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In recent years, researchers have become focused on the relationship between lipids and bone metabolism balance. Moreover, many diseases related to lipid metabolism disorders, such as nonalcoholic fatty liver disease, atherosclerosis, obesity, and menopause, are associated with osteoporotic phenotypes. It has been clinically observed in humans that these lipid metabolism disorders promote changes in osteoporosis-related indicators bone mineral density and bone mass. Furthermore, similar osteoporotic phenotype changes were observed in high-fat and high-cholesterol-induced animal models. Abnormal lipid metabolism (such as increased oxidized lipids and elevated plasma cholesterol) affects bone microenvironment homeostasis via cross-organ communication, promoting differentiation of mesenchymal stem cells to adipocytes, and inhibiting commitment towards osteoblasts. Moreover, disturbances in lipid metabolism affect the bone metabolism balance by promoting the secretion of cytokines such as receptor activator of nuclear factor-kappa B ligand by osteoblasts and stimulating the differentiation of osteoclasts. Conclusively, this review addresses the possible link between lipid metabolism disorders and osteoporosis and elucidates the potential modulatory mechanisms and signaling pathways by which lipid metabolism affects bone metabolism balance. We also summarize the possible approaches and prospects of intervening lipid metabolism for osteoporosis treatment.

Background: Osteoarthritis (OA) is a chronic degenerative joint disease that primarily affects middle-aged and elderly individuals. The decline in chondrocyte function plays a crucial role in the development of OA. Inflammasome-mediated chondrocyte pyroptosis is implicated in matrix degradation and cartilage degeneration in OA patients. Guanylate binding protein 5 (GBP5), a member of the GTPase family induced by Interferon-γ (IFN-γ), significantly influences cellular inflammatory responses, including intracellular inflammasome activation and cytokine release. However, the role of GBP5 in chondrocyte pyroptosis and OA progression remains unclear. Methods: In this study, we used tumor necrosis factor-α (TNF-α) to induce inflammation and created an OA mouse model with surgically-induced destabilization of the medial meniscus (DMM). We isolated and cultured primary chondrocytes from the knee joints of suckling C57 mice. TNF-α-stimulated primary chondrocytes served as an in vitro model for OA and underwent RNA sequencing. Chondrocytes were transfected with GBP5-overexpression plasmids and small interfering RNA and were subsequently treated with TNF-α. We assessed the expression of cartilage matrix components (COL2A1 and aggrecan), catabolic factors (MMP9 and MMP13), and NLRP3 inflammasome pathway genes (NLRP3, Caspase1, GSDMD, Pro-IL-1β, and Pro-Caspase1) using RT-qPCR and Western blotting. We analyzed the expression of GBP5, NLRP3, and Caspase1 in the cartilage of DMM-induced post-traumatic OA mice and human OA patients. Immunohistochemistry (IHC) was used to detect the expression of GBP5, NLRP3 and GSDMD in cartilage specimens from OA patients and mouse DMM models. Chondrocyte pyroptosis was assessed using flow cytometry, and the levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) were measured with ELISA. We conducted double luciferase reporter gene and chromatin immunoprecipitation (ChIP) assays to confirm the relationship between IRF1 and GBP5. Results: GBP5 expression increased in TNF-α-induced chondrocytes, as revealed by RNA sequencing. GBP5 inhibited COL2A1 and aggrecan expression while promoting the expression of MMP9, MMP13, NLRP3, Caspase1, GSDMD, Pro-IL-1β, and Pro-Caspase1. GBP5 expression also increased in the cartilage of DMM-induced post-traumatic OA mice and human OA patients. Knockout of GBP5 reduced chondrocyte injury in OA mice. GBP5 promoted chondrocyte pyroptosis and the production of IL-1β and IL-18. Additionally, we found that IRF1 bound to the promoter region of GBP5, enhancing its expression. After co-transfected with ad-IRF1 and siGBP5, the expression of pyroptosis-related genes was significantly decreased compared with ad-IRF1 group. Conclusions: The IRF1/GBP5 axis enhances extracellular matrix (ECM) degradation and promotes pyroptosis during OA development, through the NLRP3 inflammasome signaling pathway. The translational potential of this article: This study underscores the significance of the IRF1/GBP5 axis in NLRP3 inflammasome-mediated chondrocyte pyroptosis and osteoarthritic chondrocyte injury. Modulating IRF1 and GBP5 expression could serve as a novel therapeutic target for OA.

Abstract Background The N6‐methyladenosine (m6A) dynamics in the progression of intervertebral disc (IVD) aging remain largely unknown. This study aimed to explore the distribution and pattern of m6A modification in nucleus pulpous (NP) tissues of rats at different ages. Methods Histological staining and MRI were performed to evaluate the degeneration of IVD. The expression of m6A modifiers was analyzed using qRT‐PCR and western blot. Subsequently, methylated RNA immunoprecipitation next generation sequencing and RNA‐seq were conducted to identify differences in m6A methylome and transcriptome of NP tissues. Results Compared to 2‐month‐old rats, we found significant changes in the global m6A level and the expression of Mettl3 and FTO in NP tissues from 20‐month‐old rats. During the progression of NP aging, there were 1126 persistently differentially m6A peaks within 931 genes, and 51 persistently differentially expressed genes. GO and KEGG analyses showed that these m6A peaks and m6A modified genes were mainly engaged in the biological processes and pathways of intervertebral disc degermation (IDD), such as extracellular matrix metabolism, angiogenesis, inflammatory response, mTOR and AMPK signaling pathways. Meanwhile, conjoint analyses and Venn diagram revealed a total of 405 aging related genes contained significant methylation and expression levels in 20‐month‐old rats in contrast to 2‐month‐old and 10‐month‐old rats. Moreover, it was found that four aging related genes with hypermethylated modification including BUB1, CA12, Adamts1, and Adamts4 depicted differentially expressed at protein level, of which BUB1 and CA12 were decreased, while Adamts1 and Adamts4 were increased during the progression of NP aging. Conclusion Collectively, this study elucidated the distribution and pattern of m6A modification during the aging of IVD. Furthermore, the m6A modified genes were involved in the IDD related biological processes and pathways. These findings may provide novel insights into the mechanisms and therapies of IDD from the perspective of aging.

More than 50% of prostate cancer (PCa) patients have bone metastasis with osteoblastic lesions. MiR-18a-5p is associated with the development and metastasis of PCa, but it remains unclear whether it is involved in osteoblastic lesions. We first found that miR-18a-5p was highly expressed in the bone microenvironment of patients with PCa bone metastases. To address how miR-18a-5p affects PCa osteoblastic lesions, antagonizing miR-18a-5p in PCa cells or pre-osteoblasts inhibited osteoblast differentiation in vitro. Moreover, injection of PCa cells with miR-18a-5p inhibition improved bone biomechanical properties and bone mineral mass in vivo. Furthermore, miR-18a-5p was transferred to osteoblasts by exosomes derived from PCa cells and targeted the Hist1h2bc gene, resulting in Ctnnb1 up-regulation in the Wnt/β-catenin signaling pathway. Translationally, antagomir-18a-5p significantly improved bone biomechanical properties and alleviated sclerotic lesions from osteoblastic metastases in BALB/c nude mice. These data suggest that inhibition of exosome-delivered miR-18a-5p ameliorates PCa-induced osteoblastic lesions.

The anterior cruciate ligament plays an important role in maintaining the stability of the knee joint. Its injury is a common cause of articular cartilage degeneration and osteoarthritis (OA). The anterior cruciate ligament transection (ACLT) method is commonly employed to construct animal models for studying osteoarthritis pathogenesis. However, the precise mechanism of how anterior cruciate ligament injury leads to osteoarthritis is not fully understood. This study utilized finite element analysis (FEA) with human medical images to simulate the biomechanical characteristics of anterior cruciate ligament (ACL) injury. Osteoarthritis models were subsequently established in C57BL/6 mice using ACLT to explore the link between ACL injury and osteoarthritis development. The results of FEA showed that, after an anterior cruciate ligament injury, abnormal stress was concentrated in the medial and lateral of the femoral and tibial articular cartilage during knee flexion and extension. In order to better display the pathological changes of articular cartilage in the stress areas, the medial tibial cartilage was selected as a representative area to observe the continuous pathological changes of articular cartilage in ACLT-induced OA mice. The articular cartilage degeneration was most dramatic at four weeks post ACLT operation and then remained relatively stable. This study may have significant implications for the development of animal models of osteoarthritis and provide a reference for histopathological research on osteoarthritis.

Reactive oxygen burst in articular chondrocytes is a major contributor to osteoarthritis progression. Although selenium is indispensable role in the antioxidant process, the narrow therapeutic window, delicate toxicity margins, and lack of an efficient delivery system have hindered its translation to clinical applications. Herein, transcriptomic and biochemical analyses revealed that osteoarthritis was associated with selenium metabolic abnormality. A novel injectable hydrogel to deliver selenium nanoparticles (SeNPs) was proposed to intervene selenoprotein expression for osteoarthritis treatment. The hydrogels based on oxidized hyaluronic acid (OHA) cross-linked with hyaluronic acid-adipic acid dihydrazide (HA-ADH) was formulated to load SeNPs through a Schiff base reaction. The hydrogels were further incorporated with SeNPs, which exhibited minimal toxicity, mechanical properties, self-healing capability, and sustained drug release. Encapsulated with SeNPs, the hydrogels facilitated cartilage repair through synergetic effects of scavenging reactive oxygen species (ROS) and depressing apoptosis. Mechanistically, the hydrogel restored redox homeostasis by targeting glutathione peroxidase-1 (GPX1). Therapeutic outcomes of the SeNPs-laden hydrogel were demonstrated in an osteoarthritis rat model created by destabilization of the medial meniscus, including cartilage protection, subchondral bone sclerosis improvement, inflammation attenuation, and pain relief were demonstrated. These results highlight therapeutic potential of OHA/HA-ADH@SeNPs hydrogels, providing fundamental insights into remedying selenium imbalance for osteoarthritis biomaterial development.

Cervidae represents a family that is not only rich in species diversity but also exhibits a wide range of karyotypes. The controversies regarding the phylogeny and classification of Cervidae still persist. The flourishing development of the genomic era has made it possible to address these issues at the genomic level. Here, the genomes of nine species were used to explore the phylogeny and chromosomal evolutionary events of Cervidae. By conducting whole-genome comparisons, we identified single-copy orthologous genes across the nine species and constructed a phylogenetic tree based on the single-copy orthologous genes sequences, providing new insights into the phylogeny of Cervidae, particularly the phylogenetic relationship among sika deer, red deer, wapiti and Tarim red deer. Gene family analysis revealed contractions in the olfactory receptor gene family and expansions in the histone gene family across eight Cervidae species. Furthermore, synteny analysis was used to explore the chromosomal evolutionary events of Cervidae species, revealing six chromosomal fissions during the evolutionary process from Bovidae to Cervidae. Notably, specific chromosomal fusion events were found in four species of Cervus, and a unique chromosomal fusion event was identified in Muntiacus reevesi. Our study further completed the phylogenetic relationship within the Cervidae and demonstrated the feasibility of inferring species phylogeny at the whole-genome level. Additionally, our findings on gene family evolution and the chromosomal evolutionary events in eight Cervidae species lay a foundation for comprehensive research of the evolution of Cervidae.

Endochondral bone formation is an important route for bone repair. Although emerging evidence has revealed the functions of long non-coding RNAs (lncRNAs) in bone and cartilage development, the effect of lncRNAs in endochondral bone repair is still largely unknown. Here, we identified a lncRNA, named Hypertrophic Chondrocyte Angiogenesis-related lncRNA (HCAR), and proved it to promote the endochondral bone repair by upregulating the expression of matrix metallopeptidase 13 (Mmp13) and vascular endothelial growth factor α (Vegfa) in hypertrophic chondrocytes. Lnc-HCAR knockdown in hypertrophic chondrocytes restrained the cartilage matrix remodeling and decrease the CD31hiEmcnhi vessels number in a bone repair model. Mechanistically, we proved that lnc-HCAR was mainly enriched in the cytoplasm using fluorescence in situ hybridization (FISH) assay, and it acted as a molecular sponge for miR-15b-5p. Further, in hypertrophic chondrocytes, lnc-HCAR competitively bound to miR-15b-5p to increase Vegfa and Mmp13 expression. Our results proved that lncRNA is deeply involved in endochondral bone repair, which will provide a new theoretical basis for future strategies for promoting fracture healing.

Antiresorptive drugs are widely used for treatment of osteoporosis and cancer bone metastasis, which function mainly through an overall inhibition of osteoclast. However, not all osteoclasts are “bone eaters”; preosteoclasts (pOCs) play anabolic roles in bone formation and angiogenesis through coupling with osteoblasts and secreting platelet derived growth factor-BB (PDGF-BB). In this study, a bone-targeted pH-responsive nanomaterial was designed for selectively eliminating mature osteoclasts (mOCs) without affecting pOCs. Biocompatible cerium nano-system (CNS) was guided to the acidic extracellular microenvironment created by mOCs and gained oxidative enzymatic activity. Oxidative CNS decreased the viability of mOCs through accumulating intracellular reactive oxygen species and enhancing calcium oscillation. Non-acid secreting anabolic pOCs were thus preserved and kept producing PDGF-BB, which lead to mesenchymal stem cell osteogenesis and endothelial progenitor cell angiogenesis via PI3K-Akt activated focal adhesion kinase. In treating osteoporotic ovariectomized mice, CNS showed better protective effects compare with the current first line antiresorptive drug due to the better anabolic effects marked by higher level of bone formation and vascularization. We provided a novel anabolic therapeutic strategy in treating bone disorders with excessive bone resorption.

Osteoporosis is caused by an osteoclast activation mechanism. People suffering from osteoporosis are prone to bone defects. Increasing evidence indicates that scavenging reactive oxygen species (ROS) can inhibit receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis and suppress ovariectomy-induced osteoporosis. It is critical to develop biomaterials with antioxidant properties to modulate osteoclast activity for treating osteoporotic bone defects. Previous studies have shown that manganese (Mn) can improve bone regeneration, and Mn supplementation may treat osteoporosis. However, the effect of Mn on osteoclasts and the role of Mn in osteoporotic bone defects remain unclear. In present research, a model bioceramic, Mn-contained β-tricalcium phosphate (Mn-TCP) was prepared by introducing Mn into β-TCP. The introduction of Mn into β-TCP significantly improved the scavenging of oxygen radicals and nitrogen radicals, demonstrating that Mn-TCP bioceramics might have antioxidant properties. The in vitro and in vivo findings revealed that Mn2+ ions released from Mn-TCP bioceramics could distinctly inhibit the formation and function of osteoclasts, promote the differentiation of osteoblasts, and accelerate bone regeneration under osteoporotic conditions in vivo. Mechanistically, Mn-TCP bioceramics inhibited osteoclastogenesis and promoted the regeneration of osteoporotic bone defects by scavenging ROS via Nrf2 activation. These results suggest that Mn-containing bioceramics with osteoconductivity, ROS scavenging and bone resorption inhibition abilities may be an ideal biomaterial for the treatment of osteoporotic bone defect.

Osteoporotic fractures, also known as fragility fractures, are prevalent in the elderly and bring tremendous social burdens. Poor bone quality, weak repair capacity, instability, and high failure rate of internal fixation are main characteristics of osteoporotic fractures. Osteoporotic bone defects are common and need to be repaired by appropriate materials. Proximal humerus, distal radius, tibia plateau, calcaneus, and spine are common osteoporotic fractures with bone defect. Here, the consensus from the Osteoporosis Group of Chinese Orthopaedic Association concentrates on the epidemiology, characters, and management strategies of common osteoporotic fractures with bone defect to standardize clinical practice in bone repair of osteoporotic fractures.

A close relationship has been reported to exist between cadherin-mediated cell–cell adhesion and integrin-mediated cell mobility, and protein tyrosine phosphatase 1B (PTP1B) may be involved in maintaining this homeostasis. The stable residence of mesenchymal stem cells (MSCs) and endothelial cells (ECs) in their niches is closely related to the regulation of PTP1B. However, the exact role of the departure of MSCs and ECs from their niches during bone regeneration is largely unknown. Here, we show that the phosphorylation state of PTP1B tyrosine-152 (Y152) plays a central role in initiating the departure of these cells from their niches and their subsequent recruitment to bone defects. Based on our previous design of a PTP1B Y152 region-mimicking peptide (152RM) that significantly inhibits the phosphorylation of PTP1B Y152, further investigations revealed that 152RM enhanced cell migration partly via integrin αvβ3 and promoted MSCs osteogenic differentiation partly by inhibiting ATF3. Moreover, 152RM induced type H vessels formation by activating Notch signaling. Demineralized bone matrix (DBM) scaffolds were fabricated with mesoporous silica nanoparticles (MSNs), and 152RM was then loaded onto them by electrostatic adsorption. The DBM-MSN/152RM scaffolds were demonstrated to induce bone formation and type H vessels expansion in vivo. In conclusion, our data reveal that 152RM contributes to bone formation by coupling osteogenesis with angiogenesis, which may offer a potential therapeutic strategy for bone defects.

The bone is previously considered as a dominant organ involved in the processes of locomotion. However, in the past two decades, a large number of studies have suggested that the skeletal system closely coordinated with the immune system so as to result in the emerging area of ‘osteoimmunology’. In the evolution of many kinds of bone destruction-related diseases, osteoclasts could differentiate from dendritic cells, which contributed to increased expression of osteoclast-related membrane receptors and relatively higher activity of bone destruction, inducing severe bone destruction under inflammatory conditions. Numerous factors could influence the interaction between osteoclasts and dendritic cells, contributing to the pathogenesis of several bone diseases in the context of inflammation, including both immunocytes and a large number of cytokines. In addition, the products of osteoclasts released from bone destruction area serve as important signals for the differentiation and activation of immature dendritic cells. Therefore, the border between the dendritic cell-related immune response and osteoclast-related bone destruction has gradually unravelled. Dendritic cells and osteoclasts cooperate with each other to mediate bone destruction and bone remodelling under inflammatory conditions. In this review, we will pay attention to the interactions between dendritic cells and osteoclasts in physiological and pathological conditions to further understand the skeletal system and identify potential new therapeutic targets for the future by summarizing their significant roles and molecular mechanisms in bone destruction.

Activated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor κB ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages and identified four miRNAs that were highly expressed in mature osteoclast-derived sEVs. One of these miRNAs, miR-324, significantly induced osteogenic differentiation and mineralization of primary mesenchymal stem cells (MSCs) in vitro by targeting ARHGAP1, a negative regulator of osteogenic differentiation. We next fabricated an sEV-modified scaffold by coating decalcified bone matrix (DBM) with osteoclast-derived sEVs, and the pro-osteogenic regeneration activities of the sEV-modified scaffold were validated in a mouse calvarial defect model. Notably, miR-324-enriched sEV-modified scaffold showed the highest capacity on bone regeneration, whereas inhibition of miR-324 in sEVs abrogated these effects. Taken together, our findings suggest that miR-324-contained sEVs released from mature osteoclast play an essential role in the regulation of osteogenic differentiation and potentially bridge the coupling between osteoclasts and MSCs.

Abstract 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.

Abstract 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 CD31hiEmcnhi 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.

Inflammatory arthritis is a major cause of disability in the elderly. This condition causes joint pain, loss of function, and deterioration of quality of life, mainly due to osteoarthritis (OA) and rheumatoid arthritis (RA). Currently, available treatment options for inflammatory arthritis include anti-inflammatory medications administered via oral, topical, or intra-articular routes, surgery, and physical rehabilitation. Novel alternative approaches to managing inflammatory arthritis, so far, remain the grand challenge owing to catastrophic financial burden and insignificant therapeutic benefit. In the view of non-targeted systemic cytotoxicity and limited bioavailability of drug therapies, a major concern is to establish stimuli-responsive drug delivery systems using nanomaterials with on-off switching potential for biomedical applications. This review summarizes the advanced applications of triggerable nanomaterials dependent on various internal stimuli (including reduction-oxidation (redox), pH, and enzymes) and external stimuli (including temperature, ultrasound (US), magnetic, photo, voltage, and mechanical friction). The review also explores the progress and challenges with the use of stimuli-responsive nanomaterials to manage inflammatory arthritis based on pathological changes, including cartilage degeneration, synovitis, and subchondral bone destruction. Exposure to appropriate stimuli induced by such histopathological alterations can trigger the release of therapeutic medications, imperative in the joint-targeted treatment of inflammatory arthritis.

The periosteum is critical for bone healing. Studies have shown that the periosteum contains periosteal stem cells (PSCs) with multidirectional differentiation potential and self-renewal ability. PSCs are activated in early fracture healing and are committed to the chondrocyte lineage, which is the basis of callus formation. However, the mechanism by which PSCs are activated and committed to chondrocytes in bone regeneration remains unclear. Here, we show that tartrate acid phosphatase (TRAP)-positive monocytes secrete CTGF to activate PSCs during bone regeneration. The loss function of TRAP-positive monocytes identifies their specific role during bone healing. Then, the secreted CTGF promotes endochondral ossification and activates PSCs in mouse bone fracture models. The secreted CTGF enhances PSC renewal by upregulating the expression of multiple pluripotent genes. CTGF upregulates c-Jun expression through αVβ5 integrin. Then, c-Jun transcription activates the transcription of the pluripotent genes Sox2, Oct4, and Nanog. Simultaneously, CTGF also activates the transcription and phosphorylation of Smad3 through αVβ5 integrin, which is the central gene in chondrogenesis. Our study indicates that TRAP-positive monocyte-derived CTGF promotes bone healing by activating PSCs and directing lineage commitment and that targeting PSCs may be an effective strategy for preventing bone non-union.

Post-trauma osteoarthritis (PTOA) is the most common articular disease characterized by degeneration and destruction of articular cartilage (Bultink and Lems, Curr. Rheumatol Rep., 2013, 15, 328). Inflammatory response of local joint tissue induced by trauma is the most critical factor accelerating osteoarthritis (OA) progression (Sharma et al., 2019; Osteoarthritis. Cartilage, 28, 658–668). M1/M2 macrophages polarization and repolarization participates in local inflammation, which plays a major role in the progression of OA (Zhang et al., 2018; Ann. Rheum. Dis., 77, 1524–1534). The regulating effect of macrophage polarization has been reported as a potential therapy to alleviate OA progression. Synovitis induced by polarized macrophages could profoundly affect the chondrocyte and cartilage matrix (Zhang et al., 2018; Ann. Rheum. Dis., 77, 1524–1534). Generally, anti-inflammatory medications widely used in clinical practice have serious side effects. Therefore, we focus on exploring a new therapeutic strategy with fewer side effects to alleviate the synovitis. Angelicin (ANG) is traditional medicine used in various folk medicine. Previous studies have revealed that angelicin has an inhibitory effect on inflammation (Wei et al., 2016; Inflammation, 39, 1876–1882), tumor growth (Li et al., 2016; Oncology reports, 36, 3,504–3,512; Wang et al., 2017; Molecular Medicine Reports, 16, 5441–5449), DNA damage (Li et al., 2019; Exp. Ther. Med., 18, 1899–1906), and virus proliferation (Li et al., 2018; Front. Cell. Infect. Microbiol., 8, 178). But its specific effects on influencing the process of OA were rarely reported. In this study, the molecular mechanism of angelicin in vivo and in vitro was clearly investigated. Results showed that angelicin could regulate the M1/M2 ratio and function and alleviate the development of PTOA in the meanwhile. Bone marrow monocytes were isolated and induced by macrophage colony-stimulating factor (M-CSF), lipopolysaccharide (LPS) and interferon (IFN)-γ for M1 polarization and interleukin (IL)-4/IL-13 for M2 polarization. Subsequently, repolarization intervention was performed. The results indicate that angelicin can repolarize M1 toward M2 macrophages by upregulating the expression of CD9. Besides, angelicin can also protect and maintain M2 polarization in the presence of LPS/IFN-γ, and subsequently downregulate the expression of inflammatory mediators such as IL-1β and TNF-α. Mechanistically, angelicin can activate the p-STAT3/STAT3 pathway by conducting CD9/gp130 to repolarize toward M2 macrophages. These results suggest angelicin can alleviate the progression of OA by regulating M1/M2 polarization via the STAT3/p-STAT3 pathway. Therefore, angelicin may have a promising application and potential therapeutic value in OA clinical treatment.

Abstract Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment through mediating intercellular crosstalks. However, little is known about the contribution of EVs derived from cancer cells to the vicious cycle of bone metastasis. Here, we report a direct regulatory mode between tumour cells and osteoclasts in metastatic niche of prostate cancer via vesicular miRNAs transfer. Combined analysis of miRNAs profiles both in tumour‐derived small EVs (sEVs) and osteoclasts identified miR‐152‐3p as a potential osteolytic molecule. sEVs were enriched in miR‐152‐3p, which targets osteoclastogenic regulator MAFB. Blocking miR‐152‐3p in sEVs upregulated the expression of MAFB and impaired osteoclastogenesis in vitro. In vivo experiments of xenograft mouse model found that blocking of miR‐152‐3p in sEVs significantly slowed down the loss of trabecular architecture, while systemic inhibition of miR‐152‐3p using antagomir‐152‐3p reduced the osteolytic lesions of cortical bone while preserving basic trabecular architecture. Our findings suggest that miR‐152‐3p carried by prostate cancer‐derived sEVs deliver osteolytic signals from tumour cells to osteoclasts, facilitating osteolytic progression in bone metastasis.

Abstract Background Inflammatory osteolysis is a severe infectious bone disorder that occurs during orthopaedic surgery and is caused by disruptions in the dynamic balance of bone matrix homeostasis, which makes this condition a burden on surgical procedures. Developing novel therapeutic drugs about inhibiting excessive osteoclastogenesis acts as an efficient approach to preventing inflammatory bone destruction. Methods To study this, we explored the potential effects and mechanisms of compound 17 on inflammatory osteolysis in vitro. Meanwhile, a lipopolysaccharide (LPS)‐induced calvarial osteolysis mouse model was used to evaluate the protective effect of compound 17 on inflammatory bone destruction in vivo. Results In our study, we found that compound 17 could inhibit osteoclast (OC) differentiation and bone resorption during RANKL and LPS stimulation in a time‐ and dose‐dependent manner, while compounds 5 and 13 did not have the same effects. Mechanistically, compound 17 promoted phosphatase and tensin homologue (PTEN) activity by reducing PTEN ubiquitination, thereby restraining the RANKL‐induced NF‐κB pathway, resulting in the inhibition of the expression of osteoclastogenesis‐related genes and the formation of the NLRP3 inflammasome. Additionally, we also investigated whether compound 17 could negatively modulate macrophage polarization and repolarization due to its anti‐inflammatory effects. Moreover, compound 17 also plays an important role in osteoblast differentiation and mineralization. In vivo experiments showed that compound 17 could effectively protect mice from LPS‐induced inflammatory bone destruction by inhibiting osteoclastogenesis and inflammation. Conclusions Taken together, these results show that compound 17 might play protective role in inflammatory bone destruction through inhibiting osteoclastogenesis and inflammation. These findings imply a possible role of compound 17 in inflammatory osteolysis‐related diseases.

Abstract The transcription factor SOX9 is frequently amplified in diverse advanced‐stage human tumors. Its stability has been shown to be tightly controlled by ubiquitination‐dependent proteasome degradation. However, the exact underlying molecular mechanisms remain unclear. This work reports that SOX9 protein abundance is regulated by the Cullin 3‐based ubiquitin ligase KEAP1 via proteasome‐mediated degradation. Loss‐of‐function mutations in KEAP1 compromise polyubiquitination‐mediated SOX9 degradation, leading to increased protein levels, which facilitate tumorigenesis. Moreover, the loss of critical ubiquitination residues in SOX9, by either a SOX9 (ΔK2) truncation or K249R mutation, leads to elevated protein stability. Furthermore, it is shown that the KEAP1/SOX9 interaction is modulated by CKIγ‐mediated phosphorylation. Importantly, it is demonstrated that DNA damage drugs, topoisomerase inhibitors, can trigger CKI activation to restore the KEAP1/SOX9 interaction and its consequent degradation. Collectively, herein the findings uncover a novel molecular mechanism through which SOX9 protein stability is negatively regulated by KEAP1 to control tumorigenesis. Thus, these results suggest that mitigating SOX9 resistance to KEAP1‐mediated degradation can represent a novel therapeutic strategy for cancers with KEAP1 mutations.

Recently, extracellular matrix-based tissue-engineered bone is a promising approach to repairing bone defects, and the seed cells are mostly mesenchymal stem cells. However, bone remodelling is a complex biological process, in which osteoclasts perform bone resorption and osteoblasts dominate bone formation. The interaction and coupling of these two kinds of cells is the key to bone repair. Therefore, the extracellular matrix secreted by the mesenchymal stem cells alone cannot mimic a complex bone regeneration microenvironment, and the addition of extracellular matrix by preosteoclasts may contribute as an effective strategy for bone regeneration. Here, we established the mesenchymal stem cell/preosteoclast extracellular matrix -based tissue-engineered bones and demonstrated that engineered-scaffolds based on mesenchymal stem cell/ preosteoclast extracellular matrix significantly enhanced osteogenesis in a 3 mm rat femur defect model compared with mesenchymal stem cell alone. The bioactive proteins released from the mesenchymal stem cell/ preosteoclast extracellular matrix based tissue-engineered bones also promoted the migration, adhesion, and osteogenic differentiation of mesenchymal stem cells in vitro. As for the mechanisms, the iTRAQ-labeled mass spectrometry was performed, and 608 differentially expressed proteins were found, including the IGFBP5 and CXCL12. Through in vitro studies, we proved that CXCL12 and IGFBP5 proteins, mainly released from the preosteoclasts, contributed to mesenchymal stem cells migration and osteogenic differentiation, respectively. Overall, our research, for the first time, introduce pre-osteoclast into the tissue engineering of bone and optimize the strategy of constructing extracellular matrix–based tissue-engineered bone using different cells to simulate the natural bone regeneration environment, which provides new sight for bone tissue engineering.

Skeletal system is a highly dynamic system going through continuous resorption and reconstruction to maintain homeostasis, which is influenced by numerous factors. Once the balance is disrupted, various kinds of bone diseases may occur such as osteoporosis. It has been well known that ATP (adenosine triphosphate), an important signaling molecule, is important in maintaining the dynamic balance of bone matrix. ATP mainly functions through P2X receptors, a kind of ATP receptors expressed by various kinds of bone cells to regulate the whole network of skeleton system. Among P2X receptors, P2X7 plays a crucial role in bone since P2X7 is widely expressed by bone cells and the mutation of P2X7 receptor is associated with kinds of bone diseases. It’s acknowledged that P2X7 acts as a potential therapeutic target for clinical treatment of bone-related diseases but further investigations are needed for the practical application. However, since P2X7 has a complicated effect in many aspects, the exact role of P2X7 in skeleton system is ambiguous. This review discusses the function of P2X7 in bone and other cells and their general effect on skeleton system, especially focusing on the possible clinical application for bone diseases.

Background/Aims: Tissue engineering bone transplantation with bone marrow mesenchymal stem cells (BMSCs) is an effective technology to treat massive bone loss, while molecular regulation of the bone regeneration processes remains poorly understood. Here, we aimed to assess the role of interleukin-8 (IL-8) in the recruitment of host cells by seeded BMSCs and in the bone regeneration. Methods: A transwell assay was performed to examine the role of IL-8/CXCR1/CXCR2/PI3k/Akt on the migration potential of hBMSCs. The in vitro chondrogenic differentiation of hBMSCs was assessed by examination of 2 chondrogenic markers, Sox9 and type 2 collagen (COL2). mBMSCs were used in tissue engineered bone (TEB) with/without IL-8 implanted into bone defect area with CXCR2 or Akt inhibitors. Density and Masson staining of the regenerated bone were assessed. The chondrogenesis was assessed by expression levels of associated proteins, Sox9 and COL2, by RT-qPCR and by immunohistochemistry. Results: IL-8 may trigger in vitro migration of hBMSCs via CXCR2-mediated PI3k/Akt signaling pathway. IL-8 enhances osteogenesis in the TEB-implanted bone defect in mice. IL-8 induces chondrogenic differentiation of hBMSCs via CXCR2-mediated PI3k/Akt signaling pathway in vitro and in vivo. Conclusions: IL-8 enhances therapeutic effects of MSCs on bone regeneration via CXCR2-mediated PI3k/Akt signaling pathway.

Background/Aims: In the process of bone development and remodeling, the vasculature is regarded as the communicative network between the bone and neighboring tissues. Recently, it has been reported that the processes of angiogenesis and osteogenesis are coupled temporally and spatially. However, few studies reported the relationship and relevant mechanism between osteoclastogenesis and vasculogenesis. Methods: Arraystar Mouse lncRNA microarray V3.0 was firstly used to analyze the differentially expressed lncRNA genes in osteoclast different stages during osteoclastogenesis. Cell counting kit 8 (CCK-8) analysis, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, migration and tube formation assays were used to detect impact of osteoclast different stages on the proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs), respectively. Finally, transfection of AK131850 shRNA, miR-93-5p mimic and miR-93-5p inhibitor, qRT-PCR, western blotting, enzyme-linked immunosorbent assay (ELISA), fluorescence in situ hybridization (FISH) and luciferase reporter assay were carried out to dissect molecular mechanisms. Results: In this study, we found that newborn OCs (N-OC) and mature OCs (M-OC) during osteoclastogenesis significantly promoted proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs). Through lncRNA microarray and GO&pathway analysis, we found that AK131850 and co-expressed gene, vascular endothelial growth factor a (VEGFa), were significantly up-regulated in N-OC and M-OC. After inhibition of AK131850 the promoting effect of N-OC and M-OC on EPCs was reversed. Furthermore, we found that AK131850 directly competed miR-93-5p in N-OC and M-OC through sponge, thereby increasing VEGFa transcription, expression and secretion through derepressing of miR-93-5p on VEGFa. Conclusion: Our results provided the first finding that lncRNA-AK131850 sponged miR-93-5p in N-OC and M-OC during osteoclastogenesis to enhance the secretion of VEGFa, thus promoting vasculogenesis of EPCs.

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Abstract Background The recruitment of a sufficient number of endogenous mesenchymal stem cells (MSCs) is the first stage of in-situ tissue regeneration. Transforming growth factor beta-3 (TGFβ3) could recruit stem or progenitor cells and endothelial cells to participate in tissue regeneration. However, the mechanism of TGFβ3 recruiting MSCs toward bone regeneration has remained obscure. Methods We estimated the promigratory property of TGFβ3 on human bone marrow MSCs (hBMSCs) cocultured with the vascular cells (human umbilical artery smooth muscle cells or human umbilical vein endothelial cells) or not by Transwell assay. After the addition of the inhibitor (SB431542) or Smad3 siRNA, the levels of MCP1 and SDF1 in coculture medium were tested by ELISA kit, and then the migratory signaling pathway of hBMSCs induced by TGFβ3 was investigated by western blot analysis. In vivo, a 2-mm FVB/N mouse femur defect model was used to evaluate chemokine secretion, endogenous cell homing, and bone regeneration induced by scaffolds loading 1 μg TGFβ3 through qPCR, immunofluorescent staining, immunohistochemical analysis, and Micro-CT, compared to the vehicle group. Results TGFβ3 (25 ng/ml) directly showed a nearly 40% increase in migrated hBMSCs via the TGFβ signaling pathway, compared to the vehicle treatment. Then, in the coculture system of hBMSCs and vascular cells, TGFβ3 further upregulated nearly 3-fold MCP1 secretion from vascular cells in a Smad3-dependent manner, to indirectly enhance nearly more than 50% of migrated hBMSCs. In vivo, TGFβ3 delivery improved MCP1 expression by nearly 7.9-fold, recruited approximately 2.0-fold CD31+ vascular cells and 2.0-fold Sca-1+ PDGFR-α+ MSCs, and achieved 2.5-fold bone volume fraction (BV/TV) and 2.0-fold bone mineral density, relative to TGFβ3-free delivery. Conclusions TGFβ3, as a MSC homing molecule, recruited MSCs to initiate bone formation in the direct-dependent and indirect-dependent mechanisms. This may shed light on the improvement of MSC homing in bone regeneration.

Background/Aims: Osteogenic differentiation of mesenchymal stem cells (MSCs) plays a crucial role in bone regeneration and bone reparation. This complex process is regulated precisely and firmly by specific factors. Recent studies have demonstrated that miR-125b regulates osteogenic differentiation, but little is known about the molecular mechanisms of this regulation. Furthermore, how miR-125b regulates the osteogenic differentiation of MSCs still needs elucidation. Methods: In the present study, human bone marrow-derived mesenchymal stem cells (hBMSCs) were isolated and induced to osteoblasts with miR-125b inhibition or overexpression. qRT-PCR and western blot analysis were used to detect the expression of osteogenic marker genes and proteins. Alkaline phosphatase (ALP) and Alizarin Red (ARS) staining were performed to evaluate the osteoblast phenotype. TargetScan, PicTar and miRanda database were used to predict the target gene of miR-125b. Dual luciferase reporter assay and RNA interference were performed to verify the target gene. Micro-CT imaging and histochemical staining were used to investigate the bone defect repair capacity of miR-125b in vivo. Results: We observed that miR-125b was expressed at a low level during the osteogenic differentiation of hBMSCs. Then, we found that osteogenic marker genes were negatively regulated by miR-125b during the course of osteogenic differentiation, suggesting that miR-125b down regulation plays an important role in the process of osteogenic differentiation. Bioinformatics approaches using miRNA target prediction algorithms indicated that the bone morphogenetic protein type Ib receptor (BMPR1b) is a potential target of miR-125b. The results of the dual luciferase reporter assay indicated that miR-125b binds to the 3’-UTR of the BMPR1b gene. We observed that knockdown of BMPR1b by siRNA inhibited the osteogenic differentiation of hBMSCs. Furthermore, by co-transfecting cells with an miR-125b inhibitor and si-BMPR1b, we found that the osteogenic capacity of the cells transfected with miR-125b inhibitor was blocked upon knockdown of BMPR1b. In vivo, demineralized bone matrix (DBM) was composited with hBMSCs as a scaffold to repair segmental femoral defects. By inhibiting the expression of miR-125b, hBMSCs showed a better capacity to repair bone defects. Conclusions: Taken together, our study demonstrated that miR-125b regulated the osteogenic differentiation of hBMSCs by targeting BMPR1b and that inhibiting miR-125b expression could enhance the capacity of bone defect repair in vivo.

Background and Aims. Host-derived cells play crucial roles in the regeneration process of tissue-engineered constructs (TECs) during the treatment of large segmental bone defects (LSBDs). However, their identity, source, and cell recruitment mechanisms remain elusive. Methods. A complex model was created using mice by combining methods of GFP+ bone marrow transplantation (GFP-BMT), parabiosis (GFP+-BMT and wild-type mice), and femoral LSBD, followed by implantation of TECs or DBM scaffolds. Postoperatively, the migration of host BM cells was detected by animal imaging and immunofluorescent staining. Bone repair was evaluated by micro-CT. Signaling pathway repressors including AMD3100 and SP600125 associated with the migration of BM CD44+ cells were further investigated. In vitro, transwell migration and western-blotting assays were performed to verify the related signaling pathway. In vivo, the importance of the SDF-1/CXCR4-JNK pathway was validated by ELISA, fluorescence-activated cell sorting (FACS), immunofluorescent staining, and RT-PCR. Results. First, we found that host cells recruited to facilitate TEC-mediated bone repair were derived from bone marrow and most of them express CD44, indicating the significance of CD44 in the migration of bone marrow cells towards donor MSCs. Then, the predominant roles of SDF-1/CXCR4 and downstream JNK in the migration of BM CD44+ cells towards TECs were demonstrated. Conclusion. Together, we demonstrated that during bone repair promoted by TECs, BM-derived CD44+ cells were essential and their migration towards TECs could be regulated by the SDF-1/CXCR4-JNK signaling pathway.

OBJECTIVE: AO/OTA 31-A3 intertrochanteric femoral fractures have completely different fracture line directions and biomechanical characteristics compared with other types of intertrochanteric fractures. The choice of the fixation method has been a focus of dispute among orthopedic trauma surgeons. The purpose of this study was to review the outcomes of these fractures treated with a percutaneous compression plate at our institute. METHOD: Seventeen patients with AO/OTA 31-A3 intertrochanteric femoral fractures were treated with a percutaneous compression plate at our institute from January 2010 to December 2011. The clinical data and imaging results were retrospectively analyzed. RESULTS: The medical complication of popliteal vein thrombosis occurred in one patient. Sixteen patients were followed up for 12 to 21 months. Two patients had malunion and mild pain. Fracture collapse occurred in two patients, with one having head penetration. These two patients had moderate pain. There were no occurrences of nonunion or reoperation. The mean Harris hip score obtained during the last follow-up was 84.1 (61-97). Patients with a poor quality of reduction were more likely to have pain results (p = 0.001). A trend existed toward the presence of a poor quality of reduction (p = 0.05) in patients with a collapse of fracture. Patients with poor preoperative mobility were more likely to have a lower Harris hip score (p = 0.000). CONCLUSION: The percutaneous compression plate is an alternative device for the treatment of AO/OTA 31-A3 intertrochanteric femoral fractures. Good fracture reduction and an ideal placement position of the neck screw are important in the success of the device.

Erythropoietin-producing hepatocyte B4 (EphB4) has been reported to be a key molecular switch in the regulation of bone homeostasis, but the underlying mechanism remains poorly understood. In this study, we investigated the role of EphB4 in regulating the expression of periostin (POSTN) within bone marrow-derived mesenchymal stem cells (MSCs) and assessed its effect and molecular mechanism of osteogenic induction in vitro. Treatment with ephrinB2-FC significantly increased the expression of POSTN in MSCs, and the inhibition of EphB4 could abrogate this effect. In addition, osteogenic markers were upregulated especially in MSCs overexpressing EphB4. To elucidate the underlying mechanism of cross talk between EphB4 and the Wnt pathway, we detected the change in protein expression of phosphorylated-glycogen synthase kinase 3β-serine 9 (p-GSK-3β-Ser9) and β-catenin, as well as the osteogenic markers Runx2 and COL1. The results showed that GSK-3β activation and osteogenic marker expression levels were downregulated by ephrinB2-FC treatment, but these effects were inhibited by blocking integrin αvβ3 in MSCs. Our findings demonstrate that EphB4 can promote osteogenic differentiation of MSCs via upregulation of POSTN expression. It not only helps to reveal the interaction mechanism between EphB4 and Wnt pathway but also brings a better understanding of EphB4/ephrinB2 signaling in bone homeostasis.

Chondrocytes communicate with each other mainly via diffusible signals rather than direct cell-to-cell contact. The chondrogenic differentiation of mesenchymal stem cells (MSCs) is well regulated by the interactions of varieties of growth factors, cytokines, and signaling molecules. A number of critical signaling molecules have been identified to regulate the differentiation of chondrocyte from mesenchymal progenitor cells to their terminal maturation of hypertrophic chondrocytes, including bone morphogenetic proteins (BMPs), SRY-related high-mobility group-box gene 9 (Sox9), parathyroid hormone-related peptide (PTHrP), Indian hedgehog (Ihh), fibroblast growth factor receptor 3 (FGFR3), and β-catenin. Except for these molecules, other factors such as adenosine, O2 tension, and reactive oxygen species (ROS) also have a vital role in cartilage formation and chondrocyte maturation. Here, we outlined the complex transcriptional network and the function of key factors in this network that determine and regulate the genetic program of chondrogenesis and chondrocyte differentiation.

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is accurately regulated by essential transcription factors and signaling cascades. However, the precise mechanisms involved in this process still remain to be defined. MicroRNAs (miRNAs) regulate various biological processes by binding target mRNA to attenuate protein synthesis. To investigate the mechanisms for miRNAs-mediated regulation of chondrogenic differentiation, we identified that miR-145 was decreased during transforming growth factor beta 3 (TGF-β3)-induced chondrogenic differentiation of murine MSCs. Subsequently, dual-luciferase reporter gene assay data demonstrated that miR-145 targets a putative binding site in the 3'-UTR of SRY-related high mobility group-Box gene 9 (Sox9) gene, the key transcription factor for chondrogenesis. In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels. Furthermore, over-expression of miR-145 decreased mRNA levels for three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-β3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes. Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation.