Abstract Skeletal and mesenchymal cells have limited bone-forming potential due to their rarity, tendency for senescence and/or unstable osteogenic lineage commitment. We previously identified an osteopotent CXCR4⁺ stem cell population with unclear mechanism for its differentiation potential. Here, we found that the cystine transporter SLC3A1 was selectively enriched in CXCR4 + stem cells, uncovering a role for amino acid transport in regulating osteogenic fate commitment. Enforced SLC3A1 expression reprogrammed mesenchymal cells toward a stable osteogenic state while suppressing adipogenic differentiation. Mechanistically, SLC3A1-mediated cystine flux established a glutathione-dependent metabolic program that preserved mitochondrial fitness and restrained stem cell senescence. SLC3A1 also stabilized IGF-1 through suppression of ZMYND8-mediated ubiquitination, uncovering ZMYND8 as a previously unrecognized E3 ligase regulating osteogenic commitment. Cystine supplementation phenocopied the effects of SLC3A1 activation, promoting osteogenic differentiation and skeletal regeneration without genetic manipulation. In vivo , cystine administration accelerated bone regeneration and attenuated ovariectomy-induced bone loss, while analyses of a human osteoporosis cohort, osteoporotic specimens, and single-cell transcriptomic datasets revealed coordinated suppression of the SLC3A1-cystine-IGF-1 pathway in osteoporotic mesenchymal cells. Taken together, these findings establish SLC3A1-mediated cystine transport as a programmable metabolic determinant of osteogenic fate commitment and identify cystine metabolism as a therapeutically targetable axis for skeletal regeneration and osteoporosis.

BACKGROUND:Ligamentum flavum hypertrophy (LFH) is a primary contributor to lumbar spinal stenosis. However, a thorough understanding of the cellular and molecular mechanisms driving LFH fibrotic progression remains incomplete. METHODS:Single-cell RNA sequencing (scRNA-seq) was performed to construct the single-cell map of human ligamentum flavum (LF) samples. An integrated multi-omics approach, encompassing scRNA-seq, bulk RNA sequencing (bulk RNA-seq), and Mendelian randomization (MR), was applied to conduct comprehensive functional analysis. Clinical tissue specimens and animal models were employed to further confirm the multi-omics findings. RESULTS:ScRNA-seq provided a single-cell level view of the fibrotic microenvironment in LF, revealing significantly increased proportions of fibroblasts, myofibroblasts, and macrophages in LFH. Using transmission electron microscopy, single-cell gene set scoring, and MR analysis, ferroptosis was identified as a critical risk factor and pathway within LFH. Subcluster analysis of fibroblasts revealed functional heterogeneity among distinct subpopulations, highlighting the functional characteristics and the metabolic dynamics of fibroblast with a high ferroptosis score (High Ferro-score FB). The quantification of gene expression at single-cell level revealed that ferroptosis increased along with fibrosis in LFH specimens, a finding further validated in both human and mice tissue sections. Consistently, bulk RNA-seq confirmed increased proportions of fibroblasts and macrophages in LFH specimens, underscoring a strong correlation between these cell types through Spearman correlation analysis. Notably, subcluster analysis of the mononuclear phagocytes identified a specific subset of SPP1+ macrophages (SPP1+ Mac) enriched in LFH, which exhibited activation of fibrosis and ferroptosis-related metabolic pathways. Cell-cell communication analysis highlighted that SPP1+ Mac exhibited the strongest outgoing and incoming interactions among mononuclear phagocytes in the LFH microenvironment. Ligand-receptor analysis further revealed that the SPP1-CD44 axis could serve as a key mediator regulating the activity of High Ferro-score FB. Multiplex immunofluorescence confirmed substantial Collagen I deposition and reduced Ferritin Light Chain expression in regions with SPP1-CD44 co-localization in LFH specimens. CONCLUSIONS:Our findings indicated that SPP1+ Mac may contribute to LFH fibrosis by regulating ferroptosis in High Ferro-score FB through the SPP1-CD44 axis. This study enhances our understanding of the cellular and molecular mechanisms underlying LFH progression, potentially improving early diagnostic strategies and identifying new therapeutic targets.

Background: Heterotopic ossification (HO) refers to the development of bone tissue in areas other than the skeletal system. The development and maturation of the skeletal system are significantly influenced by macrophage migration inhibitory factor (MIF). The objective of this study was to examine the impact of MIF on the in vitro osteogenic differentiation and mineralization of tendon-derived stem cells (TDSCs), mediated by a positive feedback loop involving ROS/HIF-1 alpha/MIF. Methods: TDSCs were isolated and identified from the hind limbs of C57/BL6 mice. The functional and procedural roles of MIF in HO, focusing on the impact of MIF on the differentiation of TDSCs into bone-forming cells were investigated in vitro. Seventy-five mice were randomly assigned to five groups. Gene expression and histological analyses of MIF and its receptors, and determine the expression of osteogenic markers in vivo. Results: The results revealed a positive and concentration-dependent effect of MIF on the osteogenic differentiation of TDSCs. Furthermore, an ROS/HIF-1 alpha/MIF positive loop was detected in the simulated early trauma hypoxic microenvironment, resulting in a 3 to 4 folds increase in MIF expression levels. MIF was also found to enhance double the expression levels of markers associated with bone and cartilage at the site of injury, consequently facilitating the development of HO, which was thought to be associated with the activation of the Wnt/(3-catenin pathway. Conclusion: MIF, which mediates the ROS/HIF-1 alpha/MIF positive feedback loop during the hypoxic phase of HO, triggers the Wnt/(3-catenin signaling pathway to enhance the osteogenic differentiation and formation of HO in TDSCs.

Increased differentiation or activity of osteoclasts is the key pathogenic factor of postmenopausal osteoporosis (PMOP). N4‐acetylcytidine (ac4C) modification, catalyzed by Nat10, is a novel posttranscriptional mRNA modification related to many diseases. However, its impact on regulating osteoclast activation in PMOP remains uncertain. Here, we initially observed that Nat10-mediated ac4C positively correlates with osteoclast differentiation of monocytes and low bone mass in PMOP. The specific knockout of Nat10 in monocytes and remodelin, a Nat10 inhibitor, alleviates ovariectomized (OVX)-induced bone loss by downregulating osteoclast differentiation. Mechanistically, epitranscriptomic analyses reveal that the nuclear factor of activated T cells cytoplasmic 1 (Nfatc1) is the key downstream target of ac4C modification during osteoclast differentiation. Subsequently, translatomic results demonstrate that Nat10-mediated ac4C enhances the translation efficiency (TE) of Nfatc1, thereby inducing Nfatc1 expression and consequent osteoclast maturation. Cumulatively, these findings reveal the promotive role of Nat10 in osteoclast differentiation and PMOP from a novel field of RNA modifications and suggest that Nat10 can be a target of epigenetic therapy for preventing bone loss in PMOP.

As a type of epigenetic modifications, mRNA modifications regulate the metabolism of mRNAs, thereby influencing gene expression. Previous studies have indicated that dysregulation of mRNA modifications is closely associated with the occurrence and progression of bone diseases (BDs). In this study, we first introduced the dynamic regulatory processes of five major mRNA modifications and their effects on the nucleus export, stability, and translation of mRNAs. We then summarized the mechanisms of mRNA modifications involved in the development of osteoporosis, osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, fractures, osteomyelitis, and osteosarcoma. Finally, we reviewed therapeutic strategies for BDs based on the above mechanisms, focusing on regulating osteoblast and osteoclast differentiation, inhibiting cellular senescence and injury, and alleviating inflammation. This review identified mRNA modifications as potential targets for treating BDs and proposes perspectives on the diversity, targetability, and safety of mRNA-modifying therapies.

Intense inflammatory response impairs bone marrow mesenchymal stem cell (BMSC)-mediated bone regeneration, with transforming growth factor (TGF)-β1 being the most highly expressed cytokine. However, how to find effective and safe means to improve bone formation impaired by excessive TGF-β1 remains unclear. In this study, we found that the expression of orphan nuclear receptor Nr4a1, an endogenous repressor of TGF-β1, was suppressed directly by TGF-β1-induced Smad3 and indirectly by Hdac4, respectively. Importantly, Nr4a1 overexpression promoted BMSC osteogenesis and reversed TGF-β1-mediated osteogenic inhibition and pro-fibrotic effects. Transcriptomic and histologic analyses confirmed that upregulation of Nr4a1 increased the transcription of Wnt family member 4 (Wnt4) and activated Wnt pathway. Mechanistically, Nr4a1 bound to the promoter of Wnt4 and regulated its expression, thereby enhancing the osteogenic capacity of BMSCs. Moreover, treatment with Nr4a1 gene therapy or Nr4a1 agonist Csn-B could promote ectopic bone formation, defect repair, and fracture healing. Finally, we demonstrated the correlation of NR4A1 with osteogenesis and the activation of the WNT4/β-catenin pathway in human BMSCs and fracture samples. Taken together, these findings uncover the critical role of Nr4a1 in bone formation and alleviation of inflammation-induced bone regeneration disorders, and suggest that Nr4a1 has the potential to be a therapeutic target for accelerating bone healing.

Background Guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein, which is eventually localised to mitochondria and promotes the translation of cytochrome C oxidase 1 (COX1) mRNA. However, the role of the miR-19-3p/GRSF1/COX1 axis has not been investigated in an experimental subarachnoid haemorrhage (SAH) model. Thus, we investigated the role of the miR-19-3p/GRSF1/COX1 axis in a SAH-induced early brain injury (EBI) course.Methods Primary neurons were treated with oxyhaemoglobin (OxyHb) to simulate in vitro SAH. The rat SAH model was established by injecting autologous arterial blood into the optic chiasma cisterna. The GRSF1 level was downregulated or upregulated by treating the rats and neurons with lentivirus-GRSF1 shRNA (Lenti-GRSF1 shRNA) or lentivirus-GRSF1 (Lenti-GRSF1).Results The miR-19-3p level was upregulated and the protein levels of GRSF1 and COX1 were both downregulated in SAH brain tissue. GRSF1 silence decreased and GRSF1 overexpression increased the protein levels of GRSF1 and COX1 in primary neurons and brain tissue, respectively. Lenti-GRSF1 shRNA aggravated, but Lenti-GRSF1 alleviated, the indicators of neuronal injury and neurological impairment in both in vitro and in vivo SAH conditions. In addition, miR-19-3p mimic reduced the protein levels of GRSF1 and COX1 in cultured neurons while miR-19-3p inhibitor increased them. More importantly, Lenti-GRSF1 significantly relieved mitochondrial damage of neurons exposed to OxyHb or induced by SAH and was beneficial to maintaining mitochondrial integrity. Lenti-GRSF1 shRNA treatment, conversely, aggravated mitochondrial damage in neurons.Conclusion The miR-19-3p/GRSF1/COX1 axis may serve as an underlying target for inhibiting SAH-induced EBI by maintaining mitochondrial integrity.

Lumbar spinal canal stenosis is primarily caused by ligamentum flavum hypertrophy (LFH), which is a significant pathological factor. Nevertheless, the precise molecular basis for the development of LFH remains uncertain. The current investigation observed a notable increase in thrombospondin-1 (THBS1) expression in LFH through proteomics analysis and single-cell RNA-sequencing analysis of clinical ligamentum flavum specimens. In laboratory experiments, it was demonstrated that THBS1 triggered the activation of Smad3 signaling induced by transforming growth factor β1 (TGFβ1), leading to the subsequent enhancement of COL1A2 and α-SMA, which are fibrosis markers. Furthermore, experiments conducted on a bipedal standing mouse model revealed that THBS1 played a crucial role in the development of LFH. Sestrin2 (SESN2) acted as a stress-responsive protein that suppressed the expression of THBS1, thus averting the progression of fibrosis in ligamentum flavum (LF) cells. To summarize, these results indicate that mechanical overloading causes an increase in THBS1 production, which triggers the TGFβ1/Smad3 signaling pathway and ultimately results in the development of LFH. Targeting the suppression of THBS1 expression may present a novel approach for the treatment of LFH.

Background Heterotopic ossification (HO) refers to the development of bone tissue in areas other than the skeletal system. The development and maturation of the skeletal system are significantly influenced by macrophage migration inhibitory factor (MIF). The objective of this study was to examine the impact of MIF on the in vitro osteogenic differentiation and mineralization of tendon-derived stem cells (TDSCs), mediated by a positive feedback loop involving ROS/HIF-1α/MIF. Methods TDSCs were isolated and identified from the hind limbs of C57/BL6 mice. The functional and procedural roles of MIF in HO, focusing on the impact of MIF on the differentiation of TDSCs into bone-forming cells were investigated in vitro. Seventy-five mice were randomly assigned to five groups. Gene expression and histological analyses of MIF and its receptors, and determine the expression of osteogenic markers in vivo. Results The results revealed a positive and concentration-dependent effect of MIF on the osteogenic differentiation of TDSCs. Furthermore, an ROS/HIF-1α/MIF positive loop was detected in the simulated early trauma hypoxic microenvironment, resulting in a 3 to 4 folds increase in MIF expression levels. MIF was also found to enhance double the expression levels of markers associated with bone and cartilage at the site of injury, consequently facilitating the development of HO, which was thought to be associated with the activation of the Wnt/β-catenin pathway. Conclusion MIF, which mediates the ROS/HIF-1α/MIF positive feedback loop during the hypoxic phase of HO, triggers the Wnt/β-catenin signaling pathway to enhance the osteogenic differentiation and formation of HO in TDSCs.

Platelet-derived growth factor receptor α (PDGFRα) is often considered as a general marker of mesenchymal cells and fibroblasts, but also shows expression in a portion of osteoprogenitor cells. Within the skeleton, Pdgfrα+ mesenchymal cells have been identified in bone marrow and periosteum of long bones, where they play a crucial role in participating in fracture repair. A similar examination of Pdgfrα+ cells in calvarial bone healing has not been examined. Here, we utilize Pdgfrα-CreERTM;mT/mG reporter animals to examine the contribution of Pdgfrα+ mesenchymal cells to calvarial bone repair through histology and single-cell RNA sequencing (scRNA-Seq). Results showed that Pdgfrα+ mesenchymal cells are present in several cell clusters by scRNA-Seq, and by histology a dramatic increase in Pdgfrα+ cells populated the defect site at early timepoints to give rise to healed bone tissue overtime. Notably, diphtheria toxin-mediated ablation of Pdgfrα reporter+ cells resulted in significantly impaired calvarial bone healing. Our findings suggest that Pdgfrα-expressing cells within the calvarial niche play a critical role in the process of calvarial bone repair.

Peripheral neurons terminate at the surface of tendons partly to relay nociceptive pain signals; however, the role of peripheral nerves in tendon injury and repair remains unclear. Here, we show that after Achilles tendon injury in mice, there is new nerve growth near tendon cells that express nerve growth factor (NGF). Conditional deletion of the Ngf gene in either myeloid or mesenchymal mouse cells limited both innervation and tendon repair. Similarly, inhibition of the NGF receptor tropomyosin receptor kinase A (TrkA) abrogated tendon healing in mouse tendon injury. Sural nerve transection blocked the postinjury increase in tendon sensory innervation and the expansion of tendon sheath progenitor cells (TSPCs) expressing tubulin polymerization promoting protein family member 3. Single cell and spatial transcriptomics revealed that disruption of sensory innervation resulted in dysregulated inflammatory signaling and transforming growth factor–β (TGFβ) signaling in injured mouse tendon. Culture of mouse TSPCs with conditioned medium from dorsal root ganglia neuron further supported a role for neuronal mediators and TGFβ signaling in TSPC proliferation. Transcriptomic and histologic analyses of injured human tendon biopsy samples supported a role for innervation and TGFβ signaling in human tendon regeneration. Last, treating mice after tendon injury systemically with a small-molecule partial agonist of TrkA increased neurovascular response, TGFβ signaling, TSPC expansion, and tendon tissue repair. Although further studies should investigate the potential effects of denervation on mechanical loading of tendon, our results suggest that peripheral innervation is critical for the regenerative response after acute tendon injury.

Pericytes are multipotent mesenchymal precursor cells that demonstrate tissue-specific properties. In this study, by comparing human adipose tissue- and periosteum-derived pericyte microarrays, we identified T cell lymphoma invasion and metastasis 1 (TIAM1) as a key regulator of cell morphology and differentiation decisions. TIAM1 represented a tissue-specific determinant between predispositions for adipocytic versus osteoblastic differentiation in human adipose tissue-derived pericytes. TIAM1 overexpression promoted an adipogenic phenotype, whereas its downregulation amplified osteogenic differentiation. These results were replicated in vivo, in which TIAM1 misexpression altered bone or adipose tissue generation in an intramuscular xenograft animal model. Changes in pericyte differentiation potential induced by TIAM1 misexpression correlated with actin organization and altered cytoskeletal morphology. Small molecule inhibitors of either small GTPase Rac1 or RhoA/ROCK signaling reversed TIAM1-induced morphology and differentiation in pericytes. In summary, our results demonstrate that TIAM1 regulates the cellular morphology and differentiation potential of human pericytes, representing a molecular switch between osteogenic and adipogenic cell fates.

Objective Nowadays, more than 90% of people over 50 years suffer from intervertebral disc degeneration (IDD), but there are exist no ideal drugs. The aim of this study is to identify a new drug for IDD. Methods An approved small molecular drug library including 2040 small molecular compounds was used here. We found that taurocholic acid sodium hydrate (NAT) could induce chondrogenesis and osteogenesis in mesenchymal stem cells (MSCs). Then, an in vivo mouse model of IDD was established and the coccygeal discs transcriptome analysis and surface plasmon resonance analysis (SPR) integrated with liquid chromatography–tandem mass spectrometry assay (LC‐MS) were performed in this study to study the therapy effect and target proteins of NAT for IDD. Micro‐CT was used to evaluate the cancellous bone. The expression of osteogenic (OCN, RNX2), chondrogenic (COL2A1, SOX9), and the target related (ERK1/2, p‐ERK1/2) proteins were detected. The alkaline phosphatase staining was performed to estimate osteogenic differentiation. Blood routine and blood biochemistry indexes were analyzed for the safety of NAT. Results The results showed that NAT could induce chondrogenesis and osteogenesis in MSCs. Further experiments confirmed NAT could ameliorate the secondary osteoporosis and delay the development of IDD in mice. Transcriptome analysis identified 128 common genes and eight Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for NAT. SPR‐LC–MS assay detected 57 target proteins for NAT, including MAPK3 (mitogen‐activated protein kinase 3), also known as ERK1 (extracellular regulated protein kinase 1). Further verification experiment confirmed that NAT significantly reduced the expression of ERK1/2 phosphorylation. Conclusion NAT would induce chondrogenesis and osteogenesis of MSCs, ameliorate the secondary osteoporosis and delay the progression of IDD in mice by targeting MAPK3.Furthermore, MAPK3, especially the phosphorylation of MAPK3, would be a potential therapeutic target for IDD treatment.

Osteoarthritis (OA) is the most common chronic disease involving joints. The limited intrinsic regeneration potential of articular cartilage (AC) tissue poses a big challenge from a therapeutic perspective. Treatment options for OA are sparse, and investigators have turned to the use of mesenchymal stem/progenitor cells (MSCs/MPCs) or minimally manipulated tissues containing such stem-like cells for therapeutic purposes. Pericytes are present within the microvasculature and demonstrate features consistent with a cellular forerunner of culture-derived MSCs/MPCs. In this review, we provide an overview on the role of pericytes in OA pathophysiology and their potential application as an injectable cell therapy for cartilage injury and repair.

Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to reinnervation and osteogenesis is unknown. Here, we found that nerve growth factor (NGF) is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to affect their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair.

Heterotopic ossification (HO) is a pathologic process characterized by the formation of bone tissue in extraskeletal locations. The hip is a common location of HO, especially as a complication of arthroplasty. Here, we devise a first-of-its-kind mouse model of post-surgical hip HO and validate expected cell sources of HO using several HO progenitor cell reporter lines. To induce HO, an anterolateral surgical approach to the hip was used, followed by disclocation and acetabular reaming. Animals were analyzed with high-resolution roentgenograms and micro-computed tomography, conventional histology, immunohistochemistry, and assessments of fluorescent reporter activity. All the treated animals' developed periarticular HO with an anatomical distribution similar to human patients after arthroplasty. Heterotopic bone was found in periosteal, inter/intramuscular, and intracapsular locations. Further, the use of either PDGFRα or scleraxis (Scx) reporter mice demonstrated that both cell types gave rise to periarticular HO in this model. In summary, acetabular reaming reproducibly induces periarticular HO in the mouse reproducing human disease, and with defined mesenchymal cellular contributors similar to other experimental HO models. This protocol may be used in the future for further detailing of the cellular and molecular mediators of post-surgical HO, as well as the screening of new therapies.