, representing osteoclast precursors. Accordingly, ProCT arrests macrophage migration and causes inhibition of early but not late osteoclastogenesis. In conclusion, our results reveal a potential role of osteoblast-derived ProCT in the bone microenvironment that is required to limit bone resorption during iPTH.

Skeletal development and homeostasis in mammals are modulated by finely coordinated processes of migration, proliferation, differentiation, and death of skeletogenic cells originating from the mesoderm and neural crest. Numerous molecular mechanisms are involved in these regulatory processes, one of which is protein posttranslational modifications, particularly protein tyrosine phosphorylation (PYP). PYP occurs mainly through the action of protein tyrosine kinases (PTKs), modifying protein enzymatic activity, changing its cellular localization, and aiding in the assembly or disassembly of protein signaling complexes. Under physiological conditions, PYP is balanced by the coordinated action of PTKs and protein tyrosine phosphatases (PTPs). Dysregulation of PYP can cause genetic, metabolic, developmental, and oncogenic skeletal diseases. Although PYP is a reversible biochemical process, in contrast to PTKs, little is known about how this equilibrium is modulated by PTPs in the skeletal system. Whole-genome sequencing has revealed a large and diverse superfamily of PTP genes (over 100 members) in humans, which can be further divided into cysteine (Cys)-, aspartic acid (Asp)-, and histidine (His)-based PTPs. Here, we review current knowledge about the functions and regulatory mechanisms of 28 PTPs involved in skeletal development and diseases; 27 of them belong to class I and II Cys-based PTPs, and the other is an Asp-based PTP. Recent progress in analyzing animal models that harbor various mutations in these PTPs and future research directions are also discussed. Our literature review indicates that PTPs are as crucial as PTKs in supporting skeletal development and homeostasis.

Discoidin Domain Receptor 2 (DDR2) is a collagen-activated receptor kinase that, together with integrins, is required for cells to respond to the extracellular matrix. Ddr2 loss-of-function mutations in humans and mice cause severe defects in skeletal growth and development. However, the cellular functions of Ddr2 in bone are not understood. Expression and lineage analysis showed selective expression of Ddr2 at early stages of bone formation in the resting zone and proliferating chondrocytes and periosteum. Consistent with these findings, Ddr2 ⁺ cells could differentiate into hypertrophic chondrocytes, osteoblasts, and osteocytes and showed a high degree of colocalization with the skeletal progenitor marker, Gli1. A conditional deletion approach showed a requirement for Ddr2 in Gli1-positive skeletal progenitors and chondrocytes but not mature osteoblasts. Furthermore, Ddr2 knockout in limb bud chondroprogenitors or purified marrow-derived skeletal progenitors inhibited chondrogenic or osteogenic differentiation, respectively. This work establishes a cell-autonomous function for Ddr2 in skeletal progenitors and cartilage and emphasizes the critical role of this collagen receptor in bone development.

Osteoarthritis is a joint disease characterized by a poorly-defined inflammatory response that does not encompass a massive immune cell infiltration yet contributes to cartilage degradation and loss of joint mobility, suggesting a chondrocyte intrinsic inflammatory response. Using primary chondrocytes from joints of osteoarthritic mice and patients, we first show that these cells express ample pro-inflammatory markers and RANKL in an NF-κB dependent manner. The inflammatory phenotype of chondrocytes was recapitulated by exposure of chondrocytes to IL-1β and bone particles, which were used to model bone matrix breakdown products revealed to be present in synovial fluid of OA patients, albeit their role was not defined. We further show that bone particles and IL-1β can promote senescent and apoptotic changes in primary chondrocytes due to oxidative stress from various cellular sources such as the mitochondria. Finally, we provide evidence that inflammation, oxidative stress and senescence converge upon IκB-ζ, the principal mediator downstream of NF-κB, which regulates expression of RANKL, inflammatory, catabolic, and SASP genes. Overall, this work highlights the capacity and mechanisms by which inflammatory cues, primarily joint degradation products, i.e., bone matrix particles in concert with IL-1β in the joint microenvironment, program chondrocytes into an “inflammatory phenotype” which inflects local tissue damage.

Uncovering the molecular pathways that drive skeletal repair has been an ongoing challenge. Initial efforts have relied on in vitro assays to identify the key signaling pathways that drive cartilage and bone differentiation. While these assays can provide some clues, assessing specific pathways in animal models is critical. Furthermore, definitive proof that a pathway is required for skeletal repair is best provided using genetic tests. Stimulating the Hh (Hedgehog) pathway can promote cartilage and bone differentiation in cell culture assays. In addition, the application of HH protein or various pathway agonists in vivo has a positive influence on bone healing. Until recently, however, genetic proof that the Hh pathway is involved in bone repair has been lacking. Here, we consider both in vitro and in vivo studies that examine the role of Hh in repair and discuss some of the challenges inherent in their interpretation. We also identify needed areas of study considering a new appreciation for the role of cartilage during repair, the variety of cell types that may have differing roles in repair, and the recent availability of powerful lineage tracing techniques. We are optimistic that emerging genetic tools will make it possible to precisely define when and in which cells promoting Hh signaling can best promote skeletal repair, and thus, the clinical potential for targeting the Hh pathway can be realized.

The majority of the mammalian skeleton is formed through endochondral ossification starting from a cartilaginous template. Cartilage cells, or chondrocytes, survive, proliferate and synthesize extracellular matrix in an avascular environment, but the metabolic requirements for these anabolic processes are not fully understood. Here, using metabolomics analysis and genetic in vivo models, we show that maintaining intracellular serine homeostasis is essential for chondrocyte function. De novo serine synthesis through phosphoglycerate dehydrogenase (PHGDH)-mediated glucose metabolism generates nucleotides that are necessary for chondrocyte proliferation and long bone growth. On the other hand, dietary serine is less crucial during endochondral bone formation, as serine-starved chondrocytes compensate by inducing PHGDH-mediated serine synthesis. Mechanistically, this metabolic flexibility requires ATF4, a transcriptional regulator of amino acid metabolism and stress responses. We demonstrate that both serine deprivation and PHGDH inactivation enhance ATF4 signaling to stimulate de novo serine synthesis and serine uptake, respectively, and thereby prevent intracellular serine depletion and chondrocyte dysfunction. A similar metabolic adaptability between serine uptake and de novo synthesis is observed in the cartilage callus during fracture repair. Together, the results of this study reveal a critical role for PHGDH-dependent serine synthesis in maintaining intracellular serine levels under physiological and serine-limited conditions, as adequate serine levels are necessary to support chondrocyte proliferation during endochondral ossification.

In the bone marrow, B cells and bone-resorbing osteoclasts colocalize and form a specific microenvironment. How B cells functionally influence osteoclasts and bone architecture is poorly understood. Using genetically modified mice and high-throughput analyses, we demonstrate that prolonged HIF-1α signaling in B cells leads to enhanced RANKL production and osteoclast formation. In addition, deletion of HIF-1α in B cells prevents estrogen deficiency-induced bone loss in mice. Mechanistically, estrogen controls HIF-1α protein stabilization through HSP70-mediated degradation in bone marrow B cells. The stabilization of HIF-1α protein in HSP70-deficient bone marrow B cells promotes RANKL production and osteoclastogenesis. Induction of HSP70 expression by geranylgeranylacetone (GGA) administration alleviates ovariectomy-induced osteoporosis. Moreover, RANKL gene expression has a positive correlation with HIF1A expression in human B cells. In conclusion, HIF-1α signaling in B cells is crucial for the control of osteoclastogenesis, and the HSP70/HIF-1α axis may serve as a new therapeutic target for osteoporosis.

Bone remodeling is a lifelong process that gives rise to a mature, dynamic bone structure via a balance between bone formation by osteoblasts and resorption by osteoclasts. These opposite processes allow the accommodation of bones to dynamic mechanical forces, altering bone mass in response to changing conditions. Mechanical forces are indispensable for bone homeostasis; skeletal formation, resorption, and adaptation are dependent on mechanical signals, and loss of mechanical stimulation can therefore significantly weaken the bone structure, causing disuse osteoporosis and increasing the risk of fracture. The exact mechanisms by which the body senses and transduces mechanical forces to regulate bone remodeling have long been an active area of study among researchers and clinicians. Such research will lead to a deeper understanding of bone disorders and identify new strategies for skeletal rejuvenation. Here, we will discuss the mechanical properties, mechanosensitive cell populations, and mechanotransducive signaling pathways of the skeletal system.

Bone defects combined with tumors, infections, or other bone diseases are challenging in clinical practice. Autologous and allogeneic grafts are two main traditional remedies, but they can cause a series of complications. To address this problem, researchers have constructed various implantable biomaterials. However, the original pathological microenvironment of bone defects, such as residual tumors, severe infection, or other bone diseases, could further affect bone regeneration. Thus, the rational design of versatile biomaterials with integrated bone therapy and regeneration functions is in great demand. Many strategies have been applied to fabricate smart stimuli-responsive materials for bone therapy and regeneration, with stimuli related to external physical triggers or endogenous disease microenvironments or involving multiple integrated strategies. Typical external physical triggers include light irradiation, electric and magnetic fields, ultrasound, and mechanical stimuli. These stimuli can transform the internal atomic packing arrangements of materials and affect cell fate, thus enhancing bone tissue therapy and regeneration. In addition to the external stimuli-responsive strategy, some specific pathological microenvironments, such as excess reactive oxygen species and mild acidity in tumors, specific pH reduction and enzymes secreted by bacteria in severe infection, and electronegative potential in bone defect sites, could be used as biochemical triggers to activate bone disease therapy and bone regeneration. Herein, we summarize and discuss the rational construction of versatile biomaterials with bone therapeutic and regenerative functions. The specific mechanisms, clinical applications, and existing limitations of the newly designed biomaterials are also clarified.

Mechanical stimulation plays an important role in bone remodeling. Exercise-induced mechanical loading enhances bone strength, whereas mechanical unloading leads to bone loss. Increasing evidence has demonstrated that long noncoding RNAs (lncRNAs) play key roles in diverse biological, physiological and pathological contexts. However, the roles of lncRNAs in mechanotransduction and their relationships with bone formation remain unknown. In this study, we screened mechanosensing lncRNAs in osteoblasts and identified Neat1, the most clearly decreased lncRNA under simulated microgravity. Of note, not only Neat1 expression but also the specific paraspeckle structure formed by Neat1 was sensitive to different mechanical stimulations, which were closely associated with osteoblast function. Paraspeckles exhibited small punctate aggregates under simulated microgravity and elongated prolate or larger irregular structures under mechanical loading. Neat1 knockout mice displayed disrupted bone formation, impaired bone structure and strength, and reduced bone mass. Neat1 deficiency in osteoblasts reduced the response of osteoblasts to mechanical stimulation. In vivo, Neat1 knockout in mice weakened the bone phenotypes in response to mechanical loading and hindlimb unloading stimulation. Mechanistically, paraspeckles promoted nuclear retention of E3 ubiquitin ligase Smurf1 mRNA and downregulation of their translation, thus inhibiting ubiquitination-mediated degradation of the osteoblast master transcription factor Runx2, a Smurf1 target. Our study revealed that Neat1 plays an essential role in osteoblast function under mechanical stimulation, which provides a paradigm for the function of the lncRNA-assembled structure in response to mechanical stimulation and offers a therapeutic strategy for long-term spaceflight- or bedrest-induced bone loss and age-related osteoporosis.

Exposure to radiation causes DNA damage; hence, continuous surveillance and timely DNA repair are important for genome stability. Epigenetic modifications alter the chromatin architecture, thereby affecting the efficiency of DNA repair. However, how epigenetic modifiers coordinate with the DNA repair machinery to modulate cellular radiosensitivity is relatively unknown. Here, we report that loss of the demethylase ribosomal oxygenase 1 (RIOX1) restores cell proliferation and reduces cell death after exposure to ionizing radiation. Furthermore, RIOX1 depletion enhances homologous recombination (HR) repair but not nonhomologous end-joining (NHEJ) repair in irradiated bone marrow cells and oral mucosal epithelial cells. Mechanistic study demonstrates that RIOX1 removes monomethylation at K491 of cyclic GMP-AMP synthase (cGAS) to release cGAS from its interaction with the methyl-lysine reader protein SAGA complex-associated factor 29 (SGF29), which subsequently enables cGAS to interact with poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase 1 (PARP1) at DNA break sites, thereby blocking PARP1-mediated recruitment of Timeless. High expression of RIOX1 maintains cGAS K491me at a low level, which impedes HR repair and reduces cellular tolerance to ionizing radiation. This study highlights a novel RIOX1-dependent mechanism involved in the non-immune function of cGAS that is essential for the regulation of ionizing radiation-elicited HR repair.

The circadian clock participates in maintaining homeostasis in peripheral tissues, including intervertebral discs (IVDs). Abnormal mechanical loading is a known risk factor for intervertebral disc degeneration (IDD). Based on the rhythmic daily loading pattern of rest and activity, we hypothesized that abnormal mechanical loading could dampen the IVD clock, contributing to IDD. Here, we investigated the effects of abnormal loading on the IVD clock and aimed to inhibit compression-induced IDD by targeting the core clock molecule brain and muscle Arnt-like protein-1 (BMAL1). In this study, we showed that BMAL1 KO mice exhibit radiographic features similar to those of human IDD and that BMAL1 expression was negatively correlated with IDD severity by systematic analysis based on 149 human IVD samples. The intrinsic circadian clock in the IVD was dampened by excessive loading, and BMAL1 overexpression by lentivirus attenuated compression-induced IDD. Inhibition of the RhoA/ROCK pathway by Y-27632 or melatonin attenuated the compression-induced decrease in BMAL1 expression. Finally, the two drugs partially restored BMAL1 expression and alleviated IDD in a diurnal compression model. Our results first show that excessive loading dampens the circadian clock of nucleus pulposus tissues via the RhoA/ROCK pathway, the inhibition of which potentially protects against compression-induced IDD by preserving BMAL1 expression. These findings underline the importance of the circadian clock for IVD homeostasis and provide a potentially effective therapeutic strategy for IDD.

Bioprinting is an emerging additive manufacturing technology that has enormous potential in bone implantation and repair. The insufficient accuracy of the shape of bioprinted parts is a primary clinical barrier that prevents widespread utilization of bioprinting, especially for bone design with high-resolution requirements. During the last five years, the use of computer vision for process control has been widely practiced in the manufacturing field. Computer vision can improve the performance of bioprinting for bone research with respect to various aspects, including accuracy, resolution, and cell survival rate. Hence, computer vision plays a substantial role in addressing the current defect problem in bioprinting for bone research. In this review, recent advances in the application of computer vision in bioprinting for bone research are summarized and categorized into three groups based on different defect types: bone scaffold process control, deep learning, and cell viability models. The collection of printing parameters, data processing, and feedback of bioprinting information, which ultimately improves printing capabilities, are further discussed. We envision that computer vision may offer opportunities to accelerate bioprinting development and provide a new perception for bone research.

The cells of origin of neurogenic heterotopic ossifications (NHOs), which develop frequently in the periarticular muscles following spinal cord injuries (SCIs) and traumatic brain injuries, remain unclear because skeletal muscle harbors two progenitor cell populations: satellite cells (SCs), which are myogenic, and fibroadipogenic progenitors (FAPs), which are mesenchymal. Lineage-tracing experiments using the Cre recombinase/LoxP system were performed in two mouse strains with the fluorescent protein ZsGreen specifically expressed in either SCs or FAPs in skeletal muscles under the control of the Pax7 or Prrx1 gene promoter, respectively. These experiments demonstrate that following muscle injury, SCI causes the upregulation of PDGFRα expression on FAPs but not SCs and the failure of SCs to regenerate myofibers in the injured muscle, with reduced apoptosis and continued proliferation of muscle resident FAPs enabling their osteogenic differentiation into NHOs. No cells expressing ZsGreen under the Prrx1 promoter were detected in the blood after injury, suggesting that the cells of origin of NHOs are locally derived from the injured muscle. We validated these findings using human NHO biopsies. PDGFRα⁺ mesenchymal cells isolated from the muscle surrounding NHO biopsies could develop ectopic human bones when transplanted into immunocompromised mice, whereas CD56⁺ myogenic cells had a much lower potential. Therefore, NHO is a pathology of the injured muscle in which SCI reprograms FAPs to undergo uncontrolled proliferation and differentiation into osteoblasts.

Deep learning (DL) is currently revolutionizing peptide drug development due to both computational advances and the substantial recent expansion of digitized biological data. However, progress in oligopeptide drug development has been limited, likely due to the lack of suitable datasets and difficulty in identifying informative features to use as inputs for DL models. Here, we utilized an unsupervised deep learning model to learn a semantic pattern based on the intrinsically disordered regions of ~171 known osteogenic proteins. Subsequently, oligopeptides were generated from this semantic pattern based on Monte Carlo simulation, followed by in vivo functional characterization. A five amino acid oligopeptide (AIB5P) had strong bone-formation-promoting effects, as determined in multiple mouse models (e.g., osteoporosis, fracture, and osseointegration of implants). Mechanistically, we showed that AIB5P promotes osteogenesis by binding to the integrin α5 subunit and thereby activating FAK signaling. In summary, we successfully established an oligopeptide discovery strategy based on a DL model and demonstrated its utility from cytological screening to animal experimental verification.

The molecular control of osteoclast formation is still not clearly elucidated. Here, we show that a process of cell recognition mediated by Siglec15-TLR2 binding is indispensable and occurs prior to cell fusion in RANKL-mediated osteoclastogenesis. Siglec15 has been shown to regulate osteoclastic bone resorption. However, the receptor for Siglec15 has not been identified, and the signaling mechanism involving Siglec15 in osteoclast function remains unclear. We found that Siglec15 bound sialylated TLR2 as its receptor and that the binding of sialylated TLR2 to Siglec15 in macrophages committed to the osteoclast-lineage initiated cell fusion for osteoclast formation, in which sialic acid was transferred by the sialyltransferase ST3Gal1. Interestingly, the expression of Siglec15 in macrophages was activated by M-CSF, whereas ST3Gal1 expression was induced by RANKL. Both Siglec15-specific deletion in macrophages and intrafemoral injection of sialidase abrogated cell recognition and reduced subsequent cell fusion for the formation of osteoclasts, resulting in increased bone formation in mice. Thus, our results reveal that cell recognition mediated by the binding of sialylated TLR2 to Siglec15 initiates cell fusion for osteoclast formation.

Senescence impairs preosteoblast expansion and differentiation into functional osteoblasts, blunts their responses to bone formation-stimulating factors and stimulates their secretion of osteoclast-activating factors. Due to these adverse effects, preosteoblast senescence is a crucial target for the treatment of age-related bone loss; however, the underlying mechanism remains unclear. We found that mTORC1 accelerated preosteoblast senescence in vitro and in a mouse model. Mechanistically, mTORC1 induced a change in the membrane potential from polarization to depolarization, thus promoting cell senescence by increasing Ca²⁺ influx and activating downstream NFAT/ATF3/p53 signaling. We further identified the sodium channel Scn1a as a mediator of membrane depolarization in senescent preosteoblasts. Scn1a expression was found to be positively regulated by mTORC1 upstream of C/EBPα, whereas its permeability to Na⁺ was found to be gated by protein kinase A (PKA)-induced phosphorylation. Prosenescent stresses increased the permeability of Scn1a to Na⁺ by suppressing PKA activity and induced depolarization in preosteoblasts. Together, our findings identify a novel pathway involving mTORC1, Scn1a expression and gating, plasma membrane depolarization, increased Ca²⁺ influx and NFAT/ATF3/p53 signaling in the regulation of preosteoblast senescence. Pharmaceutical studies of the related pathways and agents might lead to novel potential treatments for age-related bone loss.

Diabetic osteoporosis (DOP) is the leading complication continuously threatening the bone health of patients with diabetes. A key pathogenic factor in DOP is loss of osteocyte viability. However, the mechanism of osteocyte death remains unclear. Here, we identified ferroptosis, which is iron-dependent programmed cell death, as a critical mechanism of osteocyte death in murine models of DOP. The diabetic microenvironment significantly enhanced osteocyte ferroptosis in vitro, as shown by the substantial lipid peroxidation, iron overload, and aberrant activation of the ferroptosis pathway. RNA sequencing showed that heme oxygenase-1 (HO-1) expression was notably upregulated in ferroptotic osteocytes. Further findings revealed that HO-1 was essential for osteocyte ferroptosis in DOP and that its promoter activity was controlled by the interaction between the upstream NRF2 and c-JUN transcription factors. Targeting ferroptosis or HO-1 efficiently rescued osteocyte death in DOP by disrupting the vicious cycle between lipid peroxidation and HO-1 activation, eventually ameliorating trabecular deterioration. Our study provides insight into DOP pathogenesis, and our results provide a mechanism-based strategy for clinical DOP treatment.

Prostaglandin E2 (PGE2), a major cyclooxygenase-2 (COX-2) product, is highly secreted by the osteoblast lineage in the subchondral bone tissue of osteoarthritis (OA) patients. However, NSAIDs, including COX-2 inhibitors, have severe side effects during OA treatment. Therefore, the identification of novel drug targets of PGE2 signaling in OA progression is urgently needed. Osteoclasts play a critical role in subchondral bone homeostasis and OA-related pain. However, the mechanisms by which PGE2 regulates osteoclast function and subsequently subchondral bone homeostasis are largely unknown. Here, we show that PGE2 acts via EP4 receptors on osteoclasts during the progression of OA and OA-related pain. Our data show that while PGE2 mediates migration and osteoclastogenesis via its EP2 and EP4 receptors, tissue-specific knockout of only the EP4 receptor in osteoclasts (EP4 ^LysM) reduced disease progression and osteophyte formation in a murine model of OA. Furthermore, OA-related pain was alleviated in the EP4 ^LysM mice, with reduced Netrin-1 secretion and CGRP-positive sensory innervation of the subchondral bone. The expression of platelet-derived growth factor-BB (PDGF-BB) was also lower in the EP4 ^LysM mice, which resulted in reduced type H blood vessel formation in subchondral bone. Importantly, we identified a novel potent EP4 antagonist, HL-43, which showed in vitro and in vivo effects consistent with those observed in the EP4 ^LysM mice. Finally, we showed that the Gαs/PI3K/AKT/MAPK signaling pathway is downstream of EP4 activation via PGE2 in osteoclasts. Together, our data demonstrate that PGE2/EP4 signaling in osteoclasts mediates angiogenesis and sensory neuron innervation in subchondral bone, promoting OA progression and pain, and that inhibition of EP4 with HL-43 has therapeutic potential in OA.

Core decompression (CD) with the elimination of osteonecrotic bone is the most common strategy for treating early-stage nontraumatic osteonecrosis of the femoral head (ONFH). Adjuvant treatments are widely used in combination with CD as suitable methods of therapy. Existing augmentations have to be fabricated in advance. Here, we report a novel injectable glycerin-modified polycaprolactone (GPCL) that can adapt to the shape of the CD cavity. GPCL shows great flowability at 52.6 °C. After solidification, its compressive modulus was 120 kPa at body temperature (37 °C). This excellent characteristic enables the polymer to provide mechanical support in vivo. In addition, GPCL acts as a carrier of the therapeutic agent zoledronic acid (ZA), demonstrating sustained release into the CD region. ZA-loaded GPCL was injected into ONFH lesions to treat early-stage nontraumatic cases. Compared to that in the CD group, CD+ZA-loaded GPCL injection preserved bone density and increased the collagen level in the femoral head. At the interface between the GPCL and CD tunnel wall, osteogenesis was significantly promoted. In addition, morphological evaluations revealed that the femoral heads in the CD+ZA-GPCL group exhibited improved pressure resistance. These results suggest a strategy effective to preserve the bone density of the femoral head, thus decreasing the possibility of femoral head collapse. This novel injectable polymer has, therefore, considerable potential in clinical applications.

Periodontal ligament stem cells (PDLSCs) are a key cell type for restoring/regenerating lost/damaged periodontal tissues, including alveolar bone, periodontal ligament and root cementum, the latter of which is important for regaining tooth function. However, PDLSCs residing in an inflammatory environment generally exhibit compromised functions, as demonstrated by an impaired ability to differentiate into cementoblasts, which are responsible for regrowing the cementum. This study investigated the role of mitochondrial function and downstream long noncoding RNAs (lncRNAs) in regulating inflammation-induced changes in the cementogenesis of PDLSCs. We found that the inflammatory cytokine-induced impairment of the cementogenesis of PDLSCs was closely correlated with their mitochondrial function, and lncRNA microarray analysis and gain/loss-of-function studies identified GACAT2 as a regulator of the cellular events involved in inflammation-mediated mitochondrial function and cementogenesis. Subsequently, a comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) and parallel reaction monitoring (PRM) assays revealed that GACAT2 could directly bind to pyruvate kinase M1/2 (PKM1/2), a protein correlated with mitochondrial function. Further functional studies demonstrated that GACAT2 overexpression increased the cellular protein expression of PKM1/2, the PKM2 tetramer and phosphorylated PKM2, which led to enhanced pyruvate kinase (PK) activity and increased translocation of PKM2 into mitochondria. We then found that GACAT2 overexpression could reverse the damage to mitochondrial function and cementoblastic differentiation of PDLSCs induced by inflammation and that this effect could be abolished by PKM1/2 knockdown. Our data indicated that by binding to PKM1/2 proteins, the lncRNA GACAT2 plays a critical role in regulating mitochondrial function and cementogenesis in an inflammatory environment.

Vascular regeneration is a challenging topic in tissue repair. As one of the important components of the neurovascular unit (NVU), pericytes play an essential role in the maintenance of the vascular network of the spinal cord. To date, subtypes of pericytes have been identified by various markers, namely the PDGFR-β, Desmin, CD146, and NG2, each of which is involved with spinal cord injury (SCI) repair. In addition, pericytes may act as a stem cell source that is important for bone development and regeneration, whilst specific subtypes of pericyte could facilitate bone fracture and defect repair. One of the major challenges of pericyte biology is to determine the specific markers that would clearly distinguish the different subtypes of pericytes, and to develop efficient approaches to isolate and propagate pericytes. In this review, we discuss the biology and roles of pericytes, their markers for identification, and cell differentiation capacity with a focus on the potential application in the treatment of SCI and bone diseases in orthopedics.

Osteoporosis caused by aging is characterized by reduced bone mass and accumulated adipocytes in the bone marrow cavity. How the balance between osteoblastogenesis and adipogenesis from bone marrow mesenchymal stem cells (BMSCs) is lost upon aging is still unclear. Here, we found that the RNA-binding protein Musashi2 (Msi2) regulates BMSC lineage commitment. Msi2 is commonly enriched in stem cells and tumor cells. We found that its expression was downregulated during adipogenic differentiation and upregulated during osteogenic differentiation of BMSCs. Msi2 knockout mice exhibited decreased bone mass with substantial accumulation of marrow adipocytes, similar to aging-induced osteoporosis. Depletion of Msi2 in BMSCs led to increased adipocyte commitment. Transcriptional profiling analysis revealed that Msi2 deficiency led to increased PPARγ signaling. RNA-interacting protein immunoprecipitation assays demonstrated that Msi2 could inhibit the translation of the key adipogenic factor Cebpα, thereby inhibiting PPAR signaling. Furthermore, the expression of Msi2 decreased significantly during the aging process of mice, indicating that decreased Msi2 function during aging contributes to abnormal accumulation of adipocytes in bone marrow and osteoporosis. Thus, our results provide a putative biochemical mechanism for aging-related osteoporosis, suggesting that modulating Msi2 function may benefit the treatment of bone aging.

Postmenopausal osteoporosis is a common bone metabolic disorder characterized by deterioration of the bone microarchitecture, leading to an increased risk of fractures. Recently, circular RNAs (circRNAs) have been demonstrated to play pivotal roles in regulating bone metabolism. However, the underlying functions of circRNAs in bone metabolism in postmenopausal osteoporosis remain obscure. Here, we report that circStag1 is a critical osteoporosis-related circRNA that shows significantly downregulated expression in osteoporotic bone marrow mesenchymal stem cells (BMSCs) and clinical bone tissue samples from patients with osteoporosis. Overexpression of circStag1 significantly promoted the osteogenic capability of BMSCs. Mechanistically, we found that circStag1 interacts with human antigen R (HuR), an RNA-binding protein, and promotes the translocation of HuR into the cytoplasm. A high cytoplasmic level of HuR led to the activation of the Wnt signaling pathway by stabilizing and enhancing low-density lipoprotein receptor-related protein 5/6 (Lrp5/6) and β-catenin expression, thereby stimulating the osteogenic differentiation of BMSCs. Furthermore, overexpression of circStag1 in vivo by circStag1-loaded adeno-associated virus (circStag1-AAV) promoted new bone formation, thereby preventing bone loss in ovariectomized rats. Collectively, we show that circStag1 plays a pivotal role in promoting the regeneration of bone tissue via HuR/Wnt signaling, which may provide new strategies to prevent bone metabolic disorders such as postmenopausal osteoporosis.

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Identification of regulators of osteoblastogenesis that can be pharmacologically targeted is a major goal in combating osteoporosis, a common disease of the elderly population. Here, unbiased kinome RNAi screening in primary murine osteoblasts identified cyclin-dependent kinase 5 (Cdk5) as a suppressor of osteoblast differentiation in both murine and human preosteoblastic cells. Cdk5 knockdown by siRNA, genetic deletion using the Cre-loxP system, or inhibition with the small molecule roscovitine enhanced osteoblastogenesis in vitro. Roscovitine treatment significantly enhanced bone mass by increasing osteoblastogenesis and improved fracture healing in mice. Mechanistically, downregulation of Cdk5 expression increased Erk phosphorylation, resulting in enhanced osteoblast-specific gene expression. Notably, simultaneous Cdk5 and Erk depletion abrogated the osteoblastogenesis conferred by Cdk5 depletion alone, suggesting that Cdk5 regulates osteoblast differentiation through MAPK pathway modulation. We conclude that Cdk5 is a potential therapeutic target to treat osteoporosis and improve fracture healing.

Osteoporosis is a systemic bone disease that affects more than 200 million people worldwide and is caused by the disruption of the equilibrium between osteoclastic bone resorption and osteoblastic bone formation. Sphingosine-1-phosphate (S1P) is a natural, bioactive sphingolipid that has been shown to play a major role in cardiovascular and immunological pathologies by regulating biological and cellular processes, including migration, differentiation, proliferation and survival. Recent studies also suggest a central role for S1P in bone diseases, including osteoporosis; however, the effects of S1P, particularly in bone metabolism, remain to be further elucidated. In this review, we summarize the available literature on the role of S1P in bone metabolism with a focus on osteoporosis. On the cellular level, S1P acts as an osteoclast-osteoblast coupling factor to promote osteoblast proliferation and bone formation. Moreover, the recruitment of osteoclast precursors to resorption sites is regulated by the interplay of S1P gradients and S1P receptor expression. From a clinical perspective, increasing evidence suggests that systemically elevated S1P blood levels may serve as an independent risk factor for osteoporosis-related fractures. Taken together, S1P signaling is a potential therapeutic target and may serve as a novel biomarker in patients with systemic bone disease.

Spinal cord injury (SCI) leads to loss of motor and sensory function below the injury level and imposes a considerable burden on patients, families, and society. Repair of the injured spinal cord has been recognized as a global medical challenge for many years. Significant progress has been made in research on the pathological mechanism of spinal cord injury. In particular, with the development of gene regulation, cell sequencing, and cell tracing technologies, in-depth explorations of the SCI microenvironment have become more feasible. However, translational studies related to repair of the injured spinal cord have not yielded significant results. This review summarizes the latest research progress on two aspects of SCI pathology: intraneuronal microenvironment imbalance and regenerative microenvironment imbalance. We also review repair strategies for the injured spinal cord based on microenvironment imbalance, including medications, cell transplantation, exosomes, tissue engineering, cell reprogramming, and rehabilitation. The current state of translational research on SCI and future directions are also discussed. The development of a combined, precise, and multitemporal strategy for repairing the injured spinal cord is a potential future direction.

Elevated osteoclast (OC) activity is a major contributor to inflammatory bone loss (IBL) during chronic inflammatory diseases. However, the specific OC precursors (OCPs) responding to inflammatory cues and the underlying mechanisms leading to IBL are poorly understood. We identified two distinct OCP subsets: Ly6C^hiCD11b^hi inflammatory OCPs (iOCPs) induced during chronic inflammation, and homeostatic Ly6C^hiCD11b^lo OCPs (hOCPs) which remained unchanged. Functional and proteomic characterization revealed that while iOCPs were rare and displayed low osteoclastogenic potential under normal conditions, they expanded during chronic inflammation and generated OCs with enhanced activity. In contrast, hOCPs were abundant and manifested high osteoclastogenic potential under normal conditions but generated OCs with low activity and were unresponsive to the inflammatory environment. Osteoclasts derived from iOCPs expressed higher levels of resorptive and metabolic proteins than those generated from hOCPs, highlighting that different osteoclast populations are formed by distinct precursors. We further identified the TNF-α and S100A8/A9 proteins as key regulators that control the iOCP response during chronic inflammation. Furthermore, we demonstrated that the response of iOCPs but not that of hOCPs was abrogated in tnf-α ^−/− mice, in correlation with attenuated IBL. Our findings suggest a central role for iOCPs in IBL induction. iOCPs can serve as potential biomarkers for IBL detection and possibly as new therapeutic targets to combat IBL in a wide range of inflammatory conditions.

Spondylocarpotarsal syndrome (SCT) is a rare musculoskeletal disorder characterized by short stature and vertebral, carpal, and tarsal fusions resulting from biallelic nonsense mutations in the gene encoding filamin B (FLNB). Utilizing a FLNB knockout mouse, we showed that the vertebral fusions in SCT evolved from intervertebral disc (IVD) degeneration and ossification of the annulus fibrosus (AF), eventually leading to full trabecular bone formation. This resulted from alterations in the TGFβ/BMP signaling pathway that included increased canonical TGFβ and noncanonical BMP signaling. In this study, the role of FLNB in the TGFβ/BMP pathway was elucidated using in vitro, in vivo, and ex vivo treatment methodologies. The data demonstrated that FLNB interacts with inhibitory Smads 6 and 7 (i-Smads) to regulate TGFβ/BMP signaling and that loss of FLNB produces increased TGFβ receptor activity and decreased Smad 1 ubiquitination. Through the use of small molecule inhibitors in an ex vivo spine model, TGFβ/BMP signaling was modulated to design a targeted treatment for SCT and disc degeneration. Inhibition of canonical and noncanonical TGFβ/BMP pathway activity restored Flnb ^−/− IVD morphology. These most effective improvements resulted from specific inhibition of TGFβ and p38 signaling activation. FLNB acts as a bridge for TGFβ/BMP signaling crosstalk through i-Smads and is key for the critical balance in TGFβ/BMP signaling that maintains the IVD. These findings further our understanding of IVD biology and reveal new molecular targets for disc degeneration as well as congenital vertebral fusion disorders.

Articular cartilage damage is a universal health problem. Despite recent progress, chondrocyte dedifferentiation has severely compromised the clinical outcomes of cell-based cartilage regeneration. Loss-of-function changes are frequently observed in chondrocyte expansion and other pathological conditions, but the characteristics and intermediate molecular mechanisms remain unclear. In this study, we demonstrate a time-lapse atlas of chondrocyte dedifferentiation to provide molecular details and informative biomarkers associated with clinical chondrocyte evaluation. We performed various assays, such as single-cell RNA sequencing (scRNA-seq), live-cell metabolic assays, and assays for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), to develop a biphasic dedifferentiation model consisting of early and late dedifferentiation stages. Early-stage chondrocytes exhibited a glycolytic phenotype with increased expression of genes involved in metabolism and antioxidation, whereas late-stage chondrocytes exhibited ultrastructural changes involving mitochondrial damage and stress-associated chromatin remodeling. Using the chemical inhibitor BTB06584, we revealed that early and late dedifferentiated chondrocytes possessed distinct recovery potentials from functional phenotype loss. Notably, this two-stage transition was also validated in human chondrocytes. An image-based approach was established for clinical use to efficiently predict chondrocyte plasticity using stage-specific biomarkers. Overall, this study lays a foundation to improve the quality of chondrocytes in clinical use and provides deep insights into chondrocyte dedifferentiation.

The physicochemical nature of DNA allows the assembly of highly predictable structures via several fabrication strategies, which have been applied to make breakthroughs in various fields. Moreover, DNA nanostructures are regarded as materials with excellent editability and biocompatibility for biomedical applications. The ongoing maintenance and release of new DNA structure design tools ease the work and make large and arbitrary DNA structures feasible for different applications. However, the nature of DNA nanostructures endows them with several stimulus-responsive mechanisms capable of responding to biomolecules, such as nucleic acids and proteins, as well as biophysical environmental parameters, such as temperature and pH. Via these mechanisms, stimulus-responsive dynamic DNA nanostructures have been applied in several biomedical settings, including basic research, active drug delivery, biosensor development, and tissue engineering. These applications have shown the versatility of dynamic DNA nanostructures, with unignorable merits that exceed those of their traditional counterparts, such as polymers and metal particles. However, there are stability, yield, exogenous DNA, and ethical considerations regarding their clinical translation. In this review, we first introduce the recent efforts and discoveries in DNA nanotechnology, highlighting the uses of dynamic DNA nanostructures in biomedical applications. Then, several dynamic DNA nanostructures are presented, and their typical biomedical applications, including their use as DNA aptamers, ion concentration/pH-sensitive DNA molecules, DNA nanostructures capable of strand displacement reactions, and protein-based dynamic DNA nanostructures, are discussed. Finally, the challenges regarding the biomedical applications of dynamic DNA nanostructures are discussed.

Osteoarthritis (OA) in China is gradually becoming an important scientific research area that has had a significant impact on research and development (R&D) activities in the OA field worldwide. This article summarizes the R&D progress related to OA in China in recent years. The National Natural Science Foundation of China (NSFC) is a national funding institution for basic research and plays a critical role in promoting and supporting Chinese scholars’ R&D activities. We collected and analyzed information on NSFC funding in the field of OA from 2010 to 2019, including the amount, the level and the program categories of the funded projects. The data fully demonstrate the important and positive role of the NSFC in supporting free exploration, cultivating research teams and young talent, and boosting OA R&D. In this article, we outline and discuss hot topics in focused areas, key advances in this field and the prospects for progress in OA research in China.

Toll-like receptors (TLRs) play pivotal roles in inflammation and provide important links between the immune and skeletal systems. Although the activation of TLRs may affect osteoclast differentiation and bone metabolism, whether and how TLRs are required for normal bone remodeling remains to be fully explored. In the current study, we show for the first time that TLR9^−/− mice exhibit a low bone mass and low-grade systemic chronic inflammation, which is characterized by the expansion of CD4⁺ T cells and increased levels of inflammatory cytokines, including TNFα, RANKL, and IL1β. The increased levels of these cytokines significantly promote osteoclastogenesis and induce bone loss. Importantly, TLR9 deletion alters the gut microbiota, and this dysbiosis is the basis of the systemic inflammation and bone loss observed in TLR9^−/− mice. Furthermore, through single-cell RNA sequencing, we identified myeloid-biased hematopoiesis in the bone marrow of TLR9^−/− mice and determined that the increase in myelopoiesis, likely caused by the adaptation of hematopoietic stem cells to systemic inflammation, also contributes to inflammation-induced osteoclastogenesis and subsequent bone loss in TLR9^−/− mice. Thus, our study provides novel evidence that TLR9 signaling connects the gut microbiota, immune system, and bone and is critical in maintaining the homeostasis of inflammation, hematopoiesis, and bone metabolism under normal conditions.

The functional interdependence of nerves and blood vessels is a well-established concept during tissue morphogenesis, yet the role of neurovascular coupling in proper and aberrant tissue repair is an emerging field of interest. Here, we sought to define the regulatory relationship of peripheral nerves on vasculature in a severe extremity trauma model in mice, which results in aberrant cell fate and heterotopic ossification (HO). First, a high spatial degree of neurovascular congruency was observed to exist within extremity injury associated heterotopic ossification. Vascular and perivascular cells demonstrate characteristic responses to injury, as assessed by single cell RNA sequencing. This vascular response to injury was blunted in neurectomized mice, including a decrease in endothelial proliferation and type H vessel formation, and a downregulation of key transcriptional networks associated with angiogenesis. Independent mechanisms to chemically or genetically inhibit axonal ingrowth led to similar deficits in HO site angiogenesis, a reduction in type H vessels, and heterotopic bone formation. Finally, a combination of single cell transcriptomic approaches within the dorsal root ganglia identified key neural-derived angiogenic paracrine factors that may mediate neuron-to-vascular signaling in HO. These data provide further understanding of nerve-to-vessel crosstalk in traumatized soft tissues, which may reflect a key determinant of mesenchymal progenitor cell fate after injury.

The field of research on pain originating from various bone diseases is expanding rapidly, with new mechanisms and targets asserting both peripheral and central sites of action. The scope of research is broadening from bone biology to neuroscience, neuroendocrinology, and immunology. In particular, the roles of primary sensory neurons and non-neuronal cells in the peripheral tissues as important targets for bone pain treatment are under extensive investigation in both pre-clinical and clinical settings. An understanding of the peripheral mechanisms underlying pain conditions associated with various bone diseases will aid in the appropriate application and development of optimal strategies for not only managing bone pain symptoms but also improving bone repairing and remodeling, which potentially cures the underlying etiology for long-term functional recovery. In this review, we focus on advances in important preclinical studies of significant bone pain conditions in the past 5 years that indicated new peripheral neuronal and non-neuronal mechanisms, novel targets for potential clinical interventions, and future directions of research.

Increasing evidence shows that adipokines play a vital role in the development of rheumatoid arthritis (RA). Fatty acid-binding protein 4 (FABP4), a novel adipokine that regulates inflammation and angiogenesis, has been extensively studied in a variety of organs and diseases. However, the effect of FABP4 on RA remains unclear. Here, we found that FABP4 expression was upregulated in synovial M1-polarized macrophages in RA. The increase in FABP4 promoted synovitis, angiogenesis, and cartilage degradation to exacerbate RA progression in vivo and in vitro, whereas BMS309403 (a FABP4 inhibitor) and anagliptin (dipeptidyl peptidase 4 inhibitor) inhibited FABP4 expression in serum and synovial M1-polarized macrophages in mice to alleviate RA progression. Further studies showed that constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) by TSC1 deletion specifically in the myeloid lineage regulated FABP4 expression in macrophages to exacerbate RA progression in mice. In contrast, inhibition of mTORC1 by ras homolog enriched in brain (Rheb1) disruption specifically in the myeloid lineage reduced FABP4 expression in macrophages to attenuate RA development in mice. Our findings established an essential role of FABP4 that is secreted by M1-polarized macrophages in synovitis, angiogenesis, and cartilage degradation in RA. BMS309403 and anagliptin inhibited FABP4 expression in synovial M1-polarized macrophages to alleviate RA development. Hence, FABP4 may represent a potential target for RA therapy.

Type II collagen-positive (Col2⁺) cells have been reported as skeletal stem cells (SSCs), but the contribution of Col2⁺ progenitors to skeletal development both prenatally and postnatally during aging remains unclear. To address this question, we generated new mouse models with ablation of Col2⁺ cells at either the embryonic or postnatal stages. The embryonic ablation of Col2⁺ progenitors resulted in the death of newborn mice due to a decrease in skeletal blood vessels, loss of all vertebral bones and absence of most other bones except part of the craniofacial bone, the clavicle bone and a small piece of the long bone and ribs, which suggested that intramembranous ossification is involved in long bone development but does not participate in spine development. The postnatal ablation of Col2⁺ cells resulted in mouse growth retardation and a collagenopathy phenotype. Lineage tracing experiments with embryonic or postnatal mice revealed that Col2⁺ progenitors occurred predominantly in the growth plate (GP) and articular cartilage, but a limited number of Col2⁺ cells were detected in the bone marrow. Moreover, the number and differentiation ability of Col2⁺ progenitors in the long bone and knee joints decreased with increasing age. The fate-mapping study further revealed Col2⁺ lineage cells contributed to, in addition to osteoblasts and chondrocytes, CD31⁺ blood vessels in both the calvarial bone and long bone. Specifically, almost all blood vessels in calvarial bone and 25.4% of blood vessels in long bone were Col2⁺ lineage cells. However, during fracture healing, 95.5% of CD31⁺ blood vessels in long bone were Col2⁺ lineage cells. In vitro studies further confirmed that Col2⁺ progenitors from calvarial bone and GP could form CD31⁺ vascular lumens. Thus, this study provides the first demonstration that intramembranous ossification is involved in long bone and rib development but not spine development. Col2⁺ progenitors contribute to CD31⁺ skeletal blood vessel formation, but the percentage differs between long bone and skull bone. The number and differentiation ability of Col2⁺ progenitors decreases with increasing age.

Bone remodeling replaces old and damaged bone with new bone through a sequence of cellular events occurring on the same surface without any change in bone shape. It was initially thought that the basic multicellular unit (BMU) responsible for bone remodeling consists of osteoclasts and osteoblasts functioning through a hierarchical sequence of events organized into distinct stages. However, recent discoveries have indicated that all bone cells participate in BMU formation by interacting both simultaneously and at different differentiation stages with their progenitors, other cells, and bone matrix constituents. Therefore, bone remodeling is currently considered a physiological outcome of continuous cellular operational processes optimized to confer a survival advantage. Bone remodeling defines the primary activities that BMUs need to perform to renew successfully bone structural units. Hence, this review summarizes the current understanding of bone remodeling and future research directions with the aim of providing a clinically relevant biological background with which to identify targets for therapeutic strategies in osteoporosis.

Physical mechanical stimulation can maintain and even increase bone mass. Here, we report an important role of osteocytic integrin α5 in regulating the anabolic response of bone to mechanical loading using an Itga5 conditional gene knockout (cKO) mouse model. Integrin α5 gene deletion increased apoptotic osteocytes and reduced cortical anabolic responses to tibial compression including decreased endosteal osteoblasts and bone formation, and increased endosteal osteoclasts and bone resorption, contributing to the decreased bone area fraction and biomechanical properties, leading to an enlarged bone marrow area in cKO mice. Similar disruption of anabolic responses to mechanical loading was also detected in cKO trabecular bone. Moreover, integrin α5 deficiency impeded load-induced Cx43 hemichannel opening, and production and release of PGE2, an anabolic factor, resulting in attenuated effects of the loading on catabolic sclerostin (SOST) reduction and anabolic β-catenin increase. Together, this study shows an indispensable role of integrin α5 in osteocytes in the anabolic action of mechanical loading on skeletal tissue through activation of hemichannels and PGE2-evoked gene expression. Integrin α5 could act as a potential new therapeutic target for bone loss, especially in the elderly population with impeded mechanical sensitivity.

The Gsα/cAMP signaling pathway mediates the effect of a variety of hormones and factors that regulate the homeostasis of the post-natal skeleton. Hence, the dysregulated activity of Gsα due to gain-of-function mutations (R201C/R201H) results in severe architectural and functional derangements of the entire bone/bone marrow organ. While the consequences of gain-of-function mutations of Gsα have been extensively investigated in osteoblasts and in bone marrow osteoprogenitor cells at various differentiation stages, their effect in adipogenically-committed bone marrow stromal cells has remained unaddressed. We generated a mouse model with expression of Gsα ^R201C driven by the Adiponectin (Adq) promoter. Adq-Gsα ^R201C mice developed a complex combination of metaphyseal, diaphyseal and cortical bone changes. In the metaphysis, Gsα ^R201C caused an early phase of bone resorption followed by bone deposition. Metaphyseal bone formation was sustained by cells that were traced by Adq-Cre and eventually resulted in a high trabecular bone mass phenotype. In the diaphysis, Gsα ^R201C, in combination with estrogen, triggered the osteogenic activity of Adq-Cre-targeted perivascular bone marrow stromal cells leading to intramedullary bone formation. Finally, consistent with the previously unnoticed presence of Adq-Cre-marked pericytes in intraosseous blood vessels, Gsα ^R201C caused the development of a lytic phenotype that affected both cortical (increased porosity) and trabecular (tunneling resorption) bone. These results provide the first evidence that the Adq-cell network in the skeleton not only regulates bone resorption but also contributes to bone formation, and that the Gsα/cAMP pathway is a major modulator of both functions.