Objective Spinal cord injury (SCI) triggers an inflammatory cascade that often culminates in chronic neurological deficits. This study investigates a combined stimulation approach using lipopolysaccharide (LPS), interleukin-4 (IL-4), and transforming growth factor-beta (TGF-β) to generate reparative-biased macrophages and evaluate their early effects after SCI. Methods Bone marrow-derived macrophages (BMDMs) from Wistar rats were exposed to LPS, IL-4, and TGF-β for 24 h to generate reparative-biased hybrid macrophages (Mi) in vitro. The anti-inflammatory and pro-regenerative markers of Mi were characterised by qPCR, immunocytochemistry, and proteomic analysis via mass spectrometry. In vivo, rats with a T9/10 clip-contusion-compression SCI received either Mi transplantation or direct inducer administration at the lesion site. Histological outcomes and functional recovery were assessed and compared between injured and uninjured controls over a 14-day period. Results Following 24 h induction, Mi exhibited significantly elevated mRNA and immunofluorescence expression of Arg1, as well as increased IL-10 mRNA levels, compared to naïve BMDMs in vitro, while iNOS expression remained unchanged. Bioinformatic analysis of proteomic data from Mi revealed enrichment in metabolic activity, immune modulation, and phagocytosis pathways. Fluorescent microsphere phagocytosis assays demonstrated that Mi macrophages retained robust phagocytic activity. In vivo, transplantation of Mi resulted in elevated Arg1 expression and reduced iNOS and IL-1β levels 3 days after SCI, suggesting an anti-inflammatory shift. Immunohistochemical analysis demonstrated significantly reduced lesions and demyelinated areas, as well as increased preservation of motor neurons and oligodendrocytes. Mi transplantation was also associated with reduced apoptosis and improved early functional recovery at 14 days after SCI. Conclusion This study suggests that LPS, IL-4, and TGF-β-induced Mi macrophages display a reparative-biased hybrid profile that modulates inflammation, supports early tissue preservation, and promotes functional recovery after SCI, supporting Mi transplantation as a potential cell-based therapeutic strategy for further investigation. The translational potential of this article Transplantation of this reparative-biased hybrid macrophage phenotype may provide a potential therapeutic strategy for the treatment of spinal cord injury.
Introduction Spinal cord injury (SCI) induces not only local spinal cord pathology but also distal brain dysfunction, causing multi-dimensional impairments in motor, emotional, and cognitive functions. Human umbilical cord mesenchymal stem cells (hUC-MSCs) transplantation has emerged as a promising therapeutic strategy for SCI, but whether it concurrently improves pathological alterations in both the spinal cord and brain remains unclear. Methods After establishing a mouse model of spinal cord contusion, hUC-MSCs were transplanted intrathecally starting 7 d post-injury and administered weekly for 4 wk. This study evaluated the effectiveness of hUC-MSCs transplantation in promoting neurological functional recovery in female mice with SCI through multi-dimensional behavioral tests. In addition, histopathological staining, immunofluorescence staining, and western blotting were performed to evaluate pathological changes in the spinal cord and hippocampus after hUC-MSCs transplantation. Results hUC-MSCs transplantation significantly improved hindlimb locomotor function and alleviated anxiety-like and depression-like behaviors, as well as long-term memory deficits in female mice with SCI. At the spinal cord level, hUC-MSCs increased IL-10 expression, reduced glial activation, and promoted axonal regeneration. In the hippocampus, hUC-MSCs alleviated neuroinflammation and region-specific abnormalities in synaptic remodeling and microglial synaptic pruning. Conclusion This study demonstrated that hUC-MSCs transplantation concurrently improved pathological alterations in both the spinal cord and hippocampus after SCI, providing experimental evidence for comprehensive neurorestoration following SCI. Translational potential This preclinical study demonstrated that subacute sequential hUC-MSCs transplantation is a promising therapeutic strategy for SCI. By improving both motor, emotional, and cognitive outcomes, it supports the concept of “brain-spinal co-repair” and provides a rationale for optimizing cell transplantation regimens to facilitate the clinical translation of hUC-MSC-based therapies for SCI.
The classical “vascular occlusion” model does not fully account for osteonecrosis of the femoral head (ONFH). Femoral head collapse may progress despite restored perfusion, and ischemia alone cannot adequately explain steroid-associated ONFH. We propose that ONFH involves disruption of bone-vascular-immune homeostasis, with macrophage immunometabolic reprogramming in response to hypoxic and lipotoxic stress acting as a potential driver of inflammatory repair failure. Among the metabolic mechanisms implicated in ONFH, the strongest disease-specific evidence supports roles for hypoxia, oxidative stress, disordered lipid metabolism, macrophage imbalance, and ferroptosis-associated injury. By contrast, macrophage-specific glycolytic reprogramming, remodeling of the tricarboxylic acid (TCA) cycle, epigenetic fixation, osteomac dysfunction, and cuproptosis remain less well established. Regulated cell death pathways, particularly ferroptosis and pyroptosis, may exacerbate local tissue injury by releasing damage-associated molecular patterns (DAMPs) and inflammatory mediators that disrupt type H vessel-osteogenesis coupling and shift repair toward fibrosis. Establishing causal relationships rather than correlative associations will require lineage-tracing experiments, spatial validation, metabolic flux analyses, and genetic loss-of-function studies in ONFH models. The translational potential of this article Viewing ONFH as an immunometabolic disorder identifies experimentally tractable targets beyond conventional anti-inflammatory strategies, including SLC7A11/GPX4-dependent ferroptosis defense, NLRP3 inflammasome signaling, and HIF-1α-associated macrophage metabolic adaptation. Extracellular vesicle- or biomaterial-based delivery systems designed for prolonged local retention should currently be considered experimental platforms for assessing lesion-specific target engagement in early-stage ONFH. Their clinical relevance must be established through ONFH-specific studies evaluating efficacy, safety, biodistribution, manufacturability, and long-term structural outcomes.
Background Aseptic loosening (AL) is the primary cause of prosthesis failure after total joint replacement, which is triggered by wear particle-induced macrophage activation, excessive inflammatory factor secretion and subsequent periprosthetic osteolysis. MicroRNAs (miRNAs) act as key regulators of this pathological cascade, yet the specific functional miRNAs and their precise regulatory mechanisms remain incompletely clarified. Methods mRNA-seq and miRNA-seq were performed in titanium particle (TiPs)-stimulated macrophages to screen osteolysis-associated miRNAs, with expression validation conducted in clinical periprosthetic osteolytic tissues. In vitro functional and mechanistic assays were used to verify the miR-155-5p/Gas6/Axl regulatory axis, and a mouse calvarial osteolysis model was applied for in vivo validation. Macrophage membrane-camouflaged PLGA-PEI nanoparticles loaded with antagomir-155-5p (Anta-155@MNPs) were constructed, and their local therapeutic efficacy was evaluated in murine osteolysis models. Results miR-155-5p was significantly upregulated in both TiPs-stimulated macrophages and clinical periprosthetic osteolytic tissues. Functionally, miR-155-5p promoted Stat1/p65 phosphorylation, proinflammatory cytokine production and M1 macrophage polarization by inhibiting the Gas6/Axl pathway via dual mechanisms: Mafb-mediated Gas6 transcriptional repression and Adam10-dependent Axl cleavage inhibition. In vivo, this axis exacerbated TiPs-induced calvarial osteolysis, while local Anta-155@MNPs administration markedly mitigated bone resorption and tissue inflammation. Conclusion miR-155-5p drives TiPs-induced periprosthetic osteolysis via the Gas6/Axl signaling pathway, and targeted inhibition of miR-155-5p is a promising strategy to prevent artificial joint AL. The translational potential of this article This study identifies the miR-155-5p/Gas6/Axl axis as a novel therapeutic target for AL, and the targeted Anta-155@MNPs delivery system provides a feasible preclinical strategy for clinical translation to treat wear particle-induced osteolysis.
Osteoarthritis (OA) is a complex degenerative joint disease driven by mechanical overload, inflammation, metabolic disorders, and aging. Despite substantial advances in basic and translational research, no disease-modifying OA drugs (DMOADs) have been approved for clinical use, largely due to the translational gap between preclinical experimental models and clinical practice. Experimental models are essential tools for investigating OA pathophysiology and evaluating therapeutic strategies, among which animal models play an irreplaceable role in recapitulating the in vivo joint environment. This review comprehensively evaluates current OA experimental models, with a focus on animal systems, including surgically induced structural instability, mechanically induced models, chemical induction approaches, metabolism-related models, spontaneous aging models, and genetically modified strains. We assess each model's advantages, limitations, and appropriate applications, clarifying their ability to reflect specific OA subtypes. Moreover, we summarize recent progress in in vitro platforms, ex vivo tissue explants, and emerging technologies such as organ-on-a-chip and organoid models, which provide complementary insights to animal models. This review aims to establish a rational model selection framework aligned with OA pathological features, thereby bridging the preclinical-clinical translational gap and facilitating the development of effective therapeutics. The translational potential of this article This review systematically evaluates OA experimental models and analyzes their practical utility in translational research of different OA subtypes. It establishes a reasonable framework for model selection and describes how cutting-edge bioengineered platforms complement traditional in vitro and animal studies, providing a reference for improving the predictive reliability of preclinical research and advancing studies on personalized OA therapies.
Stress fractures are overuse injuries that develops in response to repetitive loads applied to bone with normal structural integrity and is highly prevalent among physically active populations, but their underlying mechanisms remain incompletely understood. This article reviews the complex pathogenesis and healing mechanisms of stress fractures. Stress fractures develop when repetitive mechanical loading on the bone exceeds its threshold for adaptive repair, leading to the progressive accumulation of microdamage and ultimately disrupting the physiological equilibrium between bone resorption and formation. The healing of stress fractures is characterized by intramembranous ossification, a process that begins with periosteal woven bone formation to stabilize the fracture and proceeds through subsequent bone remodeling to repair the cracks. The development and repair processes of stress fractures involve dynamic alterations in cell types and tissue constituents, along with active signaling activities within and among the involved cells involved. The translational potential of this article:Future research should prioritize the use of larger animal models such as rabbits and minipigs, and the development of stress fracture models that more accurately replicate the clinical pathogenesis of stress fractures. Although the efficacy of anti-osteoporotic agents, non-steroidal anti-inflammatory drugs (NSAIDs), and low-intensity pulsed ultrasound (LIPUS) have been reported, future research should explore additional physical therapy modalities to elucidate their specific role in the management of stress fractures and underlying mechanisms. Overall, by reviewing the latest research advances in the pathogenesis and treatment of stress fractures and exploring targeted therapeutic strategies, this article holds the promise to offer novel insights into their prevention and management, thereby driving improvements and innovations in clinical treatment approaches and demonstrating significant translational potential.
Background:Rotator cuff injuries often lead to impaired shoulder function, and tendon-bone healing remains challenging due to the complex structure of the enthesis and persistent remodeling-associated pro-inflammatory signaling. Low-intensity pulsed ultrasound (LIPUS) and mild hyperthermia (MH) have shown potential in tissue repair, but the molecular mechanisms underlying their combined effects are unclear. Objective:This study aimed to investigate whether LIPUS and MH synergistically promote tendon-bone healing by regulating macrophage polarization through the TRPV1/Ca2+/CaMKII/BACH2 signaling pathway. Methods:A rabbit supraspinatus tendon-bone injury model and in vitro macrophage polarization assays were employed. Histology, immunohistochemistry, MRI, and biomechanical testing were performed to assess repair outcomes. RNA-seq with qPCR and Western blot validation was used to identify key signaling pathways. Functional assays, including Ca2+ flux detection, pharmacological inhibition, and siRNA knockdown, were conducted to examine the role of TRPV1, CaMKII, and BACH2. Results:LIPUS and MH significantly suppressed M1 polarization (CD86, iNOS) and enhanced M2 markers (CD163, Arg1), reducing ROS levels and inflammatory cytokine expression. Combined intervention yielded the most pronounced effects, showing improved fibrocartilage formation, collagen alignment, and bone remodeling, with superior biomechanical strength approaching native tissue. Transcriptomic and protein analyses revealed increased TRPV1 activation, enhanced Ca2+ influx, CaMKII phosphorylation, and promoted BACH2 nuclear translocation. Mechanistically, TRPV1 activation promoted Ca2+ entry, CaMKII activation, and BACH2 nuclear translocation, thereby inhibiting M1 polarization. Inhibition of TRPV1, Ca2+ influx, or CaMKII significantly reduced BACH2 nuclear translocation and diminished the anti-inflammatory effects. Conclusion:Combined LIPUS and MH synergistically enhance tendon--bone healing, potentially through modulating the TRPV1/Ca2+/CaMKII/BACH2 pathway, which appears to modulate macrophage polarization during the later stages of tendon-bone remodeling. The translational potential of this article:This study identifies the TRPV1/Ca2+/CaMKII/BACH2 signaling axis as a novel mechanistic target for non-invasive physical therapy in tendon-bone healing. Our findings provide a strong preclinical rationale for combining LIPUS and mild hyperthermia as a promising, non-pharmacological strategy to improve immune microenvironment and outcomes after rotator cuff repair. This paves the way for future clinical trials to translate this combinatorial biophysical therapy into adjuvant treatment protocols.
Ferroptosis is an iron-dependent, non-apoptotic form of regulated cell death driven by lethal lipid peroxidation and has emerged as a pivotal regulator of skeletal muscle physiology and pathology. This review systematically delineates the core molecular machinery of ferroptosis, including the system Xc--glutathione-GPX4 axis, dysregulated iron metabolism, and lipid peroxidation, together with key regulatory networks involving p53, Nrf2, and AMPK. We further highlight the context-dependent roles of ferroptosis in skeletal muscle: during development and regeneration, transient and moderate ferroptotic signaling may facilitate myogenesis and tissue remodeling, whereas sustained or excessive ferroptosis drives satellite cell depletion and impaired regenerative capacity. Pathologically, ferroptosis is implicated in a spectrum of muscle disorders-including sarcopenia, muscular dystrophy, sports-related injuries, and inflammatory myopathies-through mechanisms such as iron overload, oxidative stress, and mitochondrial dysfunction. Finally, we summarize emerging therapeutic strategies targeting ferroptosis, including iron chelators, GPX4 activators, natural compounds, gene-based interventions, and physical exercise, and discuss future directions toward precision medicine and combinatorial approaches. By integrating current evidence, this work provides a comprehensive framework for understanding ferroptosis in skeletal muscle homeostasis and disease and offers insights for the development of novel therapeutic interventions. The translational potential of this article:This review establishes ferroptosis as a convergent pathogenic mechanism across muscle disorders, offering a framework for patient stratification by ferrototic signatures. It synthesizes preclinical evidence for pharmacologic inhibitors, natural products, and gene-based interventions, while critically evaluating clinical feasibility, safety, and dosing. A tiered translational roadmap from biomarker validation to early-phase trials is proposed to accelerate bench-to-bedside development.
Background:Rheumatoid arthritis (RA) is driven in part by hyperactivated fibroblast-like synoviocytes (FLS) that invade articular structures. Iguratimod (IGU), a conventional synthetic DMARD, is clinically effective, but its direct molecular target and impact on synovial cell-cell crosstalk remain unclear. We aimed to elucidate how IGU regulates FLS invasiveness and inflammatory signaling, identify its upstream target within the JAK-STAT pathway, and develop a prodrug with improved pharmacokinetics while preserving disease-modifying activity. Methods:We combined in vitro assays in MH7A cells and rat RA-FLS with RNA sequencing and conditioned-medium fast-astral DIA proteomics to characterize IGU's effects on TNF-α-induced migration, invasion, and signaling. STAT1 dependence was interrogated by siRNA knockdown, phosphorylation-deficient mutant reconstitution and IFN-γ rescue. Integrated single-cell RNA-seq of RA and healthy synovium, together with CellChat analysis and complement component 3a (C3a) stimulation of THP-1-derived macrophages, was used to define FLS-macrophage crosstalk. Reverse virtual screening, molecular docking, thermal shift assays, cellular thermal shift assays, kinase assays, and molecular dynamics simulations were applied to characterize IGU-TYK2 interactions. A sulfonamide N-acyl IGU prodrug (AD811) was rationally designed and evaluated for pH-dependent stability, plasma and microsomal metabolism, pharmacokinetics, efficacy, and short-term safety in collagen-induced arthritis rats. Results:IGU suppressed TNF-α-induced FLS migration and invasion without cytotoxicity by selectively inhibiting STAT1 Y701 phosphorylation and nuclear translocation, while sparing STAT1 Y727 phosphorylation and STAT2 Y690 phosphorylation. Bulk and single-cell transcriptomic analyses revealed STAT1 hyperactivation in RA lining-layer FLS and uncovered a STAT1-C3-TNFα feedback loop in which FLS-derived C3/C3a enhances macrophage TNF-α production, thereby reinforcing FLS activation; IGU disrupted this loop by reducing STAT1 activity, C3 transcription, and C3a-driven macrophage TNF-α induction. Biochemical and biophysical studies showed that IGU directly engages the TYK2 JH2 pseudokinase domain, alters its thermal behavior, and inhibits kinase activity of a TYK2 construct containing JH2 and JH1, while not measurably inhibiting the isolated JH1 catalytic domain, consistent with JH2-dependent allosteric modulation of TYK2 output. The prodrug AD811 exhibited pH-sensitive stability, rapid plasma conversion to IGU, favorable oral bioavailability, and therapeutic efficacy, joint protection, and preliminary hepatic and gastric safety comparable to equimolar IGU in vivo. Conclusion:In FLS-centered experimental systems, IGU reduces RA-FLS invasiveness by targeting TYK2 JH2 and disrupting a STAT1-C3-TNFα feedback loop between lining-layer FLS and macrophages, thereby attenuating both intrinsic fibroblast aggressiveness and inflammatory crosstalk. The prodrug AD811 maintains these disease-modifying actions while improving pharmacokinetic properties, nominating AD811 as a promising candidate for further translational development in RA. The translational potential of this article:This study mechanistically links IGU, a clinically used csDMARD, to selective modulation of TYK2 JH2 and downstream STAT1 signaling in synovial lining-layer FLS. By showing that, in FLS-centered models, IGU disrupts a STAT1-C3-TNFα feedback loop between FLS and macrophages and thereby reduces FLS invasiveness and inflammatory crosstalk, our data provide a concrete cellular and molecular basis for its disease-modifying effects in rheumatoid arthritis. Furthermore, the rationally designed prodrug AD811 exhibits improved pharmacokinetic properties and a favorable short-term safety profile in vivo, supporting its further evaluation as a potential oral small-molecule candidate.
Bone organoids have evolved from simple osteogenic spheroids to increasingly sophisticated systems incorporating vascular networks, bone marrow niches, and multicellular interactions, yet achieving functional maturation remains limited by long-term viability, tissue organization, and metabolic homeostasis. Mitochondrial metabolic homeostasis represents a promising strategy in this regard. Mitochondria serve as both the core of energy metabolism and a vital signaling hub governing bone development, remodeling, and homeostasis. The metabolic switch from glycolysis to oxidative phosphorylation in osteogenic lineage cells, regulated by mitochondrial programming, directly determines bone matrix synthesis and mineralization. This review systematically summarizes the fundamental mechanisms of mitochondria in osteogenic differentiation, calcium signaling, and bone quality control, and highlights how mechanical cues, electromagnetic stimulation, and biomaterial microenvironments drive functional maturation of bone organoids by targeting mitochondria. Bone organoid construction should integrate mitochondrial metabolic requirements across developmental stages, ensuring energy-redox adaptation, organelle quality control, and intercellular metabolic coupling among heterogeneous cell populations. Accordingly, we propose a novel strategy for next-generation bone organoid engineering focused on mitochondrial metabolic microenvironment modulation. Active intervention in cellular energy metabolism can significantly enhance organoid maturity and physiological fidelity, providing a new theoretical framework and technical route for developing high-fidelity bone organoid models. Translational potential: This review delineates mitochondrial reprogramming as the core driver of bone organoid maturation. We highlight strategies targeting mitochondrial bioenergetics via physical cues, ions, and bioactive factors. Bridging molecular mechanisms with biomaterial design, this framework establishes standardized organoids with homeostatic resilience, advancing clinically translatable in vitro bone models.
