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Objective: To initially investigate the function of neuronal pentraxin 1 ( NPTX1 ) gene on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs)., Methods: hBMSCs were induced to undergo osteogenic differentiation, and then RNA was collected at different time points, namely 0, 3, 7, 10 and 14 d. The mRNA expression levels of key genes related with osteogenic differentiation, including runt-related transcription factor 2 ( RUNX2 ), alkaline phosphatase ( ALP ), osteocalcin ( OCN ), and NPTX1 , were detected on the basis of quantitative real-time polymerase chain reaction (qPCR) technology. In order to establish a stable NPTX1 -knockdown hBMSCs cell line, NPTX1 shRNA lentivirus was constructed and used to infect hBMSCs. ALP staining, alizarin red (AR) staining, and qPCR were employed to assess the impact of NPTX1 -knockdown on the osteogenic differentiation ability of hBMSCs., Results: The results showed that during the osteogenic differentiation of hBMSCs in vitro , the mRNA expression levels of osteogenic genes RUNX2 , ALP and OCN significantly increased compared with 0 d, while NPTX1 expression decreased markedly ( P < 0.01) as the osteogenic induction period exten-ded. At 72 h post-infection with lentivirus, the result of qPCR indicated that the knockdown efficiency of NPTX1 was over 60%. After knocking down NPTX1 in hBMSCs, RNA was extracted from both the NPTX1 -knockdown group (sh NPTX1 group) and the control group (shNC group) cultured in regular proliferation medium. The results of qPCR showed that the expression levels of osteogenic-related genes RUNX2 and osterix ( OSX ) were significantly higher in the sh NPTX1 group compared with the shNC group ( P < 0.01). ALP staining revealed a significantly deeper coloration in the sh NPTX1 group than in the shNC group at the end of 7 d of osteogenic induction. AR staining demonstrated a marked increase in mineralized nodules in the sh NPTX1 group compared with the shNC group at the end of 14 d of osteogenic induction., Conclusion: NPTX1 exerts a modulatory role in the osteogenic differentiation of hBMSCs, and its knockdown has been found to enhance the osteogenic differentiation of hBMSCs. This finding implies that NPTX1 could potentially serve as a therapeutic target for the treatment of osteogenic abnormalities, including osteoporosis.

The periodontal ligament (PDL) is a connective tissue, and PDL cells have a potential to differentiate into cementoblasts, osteoblasts, and gingival fibroblasts. This study investigated whether transcription factor c-Myb could induce differentiation of PDL cells for periodontal regeneration. PDL cells were isolated from extracted teeth and cultured. c-Myb was transfected to PDL cells using replication-deficient adenoviral vector. Differentiation of the PDL cells was analyzed by immunoblot, alkaline phosphatase activity, Alizarin red stain, and immunofluorescence analysis. Cell viability on titanium surfaces was analyzed by crystal violet stain and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PDL cells cultured in osteogenic medium showed increased production of osteogenic and cementogenic molecules. Moreover, c-Myb-transfected cells showed increased production of dentinogenic molecules, in addition to the osteogenic and cementogenic molecules, even in normal culture condition. c-Myb-transfected cells also exhibited increased autophagy and type I collagen production under nutrient deprivation. When grown on a titanium surface, c-Myb-transfected cells showed increased production of osteogenesis-, dentinogenesis-, and cementogenesis-related molecules and cell viability. Thus, these results suggest that c-Myb might play an essential role during periodontal regeneration by improving the differentiation of PDL cells, and c-Myb can be utilized for enhancing the attachment of PDL cells to dental implant surfaces., (© 2025 Scandinavian Division of the International Association for Dental Research. Published by John Wiley & Sons Ltd.)

Objectives: Tideglusib (Tx) is known for its osteogenic potential, yet its effects on the interplay between osteoblasts and M1 macrophages remain underexplored. This in vitro study aimed to isolate and evaluate both the individual and combined roles of M1 macrophages and osteoblasts in macrophage differentiation and osteoblast function, specifically focusing on how these interactions influence protein expression of osteogenesis and osteoclastogenesis in the presence or absence of Tx., Methods: Osteoblast and macrophage cells were co-cultured in direct contact for 24 and 48 h, with or without the presence of Tx. ALP activity, the expression of inflammatory-related genes using RT-qPCR, and histological analyses were performed., Results: Co-culturing osteoblasts and M1 macrophages with Tx increased alkaline phosphatase production, indicative of enhanced osteoblast activity. Histological assessments revealed that Tx treatment contributed to the stability and maintenance of cell morphology within the scaffold, suggesting a supportive environment for cell viability and function. Tx significantly reduced the expression of pro-inflammatory markers, such as TNF-α and IL-1β, in the co-culture at both 24 and 48 h Tx also effectively inhibited osteoclastogenic differentiation in macrophages, thereby diminishing their pro-inflammatory phenotype., Conclusions: Tx increased ALP activity and produced a significant up-regulation of RANKL expression, indicating enhanced osteoblast differentiation and osteoclast activation. Tx mitigates macrophage-driven inflammation., Clinical Significance: Tx may enhance bone regeneration by modulating inflammatory responses and preserving cell integrity., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Aim: Human stem cells derived from the apical papilla (SCAPs) are recognized for their multilineage differentiation potential and their capacity for functional tooth root regeneration. However, the molecular mechanisms underlying odonto/osteogenic differentiation remain largely unexplored. In this study, we utilized single-cell RNA sequencing (scRNA-seq) to conduct an in-depth analysis of the transcriptional changes associated with chemically induced osteogenesis in SCAPs., Methodology: scRNA-seq identified SCAPs as distinct subpopulations. Differentially expressed genes (DEGs) and Gene Ontology (GO) analyses were conducted to evaluate the potential function of each cluster. Pseudotime trajectory analysis was employed to elucidate the potential differentiation processes of the identified SCAP populations. To investigate the osteo/odontogenic potential of Deiodinase Iodothyronine Type 2 (DIO2) on SCAPs, we performed alkaline phosphatase staining, western blot analysis, Alizarin Red S staining and immunofluorescence staining. Additionally, SCAP components were transplanted into mouse calvarial defects to evaluate osteogenesis in vivo., Results: The analysis of cell clusters derived from our scRNA-seq data revealed a significant shift in cellular composition when cells were cultured in a mineralization induction medium compared to those cultured in a complete medium. Both groups exhibited heterogeneity, with some cells intrinsically predisposed to osteogenesis and others appearing to be primed for proliferative functions. Notably, we identified a subpopulation characterized by high expression of DIO2, which exhibited pronounced osteogenic activity during differentiation., Conclusions: Our study is the first to reveal a shift in the cellular composition of SCAPs when cultured in a mineralization induction medium compared to a complete medium. Following both in vitro and in vivo validation, the DIO2 + subpopulation exhibited the highest transcriptional similarity to osteogenic function, suggesting its potential utility in tissue regeneration applications., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Circadian rhythm disruption is thought to be associated with periodontitis, and molecular clock genes play critical roles in regulating bone homeostasis. However, the specific contribution of molecular clock genes to alveolar bone resorption caused by periodontitis is poorly understood. In this study, we introduced a novel Periodontitis Circadian Rhythm Score (PeriCRS) model that was established through machine learning using periodontal transcriptomic data from periodontitis clinical cohorts in the Gene Expression Omnibus (GEO) database. This approach revealed the potential regulatory role of circadian rhythm disruption in periodontitis and identified key molecular clock genes associated with alveolar bone destruction. Moreover, we established an experimental periodontitis model with circadian rhythm disturbance via periodontal ligation in mice exposed to a 6-h advanced LD12:12 cycle every 2 d. Our bioinformatics analysis revealed that NR1D1 , which encodes REV-ERBα, is a pivotal factor in the impact of circadian rhythm disruption on periodontitis in periodontal tissues. Next, we confirmed the abnormal expression of the molecular clock gene Rev-erbα in inflammatory periodontal tissue in mice and confirmed that circadian rhythm disruption altered REV-ERBα expression. Furthermore, the activation of REV-ERBα with the agonist SR9009 notably decreased RANKL-induced osteoclast differentiation and suppressed the expression of osteoclast-related factors. Subsequent in vivo experiments demonstrated that SR9009 mitigated alveolar bone loss caused by periodontitis. Mechanistically, we found that the IL-22-STAT3 pathway inhibited REV-ERBα expression and modulated RANKL-induced osteoclast differentiation in vitro. Our results elucidate the role of REV-ERBα in osteoclastogenesis and suggest a potential new therapeutic avenue for addressing alveolar bone resorption associated with periodontitis., 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.

Diabetes-associated periodontitis (DP) is recognized as an inflammatory disease that can lead to teeth loss. Uncontrolled chronic low-grade inflammation-induced senescence impairs the stemness of human periodontal stem cells (hPDLSCs). Sirtuin 3 (SIRT3), an NAD + -dependent deacetylase, is pivotal in various biological processes and is closely linked to aging and aging-related diseases. This study aims to explore the mechanism of SIRT3-related senescence and osteogenic differentiation of hPDLSCs under DP and explored the novelty therapeutic targets. Our study revealed that SIRT3 expression was markedly inhibited in periodontal ligament stem cells (PDLSCs) stimulated by high glucose and lipopolysaccharide. Both in vitro and in vivo, reduced SIRT3 expression accelerated cell senescence and impaired osteogenic differentiation of hPDLSCs. We demonstrated that SIRT3 binds to and deacetylates leucine-rich pentatricopeptide repeat-containing protein (LRPPRC), thereby modulating senescence. Additionally, we found that LRPPRC regulates senescence by modulating oxidative phosphorylation and oxidative stress. The activation of SIRT3 by honokiol significantly delayed senescence and promoted alveolar bone regeneration in mice after DP. Our findings indicate that the activation of SIRT3 negatively regulates hPDLSCs senescence by deacetylating LRPPRC, suggesting SIRT3 as a promising therapeutic target for DP., Competing Interests: Competing interests The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Osteoporosis (OP) is a prevalent bone disease characterized by reduced bone density and quality, increasing fragility and fracture risk. Osteoclast (OC) activity and circadian rhythm play a role in the pathogenesis of OP. Melatonin is a circadian regulator that affects bone metabolism, but its molecular mechanism has not been studied in detail. This study aimed to identify the relationship between melatonin-related genes and OP through bioinformatics methods and to verify it experimentally.We analysed microarray data from the GSE35959 dataset, identifying differentially expressed genes in OP patients. Circadian rhythm-related genes and melatonin-related genes intersect with these differentially expressed genes, highlighting that CSNK1D is a central gene. Functional enrichment, correlation and protein-protein interaction analyses were conducted. Experimental validation involved in vitro differentiation assays using RAW264.7 cells and in vivo studies with an ovariectomy-induced rat model of OP to evaluate the role of CSNK1D in osteoclastogenesis to verify its effect on OP. Differential expression analysis revealed 272 significant genes, with CSNK1D identified as central to the circadian rhythm and to melatonin and OP interplay. Functional analyses showed involvement of CSNK1D in OC differentiation and inflammatory pathways. in vitro experiments confirmed CSNK1D upregulation during OC differentiation, and small interfering RNA-mediated knockdown reduced OC marker expression and TRAP + cell formation. in vivo, CSNK1D expression is associated with bone loss in OP rats. Melatonin-related CSNK1D promotes OC differentiation and promotes the development of OP. These findings suggest CSNK1D as a potential therapeutic target for OP, offering insights into new treatment strategies integrating circadian rhythm regulation., (© 2024 The Author(s). Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Hajdu Cheney Syndrome (HCS), a monogenic disorder associated with NOTCH2 pathogenic variants, presents with neurological, craniofacial and skeletal abnormalities. Mouse models of the disease exhibit osteopenia. To determine the consequences of a HCS pathogenic variant in human cells, induced pluripotent NCRM1 and NCRM5 stem (iPS) cells harboring a NOTCH2 6949C>T mutation or null for HES1 alleles were created. Parental iPSCs, NOTCH2 6949C>T , HES1 null and control cells, free of chromosomal aberrations were cultured under conditions of neural crest, mesenchymal and osteogenic cell differentiation, or of embryoid body, hematopoietic and osteoclast cell differentiation. The expected cell phenotype was confirmed by cell surface markers and gene signature. NOTCH2 6949C>T cells displayed enhanced expression of Notch target genes demonstrating the presence of a NOTCH2 gain-of-function. There was a modest enhancement of osteogenesis in NOTCH2 6949C>T cells manifested by increased mineralized nodule formation and SP7, ALPL and BGLAP mRNA expression. There was enhanced osteoclastogenesis in NOTCH2 6949C>T cells as evidenced by increased number of osteoclasts and a transient increase in ACP5, CALCR and CTSK transcripts. Osteoblastogenesis was minimally affected by the HES1 deletion, but osteoclast differentiation was significantly impaired. In conclusion, a NOTCH2 pathogenic variant causes modest increases in osteoblastogenesis and osteoclastogenesis and HES1 is required for osteoclast differentiation in human iPS cells in vitro., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Alcoholic osteonecrosis of the femoral head (AIONFH) is caused by long-term heavy drinking, which leads to abnormal alcohol and lipid metabolism, resulting in femoral head tissue damage, and then pathological necrosis of femoral head tissue. If not treated in time in clinical practice, it will seriously affect the quality of life of patients and even require hip replacement to treat alcoholic femoral head necrosis. This study will confirm whether M2 macrophage exosome (M2-Exo) miR-122 mediates alcohol-induced BMSCs osteogenic differentiation, ultimately leading to the inhibition of femoral head necrosis. M2 macrophages were identified by flow cytometry, and the isolated exosomes were characterized by transmission electron microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). Next, miR-122 was overexpressed by transfecting miR-122 mimic, and the expression of miR-122 in M2 macrophages and their exosomes was evaluated. Subsequently, the effect of exosomal miR-122 on the osteogenic differentiation ability of BMSCs was detected, including cell proliferation, expression of osteogenic-related genes (RUNX2, BMP2, OPN, ALP), and calcium nodule formation. Finally, the therapeutic effect of M2-Exo was analyzed in a rat model of AIONFH, and bone repair and pathological damage were evaluated by Micro-CT, RT-qPCR, HE, Masson staining, and immunohistochemistry (COL I). The results showed that M2 macrophages were successfully polarized, with an average M2-Exo particle size of 156.4 nm and a concentration of 3.2E + 12 particles/mL. The expression of miR-122 in M2 macrophages is significantly higher than that in M0 macrophages, and miR-122 mimic can increase the content of miR-122 in M2-Exo. miR-122 in M2-Exo can promote osteogenic differentiation of rat bone marrow BMSCs, enhance cell viability, and increase the expression of osteogenesis-related genes. After being applied to the AIONFH rat model, the injection of M2-exo and miR-122 mimics significantly improved the repair effect of articular cartilage, alleviated pathological changes, and promoted the regeneration of bone tissue. M2-macrophage-derived exosomal miR-122 induces osteogenic differentiation of bone mesenchymal stem cells in treating AIONFH., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Legg-Calvé-Perthes disease (LCPD) involves femoral head osteonecrosis caused by disrupted blood supply, leading to joint deformity and early osteoarthritis. This study investigates the role of miRNA-223-5p in regulating hypoxia-induced apoptosis and enhancing osteogenesis in bone marrow mesenchymal stem cells (BMSCs). Utilizing a juvenile New Zealand white rabbit model of LCPD established through femoral neck ligation, we transfected BMSCs with miR-223-5p mimics, inhibitors, and controls, followed by hypoxic exposure. The impact of miR-223-5p on BMSC apoptosis was assessed using qPCR, Western blotting, and dual-luciferase reporter assays, focusing on the Wnt/β-catenin signaling pathway. In vivo, we evaluated the effects of transplanting miR-223-5p-overexpressing BMSCs into the LCPD model. Our results indicate that miR-223-5p is downregulated under hypoxic conditions. Overexpression of miR-223-5p in BMSCs inhibited hypoxia-induced apoptosis and activated the Wnt/β-catenin pathway by directly targeting CHAC2. In vivo, miR-223-5p-overexpressing BMSCs enhanced femoral head osteogenesis and reduced necrosis in the LCPD model. These findings suggest that miR-223-5p inhibits hypoxia-induced apoptosis in BMSCs by targeting CHAC2 and activating the Wnt/β-catenin pathway, proposing miR-223-5p as a promising target for improving bone repair in ischemic conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Yang 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.)

The antler is the only organ that can fully regenerate annually in mammals. However, the regulatory pattern and mechanism of gene expression and cell differentiation during this process remain largely unknown. Here, we obtain comprehensive assembly and gene annotation of the sika deer ( Cervus nippon ) genome. We construct, together with large-scale chromatin accessibility and gene expression data, gene regulatory networks involved in antler regeneration, identifying four transcription factors, MYC , KLF4 , NFE2L2, and JDP2 , with high regulatory activity across the whole regeneration process. Comparative studies and luciferase reporter assay suggest the MYC expression driven by a cervid-specific regulatory element might be important for antler regenerative ability. We further develop a model called combinatorial TF Oriented Program (cTOP), which integrates single-cell data with bulk regulatory networks and find PRDM1 , FOSL1 , BACH1 , and NFATC1 as potential pivotal factors in antler stem cell activation and osteogenic differentiation. Additionally, we uncover interactions within and between cell programs and pathways during the regeneration process. These findings provide insights into the gene and cell regulatory mechanisms of antler regeneration, particularly in stem cell activation and differentiation., (© 2025 Li et al.; Published by Cold Spring Harbor Laboratory Press.)

SCAPs (Stem Cells from Apical Papilla), derived from the apex of forming wisdom teeth, extracted from teenagers for orthodontic reasons, belong to the MSCs (Mesenchymal Stromal Cells) family. They have multipotent differentiation capabilities and are a potentially powerful model for investigating strategies of clinical cell therapies. Since autophagy-a regulated self-eating process-was proposed to be essential in osteogenesis, we investigated its involvement in the SCAP model. By using a combination of chemical and genetic approaches to inhibit autophagy, we studied early and late events of osteoblastic differentiation. We showed that blocking the formation of autophagosomes with verteporfin did not induce a dramatic alteration in early osteoblastic differentiation monitored by ALP (alkaline phosphatase) activity. However, blocking the autophagy flux with bafilomycin A1 led to ALP repression. Strikingly, the mineralization process was observed with both compounds, with calcium phosphate (CaP) nodules that remained inside cells under bafilomycin A1 treatment and numerous but smaller CaP nodules after verteporfin treatment. In contrast, deletion of Atg5 or Atg7 , two genes involved in the formation of autophagosomes and essential to trigger canonical autophagy, indicated that both genes could be involved differently in the mineralization process with a modification of the ALP activity while final mineralization was not altered.

Tooth/skeletal dysplasia, such as hypophosphatasia (HPP), has been extensively studied. However, there are few definitive treatments for these diseases owing to the lack of an in vitro disease model. Cells differentiated from patient-derived induced pluripotent stem cells (iPSCs) demonstrate a pathological phenotype. This study aimed to establish a method for fabricating hard tissue-forming cells derived from human iPSCs (hiPSCs) for the pathological analysis of tooth/skeletal dysplasia. Healthy (HLTH) adult-derived hiPSCs were cultured in a hard tissue induction medium (HM) with or without retinoic acid (RA) under 3D culture conditions, and mineralization and expression of dentinogenesis- and osteogenesis-related markers in 3D hiPSC constructs were evaluated. hiPSCs derived from patients with hypophosphatasia were also cultured in HM with RA. HLTH-derived hiPSCs formed mineralized 3D constructs and showed increased expression of dentinogenesis- and osteogenesis-related markers; addition of RA promoted the expression of these markers in hiPSC constructs. HPP-derived hiPSC constructs showed lower mineralization and expression of dentinogenesis- and osteogenesis-related markers than HLTH-derived hiPSCs, indicating an impaired ability to differentiate into odontoblasts and osteoblasts. This method for fabricating 3D hiPSC constructs allows for simultaneous assessment of dentinogenesis and osteogenesis, with HPP-derived hiPSC constructs recapitulating pathological phenotypes.

Adipose-derived mesenchymal stem cells (ASCs) are commonly employed in clinical treatment for various diseases due to their ability to differentiate into multi-lineage and anti-inflammatory/immunomodulatory properties. Preclinical studies support their use for bone regeneration, healing, and the improvement of functional outcomes. However, a deeper understanding of the molecular mechanisms underlying ASC biology is crucial to identifying key regulatory pathways that influence differentiation and enhance regenerative potential. In this study, we employed the NanoString nCounter technology, an advanced multiplexed digital counting method of RNA molecules, to comprehensively characterize differentially expressed transcripts involved in metabolic pathways at distinct time points in osteogenically differentiating ASCs treated with or without the pan-DNMT inhibitor RG108. In silico annotation and gene ontology analysis highlighted the activation of ethanol oxidation, ROS regulation, retinoic acid metabolism, and steroid hormone metabolism, as well as in the metabolism of lipids, amino acids, and nucleotides, and pinpointed potential new osteogenic drivers like AOX1 and ADH1A. RG108-treated cells, in addition to the upregulation of the osteogenesis-related markers RUNX2 and ALPL, showed statistically significant alterations in genes implicated in transcriptional control (MYCN, MYB, TP63, and IRF1), ethanol oxidation (ADH1C, ADH4, ADH6, and ADH7), and glucose metabolism (SLC2A3). These findings highlight the complex interplay of the metabolic, structural, and signaling pathways that orchestrate osteogenic differentiation. Furthermore, this study underscores the potential of epigenetic drugs like RG108 to enhance ASC properties, paving the way for more effective and personalized cell-based therapies for bone regeneration.

The osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is key for bone formation, and its imbalance leads to osteoporosis. Forkhead Box Protein G1 (FOXG1) is associated with osteogenesis, however, the effect of FOXG1 on osteogenesis of BMSCs and ovariectomy (OVX)-induced bone loss is unknown. In our study, FOXG1 expression in BMSCs increases after osteogenic induction. FOXG1 overexpression significantly increases osteoblast marker expression ALP activity, and calcium deposition, while the opposite results are observed in FOXG1 knockdown BMSCs, suggesting that FOXG1 promotes osteogenic differentiation. Additionally, autophagy promotes the differentiation process in BMSCs. We find that FOXG1 induces autophagy, and osteogenic differentiation is blocked via inhibiting FOXG1-caused autophagy, indicating that FOXG1 accelerates osteogenic differentiation via inducing autophagy. Eight-week-old female C57BL/6J mice are used in OVX models, FOXG1 overexpression decreases bone loss by increasing bone formation. Moreover, FOXG1 overexpression suppresses osteoclast differentiation. Mechanically, FOXG1 transcriptionally represses ubiquitin-specific protease14 (USP14) via binding to the USP14 promoter. USP14 overexpression prevents the promoting effect of FOXG1 on osteogenic differentiation in BMSCs. Therefore, our findings suggest that FOXG1 promotes BMSC osteogenic differentiation and inhibits osteoclast differentiation, eventually blocking OVX-induced bone loss, which may provide a promising approach for osteoporosis treatment., Competing Interests: Competing interests: The authors report no competing interest., (© 2025. The Author(s).)

Background: Static magnetic field (SMF) as an effective physical stimulus is capable of osteogenic differentiation for multiple mesenchymal stem cells, including human periodontal ligament stem cells (hPDLSCs). However, the exact molecular mechanism is still unknown. Therefore, this study intends to excavate molecular mechanisms related to SMF in hPDLSCs using functional experiments., Methods: hPDLSCs were treated with different intensities of SMF, H19 lentivirus, and Wnt/β-catenin pathway inhibitor (XAV939). Changes in osteogenic markers (Runx2, Col Ⅰ, and BMP2), Wnt/β-catenin markers (β-catenin and GSK-3β), and calcified nodules were examined using RT-qPCR, western blotting, and alizarin red staining in hPDLSCs., Results: SMF upregulated the expression of H19, and SMF and overexpressing H19 facilitated the expression of osteogenic markers (Runx2, Col Ⅰ, and BMP2), activation of the Wnt/β-catenin pathway, and mineralized sediment in hPDLSCs. Knockdown of H19 alleviated SMF function, and treatment with XAV939 limited SMF- and H19-mediated osteogenic differentiation of hPDLSCs. Notably, the expression of hsa-miR-532-3p, hsa-miR-370-3p, hsa-miR-18a-5p, and hsa-miR-483-3p in hPDLSCs was regulated by SMF, and may form an endogenous competitive RNA mechanism with H19 and β-catenin., Conclusion: SMF contributes to the osteogenic differentiation of hPDLSCs by mediating the H19/Wnt/β-catenin pathway, and hsa-miR-532-3p, hsa-miR-370-3p, hsa-miR-18a-5p, and hsa-miR-483-3p may be the key factors in it., 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.)

The calvarial bones of the infant skull are linked by transient fibrous joints known as sutures and fontanelles, which are essential for skull compression during birth and expansion during postnatal brain growth. Genetic conditions caused by pathogenic variants in FGFR2, such as Apert, Pfeiffer, and Crouzon syndromes, result in calvarial deformities due to premature suture fusion and a persistently open anterior fontanelle (AF). In this study, we investigated how Fgfr2 regulates AF closure by leveraging mouse genetics and single-cell transcriptomics. We find that AF cells, marked by the tendon/ligament factor SCX, are spatially organized into ecto- and endocranial domains that selectively differentiate into ligament, bone, and cartilage to form the posterior frontal suture. We show that AF cell differentiation is non-autonomously regulated by FGFR2 signaling in osteogenic front cells of the frontal bones, which regulate WNT signaling in neighboring AF cells by expressing the secreted WNT inhibitor Wif1. Upon loss of Fgfr2, Wif1 expression is downregulated, and AF cells fail to form the posterior frontal suture. This study identifies an FGF-WNT signaling circuit that that directs suture formation within the AF during postnatal development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

Postmenopausal osteoporosis, primarily driven by estrogen deficiency, is predominantly mediated through estrogen receptors such as ERα. However, the underlying mechanisms necessitate further investigation. In this study, we established an ERα-deficient model in rBMSCs to elucidate the role of ERα in osteogenic differentiation and miRNA expression profiles. Our findings demonstrate that knockdown of ERα inhibits osteogenic differentiation in rBMSCs, resulting in upregulation of 25 miRNAs and downregulation of 184 miRNAs, including a significant increase in the expression of miR-214-3p. Validation using qPCR, Western blotting, and bioinformatics analysis revealed that miR-214-3p negatively regulates osteogenic differentiation via the Wnt/β-catenin signaling pathway. Furthermore, we explored the potential therapeutic effects of quercetin (QUE) on rBMSCs. CCK8, alkaline phosphatase activity assays, and Alizarin Red staining demonstrated that QUE dose-dependently enhances rBMSCs proliferation, alkaline phosphatase activity, and mineralization within the concentration range of 0.1-1 μM. Importantly, QUE was found to downregulate miR-214-3p expression and activate the Wnt3a/β-catenin signaling pathway. Rescue experiments confirmed that QUE could counteract the inhibitory effects of miR-214-3p on the Wnt3a/β-catenin signaling pathway. Collectively, our study provides compelling evidence that knockdown of ERα inhibits the osteogenic differentiation of rBMSCs by affecting the miRNA expression profile, while QUE can reverse the inhibitory effect exerted by miR-214-3p on the Wnt3a/β-catenin signaling pathway, thereby offering novel insights into diagnosis, prevention, and treatment strategies for postmenopausal 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: La-related protein 7 (LARP7) is a key regulator of RNA metabolism and is thought to play a role in various cellular processes. LARP7 gene autosomal recessive mutations are the cause of Alazami syndrome, which presents with skeletal abnormalities, intellectual disabilities, and facial dysmorphisms. This study aimed to determine the role of LARP7 in modulating gene expression dynamics during osteogenesis., Methods and Results: First, the temporal expression profile of the LARP7 gene during various stages of osteogenesis was examined. Then, RNA interference-mediated knockdown of LARP7 was implemented and high-throughput RNA-seq analysis was performed in order to identify global gene expression changes associated with knockdown of LARP7. The findings show there were significant alterations in the overall gene expression profile. The observed down-regulation in extracellular matrix (ECM) component genes suggests that it might lead to impairments in the structure and function of the bone matrix. Additionally, modulation of alternative splicing events were observed, especially in the RUNX2 and SPP1, indicating the potential contribution of LARP7 to the phenotypic features observed in Alazami syndrome., Conclusion: Overall, the findings clarify the regulatory mechanisms of LARP7 in osteogenic differentiation and illuminate potential avenues for therapeutic interventions in patients with skeletal disorders., Competing Interests: Declarations. Ethical approval: Not applicable. Consent to participate: Not applicable. Consent to publish: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

Bone is a multifaceted tissue requiring orchestrated interplays of diverse cells within specialized microenvironments. Although significant progress has been made in understanding cellular and molecular mechanisms of component cells of bone, revealing their spatial organization and interactions in native bone tissue microenvironment is crucial for advancing precision medicine, as they govern fundamental signaling pathways and functional dependencies among various bone cells. In this study, we present the first integrative high-resolution map of human bone and bone marrow, using spatial and single-cell transcriptomics profiling from femoral tissue. This multi-modal approach discovered a novel bone formation-specialized niche enriched with osteoblastic lineage cells and fibroblasts and unveiled critical cell-cell communications and co-localization patterns between osteoblastic lineage cells and other cells. Furthermore, we discovered a novel spatial gradient of cellular composition, gene expression and signaling pathway activities radiating from the trabecular bone. This comprehensive atlas delineates the intricate bone cellular architecture and illuminates key molecular processes and dependencies among cells that coordinate bone metabolism. In sum, our study provides an essential reference for the field of bone biology and lays the foundation for advanced mechanistic studies and precision medicine approaches in bone-related disorders., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: Fracture is a common traumatic disease and there is a risk of delayed healing after fracture occurs. This study aimed to explore the regulatory roles and clinical implications of OIP5-AS1 in delayed fracture healing., Methods: The study included 80 normal fracture healing patients and 80 delayed fracture healing patients. RT-qPCR was used to assess the levels of OIP5-AS1 and miR-7-5p in the serum of patients and MC3T3-E1 cells. The ROC curve was utilized to evaluate the predictive value of OIP5-AS1 for delayed fracture healing. Dual-luciferase reporter assays and RIP assays were conducted to validate the interaction between OIP5-AS1 and miR-7-5p. Cell viability and apoptosis were determined by CCK-8 and flow cytometry., Results: OIP5-AS1 was abnormally elevated in the serum of delayed fracture healing patients, and OIP5-AS1 had a predictive value for delayed fracture healing. Cell experiments demonstrated that overexpression of OIP5-AS1 significantly inhibited osteogenic differentiation, reduced cell viability, and stimulated apoptosis. miR-7-5p was identified as the target miRNA of OIP5-AS1 and was negatively regulated by OIP5-AS1. Furthermore, transfecting with miR-7-5p mimic significantly alleviated the inhibitory effect of OIP5-AS1 on osteoblast activity., Conclusion: OIP5-AS1 was identified as a potential biomarker for predicting delayed fracture healing. Additionally, it could inhibit fracture healing through the sponge effect on miR-7-5p., Competing Interests: Declarations. Ethics approval and consent to participate: The study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Changde Hospital, Xiangya School of Medicine, Central South University (The First People’s Hospital of Changde city) before the study began. The written informed consent has been obtained from the participants involved. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Objective: Osteoporosis increases the risk of fragility fractures, impacting patients' lives. This study aimed to investigate whether LINC01271 was involved in the process of fragility fractures and healing, providing a new perspective for its diagnosis and treatment., Methods: This study included 94 healthy individuals, 82 patients with osteoporosis, and 85 patients with fragility fractures as subjects. RT-qPCR was used to measure the levels of LINC01271, miR-19a-3p, PIK3CA, and osteogenic differentiation markers in osteoblasts and subjects' serum. Luciferase reporter assays, RIP experiments, and RNA pull-down assays were utilized to verify the target relationships between LINC01271 and miR-19a-3p, as well as between miR-19a-3p and PIK3CA. Cell proliferation and apoptosis were assessed using CCK-8 assays and flow cytometry., Results: Compared to the healthy control group, the serum levels of LINC01271 were significantly reduced in patients with osteoporosis and fragility fractures. Furthermore, LINC01271 levels increased with time-dependent fracture healing. In vitro studies indicated that LINC01271 boosted osteoblast proliferation, inhibited apoptosis, and augmented osteogenic differences, whereas its inhibition reverses their effects. LINC01271 and PIK3CA were identified as targets of miR-19a-3p, and overexpression of miR-19a-3p could antagonize the effect of LINC01271 in promoting fracture healing., Conclusion: The results indicated that LINC01271 may play a key role in osteoblast function and fracture healing through its interaction with miR-19a-3p and regulation of PIK3CA., Competing Interests: Declarations. Ethics approval and consent to participate: The study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Affiliated Hospital of Zunyi Medical University before the study began. The participants’ right to be informed about the study was ensured and agreed to participate in the study. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into various lineages. They have also the potential to protect themselves against harmful stimuli to maintain their functional integrity. Drug resistance-related transporters such as ABCB1 (P-glycoprotein; P-gp), ABCC1 (MRP1; multidrug resistance-related Protein 1), and LRP (lung resistance protein) may protect MSCs against toxic substances such as chemotherapeutic agents. This study evaluated ABCB1, ABCC1, and LRP before and after the differentiation of MSCs derived from human amniotic fluid (AF) and bone marrow (BM). P-gp expression in both AFMSCs and BMMSCs was analyzed by immunocytochemistry, and pump function was analyzed by cell viability assay with doxorubicin (DOX) and Rhodamine 123 (Rh 123) dye exclusion. ABCB1, ABCC1, and LRP gene expression was determined by RT-PCR both before and after osteogenic and adipogenic differentiation. The MES-SA/DX5 cell line was used as a model of resistance to DOX and the overexpression of P-gp. Both AFMSCs and BMMSCs displayed a high P-gp expression, although lower than MES-SA/DX5 control cells. It was shown that both, undifferentiated AFMSCs and BMMSCs, have high cell viability in response to DOX, similar to the MES-SA/DX5 lineage. ABCB1 was less expressed in BM than in AFMSCs in undifferentiated samples, while no differences were observed in the expression of ABCC1 and LRP. AFMSCs showed an increase in ABCB1 after osteogenic differentiation, whereas BMMSCs exhibited lower ABCB1 and ABCC1 expression after osteogenic and adipogenic differentiation. The findings suggest that ABCB1, ABCC1, and LRP gene expression in AFMSCs and BMMSCs is influenced by differentiation processes and further support the concept that these transporters modulate MSC differentiation in a cell source-dependent way.

This study assessed the novel concept that osteoclast-derived Grem1 has regulatory functions in the skeletal response to calcium stress using an osteoclastic Grem1 conditional knockout (cKO) mouse model. The calcium stress was initiated by feeding cKO mutants and wildtype (WT) littermates a calcium-deficient diet for 2 weeks. Deletion of Grem1 in mature osteoclasts did not affect developmental bone growth nor basal bone turnover. In response to calcium depletion, male cKO mutants showed greater increases in osteoclastic resorption and trabecular bone loss than male WT littermates, indicating an enhanced skeletal sensitivity to calcium depletion in male mutants. The enhanced sensitivity to calcium depletion was sex-dependent, as female cKO mutants showed lower increases in osteoclastic resorption and bone loss than female WT littermates as well as male cKO mutants. The sex disparity in osteoclastic resorption response to calcium stress was intrinsic to osteoclasts since osteoclasts of male but not female cKO mutants showed greater in vitro bone resorption activity than osteoclasts of WT littermates of respective sex. Male cKO mutants displayed smaller bone formation response to calcium depletion than male WT littermates, while female mutants showed bigger bone formation response than female WT littermates, indicating that cKO mutants also displayed sex disparity in bone formation response. The sex disparity in bone formation response was not caused by intrinsic differences in osteoblasts but might be due to sex-dependent differential osteoclastic release of osteogenic factors. In summary, osteoclast-derived gremlin-1 has complicated and sex-dependent regulatory roles in skeletal response to calcium stress., Competing Interests: Declarations. Conflict of Interest: All authors (Matilda H.-C. Sheng, Charles H. Rundle, David J. Baylink, and Kin-Hing William Lau) state that they have no conflicts of interest.                                                           Human and Animal Rights and Informed Consent: The animal protocol (protocol #1298) was reviewed and approved by the Animal Care and Use Committee (IACUC) of VA Loma Linda Healthcare System as well as by the IACUC of the Loma Linda University and the Animal Care and Use Review Office (ACURO) of the US Army Medical Research and Materiel Command of the Department of Defense. In conducting research using animals, the investigators adhered to the Animal Welfare Act Regulations and other federal statures relating to animals and animal experimentation and the principles set forth in the current version of the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules. Disclaimers: The views, opinions and/or findings contained in this report are those of the authors and should not be construed as an official position, policy or decision of the US Army or the United States government, unless so designated by other documentation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

It has been well validated that chronic psychological stress leads to bone loss, but the underlying mechanism remains unclarified. In this study, we established and analyzed the chronic unpredictable mild stress (CUMS) mice to investigate the miRNA-related pathogenic mechanism involved in psychological stress-induced osteoporosis. Our result found that these CUMS mice exhibited osteoporosis phenotype that is mainly attributed to the abnormal activities of osteoclasts. Subsequently, miRNA sequencing and other analysis showed that miR-335-3p, which is normally highly expressed in the brain, was significantly downregulated in the nucleus ambiguous, serum, and bone of the CUMS mice. Additionally, in vitro studies detected that miR-335-3p is important for osteoclast differentiation, with its direct targeting site in Fos . Further studies demonstrated FOS was upregulated in CUMS osteoclast, and the inhibition of FOS suppressed the accelerated osteoclastic differentiation, as well as the expression of osteoclastic genes, such as Nfatc1, Acp5, and Mmp9 , in miR-335-3p-restrained osteoclasts. In conclusion, this work indicated that psychological stress may downregulate the miR-335-3p expression, which resulted in the accumulation of FOS and the upregulation of NFACT1 signaling pathway in osteoclasts, leading to its accelerated differentiation and abnormal activity. These results decipher a previously unrecognized paradigm that miRNA can act as a link between psychological stress and bone metabolism., Competing Interests: JZ, JL, JH, XX, RL, YZ, SL, WL No competing interests declared, (© 2024, Zhang et al.)

Osteogenic differentiation is crucial in normal bone formation and pathological calcification, such as calcific aortic valve disease (CAVD). Understanding the proteomic and transcriptomic landscapes underlying this differentiation can unveil potential therapeutic targets for CAVD. In this study, we employed RNA sequencing transcriptomics and proteomics on a timsTOF Pro platform to explore the multiomics profiles of valve interstitial cells (VICs) and osteoblasts during osteogenic differentiation. For proteomics, we utilized 3 data acquisition/analysis techniques: data-dependent acquisition (DDA)-parallel accumulation serial fragmentation (PASEF) and data-independent acquisition (DIA)-PASEF with a classic library-based (DIA) and machine learning-based library-free search (DIA-ML). Using RNA sequencing data as a biological reference, we compared these 3 analytical techniques in the context of actual biological experiments. We use this comprehensive dataset to reveal distinct proteomic and transcriptomic profiles between VICs and osteoblasts, highlighting specific biological processes in their osteogenic differentiation pathways. The study identified potential therapeutic targets specific for VICs osteogenic differentiation in CAVD, including the MAOA and ERK1/2 pathway. From a technical perspective, we found that DIA-based methods demonstrate even higher superiority against DDA for more sophisticated human primary cell cultures than it was shown before on HeLa samples. While the classic library-based DIA approach has proved to be a gold standard for shotgun proteomics research, the DIA-ML offers significant advantages with a relatively minor compromise in data reliability, making it the method of choice for routine proteomics., (© The Author(s) 2025. Published by Oxford University Press GigaScience.)

This study investigates the mechanism of PHF20 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). BMSCs from Balb/c mouse were cultured and identified through osteogenesis, adipogenesis, and flow cytometry. After osteogenic induction, the levels of OPN and OCN in BMSCs were detected by RT-qPCR. Alizarin red staining and alkaline phosphatase (ALP) staining were used to evaluate the osteogenic differentiation ability of BMSCs. PHF20, METTL14, and HOXA13 expressions were detected by RT-qPCR and Western blot. After quantitative analysis of m6A level, RNA immunoprecipitation (RIP) was performed to measure the enrichment of IGF2BP3 or m6A on HOXA13 mRNA. HOXA13 mRNA stability was assessed after actinomycin D treatment. PHF20, METT14, and HOXA13 expressions gradually increased during osteogenic differentiation of BMSCs. Suppression of PHF20 expression repressed the osteogenic differentiation of BMSCs, mainly resulted in a decrease in OPN and OCN levels, reduced mineralization, and weakened ALP activity. Mechanistically, PHF20 elevated METTL14 expression by enhancing the enrichment of H3K4me3 on its promoter, and METTL14 strengthened HOXA13 m6A methylation to maintain HOXA13 mRNA stability through IGF2BP3. In conclusion, PHF20 elevates METTL14 expression by enhancing H3K4me3 enrichment on its promoter and enhances HOXA13 mRNA stability via IGF2BP3-mediated m6A modification, thus facilitating HOXA13 expression and eventually inducing osteogenic differentiation of BMSCs., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Osteoporosis is characterized by decreased bone mass and accumulation of adipocytes in the bone marrow. The mechanism underlying the imbalance between osteoblastogenesis and adipogenesis in bone marrow mesenchymal stem cells (BMSCs) remains unclear. We found that ALG5 was significantly downregulated in BMSCs from osteoporotic specimens. ALG5 knockdown inhibited osteogenic differentiation and increased adipogenic differentiation of BMSCs. ALG5 deficiency diminished the N-glycosylation of SLC6A9, thereby altering its protein stability and disrupting SLC6A9-mediated glycine uptake in BMSCs. ALG5 overexpression by adeno-associated virus serotype 9 (rAAV9) alleviated bone loss in OVX mice. Taken together, our findings suggest a novel role for the ALG5-SLC6A9-glycine axis in the imbalance of BMSC differentiation in osteoporosis. Moreover, we identify ALG5 overexpression as a potential therapeutic strategy for treating osteoporosis., Competing Interests: Declarations. Conflict of interests: The authors declare that they have no competing interests. Ethical approval: The animal experimentation protocols received approval from the Institutional Animal Care and Use Committee of Sun Yat-Sen University. The assigned approval numbers for the experiments were 2022003644, 2023000742, and 2023001223. Additionally, collection and experimentation involving human specimens were ethically approved by the Ethics Committee of the Eighth Affiliated Hospital, Sun Yat-Sen University, with the respective approval numbers for human subject handling being 2021r037 and 2022r023. Consent for publication: All authors have read and approved the manuscript., (© 2025. The Author(s).)

Lateral Meningocele Syndrome (LMS), a disorder associated with NOTCH3 pathogenic variants, presents with neurological, craniofacial and skeletal abnormalities. Mouse models of the disease exhibit osteopenia that is ameliorated by the administration of Notch3 antisense oligonucleotides (ASO) targeting either Notch3 or the Notch3 mutation. To determine the consequences of LMS pathogenic variants in human cells and whether they can be targeted by ASOs, induced pluripotent NCRM1 and NCRM5 stem (iPS) cells harboring a NOTCH36692-93insC insertion were created. Parental iPSCs, NOTCH36692-93insC and isogenic controls, free of chromosomal aberrations as determined by human CytoSNP850 array, were cultured under conditions of neural crest, mesenchymal and osteogenic cell differentiation. The expected cell phenotype was confirmed by surface markers and a decline in OCT3/4 and NANOG mRNA. NOTCH36692-93insC cells displayed enhanced expression of Notch target genes HES1, HEY1, 2 and L demonstrating a NOTCH3 gain-of-function. There was enhanced osteogenesis in NOTCH36692-93insC cells as evidenced by increased mineralized nodule formation and ALPL, BGLAP and BSP expression. ASOs targeting NOTCH3 decreased both NOTCH3 wild type and NOTCH36692-93insC mutant mRNA by 40% in mesenchymal and 90% in osteogenic cells. ASOs targeting the NOTCH3 insertion decreased NOTCH36692-93insC by 70-80% in mesenchymal cells and by 45-55% in osteogenic cells and NOTCH3 mRNA by 15-30% and 20-40%, respectively. In conclusion, a NOTCH3 pathogenic variant causes a modest increase in osteoblastogenesis in human iPS cells in vitro and NOTCH3 and NOTCH3 mutant specific ASOs downregulate NOTCH3 transcripts associated with LMS., Competing Interests: PJ and MC are paid employees of Ionis Pharmaceuticals. Please note that the synthesis and applications of antisense oligonucleotides may be covered by patent(s) filed by Ionis Pharmaceuticals. Individuals wanting to obtain antisense oligonucleotides from Ionis Pharmaceuticals are required to contact Ionis Pharmaceuticals directly. This does not alter our adherence to PLOS ONE policies on sharing data and materials.” as detailed in http://journals.plos.org/plosone/s/competing-interests, (Copyright: © 2025 Canalis 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.)

This study assessed the feasibility of miR17 ~ 92-based antiresorptive strategy by determining the effects of conditional transgenic (cTG) overexpression of miR17 ~ 92 in myeloid cells on bone and osteoclasts. Osteoclasts of male and female cTG mutant mice each showed 3- to fivefold overexpression of miR17 ~ 92 cluster genes compared to those of age- and sex-matched wildtype (WT) littermates. Male but not female cTG mutant mice had more trabecular and cortical bones as well as lower bone resorption reflected by reduction in osteoclast number and resorbing surface. Osteoclasts of male but not female cTG mutants showed decreased bone resorption activity. Consistent with suppression of osteoclast maturation, osteoclasts of male cTG mutants were smaller, contained less nuclei, showed reduced levels of mRNA of genes associated with osteoclast differentiation and fusion, and formed more diffused actin ring. Osteoclastic overexpression of miR17 ~ 92 also increased bone formation, but the increase was much larger in males than in females. The increase in male mutants was due to higher mineral apposition rate, and conversely, it was caused by increasing bone-forming surface in female mutants. In summary, osteoclastic overexpression of miR17 ~ 92 increased bone mass through reduction in bone resorption along with coupled increase in bone formation in male-specific manner. Although the osteoclastic overexpression of miR17 ~ 92-induced suppression of bone resorption and increases in bone formation support the feasibility of miR17 ~ 92-based antiresorptive strategies, the male-specific sexual disparity in skeletal responses to osteoclastic overexpression of miR17 ~ 92 could limit its clinical utility as it may not be used in women with postmenopausal osteoporosis., Competing Interests: Declarations. Competing Interest: All authors state that they have no conflicts of interest. Human and Animal Rights and Informed Consent: All animal protocols (protocol #0047/1160) were reviewed and approved by the Animal Care and Use Committee of VA Loma Linda Healthcare System. In conducting research using animals, the investigators adhered to the Animal Welfare Act Regulations and other federal statures relating to animals and animal experimentation and the principles set forth in the current version of the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Bone remodeling maintains the robustness of the bone tissue by balancing bone resorption by osteoclasts and bone formation by osteoblasts. Although these cells together play a crucial role in bone remodeling, only a few reports are available on the common factors involved in the differentiation of the two types of cells. Here, we show family with sequence similarity 102 member A (Fam102a) as a bone-remodeling factor that positively regulates both osteoclast and osteoblast differentiation. Fam102a regulates osteoblast differentiation by controlling recombination signal binding protein for immunoglobulin κ J region-like (Rbpjl). The Fam102a-Rbpjl axis promotes the nuclear translocation of transcription factors and enhances the expression of Osterix, a transcription factor essential for osteoblast differentiation. The deletion of Fam102a or a functional mutation in Rbpjl leads to osteopenia accompanied by reduced osteoblastic bone formation. Thus, the Fam102a-Rbpjl axis plays an important role in osteoblasts and this finding provides insights into bone remodeling., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Purpose: Postmenopausal osteoporosis (PMO) is mainly concerned with the imbalance of bone resorption and bone formation. Icariin (ICA) plays a vital role in bone protection. This study investigated the mechanism of ICA in PMO rats., Methods: The rats were treated with ovariectomy (OVX) and ICA. Bone structure parameters were measured by Micro-CT. BMSCs were obtained from normal rats, OVX rats, and ICA-treated rats. BMSCs were infected with SOST overexpression lentivirus, and TWS119, an activator of Wnt pathway, was introduced for joint experiment. The binding of ERα to SOST promoter was verified. OVX/ICA rats were injected with DNA methyltransferase inhibitor 5-Aza-dC., Result: ICA increased bone mass and decreased bone marrow fat content in OVX rats. ICA facilitated osteogenic differentiation and repressed adipogenic differentiation of BMSCs. Overexpressing SOST antagonized the effect of ICA, whereas TWS119 rescued the effect of overexpressing SOST. ICA reduced SOST expression by attenuating the effect of ERα. Methylation of SOST inhibited ERα binding to SOST promoter. In vivo experiments confirmed that ICA improved bone mass and reduced bone marrow fat content by enhancing SOST methylation., Conclusion: Overall, ICA upregulated SOST methylation and inhibited the binding of ERα to SOST promoter, thereby promoting osteogenic differentiation and repressing adipogenic differentiation of BMSCs., (© 2025 John Wiley & Sons Ltd.)

Osteogenic differentiation of bone marrow stem cells (BMSCs) is essential for bone tissue regeneration and repair. However, this process is often hindered by an unstable differentiation influenced by local microenvironmental factors. While small extracellular vesicles (sEVs) derived from osteogenically induced adipose mesenchymal stem cells (ADSCs) reportedly can promote osteogenic differentiation of BMSCs, the underlying molecular mechanisms remain incompletely understood. In this study, we investigated the mRNA expression profile of ADSC-sEVs + and explored the role of specific mRNAs in the osteogenic differentiation of BMSCs. We first validated the osteogenic induction activity of ADSC-sEVs + through both in vitro and in vivo experiments. Using reverse transcription polymerase chain reaction, we compared mRNA expression between ADSC-sEVs + and ADSC-sEVs and further assessed the impact of specific mRNAs on the differentiation of BMSCs through a series of in vitro experiments. One of our key findings was that osterix mRNA was highly enriched in ADSC-sEVs + , which significantly enhanced alkaline phosphatase staining and upregulated downstream osteoblastic markers in BMSCs. Both overexpression and knockdown experiments confirmed that osterix mRNA is a critical signalling molecule that facilitates the differentiation of BMSCs into osteoblasts through ADSC-sEVs + . This finding expands our understanding of the molecular mechanisms underlying the osteogenic differentiation of BMSCs and offers a promising strategy for targeted osteoblastic differentiation in clinical applications., (© 2025 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

In this study, we explored the impact of different biomechanical loadings on lumbar spine motion segments, particularly concerning intervertebral disc degeneration (IVDD). We aimed to uncover the cellular milieu and mechanisms driving ossification in the nucleus pulposus (NP) during IVDD, a process whose underlying mechanisms have remained elusive. The study involved the examination of fresh NP tissue from the L3-S1 segment of five individuals, either with IVDD or healthy. The analysis consisted of histopathological evaluation and single-cell RNA sequencing. To further validate the impact of biomechanical loading on IVDD, particularly on the CITED4 + METRN + NP chondrocytes and the bone-fat balance mechanism, a retrospective analysis was conducted using paraffin-embedded NP samples from patients. A distinct subset of CITED4 + METRN+ chondrocytes in the degenerated NP that were influenced by biomechanical loading was identified. These cells were evaluated for their potential as diagnostic biomarkers. Pseudotemporal analysis indicated that inflammation and repair processes were integral to NP ossification. Notably, the L4/5 and L5/S1 segments with severe IVDD showed pronounced ossification and heightened lipogenic metabolism. Cell communication analysis sheds light on the roles of bone-fat balance proteins and various ossification genes. Additionally, immunohistochemistry and immunofluorescence confirmed that biomechanical loading intensified IVDD by fostering osteogenic differentiation, mediated by macrophage migration inhibitory factor (MIF)-regulated bone-fat balance. This research reveals the microenvironmental factors of IVDD NP ossification under biomechanical loading, highlighting the role of bone-fat imbalance. These insights significantly enhance the understanding of IVDD pathogenesis and pave the way for innovative therapeutic approaches., (© 2025 Wiley Periodicals LLC.)

MicroRNAs (miRNAs) modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. Defects in bone homeostasis result in common age-related diseases including osteoporosis. Serum levels of miR-324-3p positively correlate with several features of bone maintenance. In contrast here, using in vivo micro-computed tomography and histology, global miR-324-null mice demonstrated increased bone mineral density and both trabecular and cortical thickness, with effect magnitudes increasing with age. The bone marrow of miR-324-null mice had reduced lipid content while TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation. Ex vivo assays showed that the high bone mass phenotype of miR-324-null mice resulted from both increased osteoblast activity and decreased osteoclastogenesis. RNA-seq analysis of osteoblasts, osteoclasts and bone marrow macrophages and target validation assays identified that the osteoclast fusion regulator Pin1 and the master osteogenic regulator Runx2 were targets of miR-324-5p in osteoclast lineage cells and osteoblasts, respectively. Indeed, in vitro Runx2 overexpression recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo by the loss of miR-324. Overall, these data demonstrate the importance of miR-324 in bone homeostasis by regulating aspects of both bone formation and remodelling. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Macrophages play a pivotal role in regulating inflammatory response in periodontitis, a condition characterized by excessive osteoclast differentiation. This study aimed to investigate whether exosomes derived from M2 macrophages regulate osteoclast differentiation and to identify the underlying molecular mechanisms. Exosomes were isolated from M2 macrophages and used to treat osteoclasts. Osteoclastogenesis was assessed using tartrate-resistant acid phosphatase staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The molecular mechanism was evaluated using microarray analysis, RT-qPCR, dual-luciferase reporter analysis, and RNA pull-down assay. The results showed that exosomes from M2 macrophages inhibited receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclast differentiation. Additionally, miR-1227-5p expression in osteoclasts was increased after treatment with exosomes, and inhibition of miR-1227-5p counteracted the suppressive effects of exosomes on osteoclastogenesis. Moreover, OSCAR is a target of miR-1227-5p. In conclusion, exosomal miR-1227-5p suppresses osteoclast differentiation, potentially via targeting OSCAR. These findings provide new insights into the pathogenesis of periodontitis., 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 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The development of bone implants through bioinspired immobilization of growth factors remains a key issue in the generation of biological interfaces, especially in enhancing osteodifferentiation ability. In this study, we developed a strategy for surface functionalization of poly(lactide-glycolide) (PLGA) and hydroxyapatite (HA) composite substrates through site-specific conjugation of bone morphogenetic protein 2 containing 3,4-hydroxyphenalyalanine (DOPA-BMP2) mediated by tyrosinase and sortase A (SrtA). Firstly, the growth factor BMP2-LPETG containing LPETG motif was successfully expressed in Escherichia coli through recombinant DNA technology. The excellent binding affinity of binding growth factor (DOPA-BMP2) was achieved by converting the tyrosine residue (Y) of YKYKY-GGG peptide into DOPA (X) by tyrosinase, which bound to the substrates. Then its GGG motif was specifically bound to the end of BMP2-LPETG mediated by SrtA. Therefore, the generated bioactive DOPA-BMP2/PLGA/HA substrates significantly promoted the osteogenic differentiation of MC3T3-E1 cells. Thanks to this microbial-assisted engineering approach, our work presents a facile and highly site-specific strategy to engineer biomimetic materials for orthopedics and dentistry by effectively delivering growth factors, peptides, and other biomacromolecules., (© 2024 Wiley Periodicals LLC.)

Osteogenic differentiation of mesenchymal stem cells (MSCs) plays a pivotal role in the pathogenesis and treatment of bone-related conditions such as osteoporosis and bone regeneration. While the WW domain-containing coiled-coil adaptor (WAC) protein is primarily associated with transcriptional regulation and autophagy, its involvement in MSC osteogenesis remains unclear. Here, the data reveal that the levels of WAC are diminished in both osteoporosis patients and osteoporosis mouse models. It plays a pivotal function in facilitating MSC osteogenesis and enhancing new bone formation both in vitro and in vivo. Mechanistically, WAC promotes MSC osteogenesis by protecting PINK1, a crucial initiator of mitophagy, from ubiquitination-dependent degradation thereby activating mitophagy. Interestingly, WAC interacts with the TM domains of PINK1 and prevents the K137 site from ubiquitination modification. The study elucidates the mechanism by which WAC modulates MSC osteogenesis, binds to PINK1 to protect it from ubiquitination, and identifies potential therapeutic targets for osteoporosis and bone defect repair., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

The nuclear receptor subfamily 4 group A member 1 ( NR4A1 ) gene plays a crucial role in both osteoporosis and adipogenesis. The present study investigated the mechanisms by which NR4A1 influences osteoblastogenesis and adipogenesis in human bone marrow‑derived mesenchymal stem cells (BMD‑MSCs). NR4A1 was overexpressed or knocked down in mouse MC3T3‑E1 osteoblast cells and 3T3‑L1 adipocyte cells, as well as in PCS‑500‑012, a BMD‑MSC line. The alkaline phosphatase (ALP) assay and Alizarin Red S staining were performed using MC3T3‑E1 and BMD‑MSCs to assess ALP activity and mineralization, while Oil Red O staining was used to assess the lipid content in 3T3‑L1 cells and BMD‑MSCs. Total RNA was isolated from control, NR4A1 ‑overexpressing and NR4A1 small interfering RNA (siRNA; siNR4A1 )‑treated BMD‑MSCs. RNA sequencing (RNA‑seq) was performed to identify differentially expressed genes, followed by ingenuity pathway analysis (IPA) to determine the role of NR4A1 in osteoblastogenesis and adipogenesis. NR4A1 or Nr4a1 knockdown tended to increase ALP activity and significantly increased calcification in BMD‑MSCs (P<0.005) and MC3T3‑E1 cells (P<0.005), respectively. By contrast, NR4A1 or Nr4a1 overexpression significantly decreased ALP activity and calcification. NR4A1 or Nr4a1 knockdown and overexpression significantly decreased and increased adipogenesis, respectively, in BMD‑MSCs (P<0.005 and <0.05, respectively) and 3T3‑L1 cells (P<0.005 in both). Treatments of BMD‑MSCs with an NR4A1 antagonist, 1,1‑bis(3'‑indolyl)‑1‑(p‑hydroxyphenyl) methane and siNR4A1 showed similar results. RNA‑seq and IPA in control, NR4A1 knockdown and NR4A1 overexpressing cells indicated that Notch signaling mediated the effects of NR4A1 in osteoblastogenesis and adipogenesis. Expression of mastermind‑like transcriptional coactivator 3 was reduced in the Notch signaling pathway in cells treated with siNR4A1 . In conclusion, NR4A1 suppressed osteoblastogenesis and promotes adipogenesis in human BMD‑MSCs. The present study also suggested that NR4A1 plays a role in the progression of osteoporosis and adipogenesis by modulating the Notch signaling cascade.

Atrophic nonunion is a chronic disease without effective medications. Here, high-throughput mRNA sequencing was used to explore the novel targets in atrophic nonunion. AKR1B10, a member of aldo-keto reductase family 1, is upregulated in atrophic nonunion tissues. There are currently no studies to reveal the role of AKR1B10 in atrophic nonunion. We used rat bone marrow-derived mesenchymal stem cells (BMSCs) to explore the effect of AKR1B10 on the osteogenic differentiation and autophagy. In vivo, we implanted collagen sponges loaded with LV-shAKR1B10-transduced BMSCs into rat fractured femurs to explore the role of AKR1B10 in fracture healing. The results showed that AKR1B10 reduced the activity of ALP, suppressed the expression of COL1A1, RUNX2 and OCN, and inhibited calcification deposition in osteogenically differentiated BMSCs. AKR1B10 reduced the expression of LC3II, decreased the number of autophagosomes, and promoted the expression of p62. In addition, the promoting effect of AKR1B10 knockdown on osteogenic differentiation of BMSCs was attenuated by 3-MA treatment. Implantation of collagen sponges found that knockdown of AKR1B10 promoted bone fracture healing. In conclusion, AKR1B10 inhibited the osteogenic differentiation and autophagy, and delayed the bone fracture healing. These results provide a new perspective on revealing the role of AKR1B10 in nonunion and may also provide a new therapeutic target for the treatment of nonunion., Competing Interests: Declaration of competing interest The authors have no competing interests to declare that are relevant to the content of this article, (Copyright © 2024. Published by Elsevier Inc.)

The regenerative capacity of mesenchymal stem cells (MSCs) is now attributed to their ability to release paracrine factors into the extracellular matrix that boost tissue regeneration, reduce inflammation and encourage healing. Understanding the MSC secretome is crucial for shifting the prototypic conventional cell-based therapies to cell-free regenerative treatments. This systematic review aimed to analyse the functional annotations of the secretome of human adult adipose tissue and bone marrow MSCs and unveil the gene clusters responsible for bone formation. Bioinformatics tools were used to identify the biological processes, molecular functions, hallmarks and KEGG pathways of adipose and bone marrow MSC secretome proteins. We found a substantial overlap in the functional annotations and protein compositions of both adipose and bone marrow MSC secretome indicating that MSC source may be noninfluencial with regards to tissue regeneration. Additionally, a novel network pharmacology-based analysis of the secreted proteins revealed that the commonly secreted proteins within a single source interact with multiple drugable targets of bone diseases and regulate various KEGG pathway. This study unravels the secretome profile of human adult adipose and bone marrow MSCs based on the current literature and provides valuable insights into the therapeutic use of the MSC secretome for cell-free therapies., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in SOST can lead to various monogenic bone diseases. Its paralog, SOSTDC1, shares 55 % protein sequence homology and belongs to the BMP antagonist class. Sostdc1-/- mice exhibit distinct effects on cortical and trabecular bone. Genetic polymorphisms in SOSTDC1 impacting peak bone mass makes SOSTDC1 gene, a candidate for influencing BMD variation in humans. SOSTDC1 is upregulated in bone loss conditions, altering BMP-responsive genes and signaling modulators, suggesting its dual BMP/Wnt antagonist role may enhance both pathways. Overexpression of SOSTDC1 confirmed its role as an osteogenic antagonist. Glycine max (Soy)-derived miR4352b, identified for cross-kingdom applications, precisely targets SOSTDC1, a key regulator of bone. SOSTDC1 competitively binds to BMP2 receptor, BMPR1A. Gma-miR4352b suppresses SOSTDC1 expression, enhancing osteogenesis and countering SOSTDC1's inhibition of osteogenic potential. Modeling estrogen deficiency to mimic elevated SOSTDC1 levels, we observed an inverse correlation with SOSTDC1 expression, while serum BMP2 and PINP levels increased following gma-miR4352b supplementation. In fracture healing, SOSTDC1's crucial role becomes evident in conditions of delayed fracture healing. As healing progresses, SOSTDC1 expression decreases. Gma-miR4352b, compared to scrambled miRNA, remarkably promotes callus formation, achieving 68 % healing by day 10, surpassing the scrambled group at 44 %. By the day 13, the treatment group exhibits advanced healing, challenging to find the callus, while the scrambled group maintains a healing rate similar to day10. The accelerated healing in the treatment group underscores the importance of SOSTDC1 in influencing early fracture healing, potentially through the activation of both BMP2 and Wnt signaling pathways., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ritu Trivedi reports administrative support and equipment, drugs, or supplies were provided by CSIR-Central Drug Research Institute. Ritu Trivedi reports a relationship with CSIR-Central Drug Research Institute that includes: employment. None 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 Elsevier B.V. All rights reserved.)

Craniofacial morphogenesis depends on complex cell fate decisions during the differentiation of post-migratory cranial neural crest cells. Molecular mechanisms of cell differentiation of mesenchymal cells to developing bones, cartilage, teeth, tongue, and other craniofacial tissues are still poorly understood. We performed single-cell transcriptomic analysis of craniofacial mesenchymal cells derived from cranial NCCs in mouse embryo. Using FACS sorting of Wnt1-Cre2 progeny, we carefully mapped the cell heterogeneity in the craniofacial region during the initial stages of cartilage and bone formation. Transcriptomic data and in vivo validations identified molecular determinants of major cell populations involved in the development of lower and upper jaw, teeth, tongue, dermis, or periocular mesenchyme. Single-cell transcriptomic analysis of Meis2-deficient mice revealed critical gene expression differences, including increased osteogenic and cell adhesion markers. This leads to affected mesenchymal cell differentiation and increased ossification, resulting in impaired bone, cartilage, and tongue formation., 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 The Author(s). Published by Elsevier Inc. All rights reserved.)

Iroquois homeobox gene 3 (Irx3) and Irx5 encode transcription factors that play crucial roles in limb development and bone formation. Previous studies using knockout mice have revealed a role of Irx3 and Irx5 in osteogenesis in young adult mice. However, whether these genes are also essential for bone homeostasis in adulthood and contribute to bone diseases remain poorly understood. Osteoporosis is a disease characterized by lower bone mineral density and disrupted bone microarchitecture, typically occurs in postmenopausal women. Here, we demonstrate that Irx3/5 dHet mice with a half-reduction of Irx3 and Irx5 dosage serve as a novel model of osteoporosis. By micro-computed tomography, we found that Irx3/5 dHet mice exhibited sex-dependent bone loss patterns. While male Irx3/5 dHet mice progressively lost trabecular microstructures with aging, female mutants exhibited lower bone mineral density (BMD) and bone volume fraction (BV/TV) at early adulthood (9-15 weeks old) but without further loss later at 1 year of age. Bone marrow adipocytes are known to be elevated at the expenses of lower osteogenesis in osteoporotic bone marrow. Surprisingly, we found sex-dependent changes in adipogenesis at the age of skeletal maturity that bone marrow adipocytes were reduced in female Irx3/5 dHet mice along with deteriorated osteogenesis, while male mice exhibited elevated adipogenesis. In summary, we reported a novel genetic model for osteoporosis-like phenotypes, highlighting sex-dependent bone mineral density and bone marrow adipocyte characteristics., Competing Interests: Declaration of competing interest The author(s) declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Medication-related osteonecrosis of the jaw (MRONJ) is an intractable form of osteonecrosis of the jaw that rarely occurs in patients using bone resorption inhibitors such as bisphosphonates (BPs). Then, extracellular vesicles (EVs) carry various signaling molecules, such as mRNAs, microRNAs (miRNAs), and proteins, and have attracted attention as intercellular communication tools. Recently, the role of EVs in communication between osteoclasts and surrounding bone cells has been confirmed. This study aimed to elucidate the effects of EVs derived from osteoclasts treated with zoledronic acid (ZA), one of the BPs on osteoclast function. EVs were isolated by ultracentrifugation of the culture supernatant of osteoclasts treated with ZA, and miRNAs were extracted from these EVs. Tartrate-resistant acid phosphatase staining of the ZA treated osteoclasts showed reduced osteoclastogenesis. In addition, pit assay showed that ZA significantly decreased the bone resorption capacity of osteoclasts. miRNA-seq analysis identified 11 upregulated and 5 downregulated differentially expressed genes (DEGs) in the miRNA of EVs derived from ZA-treated osteoclasts compared to EVs derived from osteoclasts not treated with ZA. qRT-PCR analysis confirmed the amount of these specific miRNAs, with miR-146a-5p, and miR-322-3p being significantly upregulated by ZA. Overexpression of miR-146a-5p in osteoclasts inhibited osteoclastogenesis and decreased the mRNA expression of osteoclast markers. In addition, Traf6 was identified as a candidate target gene of miR-146a-5p in several miRNA databases. Indeed, the overexpression of miR-146a-5p decreased the expression level of Traf6 in osteoclasts. Additionally, overexpression of miR-322-3p in the pre-osteoblast, MC3T3-E1 cells, resulted in a significant increase in the mRNA expression levels of Sp7. Our data indicate that BPs attenuate osteoclastogenesis by simultaneously altering the characteristics of osteoclast-derived EVs. Overexpression of miR-146a-5p and miR-322-3p influences osteoclast differentiation, and Traf6 is a target gene of miR-146a-5p. On the other hand, Overexpression of miR-322-3p affects osteoblast differentiation. We suggest that ZA-treated osteoclast-derived EVs may play an important role in osteoclast function and bone resorption., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To explore the potential mechanisms which O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulates osteogenesis, a publicly RNA-seq dataset was re-analyzed with literature-mining and showed the primary targets of O-GlcNAcylation in osteoblasts are mitochondria/cytoskeleton. Although the O-GlcNAcylation-regulated mitochondria/cytoskeleton has been extensively studied, its specific role during osteogenesis remains unclear. To address this, we knocked out Ogt (Ogt-KO) in MC3T3-E1 osteoblastic cells. Then, significantly reduced osteoblast differentiation, motility, proliferation, mitochondria-endoplasmic reticulum (Mito-ER) coupling, volume of ER, nuclear tubulins, and oxygen metabolism were observed in Ogt-KO cells. Through artificial intelligence (AI)-predicted cellular structures, the time-lapse live cells imaging with reactive-oxygen-species/hypoxia staining showed that lower cell proliferation and altered oxygen metabolism in the Ogt-KO cells were correlated with the Mito-ER coupling. Bioinformatics analysis, combined with correlated mRNA and protein expression, suggested that Ezh2 and its downstream targets (Opa1, Gsk3a, Wnt3a, Hif1a, and Hspa9) may be involved in O-GlcNAcylation-regulated Mito-ER coupling, ultimately impacting osteoblast differentiation. In conclusion, our findings indicate that O-GlcNAcylation-regulated osteoblast differentiation is linked to morphological changes in mitochondria, cytoskeleton, and ER, with Ezh2 potentially playing a crucial role., (© 2024 International Union of Biochemistry and Molecular Biology.)

Extracellular matrix (ECM) stiffness is a major driver of stem cell fate. However, the involvement of the three-dimensional (3D) genomic reorganization in response to ECM stiffness remains unclear. Here, we generated comprehensive 3D chromatin landscapes of mesenchymal stem cells (MSCs) exposed to various ECM stiffness. We found that there were more long-range chromatin interactions, but less compartment A in MSCs cultured on stiff ECM than those cultured on soft ECM. However, the switch from compartment B in MSCs cultured on soft ECM to compartment A in MSCs cultured on stiff ECM included genes encoding proteins primarily enriched in cytoskeleton organization. At the topologically associating domains (TADs) level, stiff ECM tends to have merged TADs on soft ECM. These merged TADs on stiff ECM include upregulated genes encoding proteins enriched in osteogenesis, such as SP1, ETS1, and DCHS1, which were validated by quantitative real-time polymerase chain reaction and found to be consistent with the increase of alkaline phosphatase staining. Knockdown of SP1 or ETS1 led to the downregulation of osteogenic marker genes, including COL1A1, RUNX2, ALP, and OCN in MSCs cultured on stiff ECM. Our study provides an important insight into the stiff ECM-mediated promotion of MSC differentiation towards osteogenesis, emphasizing the influence of mechanical cues on the reorganization of 3D genome architecture and stem cell fate., 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 Ltd. All rights reserved.)

Background/aim: Gangliosides regulate bone formation and resorption. Bone formation is reduced in mice lacking ganglioside GM2/GD2 synthase due to a decrease in osteoblasts. However, the effects of the loss of complex gangliosides by the deletion of both GM2/GD2 and GD3 synthases are unknown. Therefore, we investigated whether deletion of complex gangliosides in mice affected bone metabolism., Materials and Methods: Twenty-six double-knockout mice lacking both GM2/GD2 and GD3 synthases (dKO) and 30 wild-type (WT) mice as controls were used. The mass of cancellous bone and bone strength in femurs were determined using three-dimensional micro-computed tomography and three-point bending test, respectively. Bone formation and resorption were assessed using histomorphometrical analysis with hematoxylin and eosin, and tartrate-resistant acid phosphatase (TRAP), respectively. Osteoblast proliferation was determined by bromodeoxyuridine assay and the differentiation into osteoclasts by TRAP staining; mRNA levels of osteoclast differentiation markers [nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1); Trap; and cathepsin K (Ctsk)] were also determined., Results: Bone mass increased in dKO mice, while bone formation and resorption decrease. In terms of bone strength, breaking displacement significantly increased in dKO mice. Furthermore, the proliferation of osteoblasts was suppressed, and the number of TRAP-positive multinucleated cells was reduced in dKO mice. Treatment with receptor activator of NF-[Formula: see text]B ligand significantly reduced Nfatc1, Trap and Ctsk mRNA levels in macrophages from dKO mice., Conclusion: Bone formation and resorption were reduced by the deletion of genes for complex gangliosides. The slight increase in bone strength in dKO mice may be due to the cancellous bone volume increase in these mice., (Copyright © 2025, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Ankylosing spondylitis (AS) is an autoimmune disease characterized by dysfunction of the immune system, which leads to chronic inflammation and progressive ossification of spinal ligaments. The precise pathogenesis of this condition remains unclear, thereby impeding the development of effective treatments., Methods: We analyzed the GSE25101 dataset and identified the aberrant expression and potential pathogenic role of TXN. This was achieved through a combination of histopathological testing, quantitative PCR (qPCR), Western blotting (WB), and ELISA to elucidate its aberrant expression and distribution. The pathogenic role of TXN was further clarified by employing lentiviral infection, plasmid transfection, electron microscopy, ALP and ARS staining, WB, and qPCR. Finally, the osteogenic role of TXN in the ligaments of AS mice was further elucidated through in vivo plasmid transfection., Results: Through an analysis of the public database GSE25101, we identified abnormal transcription of thioredoxin (TXN) in peripheral blood mononuclear cells. Subsequent experiments utilizing ELISA, qPCR, immunohistochemistry, and immunofluorescence revealed a potential link between TXN expression and pathological ossification. KEGG and GO analyses, along with the application of lentivirus and plasmid treatments on mesenchymal stem cells (MSCs), indicated that TXN promotes the osteogenic differentiation of MSCs by activating their autophagy levels. Animal experiments further corroborated the role of TXN in facilitating pathological ossification of ligaments in mice., Conclusions: Abnormal TXN expression in the immune system is closely associated with the development of pathological ossification in ankylosing spondylitis. Future research should aim to elucidate the underlying mechanisms, with the inhibition of TXN production emerging as a promising strategy for preventing spinal ligament ossification in this condition., 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Intermuscular bones (IBs) are segmental intramembranous ossifications located within myosepta. They share similarities with tendon ossification, a form of heterotopic ossification (HO). The mechanisms underlying IB formation remain incompletely understood., Results: In this study, we systematically analyzed transcriptome data across multiple tissues, species, time points, and resolutions in teleosts. First, we identified IB-specific expression genes using the tau index method. Through cross-species comparisons of the tendon development process, we discovered that candidate genes were primarily enriched in extracellular matrix organization, ossification, regulation of angiogenesis, and other related processes. We also revealed that some of these candidate genes are abnormally expressed in runx2b -/- zebrafish, which lack IBs. To clarify the trajectory of cell differentiation during IB formation, we demonstrated that myoseptal stem cells differentiate into osteoblasts, fibroblasts, and tenocytes in runx2b +/+ zebrafish. However, in runx2b -/- zebrafish, the differentiation of myoseptal stem cell into osteoblast was inhibited, while differentiation into clec3bb + tenocyte and fibroblast was enhanced. Additionally, runx2b deficiency led to the upregulation of clec3bb expression in the clec3bb + tenocyte cluster. Notably, a compensatory effect was observed in cell differentiation and gene expression in runx2b -/- zebrafish, suggesting that runx2b and the candidate genes, such as clec3bb, were involved in the gene network of IB development., Conclusions: The results elucidate cell differentiation process during tendon ossification in teleosts and identify the key factor clec3bb involved in this process. These findings provide a foundation for understanding tendon ossification in teleosts and for further research on tendon ossification in mammals., Competing Interests: Declarations. Ethics approval and consent to participate: All experimental protocols in this study were approved by the Animal Experimental Ethical Inspection of Laboratory Animal Center, Huazhong Agricultural University, Wuhan, China (HZAUFI-2024–0004). All efforts were made to minimize the suffering of the fish. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)