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Osteoporosis is a common degenerative skeletal disease among older people, especially postmenopausal women. Bone marrow mesenchymal stem cells (BMSCs), the progenitors of osteoblasts, are essential to the pathophysiology of osteoporosis. Herein, targeting miRNAs with differential expression in dysfunctional BMSCs was accomplished by bioinformatics analysis based on public databases. Target mRNAs were predicted and applied for signaling pathway and function enrichment annotations. In vitro and in vivo effects of selected miRNA on BMSC proliferation and osteogenesis were investigated, the putative binding between selected miRNA and predicted target mRNA was verified, and the co-effects of the miRNA/mRNA axis on BMSCs were determined. miRNA 665 (miR-665) was down-regulated in osteoporotic BMSCs compared with normal BMSCs and elevated in BMSCs experiencing osteogenic differentiation. In BMSCs, miR-665 overexpression promoted cell proliferation and osteogenic differentiation. miR-665 targeted the Wnt signaling inhibitor sclerostin (SOST) and inhibited SOST mRNA and protein expression. SOST overexpression inhibited BMSC cell proliferation and osteogenic differentiation. When co-transduced to BMSCs, SOST knockdown significantly reversed the effects of miR-665 on BMSCs. In ovariectomy (OVX)-induced osteoporosis model mice, OVX remarkably decreased bone mass, whereas miR-665 overexpression partially improved OVX-induced bone mass loss. miR-665 was down-regulated in osteoporotic BMSCs and up-regulated in osteogenically differentiated BMSCs. In conclusion, the miR-665/SOST axis modulates BMSC proliferation, osteogenic differentiation, and OVX-induced osteoporosis in mice, possibly through Wnt signaling., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Osteoporosis manifests through adipocyte accrual and osteoblast diminution within bone marrow. However, the precise mechanisms driving the shift from osteogenesis to adipogenesis in bone marrow mesenchymal stem cells (BMSCs) remain largely undefined. In this study, we harnessed the power of bioinformatic tools to analyze gene expression patterns of BMSCs during adipogenic differentiation and osteoporosis using the data from Gene Expression Omnibus (GEO) repositories (GSE113253 and GSE35956), complemented by in vitro and in vivo experiments to validate the findings. Five distinct expression profiles of differentially expressed genes across the adipogenic timeline were identified. The initial phase is marked by ribosome biogenesis and rRNA processing, which is followed by the metabolism of organic acids and processing of inorganic ions. In contrast, the terminal phase is characterized by lipid transport, accumulation, and metabolism, alongside inorganic cation metabolism, thereby underscoring unique transcriptional signatures during the early and late stages of adipogenic differentiation. In BMSCs derived from osteoporotic samples, there is a notable decline in cellular proliferation and a diminished osteogenic capacity. Critically, the genes common to both adipogenesis and osteoporosis in BMSCs are predominantly involved in the negative regulation of Wnt signaling and cellular proliferation. Key genes including SOCS1, MYC, CEBPB, FYN, AXIN2, and RXRA are identified and show downregulation in BMSCs from aged mice. Subsequent in vitro experiments have validated the regulatory influence of RXRA on both adipogenic and osteogenic differentiations of BMSCs, highlighting its crucial role as a central modulator in bone formation and the pathophysiology of osteoporosis., (© 2024 John Wiley & Sons, Ltd.)

Recently, exosomes that are derived from bone marrow mesenchymal stem cells (BMSCs) have garnered considerable interest due to their significant roles in the processes of bone regeneration and repair. Among the various molecular components present within these exosomes, miR-668-3p has emerged as a pivotal microRNA that may be instrumental in modulating the function and proliferation of osteoblasts, the cells responsible for bone formation. The primary objective of this research was to examine the enhancing effects of BMSC-derived exosomes that are enriched with miR-668-3p on the advancement of osteoblasts in the context of osteonecrosis of the femoral head. Furthermore, the study aimed to analyze how the expression of specific exosomal proteins, namely CD63 and CD9, influences this biological process. To conduct the investigation, BMSCs were isolated from healthy rat models, followed by the extraction of their secreted exosomes. The subsequent phase of the study involved assessing the proliferation and differentiation of osteoblasts by introducing the exosomes enriched with miR-668-3p into an experimental setup representing osteonecrosis of the femoral head. The findings revealed that exosomes derived from BMSCs, which contained miR-668-3p, significantly enhanced the proliferation of osteoblasts as well as the expression of key osteogenic marker genes. Notably, the levels of CD63 and CD9 proteins were markedly increased in the treated groups, indicating that the mechanisms underlying this promotion might involve cell adhesion and the endocytic uptake of exosomes., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The present study aims to investigate the role of interferon regulatory factor 4 (IRF4) in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and analyze the underlying signaling of these processes. In this study, IRF4 is upregulated in periodontitis periodontal ligament tissues, as compared to healthy periodontal ligament tissues. IRF4 knockdown increases cell proliferation, decreases levels of tumor necrosis factor-alpha, interleukin-6, and interleukin-8, enhances osteogenic activity, and increases the expression of RUNX family transcription factor 2, Collagen I, and Osteocalcin in PDLSCs. The opposite results are observed in IRF4 overexpressed PDLSCs. Additionally, GSEA shows that IRF4 activates the interleukin-18 (IL-18) signaling pathway. The expressions of IL-18, B-cell translocation gene 2, interleukin-1beta, and caspase-3 are decreased by IRF4 knockdown, while increased by IRF4 overexpression. IL-18 overexpression eliminates the promoting effect of IRF4 knockdown on osteogenic differentiation of PDLSCs. In conclusion, IRF4 suppresses osteogenic differentiation of PDLSCs by activating the IL-18 signaling pathway., (© 2024 Wiley‐VCH GmbH.)

The Retinoblastoma (RB1) gene plays a pivotal role in osteogenic differentiation. Our previous study, employing temporal gene expression analysis using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), revealed the deregulation of osteogenic differentiation in patient-derived heterozygous RB1 mutant orbital adipose-derived mesenchymal stem cells (OAMSCs). The study revealed increased Alizarin Red staining, suggesting heightened mineralization without a corresponding increase in osteogenic lineage-specific gene expression. In this study, we performed high-throughput RNA sequencing on RB1 +/+ and RB1 +/- patient-derived OAMSCs differentiated towards the osteogenic lineage to investigate the pathways and molecular mechanisms. The pathway analysis revealed significant differences in cell proliferation, DNA repair, osteoblast differentiation, and cancer-related pathways in RB1 +/- OAMSC-derived osteocytes. These findings were subsequently validated through functional assays. The study revealed that osteogenic differentiation is increased in RB1 +/- cells, along with enhanced proliferation of the osteocytes. There were delayed but persistent DNA repair mechanisms in RB1 +/- osteocytes, which were sufficient to maintain genomic integrity, thereby preventing or delaying the onset of tumors. This contrasts with our earlier observation of increased mineralization without corresponding gene expression changes, emphasizing the importance of high-throughput analysis over preselected gene set analysis in comprehending functional assay results., (Copyright © 2024 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Introduction: Heterotopic ossification of the tendon and ligament (HOTL) is a chronic progressive disease that is usually accompanied by thickening and ossification of ligaments and high osteogenic activity of the surrounding ligament tissue. However, the molecular mechanism of maintaining the cellular phenotype of HOTL remains unclear., Materials and Methods: We first constructed a model of HOTL, Enpp1 flox/flox /EIIa-Cre mice, a novel genetic mouse system. Imaging, histological, and cell-level analyses were performed to investigate the progressive ossification of the posterior longitudinal ligament, Achilles tendons, and degeneration joints caused by Enpp1 deficiency., Results: The results indicate that Enpp1 deficiency led to markedly progressive heterotopic ossification (HO), especially spine, and Achilles tendons, and was associated with progressive degeneration of the knees. The bone mass was decreased in the long bone. Furthermore, fibroblasts from Enpp1 flox/flox /EIIa-Cre mice had greater osteogenic differentiation potential following induction by osteogenesis, accompanied by enhanced hedgehog (Hh) signaling. In addition, fibroblast cells show senescence, and aggravation of the senescence phenotype by further osteogenic induction., Conclusion: Our study indicated that with increasing age, mutations in Enpp1 promote ectopic ossification of spinal ligaments and endochondral ossification in tendons and further aggravate knee degeneration by upregulating hedgehog signaling., Competing Interests: Declarations. Conflict of interest: The authors declared no potential conflicts of interest to this article's research, authorship and publication. Ethical approval: The studies were reviewed and approved by the Institutional Animal Care and Use Committee, Sun Yat-Sen University (No. 2020000147)., (© 2024. The Author(s).)

Background: Zoledronic acid (ZOL) is a type of bisphosphonate with good therapeutic effects on orthopaedic diseases. However, the pharmacological functions of ZOL on steroid-induced avascular necrosis of femoral head (SANFH) and the underlying mechanism remain unclear, which deserve further research., Methods: SANFH models both in vivo and in vitro were established by dexamethasone (Dex) stimulation. Osteoclastogenesis was examined by TRAP staining. Immunofluorescence was employed to examine autophagy marker (LC3) level. Cell apoptosis was analyzed by TUNEL staining. The interaction between Foxhead box D3 protein (FOXD3) and Annexin A2 (ANXA2) promoter was analyzed using ChIP and dual luciferase reporter gene assays., Results: Dex aggravated osteoclastogenesis and induced osteoclast differentiation and autophagy in vitro, which was abrogated by ZOL treatment. PI3K inhibitor LY294002 abolished the inhibitory effect of ZOL on Dex-induced osteoclast differentiation and autophagy. FOXD3 overexpression neutralized the downregulation effects of ZOL on Dex-induced osteoclasts by transcriptionally activating ANXA2. ANXA2 knockdown reversed the effect of FOXD3 overexpression on ZOL-mediated biological effects in Dex-treated osteoclasts. In addition, ZOL improved SANFH symptoms in rats., Conclusion: ZOL alleviated SANFH through regulating FOXD3 mediated ANXA2 transcriptional activity and then promoting PI3K/AKT/mTOR pathway, revealing that FOXD3 might be a target for ZOL in SANFH treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Human embryonic bone and joint formation is determined by coordinated differentiation of progenitors in the nascent skeleton. The cell states, epigenetic processes and key regulatory factors that underlie lineage commitment of these cells remain elusive. Here we applied paired transcriptional and epigenetic profiling of approximately 336,000 nucleus droplets and spatial transcriptomics to establish a multi-omic atlas of human embryonic joint and cranium development between 5 and 11 weeks after conception. Using combined modelling of transcriptional and epigenetic data, we characterized regionally distinct limb and cranial osteoprogenitor trajectories across the embryonic skeleton and further described regulatory networks that govern intramembranous and endochondral ossification. Spatial localization of cell clusters in our in situ sequencing data using a new tool, ISS-Patcher, revealed mechanisms of progenitor zonation during bone and joint formation. Through trajectory analysis, we predicted potential non-canonical cellular origins for human chondrocytes from Schwann cells. We also introduce SNP2Cell, a tool to link cell-type-specific regulatory networks to polygenic traits such as osteoarthritis. Using osteolineage trajectories characterized here, we simulated in silico perturbations of genes that cause monogenic craniosynostosis and implicate potential cell states and disease mechanisms. This work forms a detailed and dynamic regulatory atlas of bone and cartilage maturation and advances our fundamental understanding of cell-fate determination in human skeletal development., Competing Interests: Competing interests: C.D.B. is a founder of Mestag Therapeutics. S.A.T. is a scientific advisory board member of ForeSite Labs, OMass Therapeutics, Qiagen, a co-founder and equity holder of TransitionBio and EnsoCell Therapeutics, a non-executive director of 10x Genomics and a part-time employee of GlaxoSmithKline. Sarah also holds equity in TransitionBio and options for equity in Element Biosciences, where she is a former SAB member.  All other authors declare no competing interests., (© 2024. The Author(s).)

Postmenopausal osteoporosis (PMOP) is a metabolic disorder characterized by the loss of bone density, which increases the risk of developing complications such as fractures. A pivotal factor contributing to the onset of PMOP is the diminished osteogenic differentiation capacity of bone marrow mesenchymal stem cells (BMSCs). MicroRNAs (miRNAs) play a substantial role in this process; however, their specific impact on regulating BMSCs osteogenesis remains unclear. Studies have evidenced a reduced expression of miR-18a-5p in PMOP, and concomitantly, our observations indicate an augmented expression of miR-18a-5p during the osteogenic differentiation of BMSCs. This investigation seeks to elucidate the regulatory influence of miR-18a-5p on BMSC osteogenic differentiation and the underlying mechanisms. In vitro experiments demonstrated that the overexpression of miR-18a-5p facilitated the osteogenic differentiation of BMSCs, while the downregulation of miR-18a-5p yielded converse outcomes. Mechanistically, We employed bioinformatics techniques to screen out the target gene Notch2 of miR-18a-5p. Subsequently, dual-luciferase reporter gene assays and rescue experiments substantiated that miR-18a-5p promotes BMSC osteogenic differentiation by suppressing Notch2. Finally, miR-18a-5p was overexpressed via adenovirus injection into the femoral bone marrow cavity, with results demonstrating its capability to enhance osteogenic differentiation and alleviate PMOP symptoms. Our findings disclose that miR-18a-5p fosters osteogenic differentiation of BMSC by inhibiting Notch2, thereby offering novel targets and strategies for PMOP treatment., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aim: The regenerative capacity of dental pulp relies on the odonto/osteogenic differentiation of dental pulp cells (DPCs), but dynamic microenvironmental changes hinder the process. Bone morphogenetic protein 9 (BMP9) promotes differentiation of DPCs towards an odonto/osteogenic lineage, forming dentinal-like tissue. However, the molecular mechanism underlying its action remains unclear. This study investigates the role of DLX6 antisense RNA 1 (DLX6-AS1) in odonto/osteogenic differentiation induced by BMP9., Methodology: Custom RT 2 profiler PCR array, quantitative Real-Time PCR (qRT-PCR) and western blots were used to investigate the expression pattern of DLX6-AS1 and its potential signal axis. Osteogenic ability was evaluated using alkaline phosphatase and alizarin red S staining. Interactions between lncRNA and miRNA, as well as miRNA and mRNA, were predicted through bioinformatic assays, which were subsequently validated via RNA immunoprecipitation and dual luciferase reporter assays. Student's t-test or one-way ANOVA with post hoc Tukey HSD tests were employed for data analysis, with a p-value of less than .05 considered statistically significant., Results: DLX6-AS1 was upregulated upon BMP9 overexpression in DPCs, thereby promoting odonto/osteogenic differentiation. Additionally, miR-128-3p participated in BMP9-induced odonto/osteogenic differentiation by interacting with the downstream signal MAPK14. Modifying the expression of miR-128-3p and transfecting pcMAPK14/siMAPK14 had a rescue impact on odonto/osteogenic differentiation downstream of DLX6-AS1. Lastly, miR-128-3p directly interacted with both MAPK14 and DLX6-AS1., Conclusions: DLX6-AS1 could regulate the odonto/osteogenic differentiation of DPCs under the control of BMP9 through the miR-128-3p/MAPK14 axis., (© 2024 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Osteogenic differentiation is a crucial process in the formation of the skeleton and the remodeling of bones. It relies on a complex system of signaling pathways and transcription factors, including Runt-related transcription factor 2 (RUNX2). Non-coding RNAs (ncRNAs) control the bone-specific transcription factor RUNX2 through post-transcriptional mechanisms to regulate osteogenic differentiation. The most research has focused on microRNAs (miRNAs) and long ncRNAs (lncRNAs) in studying how they regulate RUNX2 for osteogenesis in both normal and pathological situations. This article provides a concise overview of the recent advancements in understanding the critical roles of lncRNA/miRNA/axes in controlling the expression of RUNX2 during bone formation. The possible application of miRNAs and lncRNAs as therapeutic agents for the treatment of disorders involving the bones and bones itself is also covered., Competing Interests: Declaration of competing interest The authors affirm that they do not have any conflicts of interest., (Copyright © 2024 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Several neuropeptides present in bone tissues, produced by nerve fibers and bone cells, have been reported to play a role in regulating the fine-tuning of osteoblast and osteoclast functions to maintain bone homeostasis. This study aims to characterize the influence of the neuropeptide vasoactive intestinal peptide (VIP) on the differentiation process of human mesenchymal stem cells (MSCs) into osteoblasts and on their anabolic function. We describe the mRNA and protein expression profile of VIP and its receptors in MSCs as they differentiate into osteoblasts, suggesting the presence of an autocrine signaling pathway in these cells. Our findings reveal that VIP enhances the expression of early osteoblast markers in MSCs under osteogenic differentiation and favors both bone matrix formation and proper cytoskeletal reorganization. Finally, our data suggest that VIP could be exerting a direct modulatory role on the osteoblast to osteoclast signaling by downregulating the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio. These results highlight the potential of VIP as an osteoinductive differentiation factor, emerging as a key molecule in the maintenance of human bone homeostasis., (© 2024 The Authors. BioFactors published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Osteoarthritis (OA) is the most common joint disease with high prevalence and incidence. Increasing reports has indicated that circular RNAs (circRNAs) are implicated in OA progression. Nevertheless, the roles and functions of most circRNAs in OA remain to be elucidated. In this study, we emphatically discussed circ-IQGAP1 (circ_0104873) in OA. Firstly, we discovered that circ_0104873 was dramatically overexpressed during osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Several functional assays demonstrated that circ_0104873 inhibition repressed BMSCs proliferation and osteogenic differentiation. Moreover, mechanism assays also revealed that circ_0104873 sponged microRNA-875-5p (miR-875-5p) to up-regulate notch receptor 3 (NOTCH3), thereby activating the Notch signaling pathway. Rescue assays disclosed that circ_0104873 contributed to the development of OA via targeting miR-875-5p/NOTCH3 axis. In conclusion, circ_0104873 promoted the progression of OA by miR-875-5p/NOTCH3/Notch signaling pathway, which might provide a promising target for OA treatment., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The transcription factor Runx2 plays a crucial role in regulating osteogenic differentiation and skeletal development. This factor not only controls the expression of genes involved in bone formation, but also interacts with signaling pathways such as the Notch pathway, which are essential for body development. However, studies have produced conflicting results regarding the relationship between Runx2 and the Notch pathway. Some studies suggest a synergistic interaction between these molecules, while others suggest an inhibitory one, for example, the interplay between Notch signaling, Runx2, and vitamin D3 in osteogenic differentiation and bone remodeling. The findings suggest a complex relationship between Notch signaling and osteogenic differentiation, with ongoing research needed to clarify the mechanisms involved and resolve existing contradictions regarding role of Notch in this process. Additionally, there is increasing evidence of contradictory roles for Runx2 in various tissues and organs, both under normal conditions and in pathological states. This diversity of roles makes Runx2 a potential therapeutic target, offering new directions for research. In this review, we have discussed the mechanisms of osteogenic differentiation and the important role of Runx2 in this process. We have also examined its relationship with different signaling pathways. However, there are still many uncertainties and inconsistencies in our current understanding of these interactions. Additionally, given that Runx2 is also involved in numerous other events in various tissues, we have tried to comprehensively examine its functions outside the skeletal system.

Osteoblasts, the key cells responsible for bone formation and the maintenance of skeletal integrity, originate from a diverse array of progenitor cells. However, the mechanisms underlying osteoblast differentiation from these multiple osteoprogenitors remain poorly understood. To address this knowledge gap, we developed a comprehensive framework to investigate osteoblast differentiation at multiple scales, encompassing cells, genes, and gene modules. We constructed a reference atlas focused on differentiation, which incorporates various osteoprogenitors and provides a seven-level cellular taxonomy. To reconstruct the differentiation process, we developed a model that identifies the transcription factors and pathways involved in differentiation from different osteoprogenitors. Acknowledging that covariates such as age and tissue type can influence differentiation, we created an algorithm to detect differentially expressed genes throughout the differentiation process. Additionally, we implemented methods to identify conserved pseudotemporal gene modules across multiple samples. Overall, our framework systematically addresses the heterogeneity observed during osteoblast differentiation from diverse sources, offering novel insights into the complexities of bone formation and serving as a valuable resource for understanding osteogenesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Han et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human blood vessel walls show concentric layers, with the outermost tunica adventitia harboring mesenchymal progenitor cells. These progenitor cells maintain vessel homeostasis and provide a robust cell source for cell-based therapies. However, human adventitial stem cell niche has not been studied in detail. Here, using spatial and single-cell transcriptomics, we characterized the phenotype, potential, and microanatomic distribution of human perivascular progenitors. Initially, spatial transcriptomics identified heterogeneity between perivascular layers of arteries and veins and delineated the tunica adventitia into inner and outer layers. From this spatial atlas, we inferred a hierarchy of mesenchymal progenitors dictated by a more primitive cell with a high surface expression of CD201 (PROCR). When isolated from humans and mice, CD201 Low expression typified a mesodermal committed subset with higher osteogenesis and less proliferation than CD201 High cells, with a downstream effect on canonical Wnt signaling through DACT2. CD201 Low cells also displayed high translational potential for bone tissue generation., Competing Interests: Declaration of interests A.W.J. declared scientific advisory board for Novadip LLC, consultant for Lifesprout LLC and Novadip LLC, and Editorial Board of Stem Cells, Bone Research, and American Journal of Pathology. K.B. declared consultant for Dilon Technologies. This arrangement has been reviewed and approved by Johns Hopkins University in accordance with its conflict of interest policies., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background/purpose: The use of human dental pulp stem cells (hDPSCs) as autologous stem cells for tissue repair and regenerative techniques is a significant area of global research. The objective of this study was to investigate the effects of long-term in vitro culture on the multidifferentiation potential of hDPSCs and the potential molecular mechanisms involved., Materials and Methods: The tissue block method was used to extract hDPSCs from orthodontic-minus-extraction patients, which were then expanded and cultured in vitro for 12 generations. Stem cells from passages three, six, nine, and twelve were selected. Flow cytometry was used to detect the expression of stem cell surface markers, and CCK-8 was used to assess cell proliferation. β-Galactosidase staining was employed to detect cellular senescence, Alizarin Red S staining to assess osteogenic potential, and Oil Red O staining to evaluate lipogenic capacity. RNA sequencing (RNA-seq) was conducted to identify differentially expressed genes in DPSCs and investigate their potential mechanisms., Results: With increasing passage numbers, pulp stem cells showed an increase in senescence and a decrease in proliferative capacity and osteogenic-lipogenic multidifferentiation potential. The expression of stem cell surface markers CD34 and CD45 was stable, whereas the expression of CD73, CD90, and CD105 decreased with increasing passages. According to the RNA-seq analysis, the differentially expressed genes CFH, WNT16, HSD17B2, IDI1, and COL5A3 may be associated with stem cell senescence., Conclusion: Increased in vitro expansion induced cellular senescence in pulp stem cells, which resulted in a reduction in their proliferative capacity and osteogenic-lipogenic differentiation potential. The differential expression of genes such as CFH, WNT16, HSD17B2, IDI1, and COL5A3 may represent a potential mechanism for the induction of cellular senescence in pulp stem cells., (© 2024. The Author(s).)

Human bone marrow mesenchymal stem cells (hBMSCs) are adult stem cells residing in the bone marrow, characterized by their capacity for multi-directional differentiation, self-renewal, migration, and engraftment. Serving as seed cells, BMSCs play a pivotal role in the regeneration of bone defects. Hence, investigating the transcription factors and signaling pathways involved in the regulation of osteogenic differentiation in BMSCs holds significant importance. Recent research has unveiled that certain circular RNAs (circRNAs) can function as molecular sponges, influencing the osteogenic differentiation process of mesenchymal stem cells. However, many circRNAs remain undiscovered, and their precise mechanisms remain elusive. Therefore, the objective of this study is to construct an osteogenic differentiation-related circRNA-miRNA-mRNA network in hBMSCs. Subsequently, through bioinformatics analysis, we constructed a ceRNA network related to the osteogenic differentiation ability of hBMSCs, comprising 22 circRNAs, 17 miRNAs, and 15 mRNAs. The potential circRNA-miRNA-mRNA axes, including the role of hsa_circ_0001600 in promoting the osteogenic differentiation of hBMSCs through the targeted regulation of hsa-miR-542-3p, were validated through in vitro experiments., (© 2024. The Author(s).)

Cellular communication network factor 2 (CCN2) is a secreted extracellular matrix-associated protein, and its aberrantly increased expression has been implicated in a diversity of diseases involving pathological processes of fibrosis, chronic inflammation, or tissue injury, which has promoted the evaluation of CCN2 as therapeutic targets for multiple disorders. However, human phenotypes associated with CCN2 deficiency have remained enigmatic; variants in CCN2 have not yet been associated with a human phenotype. Here, we collected families diagnosed with spondyloepimetaphyseal dysplasia (SEMD), and screened candidate pathogenic genes for families without known genetic causes using next-generation sequencing. We identified a monoallelic variant in signal peptide of CCN2 (NM_001901.2: c.65 G > C [p.Arg22Pro]) as the cause of SEMD in 14 subjects presenting with different degree of short stature, premature osteoarthritis, and osteoporosis. Affected subjects showed decreased serum CCN2 levels. Cell lines harboring the variant displayed decreased amount of CCN2 proteins in culture medium and an increased intracellular retention, indicating impaired protein secretion. And the variant weakened the stimulation effect of CCN2 on osteogenesis of bone marrow mesenchymal stem cells. Zebrafish ccn2a knockout model and osteoblast lineage-specific Ccn2-deficient mice (Ccn2 fl/fl ;Prx1 Cre ) partially recapitulated the phenotypes including low bone mass observed in affected subjects. Pathological mechanism implicated in the skeletal abnormality in Ccn2 fl/fl ;Prx1 Cre mice involved decreased bone formation, increased bone resorption, and abnormal growth plate formation. Collectively, our study indicate that monoallelic variants in CCN2 lead to a human inherited skeletal dysplasia, and highlight the critical role of CCN2 in osteogenesis in human., (© 2024. The Author(s).)

Bone marrow and teeth contain mesenchymal stem cells (MSCs) that could be used for cell-based regenerative therapies. MSCs from these two tissues represent heterogeneous cell populations with varying degrees of lineage commitment. Although human bone marrow stem cells (hBMSCs) and human dental pulp stem cells (hDPSCs) have been extensively studied, it is not yet fully defined if their adipogenic potential differs. Therefore, in this study, we compared the in vitro adipogenic differentiation potential of hDPSCs and hBMSCs. Both cell populations were cultured in adipogenic differentiation media, followed by specific lipid droplet staining to visualise cytodifferentiation. The in vitro differentiation assays were complemented with the expression of specific genes for adipogenesis and osteogenesis-dentinogenesis, as well as for genes involved in the Wnt and Notch signalling pathways. Our findings showed that hBMSCs formed adipocytes containing numerous and large lipid vesicles. In contrast to hBMSCs, hDPSCs did not acquire the typical adipocyte morphology and formed fewer lipid droplets of small size. Regarding the gene expression, cultured hBMSCs upregulated the expression of adipogenic-specific genes (e.g., PPARγ2 , LPL , ADIPONECTIN ). Furthermore, in these cells most Wnt pathway genes were downregulated, while the expression of NOTCH pathway genes (e.g., NOTCH1 , NOTCH3 , JAGGED1 , HES5 , HEY2 ) was upregulated. hDPSCs retained their osteogenic/dentinogenic molecular profile (e.g., RUNX2 , ALP , COLIA1 ) and upregulated the WNT-specific genes but not the NOTCH pathway genes. Taken together, our in vitro findings demonstrate that hDPSCs are not entirely committed to the adipogenic fate, in contrast to the hBMSCs, which are more effective to fully differentiate into adipocytes.

Background: The ligamentum flavum (LF) is an important anatomical structure of the spine. Ossification of the LF (OLF) has become the leading cause of thoracic spinal stenosis. Circular RNAs (circRNAs) and N6-methyladenosine (m6A) modification are reported to be associated with several human diseases. However, the role of circRNAs and m6A modification in the pathogenesis of OLF has not been fully investigated. Here, we aimed to explore the vital function of circRNAs and m6A modification in OLF., Materials and Methods: We analysed the circRNA expression of 4 OLF tissues and 4 normal LF tissues using bioinformatic analysis and identified circCDK14 for further analysis. We investigated the effects of circCDK14 on the osteogenic differentiation of LF cells. We observed that circCDK14 regulated its target genes by binding to miRNAs as a miRNA sponge. Moreover, the circRNA pull-down assay indicated that RNA-binding proteins might regulate the expression of circCDK14 via m6A modification., Results: CircCDK14 was significantly upregulated in OLF tissues compared to normal LF tissues. Overexpression of circCDK14 promoted the osteogenic differentiation of LF cells. Mechanistically, CircCDK14 promoted the expression of ALF transcription elongation Factor 4 (AFF4) by serving as a sponge for miR-93-5p. Moreover, Wilms tumour 1-associated protein (WTAP) increased the stability of circCDK14 via N6-methyladenosine modification., Conclusion: The m6A-modified CircCDK14 binding to miR-93-5p played an important role in the osteogenesis of LF cells by targeting AFF4, providing a promising therapeutic target for OLF., (© 2024. The Author(s).)

Mesenchymal stem cells (MSCs) have gained tremendous interest due to their overall potent pro-regenerative and immunomodulatory properties. In recent years, various in vitro and preclinical studies have investigated different priming ("licensing") approaches to enhance MSC functions for specific therapeutic purposes. In this study, we primed bone marrow-derived human MSCs (hMSCs) with an inflammation cocktail designed to mimic the elevated levels of inflammatory mediators found in serum of patients with severe injuries, such as bone fractures. We observed a significantly enhanced osteogenic differentiation potential of primed hMSCs compared to untreated controls. By RNA-sequencing analysis, we identified the immediate early response 3 (IER3) gene as one of the top-regulated genes upon inflammatory priming. Small interfering RNA knockdown experiments established IER3 as a novel positive regulator of osteogenic differentiation. Mechanistic analysis further revealed that IER3 deletion significantly downregulated bone marrow stromal cell antigen 2 (BST2) expression and extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in hMSCs, suggesting that IER3 regulates osteogenic differentiation through BST2 and ERK1/2 signaling pathway activation. On the basis of these findings, we propose IER3 as a novel therapeutic target to promote hMSC osteoblastogenesis, which might be of high clinical relevance, for example, in patients with osteoporosis or compromised fracture healing., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Our previous research revealed a close association between the acetylation of peroxisome proliferator-activated receptor γ (PPARγ) histone H3K27 and the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). We preliminarily explored the epigenetic mechanism of steroid-induced avascular necrosis of the femoral head (SANFH) development, but the specific histone deacetylase (HDAC) involved in this regulatory process remains unknown. In this study, we combined cell, animal, and clinical specimen experiments to screen for specific HDAC genes that could regulate BMSC adipogenic differentiation and to explore their roles. The results showed that dexamethasone (DEX) significantly exacerbated the imbalance between the adipogenic and osteogenic differentiation of BMSCs, and there were differences in HDAC expression in the adipogenic differentiation cell models, with histone deacetylase 10 (HDAC10) showing the most significant decrease in expression. Subsequent use of a chromatin immunoprecipitation assay kit and quantitative polymerase chain reaction (ChIP‒qPCR) revealed a decrease in HDAC10 expression at predicted potential sites within the PPARγ promoter, indicating a significant decrease in HDAC10 enrichment in the PPARγ promoter region of BMSCs, thereby promoting sustained PPARγ expression. Additionally, immunohistochemistry of samples collected from mice and humans with SANFH and normal femoral heads revealed an imbalance between adipogenic and osteogenic differentiation in the necrotic area of femoral heads, with a significant decrease in the relative expression of HDAC10 in the necrotic area of femoral heads with SANFH. In summary, we speculate that HDAC10 affects the progression of SANFH by regulating BMSC adipogenic differentiation, a process possibly related to PPARγ histone acetylation. These findings provide a promising direction for the treatment of SANFH., (© 2024. The Author(s).)

Human dental pulp stem cells (HDPSCs) showed an age-dependent decline in proliferation and differentiation capacity. Decline in proliferation and differentiation capacity affects the dental stromal tissue homeostasis and impairs the regenerative capability of HDPSCs. However, which age-correlated proteins regulate the senescence of HDPSCs remain unknown. Our study investigated the proteomic characteristics of HDPSCs isolated from subjects of different ages and explored the molecular mechanism of age-related changes in HDPSCs. Our study showed that the proliferation and osteogenic differentiation of HDPSCs were decreased, while the expression of aging-related genes (p21, p53) and proportion of senescence-associated β-galactosidase (SA-β-gal)-positive cells were increased with aging. The bioinformatic analysis identified that significant proteins positively correlated with age were enriched in response to the mammalian target of rapamycin (mTOR) signaling pathway (ILK, MAPK3, mTOR, STAT1, and STAT3). We demonstrated that OSU-T315, an inhibitor of integrin-linked kinase (ILK), rejuvenated aged HDPSCs, similar to rapamycin (an inhibitor of mTOR). Treatment with OSU-T315 decreased the expression of aging-related genes (p21, p53) and proportion of SA-β-gal-positive cells in HDPSCs isolated from old (O-HDPSCs). Additionally, OSU-T315 promoted the osteoblastic differentiation capacity of O-HDPSCs in vitro and bone regeneration of O-HDPSCs in rat calvarial bone defects model. Our study indicated that the proliferation and osteoblastic differentiation of HDPSCs were impaired with aging. Notably, the ILK/AKT/mTOR/STAT1 signaling pathway may be a major factor in the regulation of HDPSC senescence, which help to provide interventions for HDPSC senescence., (© The Author(s) 2024. Published by Oxford University Press.)

Osteoporosis, a common bone disease in older individuals, involves the progression influenced by N6-methyladenosine (m6A) modification. This study aimed to elucidate the effects of VDAC3 m6A modification on human bone mesenchymal stromal cell (BMSC) senescence and osteogenic differentiation. BMSCs were treated with etoposide to induce senescence. Senescence was assessed by β-galactosidase staining and quantitative real-time PCR (qPCR), and osteogenic differentiation was evaluated using Western blot, alkaline phosphatase, and alizarin red S staining. VDAC3 and ALKBH5 expression were quantified by qPCR, and their interaction was assessed by RNA immunoprecipitation (RIP) and luciferase reporter assay. m6A methylation was analyzed using the Me-RIP assay. VDAC3 expression was significantly decreased in etoposide-treated BMSCs (1.00 ± 0.13 vs. 0.26 ± 0.06). VDAC3 overexpression reduced etoposide-induced senescence and promoted osteogenic differentiation. ALKBH5 overexpression inhibited VDAC3 m6A modification (1.00 ± 0.095 vs. 0.233 ± 0.177) and its stability. ALKBH5 knockdown decreased etoposide-induced senescence and promoted osteogenic differentiation, effects that were reversed by VDAC3 knockdown. YTHDF1 was identified as the m6A methylation reader, and its overexpression inhibited VDAC3 stability. We demonstrated that ALKBH5 inhibited osteogenic differentiation of etoposide-induced senescent cells through the inhibition of VDAC3 m6A modification, and YTHDF1 acted as the m6A methylation reader. These findings provide a novel theoretical basis for the treatment of osteoporosis., (© 2024. The Author(s).)

Bone contains multiple pools of skeletal stem/progenitor cells (SSPCs), and SSPCs in periosteal compartments are known to exhibit higher regenerative potential than those in BM and endosteal compartments. However, the in vivo identity and hierarchical relationships of periosteal SSPCs (P-SSPCs) remain unclear due to a lack of reliable markers to distinguish BM SSPCs and P-SSPCs. Here, we found that periosteal mesenchymal progenitor cells (P-MPs) in periosteum can be identified based on Postn-CreERT2 expression. Postn-expressing periosteal subpopulation produces osteolineage descendants that fuel bones to maintain homeostasis and support regeneration. Notably, Postn+ P-MPs are likely derived from Gli1+ skeletal stem cells (SSCs). Ablation of Postn+ cells results in impairments in homeostatic cortical bone architecture and defects in fracture repair. Genetic deletion of Igf1r in Postn+ cells dampens bone fracture healing. In summary, our study provides a mechanistic understanding of bone regeneration through the regulation of region-specific Postn+ P-MPs.

Background: Mesenchymal stromal cells (MSCs) are recognized for their potential in regenerative medicine, attributed to their multipotent differentiation capabilities and immunomodulatory properties. Despite this potential, the classification and detailed characterization of MSCs, especially those derived from specific tissues like the pancreas, remains challenging leading to a proliferation of terminology in the literature. This study aims to address these challenges by providing a thorough characterization of human pancreatic islets-derived mesenchymal stromal cells (hPD-MSCs)., Methods: hPD-MSCs were isolated from donor islets using enzymatic digestion, immortalized through lentiviral transduction of human telomerase reverse transcriptase (hTERT). Cells were characterized by immunostaining, flow cytometry and multilineage differentiation potential into adipogenic and osteogenic lineages. Further a transcriptomic analysis was done to compare the gene expression profiles of hPD-MSCs with other mesenchymal cells., Results: We show that hPD-MSCs express the classical MSC features, including morphological characteristics, surface markers expression (CD90, CD73, CD105, CD44, and CD106) and the ability to differentiate into both adipogenic and osteogenic lineages. Furthermore, transcriptomic analysis revealed distinct gene expression profiles, showing notable similarities between hPD-MSCs and pancreatic stellate cells (PSCs). The study also identified specific genes that distinguish hPD-MSCs from MSCs of other origins, including genes associated with pancreatic function (e.g., ISL1) and neural development (e.g., NPTX1, ZNF804A). A novel gene with an unknown function (ENSG00000286190) was also discovered., Conclusions: This study enhances the understanding of hPD-MSCs, demonstrating their unique characteristics and potential applications in therapeutic strategies. The identification of specific gene expression profiles differentiates hPD-MSCs from other mesenchymal cells and opens new avenues for research into their role in pancreatic function and neural development., (© 2024. The Author(s).)

Background and Aims: Valve interstitial cells (VICs) undergo a transition to intermediate state cells before ultimately transforming into the osteogenic cell population, which is a pivotal cellular process in calcific aortic valve disease (CAVD). Herein, this study successfully delineated the stages of VIC osteogenic transformation and elucidated a novel key regulatory role of lumican (LUM) in this process., Methods: Single-cell RNA-sequencing (scRNA-seq) from nine human aortic valves was used to characterize the pathological switch process and identify key regulatory factors. The in vitro, ex vivo, in vivo, and double knockout mice were constructed to further unravel the calcification-promoting effect of LUM. Moreover, the multi-omic approaches were employed to analyse the molecular mechanism of LUM in CAVD., Results: ScRNA-seq successfully delineated the process of VIC pathological transformation and highlighted the significance of LUM as a novel molecule in this process. The pro-calcification role of LUM is confirmed on the in vitro, ex vivo, in vivo level, and ApoE-/-//LUM-/- double knockout mice. The LUM induces osteogenesis in VICs via activation of inflammatory pathways and augmentation of cellular glycolysis, resulting in the accumulation of lactate. Subsequent investigation has unveiled a novel LUM driving histone modification, lactylation, which plays a role in facilitating valve calcification. More importantly, this study has identified two specific sites of histone lactylation, namely, H3K14la and H3K9la, which have been found to facilitate the process of calcification. The confirmation of these modification sites' association with the expression of calcific genes Runx2 and BMP2 has been achieved through ChIP-PCR analysis., Conclusions: The study presents novel findings, being the first to establish the involvement of lumican in mediating H3 histone lactylation, thus facilitating the development of aortic valve calcification. Consequently, lumican would be a promising therapeutic target for intervention in the treatment of CAVD., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Gain-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) can cause high-bone-mass (HBM) phenotype, with 19 identified mutations so far. The A242T mutation in LRP5 has been found in 9 families, making it one of the most prevalent mutations. However, the correlation between the A242T mutation and HBM phenotype remains unverified in animal models. This study aimed to investigate the bone properties in a new transgenic mouse model carrying the LRP5 A241T missense mutation, equivalent to A242T in humans. Heterozygous Lrp5 A241T mice were generated using CRISPR/Cas9 genome editing. Body weight increased with age from 4 to 16 weeks, higher in males than females, with no difference between Lrp5 A241T mice and wild-type control. Micro-CT showed slightly longer femur and notably elevated trabecular bone mass of the femur and fifth lumbar spine with higher bone mineral density, bone volume fraction, and trabecular thickness in Lrp5 A241T mice compared to wild-type mice. Additionally, increased cortical bone thickness and volume of the femur shaft and skull were observed in Lrp5 A241T mice. Three-point bending tests of the tibia demonstrated enhanced bone strength properties in Lrp5 A241T mice. Histomorphometry confirmed that the A241T mutation increased bone formation without affecting osteoblast number and reduced resorption activities in vivo. In vitro experiments indicated that the LRP5 A241T mutation enhanced osteogenic capacity of osteoblasts with upregulation of the Wnt signaling pathway, with no significant impact on the resorptive activity of osteoclasts. In summary, mice carrying the LRP5 A241T mutation displayed high bone mass and quality due to enhanced bone formation and reduced bone resorption in vivo, potentially mediated by the augmented osteogenic potential of osteoblasts. Continued investigation into the regulatory mechanisms of its bone metabolism and homeostasis may contribute to the advancement of novel therapeutic strategies for bone disorders., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Guoli Yang reports financial support was provided by National Natural Science Foundation of China. Ke Yu reports financial support was provided by National Natural Science Foundation of China. Guoli Yang reports financial support was provided by Science Technology Department of Zhejiang Province. Zhiwei Jiang reports financial support was provided by National Natural Science Foundation of China. Ying Wang reports financial support was provided by National Natural Science Foundation of China. Ke Yu reports financial support was provided by China Postdoctoral Science Fundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Introduction and Aims: Specific circular RNAs (circRNAs) have been proven to play crucial roles in osteogenesis in vitro and in vivo. This study aims to identify a certain circRNA involved in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and explore its regulatory role., Methods: The expression of 5 candidate circRNAs (circ_0026344, circ_ACAP2, circ_0003764, circ_0008259, and circ_0060731) was detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) after PDLSCs were cultured in the osteogenic induction medium or medium supplemented with tumour necrosis factor-α (TNF-α, 10 ng/mL) for 3 and 7 days. The circRNA significantly decreased in both 3 and 7 days of osteogenic induction in PDLSCs and markedly increased in TNF-α-induced PDLSCs for 3 and 7 days screened. Identified circRNA was knocked down or overexpressed, and the effect on the osteogenic differentiation of PDLSCs was investigated by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, and alizarin red S (ARS) staining. Cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were applied to detect the effect of the circRNA on the proliferation of PDLSCs., Results: qRT-PCR results showed that the expression of circ_0003764 was significantly decreased when PDLSCs were cultured in the osteogenic induction medium for 3 or 7 days, whereas it was dramatically increased in TNF-α-induced PDLSCs. Knockdown of circ_0003764 promoted the expression of the osteogenesis-related genes (RUNX2, ALP, OCN) and proteins (RUNX2, OCN), enhanced the ALP activity, and elevated the mineralization by PDLSCs, as shown by ARS staining. However, with the overexpression of circ_0003764, the osteogenic differentiation capacity of PDLSCs was significantly reduced. The CCK-8 and EdU results indicated that circ_0003764 could inhibit the proliferation of PDLSCs., Conclusion: Circ_0003764 is involved in the osteogenesis process and inhibits the osteogenic differentiation and proliferation of PDLSCs., Clinical Relevance: This study indicates that circ_0003764 can serve as a diagnostic and therapeutic target in bone regeneration-related diseases treated by PDLSCs-based tissue engineering., Competing Interests: Conflict of interest None disclosed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is important for human bone formation. Long non-coding RNAs (lncRNAs) are critical regulators in osteogenic differentiation. This study aimed to explore the function and mechanisms of long intergenic non-protein coding RNA 963 (LINC00963) in affecting osteogenesis. Cell differentiation was assessed by alkaline phosphatase (ALP) activity detection and ALP staining assay. Meanwhile, levels of osteogenic marker genes, including RUNX family transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN), were detected by RT-qPCR and western blot. Cell proliferation and apoptosis were measured using CCK-8 assay and flow cytometry analysis. RNA immunoprecipitation (RIP), RNA pull-down and luciferase reporter assays were used to investigate the interaction between genes. LINC00963 expression was down-regulated in hBMSCs treated with osteogenic induction. LINC00963 overexpression inhibited hBMSCs differentiation, proliferation, and elevated apoptosis. LINC00963 acted as a competing endogenous RNA (ceRNA) to interact with miR-10b-5p and thereby regulated the expression level of Ras-related protein Rap-2a (RAP2A). LINC00963 regulated RAP2A to inhibit the level of phosphorylated AKT (p-AKT). LINC00963 inhibited hBMSCs differentiation, proliferation, and elevated apoptosis via the miR-10b-5p/RAP2A/AKT signaling, which might help improve the treatment of osteoporosis., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Calcium oxalate (CaOx) kidney stones are common and recurrent, lacking pharmacological prevention. Randall's plaques (RPs), calcium deposits in renal papillae, serve as niduses for some CaOx stones. This study explores the role of osteogenic-like cells in RP formation resembling ossification. CaP crystals deposit around renal tubules, interstitium, and blood vessels in RP tissues. Human renal interstitial fibroblasts (hRIFs) exhibit the highest osteogenic-like differentiation potential compared to chloride voltage-gated channel Ka positive tubular epithelial cells, aquaporin 2 positive collecting duct cells, and vascular endothelial cells, echoing the upregulated osteogenic markers primarily in hRIFs within RP tissues. Utilizing RNA-seq, osteomodulin (OMD) is found to be upregulated in hRIFs within RP tissues and hRIFs following osteogenic induction. Furthermore, OMD colocalizes with CaP crystals and calcium vesicles within RP tissues. OMD can enhance osteogenic-like differentiation of hRIFs in vitro and in vivo. Additionally, crystal deposits are attenuated in mice with Omd deletion in renal interstitial fibroblasts following CaOx nephrocalcinosis induction. Mechanically, a positive feedback loop of OMD/BMP2/BMPR1A/RUNX2/OMD drives hRIFs to adopt osteogenic-like fates, by which OMD induces osteogenic-like microenvironment of renal interstitium to participate in RP formation. We identify OMD upregulation as a pathological feature of RP, paving the way for preventing CaOx stones., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Recent research has revealed several important pathways of epigenetic regulation leading to transcriptional changes in bone cells. Rest Corepressor 2 (Rcor2) is a coregulator of Lysine-specific histone demethylase 1 (Lsd1), a demethylase linked to osteoblast activity, hematopoietic stem cell differentiation and malignancy of different neoplasms. However, the role of Rcor2 in osteoblast differentiation has not yet been examined in detail. We have previously shown that Rcor2 is highly expressed in mesenchymal stromal cells (MSC) and particularly in the osteoblastic lineage. The role of Rcor2 in osteoblastic differentiation in vitro was further characterized and we demonstrate here that lentiviral silencing of Rcor2 in MC3T3-E1 cells led to a decrease in osteoblast differentiation. This was indicated by decreased alkaline phosphatase and von Kossa stainings as well as by decreased expression of several osteoblast-related marker genes. RNA-sequencing of the Rcor2-downregulated MC3T3-E1 cells showed decreased repression of Rcor2 target genes, as well as significant upregulation of majority of the differentially expressed genes. While the heterozygous, global loss of Rcor2 in vivo did not lead to a detectable bone phenotype, conditional deletion of Rcor2 in limb-bud mesenchymal cells led to a moderate decrease in cortical bone volume. These findings were not accentuated by challenging bone formation by ovariectomy or tibial fracture. Furthermore, a global deletion of Rcor2 led to decreased white adipose tissue in vivo and decreased the capacity of primary cells to differentiate into adipocytes in vitro. The conditional deletion of Rcor2 led to decreased adiposity in fracture callus. Taken together, these results suggest that epigenetic regulation of mesenchymal stromal cell differentiation is mediated by Rcor2, which could thus play an important role in defining the MSC fate., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to investigate the effects of Shuanglongjiegu pill (SLJGP) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explore its mechanism based on miR-217/RUNX2 axis. Results found that drug-containing serum of SLJGP promoted BMSCs viability with a dose-dependent effect. Under osteogenic differentiation conditions, SLJGP promoted the expression of ALP, OPN, BMP2, RUNX2, and the osteogenic differentiation ability of BMSCs. In addition, SLJGP significantly reduced miR-217 expression, and miR-217 directly targeted RUNX2. After treatment with miR-217 mimic, the promoting effects of SLJGP on proliferation and osteogenic differentiation of BMSCs were significantly inhibited. MiR-217 mimic co-treated with pcDNA-RUNX2 further confirmed that the miR-217/RUNX2 axis was involved in SLJGP to promote osteogenic differentiation of BMSCs. In addition, analysis of Wnt/β-catenin pathway protein expression showed that SLJGP activated the Wnt/β-catenin pathway through miR-217/RUNX2. In conclusion, SLJGP promoted osteogenic differentiation of BMSCs by regulating miR-217/RUNX2 axis and activating Wnt/β-catenin pathway., (© 2024. The Author(s).)

Objective: Periodontitis can lead to the destruction of periodontal tissues and potentially tooth loss. Numerous periodontal tissue-derived cells display osteogenic differentiation potential. The presence of differentially expressed long non-coding RNAs (lncRNAs) in these cells indicate their ability to regulate the process of osteogenic differentiation. We aim to elucidate the various lncRNA-mediated regulatory mechanisms in the osteogenic differentiation of periodontal tissue-derived cells in the field of periodontitis at epigenetic modification, transcriptional, and post-transcriptional levels., Subjects and Methods: We systematically searched the PubMed, Web of Science, and ScienceDirect databases to identify relevant literature in the field of periodontitis discussing the role of lncRNAs in regulating osteogenic differentiation of periodontal tissue-derived cells. The identified literature was subsequently summarized for comprehensive review., Results: In this review, we have comprehensively summarized the regulatory mechanisms of lncRNAs in the osteogenic differentiation of periodontal tissue-derived cells in the field of periodontitis and discussed how these lncRNAs provide novel perspectives for understanding the pathogenesis and progression of periodontitis., Conclusion: These results indicate the pivotal role of lncRNAs as regulators in the osteogenic differentiation of periodontal tissue-derived cells, providing a solid basis for future investigations on the role of lncRNAs in the periodontitis field., (© 2024 Wiley Periodicals LLC.)

Splicing and endoplasmic reticulum (ER)-proteostasis are two key processes that ultimately regulate the functional proteins that are produced by a cell. However, the extent to which these processes interact remains poorly understood. Here, we identify SNRPB and other components of the Sm-ring, as targets of the unfolded protein response and novel regulators of export from the ER. Mechanistically, The Sm-ring regulates the splicing of components of the ER export machinery, including Sec16A, a component of ER exit sites. Loss of function of SNRPB is causally linked to cerebro-costo-mandibular syndrome (CCMS), a genetic disease characterized by bone defects. We show that heterozygous deletion of SNRPB in mice resulted in bone defects reminiscent of CCMS and that knockdown of SNRPB delays the trafficking of type-I collagen. Silencing SNRPB inhibited osteogenesis in vitro, which could be rescued by overexpression of Sec16A. This rescue indicates that the role of SNRPB in osteogenesis is linked to its effects on ER-export. Finally, we show that SNRPB is a target for the unfolded protein response, which supports a mechanistic link between the spliceosome and ER-proteostasis. Our work highlights components of the Sm-ring as a novel node in the proteostasis network, shedding light on CCMS pathophysiology., (© 2024. The Author(s).)

Sad and UNC84 domain 1 (SUN1) is a kind of nuclear envelope protein with established involvement in cellular processes, including nuclear motility and meiosis. SUN1 plays an intriguing role in human adipose-derived stem cells (hASCs) differentiation; however, this role remains largely undefined. This study was undertaken to investigate the role of SUN1 in hASCs differentiation, as well as its underlying mechanisms. Employing siRNAs, we selectively downregulated SUN1 and CD36 expression. Microtubules were depolymerized using nocodazole, and PPARγ was activated using rosiglitazone. Western blotting was performed to quantify SUN1, PPARγ, α-tubulin, CD36, OPN, and adiponectin protein expression levels. Alkaline phosphatase and Oil red O staining were used to assess osteogenesis and adipogenesis, respectively. Downregulated SUN1 expression increased osteogenesis and decreased adipogenesis in hASCs, concomitant with upregulated α-tubulin expression and downregulated CD36 expression, alongside reduced nuclear localization of PPARγ. Microtubule depolymerization increased CD36 expression. Rescue experiments indicated that microtubule depolymerization counteracted the downregulated SUN1-induced phenotypic changes. This study demonstrates that SUN1 influences the differentiation of hASCs towards osteogenic and adipogenic lineages, indicating its essential role in cell fate., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

AMP-activated protein kinase (AMPK) is a typical sensor of intracellular energy metabolism. Our previous study revealed the role of activated AMPK in the suppression of osteogenic differentiation and traumatic heterotopic ossification, but the underlying mechanism remains poorly understood. The E3 ubiquitin ligase Smurf1 is a crucial regulator of osteogenic differentiation and bone formation. We report here that Smurf1 is primarily SUMOylated at a C-terminal lysine residue (K324), which enhances its activity, facilitating ALK2 proteolysis and subsequent bone morphogenetic protein (BMP) signaling pathway inhibition. Furthermore, SUMOylation of the SUMO E3 ligase PIAS3 and Smurf1 SUMOylation was suppressed during the osteogenic differentiation and traumatic heterotopic ossification. More importantly, we found that AMPK activation enhances the SUMOylation of Smurf1, which is mediated by PIAS3 and increases the association between PIAS3 and AMPK. Overall, our study revealed that Smurf1 can be SUMOylated by PIAS3, Furthermore, Smurf1 SUMOylation mediates osteogenic differentiation and traumatic heterotopic ossification through suppression of the BMP signaling pathway. This study revealed that promotion of Smurf1 SUMOylation by AMPK activation may be implicated in traumatic heterotopic ossification treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aims: N6-Methyladenosine (m 6 A) has been confirmed to play a dynamic role in osteoporosis and bone metabolism. However, whether m 6 A is involved in the osteogenic differentiation of human periodontal ligament cells (hPDLCs) remains unclear. The present study aimed to verify the role of methyltransferase-like 3 (METTL3)-mediated m 6 A modification in the osteogenic differentiation of hPDLCs., Methods: The METTL3, Runx2, Osx, and YAP mRNA expression was determined by qPCR. METTL3, RUNX2, OSX, YTHDF1, YAP, IGF2BP1, and eIF3a protein expression was measured by Western blotting and immunofluorescence assays. The levels of m 6 A modification were evaluated by methylated RNA immunoprecipitation (MeRIP) and dot blot analyses. MeRIP-seq and RNA-seq were used to screen potential candidate genes. Nucleic acid and protein interactions were detected by immunoprecipitation. Alizarin red staining was used to evaluate the osteogenic differentiation of hPDLCs. Gene transcription and promoter activities were assessed by luciferase reporter assays (n ≥ 3)., Results: The expression of METTL3 and m 6 A modifications increased synchronously with the osteogenic differentiation of hPDLCs (p = .0016). YAP was a candidate gene identified by MeRIP-seq and RNA-seq, and its mRNA and protein expression levels were simultaneously increased. METTL3 increased the m 6 A methylated IGF2BP1-mediated stability of YAP mRNA (p = .0037), which in turn promoted osteogenic differentiation (p = .0147). Furthermore, METTL3 increased the translation efficiency of YAP by recruiting YTHDF1 and eIF3a to the translation initiation complex (p = .0154), thereby promoting the osteogenic differentiation of hPDLCs (p = .0012)., Conclusion: Our study revealed that METTL3-initiated m 6 A mRNA methylation promotes osteogenic differentiation of hPDLCs by increasing IGF2BP1-mediated YAP mRNA stability and recruiting YTHDF1 and eIF3a to the translation initiation complex to increase YAP mRNA translation. Our findings reveal the mechanism of METTL3-mediated m 6 A modification during hPDLC osteogenesis, providing a potential therapeutic target for periodontitis and alveolar bone defects., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: This study investigated the miRNA expression profile in Notch-activated human dental stem pulp stem cells (DPSCs) and validated the functions of miRNAs in modulating the odonto/osteogenic properties of DPSCs., Methods: DPSCs were treated with indirect immobilized Jagged1. The miRNA expression profile was examined using NanoString analysis. Bioinformatic analysis was performed, and miRNA expression was validated. Odonto/osteogenic differentiation was examined using alkaline phosphatase staining, Alizarin Red S staining, as well as odonto/osteogenic-related gene and protein expression., Results: Fourteen miRNAs were differentially expressed in Jagged1-treated DPSCs. Pathway analysis revealed that altered miRNAs were associated with TGF-β, Hippo, ErbB signalling pathways, FoxO and Ras signalling. Target prediction analysis demonstrated that 7604 genes were predicted to be targets for these altered miRNAs. Enrichment analysis revealed relationships to various DNA bindings. Among differentially expressed miRNA, miR-296-3p and miR-450b-5p were upregulated under Jagged1-treated conditions. Overexpression of miR-296-3p and miR-450b-5p enhanced mineralization and upregulation of odonto/osteogenic-related genes, whereas inhibition of these miRNAs revealed opposing results. The miR-296-3p and miR-450b-5p inhibitors attenuated the effects of Jagged1-induced mineralization in DPSCs., Conclusions: Jagged-1 promotes mineralization in DPSCs that are partially regulated by miRNA. The novel understanding of these miRNAs could lead to innovative controlled mechanisms that can be applied to modulate biology-targeted dental materials., (© 2024 Wiley Periodicals LLC.)

Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation as the essential functional component of primary cilia. The effects of IFT80 on early bone healing of extraction sockets have not been well studied. To investigate whether deletion of Ift80 in alveolar bone-derived mesenchymal stem cells (aBMSCs) affected socket bone healing, we generated a mouse model of specific knockout of Ift80 in Prx1 mesenchymal lineage cells (Prx1 Cre ;IFT80 f/f ). Our results demonstrated that deletion of IFT80 in Prx1 lineage cells decreased the trabecular bone volume, ALP-positive osteoblastic activity, TRAP-positive osteoclastic activity, and OSX-/COL I-/OCN-positive areas in tooth extraction sockets of Prx1 Cre ; IFT80 f/f mice compared with IFT80 f/f littermates. Furthermore, aBMSCs from Prx1 Cre ; IFT80 f/f mice showed significantly decreased osteogenic markers and downregulated migration and proliferation capacity. Importantly, the overexpression of TAZ recovered significantly the expressions of osteogenic markers and migration capacity of aBMSCs. Lastly, the local administration of lentivirus for TAZ enhanced the expression of RUNX2 and OSX and promoted early bone healing of extraction sockets from Prx1 Cre ; IFT80 f/f mice. Thus, IFT80 promotes osteogenesis and early bone healing of tooth sockets through the activation of TAZ/RUNX2 pathway., (© 2024 Wiley Periodicals LLC.)

With the aging of the global demographic, the prevention and treatment of osteoporosis are becoming crucial issues. The gradual loss of self-renewal and osteogenic differentiation capabilities in bone marrow stromal cells (BMSCs) is one of the key factors contributing to osteoporosis. To explore the regulatory mechanisms of BMSCs differentiation, we collected bone marrow cells of femoral heads from patients undergoing total hip arthroplasty for single-cell RNA sequencing analysis. Single-cell RNA sequencing revealed significantly reduced CRIP1 (Cysteine-Rich Intestinal Protein 1) expression and osteogenic capacity in the BMSCs of osteoporosis patients compared to non-osteoporosis group. CRIP1 is a gene that encodes a member of the LIM/double zinc finger protein family, which is involved in the regulation of various cellular processes including cell growth, development, and differentiation. CRIP1 knockdown resulted in decreased alkaline phosphatase activity, mineralization and expression of osteogenic markers, indicating impaired osteogenic differentiation. Conversely, CRIP1 overexpression, both in vitro and in vivo, enhanced osteogenic differentiation and rescued bone mass reduction in ovariectomy-induced osteoporosis mice model. The study further established CRIP1's modulation of osteogenesis through the Wnt signaling pathway, suggesting that targeting CRIP1 could offer a novel approach for osteoporosis treatment by promoting bone formation and preventing bone loss., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Fracture management largely relies on the bone's inherent healing capabilities and, when necessary, surgical intervention. Currently, there are limited osteoinductive therapies to promote healing, making targeting skeletal stem/progenitor cells (SSPCs) a promising avenue for therapeutic development. A limiting factor for this approach is our incomplete understanding of the molecular mechanisms governing SSPCs' behavior. We have recently identified that the Leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6) is expressed in sub-populations of SSPCs, and is required for maintaining bone volume during adulthood and for proper fracture healing. Lgr family members (Lgr4-6) are markers of stem cell niches and play a role in tissue regeneration primarily by binding R-Spondin (Rspo1-4). This interaction promotes canonical Wnt (cWnt) signaling by stabilizing Frizzled receptors. Interestingly, our findings here indicate that Lgr6 may also influence cWnt-independent pathways. Remarkably, Lgr6 expression was enhanced during Bmp-mediated osteogenesis of both human and murine cells. Using biochemical approaches, RNA sequencing, and bioinformatic analysis of published single-cell data, we found that elements of BMP signaling, including its target gene, pSMAD, and gene ontology pathways, are downregulated in the absence of Lgr6. Our findings uncover a molecular interdependency between the Bmp pathway and Lgr6, offering new insights into osteogenesis and potential targets for enhancing fracture healing., Competing Interests: Declaration of competing interest The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Human adipose-derived stem cells (hASCs) are commonly used in bone tissue regeneration. The N6-methyladenosine (m 6 A) modification has emerged as a novel regulatory mechanism for gene expression, playing a critical role in osteogenic differentiation of stem cells. However, the precise role and mechanism of alkylation repair homolog 5 (ALKBH5) in hASC osteogenesis remain incompletely elucidated and warrant further investigation. Herein, we employed methylated RNA immunoprecipitation sequencing, RNA sequencing, and weighted gene coexpression network analysis to identify a key long noncoding RNA (lncRNA) in hASCs: lncRNA AK311120. Functional experiments demonstrated that lnc-AK311120 promoted the osteogenic differentiation of hASCs, while a mutation at the m 6 A central site A of lnc-AK311120 was found to decrease the level of m 6 A modification. The osteogenic effect of ALKBH5 was confirmed both in vitro and in vivo using a mandibular defect model in nude mice. Subsequent investigations revealed that knockdown of ALKBH5 resulted in a significant increase in the m 6 A modification level of lnc-AK311120, accompanied by a downregulation in the expression level of lnc-AK311120. Additional rescue experiments demonstrated that overexpression of lnc-AK311120 could restore the phenotype after ALKBH5 knockdown. We observed that AK311120 interacted with the RNA-binding proteins DExH-Box helicase 9 (DHX9) and YTH domain containing 2 (YTHDC2) to form a ternary complex, while mitogen-activated protein kinase kinase 7 (MAP2K7) served as the shared downstream target gene of DHX9 and YTHDC2. Knockdown of AK311120 led to a reduction in the binding affinity between DHX9/YTHDC2 and the target gene MAP2K7. Furthermore, ALKBH5 facilitated the translation of MAP2K7 and activated the downstream JNK signaling pathway through the AK311120-DHX9-YTHDC2 complex, without affecting its messenger RNA level. Collectively, we have investigated the regulatory effect and mechanism of ALKBH5-mediated demethylation of lncRNA in hASC osteogenesis for the first time, offering a promising approach for bone tissue engineering., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Weightlessness osteoporosis, which progresses continuously and has limited protective effects, has become one of the major problems that need to be solved in manned spaceflight. Our study aims to investigate the regulatory role of PHF8 in disuse osteoporosis by observing the expression of PHF8 in bone marrow mesenchymal stem cells (BMSCs) under simulated weightlessness conditions. Therefore, we used the model of ground-based microgravity simulated by disuse osteoporosis patients and tail suspension in mice to simulate microgravity in vivo, and measured the expression of PHF8 in bone tissue. Subsequently, we used the 2D gyroscope to simulate the weightless effect on bone marrow mesenchymal stem cells. In the weightless condition, we detected the proliferation, apoptosis, osteogenesis, and osteogenic differentiation functions of BMSCs. We also detected the expression of osteogenic-related transcription factors after knocking down and overexpressing PHF8. Our results show that the weightless effect can inhibit the proliferation, osteogenesis, and osteogenic differentiation functions of BMSCs, while enhancing their apoptosis; and overexpression of PHF8 can partially alleviate the osteoporosis caused by simulated weightlessness, providing new ideas and clues for potential drug targets to prevent weightlessness and disuse osteoporosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Osteoporosis is a metabolic bone disease. β-Catenin is associated with fractures. Jian-Pi-Bu-Shen (JPBS) can promote the healing of osteoporotic fractures (OPF). However, the mechanism of β-catenin-mediated skeletal muscle satellite cells (SMSCs) in OPF by the JPBS is unclear. SMSCs were isolated and divided into five groups. The results showed that the survival rate of SMSCs was significantly higher in the low, medium, and high dose JPBS-containing serum groups after 7 days of incubation. The ALP activity and the number of SMSCs mineralized in the JPBS-containing serum intervention group were elevated. Axin, GSK-3β, β-catenin siRNAs were constructed and transfected into cells. Transfection of siRNAs reduced Axin, GSK-3β, and β-catenin expressions, respectively. β-Catenin-siRNA reversed ALP activity, the number of SMSCs mineralized, and the expression of β-catenin, BMP2, Runx2, COL-I, SP7/Ostrix, Osteocalcin, and BMP-7. Transcriptomic results suggested that the TNF signaling pathway associated with OPF was enriched. SD rats were subjected to the construction of OPF model by removing the ovaries. JPBS decreased the levels of PINP, ALP, CTX, and NTX through β-catenin in OPF rats, while increasing Runx2, β-catenin expressions through β-catenin at the broken end of fractures. Moreover, JPBS decreased BMC, BMD, and BV/TV and improved pathological damage through β-catenin in OPF rats. JPBS decreased the expression of Axin, GSK-3β mRNA, and protein, but increased the expressions of β-catenin, Pax7, COL-II, COL-II, BMP2, and Runx2 through β-catenin in OPF rats. In conclusion, JPBS inhibits Axin/GSK-3β expression, activates the β-catenin signaling, and promotes the osteogenic differentiation of SMSCs., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The Hox gene plays a crucial role in the bone development, determining their structure and morphology. Limb bone grafts expressing Hox positive genes are commonly used for free transplantation to repair Hox negative mandibular critical bone defects. However, the specific role of original Hox genes in newly formed bone during the cross-layer bone grafting healing process remains unexplored. Our findings demonstrate that femurs ectopically grafted into the mandibular environment retained a significant ability to differentiate into cartilage and form cartilaginous callus, which may be a key factor contributing to differences in bone graft healing. Hoxc10, an embryonic layer-specific genes, regulates cartilage formation during bone healing. Mechanistically, we observed Hoxc10 retention in co-cultured femoral BMSCs. Knocking out Hoxc10 narrows the bone gap and reduces cartilage formation. In summary, we reveal Hoxc10's 'positional memory' after adult cross-layer bone graft, influencing the outcomes of autologous bone graft., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Neuropilin-1 (NRP1) is a single transmembrane glycoprotein involved in a variety of physiological events. However, the exact mechanisms by which NRP1 regulates dental pulp stem cells (DPSCs) to differentiate toward an osteo/odontogenic phenotype are poorly understood. Here, we determined the significantly increased expression of full-length NRP1 and glycosaminoglycan (GAG)-modified NRP1 during osteo/odontogenesis in DPSCs. NRP1 was confirmed to promote alkaline phosphatase (ALP) activity, mineralized nodule deposition, protein and mRNA expression of Runx2, DSPP and DMP1 in DPSCs via the loss-of-function and gain-of-function approaches. Further, a non-GAG-modified NRP1 mutant (NRP1 S 612 A) was generated and the suppression of osteo/odontogenic differentiation was observed in the NRP1 S 612 A overexpression cells. Knockdown of the adaptor protein shroom3 resulted in the inhibition of osteo/odontogenesis. The protein-protein interaction network, the protein-protein docking and confocal analyses indicated the interactions between NRP1 and shroom3. Furthermore, immunoprecipitation followed by western analysis confirmed the binding of NRP1 to shroom3, but overexpression of NRP1 S 612 A greatly influenced the recruitment of shroom3 by NRP1. These results provide strong evidence that NRP1 is a critical regulator for osteo/odontogenesis through interacting with shroom3. Moreover, our results indicate that NRP1 S 612 A attenuates osteo/odontogenesis, suggesting that GAG modification is essential for NRP1 in DPSCs., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Early B cell factor 1 (EBF1) is a transcription factor expressed by multiple lineages of stromal cells within the bone marrow. While cultures of Ebf1-deficient cells have been demonstrated to have impaired differentiation into either the osteoblast or adipogenic lineage in vitro by several groups, in vivo there has been a nominal consequence of the loss of EBF1 on skeletal development. In this study we used Prx-cre driven deletion of Ebf1 to eliminate EBF1 from the entire mesenchymal lineage of the skeleton and resolve this discrepancy. We report here that EBF1 is expressed primarily in the Mesenchymal Stem and Progenitor Cell (MSPC)-Adipo, MSPC-Osteo, and the Early Mesenchymal Progenitors, and that loss of EBF1 has a plethora of consequences to maintenance of the skeleton throughout adulthood. Stroma from the Prx-cre;Ebf1 fl/fl bones had impaired osteogenic differentiation, an age-dependent loss of CFU-F, and elevated senescence accompanying Ebf1-deletion. New bone formation was reduced after 3 months, and resulted in a quiescent bone environment with fewer osteoblasts and an accompanied reduction in osteoclast-mediated remodeling. Consequently, bones were less ductile at a younger age, and deletion of EBF1 dramatically impaired fracture repair. Disruption of EBF1 in perivascular populations also rearranged the vascular network within these bones and disrupted cytokine signaling from key hematopoietic niches resulting in anemia, reductions in B cells, and myeloid skewing of marrow hematopoietic lineages. Mechanistically we observed disrupted BMP signaling within Ebf1-deficient progenitors with reduced SMAD1-phosphorylation, and elevated secretion of the soluble BMP-inhibitor Gremlin from the MSPC-Adipo cells. Ebf1-deficient progenitors also exhibited posttranslational suppression of glucocorticoid receptor expression. Together, these results suggest that EBF1 signaling is required for mesenchymal progenitor mobilization to maintain the adult skeleton, and that the primary action of EBF1 in the early mesenchymal lineage is to promote proliferation, and differentiation of these perivascular cells to sustain a healthy tissue., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this manuscript., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Regular physical activity is widely recognized for reducing the risk of various disorders, with skeletal muscles playing a key role by releasing biomolecules that benefit multiple organs and tissues. However, many individuals, particularly the elderly and those with clinical conditions, are unable to engage in physical exercise, necessitating alternative strategies to stimulate muscle cells to secrete beneficial biomolecules. Histone acetylation and deacetylation significantly influence exercise-induced gene expression, suggesting that targeting histone deacetylases (HDACs) could mimic some exercise responses. In this study, we explored the effects of the HDAC inhibitor Trichostatin A (TSA) on human skeletal muscle myoblasts (HSMMs). Our findings showed that TSA-induced hyperacetylation enhanced myotube fusion and increased the secretion of extracellular vesicles (EVs) enriched with miR-873-3p. These TSA-EVs promoted osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs) by targeting H2 calponin (CNN2). In vivo, systemic administration of TSA-EVs to osteoporosis mice resulted in significant improvements in bone mass. Moreover, TSA-EVs mimicked the osteogenic benefits of exercise-induced EVs, suggesting that HDAC inhibition can replicate exercise-induced bone health benefits. These results demonstrate the potential of TSA-induced muscle-derived EVs as a therapeutic strategy to enhance bone formation and prevent osteoporosis, particularly for individuals unable to exercise. Given the FDA-approved status of various HDAC inhibitors, this approach holds significant promise for rapid clinical translation in osteoporosis treatment., (© 2024. The Author(s).)