ABSTRACT Purpose M2 macrophage-to-myofibroblast transformation (MMT) contributes to fibrosis. The role of interferon regulatory factor 4 (IRF4), a key transcription factor in M2 macrophage polarization, in M2 MMT remains unclear. This study aimed to elucidate the role of IRF4 in M2 MMT. Materials and Methods An in vitro M2 MMT model was established using RAW264.7 macrophages stimulated with transforming growth factor-β1. Cellular phenotypic shift and extracellular matrix (ECM)-associated proteins were detected. The regulatory role of Peli3 in IRF4 ubiquitination modification was assessed. In a full-thickness skin defect mouse model, the regulatory effects of PELI3 on scar fibrosis and the MMT process were evaluated through AAV-mediated overexpression of PELI3. Results In M2 MMT, IRF4 protein levels were significantly upregulated while ubiquitination levels decreased. Knockdown of IRF4 inhibited M2 polarization and M2 MMT and reduced expression of ECM-associated proteins. Peli3 was significantly downregulated in M2 MMT, and its overexpression suppressed IRF4 protein levels, disrupting M2 MMT and ECM deposition. To directly validate the role of IRF4 in M2 MMT, functional rescue experiments were conducted in PELI3-overexpressing cells. The results demonstrated that overexpressing IRF4 partially reversed the MMT inhibition induced by PELI3 overexpression. Peli3 accelerated IRF4 degradation by promoting its ubiquitin modification. In vivo, Peli3 overexpression reduced fibrosis and MMT. Conclusions IRF4 promotes M2 MMT and fibrosis progression, with its stability regulated by Peli3-mediated ubiquitination.

ABSTRACT Purpose After anterior cruciate ligament reconstruction surgeries, tendon grafts lose their mechanical integrity in vivo. Both auto- and allografts contain dead cells, and remaining viable cells are restricted temporarily from abundant nutrient access. To evaluate a possible connection, nutrient deprivation was hypothesized to reduce cell viability, compromising tissue mechanics as cells adopt a catabolic state. Materials and Methods Taking inspiration from the graft environment, collagenous tendon-mimics with tendon-derived cells were reconstituted in vitro. Subsequently, tendon-mimics were cultured at varying glucose and serum concentrations, ranging from complete medium (5 mM glucose, 2.5% serum), to mild deprivation (0.5 mM glucose, no serum), extreme deprivation (0 mM glucose, no serum), and a frozen control group (0 mM glucose, no serum). Results In the complete medium group, cell viability remained stable, improving tendon-mimic mechanics. Tendon cells were resilient to mild nutrient deprivation, maintaining viability. However, at extreme nutrient deprivation or tissue freezing, cell viability dropped significantly. Surprisingly, mechanical properties remained unaltered in all the groups, except the complete medium group. MMP8 secretion and activation confirmed a catabolic potential only in the complete medium group, while no further MMPs were secreted or activated in the nutrient-deprived groups. This suggests that viable cells that are exposed to a nutrient deprived environment likely lack the energy to exert contractile forces or produce/activate MMPs. Conclusions This study revealed that tendon-derived cells do not compromise collagenous tendon-mimic integrity under nutrient-deprived conditions in an in vitro model, highlighting the resilience of tenocytes in maintaining tissue structure.

ABSTRACT Osteoarthritis (OA) is a complex, multifactorial joint disease and a leading contributor to global disability. Despite its high prevalence and socioeconomic burden, no curative or preventive therapies currently exist. The ability to detect early OA, or even “pre-OA” could provide the opportunity for earlier interventions. Current conventional imaging modalities such as radiography and Magnetic resonance imaging (MRI) are limited by their inability to detect early pathophysiological molecular changes. This review highlights the potential of positron emission tomography (PET) imaging to transform the diagnosis and therapeutic monitoring of OA. PET has emerged as a transformative tool capable of visualizing early metabolic, inflammatory, and cellular alterations. But while current clinical PET imaging with [18F]Fluorodeoxyglucose ([18F]-FDG) and [18F] Sodium fluoride ([18F]-NaF) can assess synovial inflammation and subchondral bone remodeling, their lack of specificity hinders further advances. We also review the recent development of radiotracers targeting specific immune and mesenchymal cell populations, such as translocator protein (TSPO) and fibroblast activation protein inhibitor (FAPI) that have demonstrated potential for characterizing the inflammatory endotype in OA and monitoring treatment response. Given the lack of validated cell-specific tracers, limited studies in early-stage or asymptomatic OA, and few longitudinal data sets, future research should prioritize development and validation of pathophysiology-specific tracers and incorporation of PET into longitudinal and interventional studies. This evolving field holds promise not only for advancing OA preclinical research but also for informing precision diagnostics and early therapeutic strategies in clinical practice.

ABSTRACT Epigenetic mechanisms are implicated in osteoarthritis (OA) as they regulate the expression of several key genes involved in OA disease progression. This mini-review highlights major epigenetic studies in OA from the past 25 years, focusing on mechanistic and therapeutic perspectives. We discuss how DNA methylation, histone modifications, and non-coding RNAs (ncRNAs) impact OA, highlighting preclinical studies targeting epigenetic mechanisms in mouse models. Indeed, existing studies demonstrate that DNA methylation regulates the expression of OA-related genes through DNA methyltransferases, and targeting their activity has shown promise in restoring cartilage homeostasis. EZH2 and DOT1L are key methyltransferases involved in histone methylation with opposing roles in OA: high EZH2 promotes disease progression and is a potential therapeutic target, whereas DOT1L exerts protective effects, partly by suppressing Wnt signaling. Additionally, targeting enzymes that catalyze histone acetylation (PCAF, BRD4) and deacetylation (HDAC1/2) has demonstrated therapeutic potential in preclinical OA models. ncRNAs—including miRNAs, circRNAs, and lncRNAs—regulate gene expression in OA tissues at multiple levels. Several miRNAs (e.g. miR-17, miR-27b-3p) influence cartilage homeostasis and OA pathogenesis, while circRNAs (e.g. circPDE4B) and lncRNAs (e.g. ELDR) have emerged as important disease regulators, offering new therapeutic avenues. Despite significant advancements in OA-related epigenetic mechanisms, clinical translation remains challenging due to the complexity of epigenetic regulation, patient heterogeneity, and limited success of preclinical studies. Importantly, epigenetic alterations are often context-specific, necessitating nuanced interpretation to accurately discern their role in OA. Future research should prioritize identifying specific epigenetic markers linked to clinical outcomes (e.g. structural changes, functional impairment, pain) and developing more selective and potent epigenetic modulators for therapeutic use.

ABSTRACT Background Chondrocytes, the only native cell type in cartilage, use mechanosensitive ion channels such as Transient Receptor Potential Vanilloid 4 (TRPV4) and PIEZO1 to transduce mechanical forces into transcriptomic changes that regulate cell behavior under both physiologic and pathologic conditions. Recent work has identified and characterized the differentially expressed genes (DEGs) that are upregulated following TRPV4 or PIEZO1 activation, but the transcriptomic systems downregulated by these ion channels also represent an important aspect of the chondrocyte regulatory process that remains poorly studied. Methods Here, we utilized previously established bulk RNAsequencing libraries to analyze the transcriptomes downregulated by activation of TRPV4 and PIEZO1 through differential gene expression analysis (using DESeq2), Gene Ontology, RT-qPCR, and Weighted Gene Correlation Network Analysis (WGCNA). Results TRPV4 and PIEZO1 activations downregulated largely unique sets of DEGs, though the set of DEGs downregulated by TRPV4 exhibited a notable overlap with genes downregulated by treatment with inflammatory mediator Interleukin-1 (IL-1). The DEG set downregulated by PIEZO1 activation included genes associated with the G2/M cell cycle checkpoint, a system that checks cells for DNA damage prior to entry into mitosis, and this result was confirmed with RT-qPCR. WGCNA revealed modules of gene regulation negatively correlated with TRPV4, PIEZO1, and IL-1, outlining how these downregulated DEGs may interact to form gene regulatory networks (GRNs). Conclusion This study complements previous work in describing the full mechanosensitive transcriptome (or “mechanome”) of differential gene expression in response to activation of mechanosensitive ion channels TRPV4 and PIEZO1 Q2 and suggests potential avenues for future therapeutic treatment design.

ABSTRACT Background Methyltransferase-like 3 (METTL3) is implicated in human diseases, including osteoporosis (OP). In this study, we aimed to explore the functions and mechanisms of METTL3 in OP using bone marrow mesenchymal stem cells (BMSCs). Methods The identification of BMSCs-derived exosomes was conducted by transmission electron microscope (TEM), Nanoparticle Tracking Analysis (NTA) and western blot. The osteogenic differentiation of osteoblasts (hFOB1.19) was analyzed by Alizarin red staining assay, Alkaline phosphatase (ALP) staining assay and western blot. The relationship between METTL3 and SMAD family member 5 (SMAD5) was analyzed by Methylated RNA Immunoprecipitation (MeRIP) assay and dual-luciferase reporter assay. Results BMSCs-derived exosomes (BMSC-Exos) promoted the osteogenic differentiation and elevated METTL3 expression in hFOB1.19 cells. Exosomal METTL3 knockdown repressed the osteogenic differentiation in hFOB1.19 cells. METTL3 could stabilize and regulate SMAD5 expression by N6-methyladenosine (m6A) modification. Moreover, SMAD5 overexpression restored exosomal METTL3 knockdown-mediated effect on the osteogenic differentiation in hFOB1.19 cells. Conclusion BMSCs-derived exosomal METTL3 mediated the m6A methylation of SMAD5 to facilitate osteogenic differentiation of hFOB1.19 cells.

ABSTRACT Introduction The cervix plays important mechanical roles in pregnancy and regulating the timing of parturition. Dysfunction of the cervix is implicated in disorders of parturition including spontaneous preterm birth, failed induction of labor and post term pregnancies. To address these disorders, it is imperative to first understand the function of the cervix throughout a normal pregnancy. However, current knowledge on the response of the cervix to mechanical fatigue and the underlying microstructural changes throughout a pregnancy is lacking. Methods In this study, mechanical fatigue properties were measured at different stages of pregnancy using uniaxial fatigue testing that simulated circumferential hoop stresses in the cervix. Collagen microstructure was quantified using second harmonic generation imaging and three-dimensional orientation analysis. Results The stiffness and modulus of the cervix during fatigue testing were dramatically reduced in all stages of pregnancy, and pregnant samples experienced greater peak strain before failure. All mechanical properties recovered postpartum despite persistent changes in cervix size. Microstructural analysis demonstrated increased local collagen alignment in postpartum samples, which may indicate a mechanism that serves to improve material properties after childbirth. Discussion Altogether, conclusions from this study enhance our understanding of how properties of the cervix change with pregnancy and lay the foundation for future work investigating how alterations from this healthy function can lead to spontaneous preterm birth and other reproductive complications.

ABSTRACT Post-traumatic osteoarthritis (PTOA) is a common and debilitating problem following meniscal injury, which may lead to pain, loss of function, and early joint failure. Over the past 25 years, clinical, laboratory, and translational studies have greatly improved our understanding of PTOA pathogenesis and prevention. Clinical studies have established the benefit of meniscal preservation in preventing PTOA, leading to a significant increase in meniscus repair. Similarly, improved understanding of the biomechanical importance of the meniscal root attachment has increased focus on the detection and treatment of meniscal root injuries. Laboratory studies have demonstrated a preliminary mechanistic pathway of PTOA development following meniscal injury, whereby injury and altered joint loading stimulate a pro-inflammatory response that leads to both articular cartilage breakdown and impaired meniscal healing. In vitro evidence suggests that mechanical loading of the meniscus may ameliorate this catabolic response, with implications for treatment and rehabilitation protocols. Numerous animal models have emerged, allowing for in vivo assessment of PTOA initiation and offering a platform to test potential therapeutic targets. Despite these advances, meniscal repair remains imperfect and is not always possible, and investigations translating laboratory findings to the human setting have been limited. Future directions include further characterizing the immune and cellular responses to meniscal injury, investigating therapies to target the pro-inflammatory cascade and enhance meniscal healing, and developing new models to better distinguish PTOA pathogenesis in human subjects. Continued laboratory, translational, and clinical research efforts are required to identify treatment strategies to reduce the burden of PTOA after meniscal injury.

ABSTRACT Background Steroid-induced osteonecrosis of the femoral head (SONFH) is a metabolic disorder that leads to structural changes, collapse of the femoral head, and joint dysfunction. This study investigates the role of interferon regulatory factor 8 (IRF8) in osteocyte apoptosis in SONFH, so as to find new targets for the treatment of SONFH. Methods Murine long bone osteocyte-Y4 cells were cultured and treated with dexamethasone to establish SONFH cell models. si-IRF8 was transfected into the cells. The expression levels of IRF8, B cell leukemia/lymphoma 2 (Bcl-2), BCL2 associated X (Bax), zinc finger protein 667 (ZNF667), and miR-181a-5p were detected. Cell apoptosis and viability were detected. The enrichment of IRF8 on the miR-181a-5p promoter was assayed. The binding relationship between IRF8 and miR-181a-5p promoter, and between miR-181a-5p and ZNF667 3’UTR sequence was verified. Combined experiments with miR-181a-5p knockdown or ZNF667 overexpression were performed to observe the changes in cell apoptosis. Results IRF8 and ZNF667 were increased in SONFH cells and miR-181a-5p was decreased. Inhibition of IRF8 increased SONFH cell viability and reduced apoptosis. Mechanistically, IRF8 was enriched in the miR-181a-5p promoter to inhibit miR-181a-5p and miR-181a-5p targeted and inhibited ZNF667. miR-181a-5p knockdown or ZNF667 overexpression could alleviate the inhibitory effect of IRF8 down-regulation on osteocyte apoptosis in SONFH. Conclusion IRF8 was enriched in the miR-181a-5p promoter to inhibit miR-181a-5p, thus promoting ZNF667 levels and increasing osteocyte apoptosis in SONFH, which may be a new theoretical basis for the treatment of SONFH.

ABSTRACT Background Lysophosphatidic acid (LPA), a simple bioactive lysophospholipid, has been reported to regulate bone homeostasis and bone remodeling. This study aimed to elucidate the function and intrinsic mechanism of LPA in osseointegration in murine models. Method We constructed immediate implant models in murine maxillae. Micro-CT, H&E staining, and PCR assays were performed to evaluate the effects of LPA on osseointegration. Furthermore, Prx1-Cre;Yapf/f mice and Sp7-Cre;Yapf/f mice were generated to investigate the role of YAP on LPA-induced osseointegration. Result In this study, we identified that LPA might promote bone deposition on the tissue-implant interface and improve osseointegration. In addition, conditional knockout of YAP from MCSs and pre-osteoblasts blunts LPA-induced osteogenesis and osseointegration in mice. Conclusion Our data demonstrated that LPA-YAP signaling is particularly important to regulate osseointegration, which expands our understanding of LPA and provide the potential of LPA to be used in osseointegration.