Osteoarthritis and Cartilage

ObjectiveThe Agc1CreERT2 mouse line is a powerful tamoxifen-inducible genetic tool used for conditional gene manipulation specifically in cartilage. The aim of this study was to investigate the effects of aggrecan hypomorphism on the progression of post-traumatic osteoarthritis (PT-OA) in Agc1CreERT2 mice. MethodsProteoglycan content in cartilage samples from the knees of E18.5 embryos extracts were quantified by sulfated glycosaminoglycan (sGAG) assay. Destabilization of the medial meniscus (DMM) surgery was performed to induce PT-OA in 12-week-old wild-type, heterozygous Agc1CreERT2/+ and homozygous Agc1CreERT2/CreERT2 mice. Progression of OA was assessed at 4-, 8-, and 12-weeks post-DMM by OARSI, synovitis and osteophyte maturation histopathology scores and micro-computed tomography ({micro}CT). Aggrecan deposition, cartilage matrix-degrading proteases, aggrecan and collagen II degradation neoepitopes were investigated by immunohistochemical staining, and serum C-terminal cross-linked telopeptide of type II collagen (CTX-II) levels by an enzyme-linked immunosorbent assay (ELISA). Chondrocyte apoptosis was analyzed with the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The biomechanical properties of articular cartilage (AC) were investigated with indentation-type atomic force microscopy (IT-AFM). ResultsBefore DMM, homozygous Agc1CreERT2/CreERT2 mice had reduced sGAG and aggrecan levels and increased cartilage stiffness. After DMM, they exhibited increased cartilage degradation, synovitis, osteophyte formation and meniscus mineralization, chondrocyte apoptosis and cartilage stiffness compared to wild-type mice. Immunohistochemistry demonstrated increased expression of the aggrecanase ADAMTS-5, the metalloproteinase MMP-13, the aggrecan degradation neoepitope NITEGE and the collagen degradation neoepitope C1,2C in AC. ELISA also revealed elevated serum CTX-II levels. Heterozygous Agc1CreERT2/+ mice also exhibited accelerated PT-OA compared with wild-type mice, characterized by elevated CTX-II levels at 4-weeks, increased synovitis, osteophyte and soft tissue mineralization at 8-weeks, and more severe cartilage degeneration at 12-weeks post-DMM. ConclusionBoth homozygous and heterozygous Agc1CreERT2 mice exhibit increased susceptibility to PT-OA, underscoring the importance of physiological aggrecan expression in maintaining joint homeostasis and regulating joint pathophysiology. These findings indicate that Agc1CreERT2/+ mice are not phenotypically neutral in the DMM model and that this intrinsic susceptibility should be considered when interpreting studies employing inducible, cartilage-specific gene deletion.

BackgroundPost-traumatic osteoarthritis (PT-OA) is a debilitating condition with significant unmet clinical need. Mesenchymal stem cells (MSCs) represent promising candidates for the treatment of cartilage conditions, owing to their immunomodulatory and regenerative capacities. However, the marked heterogeneity of MSC preparations remains a major challenge for product standardization and prediction of therapeutic efficacy. We previously identified integrin 10{beta}1 as a marker for the selection of a more homogenous MSC preparation, with cartilage repair potential in vivo. In this study, we evaluated the therapeutic efficacy of human MSCs selected for high integrin 10{beta}1 expression in a murine PT-OA model, and compared their effects with those of unselected MSCs. MethodsUnselected or integrin 10-selected human bone marrow MSCs were characterized by flow cytometry and differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Cells encapsulated into fibrin gel were applied intra-articularly at the time of surgery in the destabilization of the medial meniscus (DMM) mouse model of PT-OA. Eight weeks after DMM induction, severity of cartilage damage was assessed on Safranin O-stained sections using the OARSI scoring system. Synovitis, periarticular chondrogenesis, and osteophyte formation were evaluated histologically. OA-associated proteases, extracellular matrix degradation markers, and apoptosis were analyzed by immunohistochemistry, ELISA, and TUNEL assay. Persistence of transplanted human cells was assessed by PCR. ResultsIntegrin 10{beta}1-selected MSCs showed the characteristic MSCs immunophenotype and trilineage differentiation capacity. In vivo, treatment with integrin 10-selected MSCs significantly attenuated PT-OA-induced articular cartilage degeneration compared with both unselected MSCs and vehicle-treated controls. Further histopathological analyses revealed tendency toward reduced synovitis and periarticular chondrogenesis. Moreover, integrin 10-selected MSC treatment was associated with modest reductions in apoptotic activity and decreased expression of OA-related proteases and extracellular matrix degradation markers. Lastly, human cells were not detectable in joint tissues at the study endpoint. ConclusionsIntegrin 10-selected MSCs demonstrated superior chondroprotective effects compared with unselected MSCs, highlighting their potential as a standardized and efficacious cell therapy for PT-OA. These findings further validate the feasibility and safety of selection and in vivo administration of MSCs with high expression of integrin 10.

ObjectiveThe repair response to focal osteochondral injuries frequently fails to truly restore native osteochondral tissue, predisposing the joint to the likelihood of progressive degeneration and post-traumatic osteoarthritis. The biological mechanisms governing the earliest stages of repair in these tissues remain poorly understood, limiting the development of effective regenerative therapies. We therefore aimed to define the early cellular and spatial organisation of repair in a reproducible murine osteochondral injury model by integrating single cell spatial transcriptomics across the whole joint with longitudinal structural imaging and histological analyses. DesignA reproducible, non-critical osteochondral injury was created in the trochlear groove of female C57BL/6 mice. Structural repair was assessed using a multimodal approaching comprising quantitative histology, immunophenotyping, longitudinal magnetic resonance imaging (MRI) and micro-computed tomography ({micro}CT), while whole-joint Xenium spatial transcriptomics at days 3 and 7 defined the cellular and molecular organisation of the early repair response. ResultsSpatial transcriptomics demonstrated that the first week after injury is characterised by the emergence of anatomically distinct immune, vascular and stromal microenvironments across the synovial joint. Resolution of the early inflammatory response was accompanied by regional organisation of repair-associated stromal populations by day 7 after injury. The synovium preferentially supported matrix-associated fibro-chondrocyte-like cells, whereas the osteochondral injury itself retained stress-responsive stromal states with comparatively limited representation of matrix-associated populations. These findings indicate that distinct anatomical niches within the joint are associated with transcriptionally distinct stromal cell phenotypes during early repair. Longitudinal MRI and {micro}CT and histological analysis, demonstrated that these early spatial differences in cell phenotype were associated with progressive restoration of osteochondral architecture, with more effective regeneration of subchondral bone and limited restoration of native articular cartilage. ConclusionsThis study provides, to our knowledge, the first spatially resolved transcriptomic analysis of the early osteochondral repair response to injury across the whole synovial joint. Our findings demonstrate that the first week after injury establishes spatially organised immune, vascular and stromal cell microenvironments. Furthermore, these data suggest that incomplete cartilage repair may reflect an initial failure to establish and sustain matrix-associated stromal cellular states within the injury niche. These findings identify the early repair microenvironment as a critical determinant of tissue regeneration and provide a rationale for regenerative strategies that target repair with spatial and temporal precision.

Background: Knee osteoarthritis (OA) is characterized by progressive joint degradation and altered gait biomechanics. Inter-limb load asymmetry during daily living tasks may accelerate joint deterioration, particularly when combined with physical fatigue. This study investigates the relationship between daily physical fatigue and inter-limb load asymmetry patterns during functional activities in individuals with unilateral knee OA. Objective: To evaluate the impact of fatigue on inter-limb load asymmetry and joint loading rates during daily living tasks in patients with unilateral knee osteoarthritis, and to identify biomechanical and clinical factors associated with load distribution patterns. Methods: One hundred eighteen participants with unilateral knee OA (mean age 62.6 (SD 7.4) years, BMI 29.9 (SD 4.2) kg/m2) completed biomechanical assessments during non-fatigued and fatigued conditions. Ground reaction force measurements were obtained during functional tasks including stair climbing, sit-to-stand transitions, and level walking. Load asymmetry was quantified as the inter-limb difference in peak vertical ground reaction forces. Statistical analysis was conducted using Python (v3.9), including paired t-tests, analysis of variance, and linear regression modeling. Results: Participants demonstrated significantly increased load asymmetry under fatigue conditions (mean difference 8.7% (SD 12.3) %, p < 0.001). Fatigue-induced asymmetry was task-dependent, with stair negotiation showing the greatest increase (11.2% (SD 14.1) %). Peak loading rates on the affected knee were inversely correlated with time-to-peak load during fatigued conditions (r = -0.68, p < 0.001). Disease severity (WOMAC score) and age significantly predicted asymmetry changes (R2 = 0.52, p < 0.001). Conclusions: Physical fatigue exacerbates inter-limb load asymmetry in unilateral knee OA, potentially accelerating joint degeneration. Task-specific interventions targeting fatigue management and load redistribution may improve long-term outcomes. Future longitudinal studies should examine the temporal relationship between acute biomechanical changes and progressive structural joint damage. Keywords: Knee osteoarthritis, load asymmetry, fatigue, biomechanics, ground reaction forces, functional tasks.

Objective: Kindlin-2, encoded by FERMT2, is a focal adhesion protein essential for cartilage homeostasis and mechanotransduction. While loss of Kindlin-2 in mice induces osteoarthritis (OA)-like pathology, the upstream mechanisms regulating its expression in human cartilage remain unknown. Methods: A bioinformatics pipeline was applied to the FERMT2 promoter to predict transcriptional regulators, followed by network and enrichment analyses. Predicted candidates were validated by siRNA knockdown in the human C28/I2 chondrocyte cell line and in primary OA chondrocytes. Pathway enrichment highlighted hypoxia-related regulators, which were further tested by hypoxia mimetic treatment (IOX2) and culture under low oxygen (1% O2). Gene expression changes were quantified by qPCR, and effects on extracellular matrix (ECM) markers were assessed. Results: In silico analysis identified 21 candidate transcription factors, of which GATA1, MEF2A, and RBPJ were validated as regulators of FERMT2. Silencing of GATA1 and MEF2A reduced FERMT2 expression, whereas RBPJ knockdown increased FERMT2 in both C28/I2 cells and primary OA chondrocytes. However, RBPJ silencing also reduced ACAN and increased COL1A1, suggesting detrimental effects on ECM homeostasis. Enrichment analysis revealed a strong association between FERMT2 regulation and hypoxia pathways, supported by conserved hypoxia response elements in the promoter. Experimentally, both IOX2 and 1% O2 significantly upregulated FERMT2 expression in primary chondrocytes. Hypoxia also increased anabolic ECM genes and reduced catabolic enzymes, but these effects occurred independently of Kindlin-2. Conclusions: This study identifies hypoxia as a novel regulator of kindlin-2 expression in human cartilage, providing new insight into the upstream control of this molecule in osteoarthritis. While additional transcription factors such as GATA1, MEF2A, and RBPJ contribute to FERMT2 regulation, only hypoxia consistently enhanced FERMT2 expression without adverse ECM effects.

Even though osteoarthritis is considered a disease of the whole joint, integrated analysis of articular calcified cartilage (ACC) behaviour with adjacent tissues remains a persistent limitation. Herein, we performed synchrotron X-ray computed tomography in intact loaded joints and coupled anatomical analysis with digital volume correlation to examine whether microarchitectural ACC features serve as predictive mechano-biomarkers of osteoarthritis. We reveal that male osteoarthritis-prone STR/Ort mice have thicker yet more porous ACC that contains larger but less spherical chondrocyte lacunae than their healthy parental-control CBA mice. This was linked to asymmetrical distribution of tensile and compressive strains generated under physiological loads in the ACC and underlying subchondral bone in tibial epiphyses of STR/Ort mice. Together these data suggest that murine ACC microarchitecture has a mechano-predictive role in age-related osteoarthritis.

Objective: Toll like receptor (TLR) activation by cartilage derived damage-associated molecular patterns contributes to osteoarthritis (OA) pathogenesis, but the role of synovial fibroblasts in this process remains incompletely understood. We investigated the TLR responsiveness of human OA synovial fibroblasts and determined how TLR activation influences fibroblast-mediated regulation of chondrocyte function. Design: Primary synovial fibroblasts isolated from OA patients were characterized for TLR expression and stimulated with agonists targeting TLR1/2 to TLR9. Inflammatory mediator production, matrix metalloproteinase expression, mitochondrial respiration, and transcriptomic responses were assessed. Functional effects on cartilage homeostasis were evaluated using autologous chondrocyte spheroid co-cultures. Results: Approximately 50% of cells within OA synovial membranes were fibroblasts (CD45-CD31-PDPN+). Synovial fibroblasts expressed multiple TLRs at both the transcript and protein levels. Activation of TLR1/2, TLR4, TLR5, and TLR2/6 induced robust expression of IL-6, IL-8, G-CSF, MMP3, and MMP10, with minimal effects on mitochondrial respiratory function. Transcriptomic analysis revealed activation of inflammatory pathways together with enrichment of antigen presentation and protein translation programs. In autologous co-culture, TLR1/2-activated synovial fibroblasts promoted inflammatory and catabolic gene expression, suppressed anabolic gene expression, and impaired chondrocyte spheroid growth. Conclusions: Human OA synovial fibroblasts are highly responsive to TLR activation and acquire a pro-inflammatory, cartilage-degrading phenotype that directly impairs chondrocyte homeostasis. These findings identify synovial fibroblasts as key effectors of innate immune signalling within the OA joint and support targeting shared TLR-mediated pathways in joint-resident cells as a potential disease-modifying therapeutic strategy.

ObjectiveCellular senescence has been shown to underlie many age-related diseases, including osteoarthritis (OA). In addition to age, biological sex is an OA risk factor with females at greater risk of hand and knee OA. We profiled the senescence burden in OA human synovial fibroblasts while accounting for these factors to understand how senescence may contribute to the increased burden of OA in females. MethodsSynovial fibroblasts were isolated from tissue obtained at knee arthroplasty for OA from 10 male and 10 female donors. Single cell multiplexed immunofluorescence imaging was used to profile the senescence burden in samples age-matched to account for the differences in chronological age. Clustering was performed using stability and generalizability scoring. ResultsIndependent of chronological age, OA synovial fibroblasts from female donors showed higher levels of senescence associated proteins p16, p21, p53, phospho-p65, IL-6, and IL-8. Assessment of oxidative stress associated proteins NRF2, SEPP1, NQO1 and TXNIP indicated a lower capacity for female cells to respond to oxidative stress. Clustering analysis revealed male and female enriched clusters. The female-enriched clusters showed higher levels of senescence-associated proteins and an increased oxidative stress response. ConclusionsOA synovial fibroblasts from female donors demonstrated higher levels of senescence associated markers, lower ability to respond to oxidative stress, and increased senescence with increasing age. These findings indicate that female synovial fibroblasts are more likely to show markers of senescence and oxidative stress, suggesting senescence can contribute to the increased incidence of osteoarthritis in women.

BackgroundKnee osteoarthritis (KOA) is a prevalent degenerative joint disease in which synovial inflammation and fibrosis are closely linked to pain, stiffness, and functional limitation. Growing evidence suggests that metabolic dysregulation, particularly in lipid metabolism, is involved in KOA pathogenesis, but the underlying mechanisms remain incompletely defined. MethodsSprague Dawley rats underwent bilateral anterior cruciate ligament transection to establish a KOA model; sham-operated rats served as controls. RNA sequencing of synovial tissues was performed to identify differentially expressed genes (DEGs) and enriched pathways, followed by GO/KEGG and GSEA analyses. In vivo, adeno-associated virus vectors were used to overexpress or knock down PPAR-{gamma} and phosphoenolpyruvate carboxykinase 1 (PCK1) via intra-articular injection. Ex vivo, primary rat fibroblast-like synoviocytes (FLSs) were stimulated with IL-1{beta} and transfected with PPAR-{gamma} or PCK1 siRNA/overexpression plasmids. synovitis and fibrosis were evaluated by HE, Masson, and Sirius Red staining, immunofluorescence, ELISA, RT-qPCR, and Western blotting. ResultsRNA-seq revealed 621 up-regulated and 228 down-regulated genes in KOA synovium versus sham, with DEGs significantly enriched in PPAR signaling, adipocytokine, and AMPK pathways. Metabolism-related genes including Fabp5, Plin1, Adipoq, Lep, and Pck1 were up-regulated. GSEA indicated downregulation of PPAR-{gamma} signaling in KOA synovium. In vivo and ex vivo, PPAR-{gamma} expression was reduced in KOA, whereas PCK1, FABP5, and ADIPOQ were increased. PPAR-{gamma} overexpression alleviated synovial inflammation, collagen I deposition, and fibrosis, and suppressed FABP5, ADIPOQ, and PCK1 expression; PPAR-{gamma} knockdown produced the opposite effects. Functional studies showed that PCK1 overexpression aggravated synovial inflammatory cell infiltration and fibrosis, elevated IL-1{beta}, IL-18, and TGF-{beta}, and decreased TIMP1 levels in serum, synovial tissue, and FLSs supernatants, whereas PCK1 silencing reversed these changes. ConclusionsThe PPAR-{gamma}/PCK1 metabolic axis modulates synovitis and fibrosis in KOA. Downregulation of PPAR-{gamma} and consequent upregulation of PCK1 promote synovitis and fibrotic remodeling. These findings identify the PPAR-{gamma}/PCK1 pathway as a potential therapeutic target for KOA.

Objective: To determine how fibronectin fragment (Fn-f), a matrikine that induces an osteoarthritis (OA)-like phenotype, reshapes chondrocyte states and transcriptional programs at single-cell resolution. Design: Primary human ankle chondrocytes from two cadaveric donors without OA were treated with PBS or purified recombinant Fn-f for 18 hours and profiled by single-cell RNA sequencing. Transcriptional states were characterized by marker genes and functional enrichment, compared with classifications used for OA chondrocytes, and evaluated for Fn-f-dependent changes in abundance and transcriptional programs. Results: Five transcriptionally distinct populations were identified with features that matched previous classifications of effector, inflammatory, pre-hypertrophic/fibrochondrocyte, reparative, and homeostatic chondrocytes. Fn-f markedly redistributed cells among these states, enriching inflammatory chondrocytes while reducing effector and reparative populations. Within transcriptional states, Fn-f differentially altered matrix/cartilage, inflammatory, and stress/immediate-early programs. These responses did not uniformly correspond to assigned subtype identities, demonstrating that transcriptional programs can be dynamically deployed across chondrocyte states. Conclusions: Controlled perturbations of healthy chondrocytes complement observational OA atlases by resolving stimulus-dependent changes in chondrocyte state abundance and transcriptional programs. Discordance between subtype annotations and dynamic functional programs further highlights the need for harmonized definitions of chondrocytes across phenotypic states.

Abstract Chondrocytes in native articular cartilage are enclosed within a collagen VI (COL VI)-rich pericellular matrix (PCM), forming functional units known as chondrons. However, enzymatic isolation disrupts the PCM, and the functional consequences of its loss and restoration remain poorly understood. In this study, primary human chondrocytes were cultured in alginate beads to promote PCM reconstitution and subsequently recovered as reconstituted chondrons. Chondrocytes and reconstituted chondrons were encapsulated in gelatin methacryloyl hydrogels and compared under inflammatory and mechanical stimulation. Alginate preconditioning generated chondron-like units with a distinct COL VI-positive PCM that was retained after transfer to three-dimensional culture. Under inflammatory conditions, reconstituted chondrons exhibited reduced inflammatory, catabolic, neuroinflammatory, and angiogenic responses compared with isolated chondrocytes at both gene and protein levels. Under interleukin-1{beta} stimulation, mechanical loading further increased inflammatory gene expression in chondrocytes in a donor-dependent manner, whereas responses remained comparatively limited in reconstituted chondrons. Conditioned medium from reconstituted chondrons was also associated with lower capsaicin- and potassium chloride-evoked calcium responses in human induced pluripotent stem cell-derived sensory neurons than corresponding chondrocyte-conditioned medium. These findings demonstrate that reconstitution of a COL VI-rich PCM restores a chondron-like pericellular microenvironment that attenuates inflammatory activation and buffers load-associated inflammatory amplification, while exploratory sensory neuron experiments suggest downstream modulation of neuronal responsiveness. PCM restoration may therefore provide a promising strategy for cartilage tissue engineering and regenerative applications.

Osteoarthritis is a leading cause of chronic pain and disability, which lacks disease-modifying treatment. Given the complex multi-tissue and multifactorial drivers behind disease progression, effective treatments will require simultaneously targeting several mechanisms underlying joint degeneration and pain. Here, we developed and evaluated a combinatorial gene therapy, consisting of a high-capacity adenoviral vector carrying two therapeutic genes to target distinct pathological mechanisms: inflammation (IL-1Ra) and chondrocyte health (PRG4). Intra-articular delivery of this treatment improved functional, structural, and pain outcomes in murine and equine osteoarthritis models. In addition, treatment normalized inflammatory environments in joint tissues, as well as in the dorsal root ganglia (DRG) known to harbor joint-innervating sensory neurons. Moreover, gene therapy reversed OA-induced molecular signatures of neural hyperexcitability, suggesting amelioration of peripheral sensitization. Collectively, these findings support combinatorial gene therapy as a promising treatment for osteoarthritis, while identifying neuroinflammatory signatures for correction of disease progression and pain. One Sentence SummaryA single intra-articular injection of a combinatorial gene therapy slows OA progression and reduces pain in small and large animal models.

Knee Osteoarthritis (KOA) is a progressive whole-joint disease without approved disease modifying OA drugs (DMOADs). Effective treatments have been hindered by multiple layers of heterogeneity, including diverse disease etiology and patient-to-patient variability. Here, we present a scalable, KOA patient-derived (PD) cartilage-on-a-chip (CartChip) model integrating end-stage KOA cartilage tissue explants on a microengineered platform that, under mechanical overloading and hyperinflammatory stressors, mimics different KOA etiologies. These stressors drove distinct multivariable model features, including changes in a curated panel of anabolic and catabolic genes, extracellular matrix protein and soluble factors. Exploratory analysis of coordinated model readouts identified stressor-agnostic and -specific KOA disease signatures. Despite using KOA tissue, the model showed improvements to dexamethasone, a symptom-modifying, anti-inflammatory KOA treatment. The model responses to dexamethasone were dependent on both stressor and donor heterogeneity. Exploratory groupings of coordinated model readouts provided proof-of-concept for predicting categories of patient responsiveness to test therapeutics. Annotating patient data provided additional donor-dependent contexts for interpreting model responsiveness. PD-CartChip provides a powerful research platform to potentially surmount the donor and stressor heterogeneity barrier in developing DMOADs.

Introduction: Osteoarthritis (OA) is a degenerative joint condition characterized by chronic pain and the need for pain management. Locally targeting the endocytotic AP2 complex in nociceptors presents a potential strategy for providing sustained pain relief in individuals with OA. Objective: We investigated whether pain behavior associated with OA can be mitigated by genetically silencing the AP2alpha2 subunit of the AP2 complex in nociceptors and by pharmacologically inhibiting the AP2 complex through the intraarticular administration of a small lipidated decoy peptide. Method: Monoiodoacetate (MIA) was employed to induce knee joint OA in mice and rats. Pain behavior was assessed using dynamic weight-bearing and von Frey filaments. Upon confirmation of established OA pain behavior, in vivo AP2alpha2 genetic knockdown in mice was achieved through sciatic nerve transfection of a targeting AP2alpha2 short hairpin RNA (shRNA). To pharmacologically target endocytosis, a single intraarticular injection of peptide was administered into the arthritic knee of rats. The injection contained either the AP2 inhibitor peptide or a scrambled peptide control. Results: Pain behavior was significantly reduced after both genetic and pharmacological disruption of AP2-driven endocytosis. Animals treated with the Ap2 inhibitor peptide exhibited reduced pain behavior throughout the 28-day assay period. Following the completion of behavioral testing, arthritic knee joints and contralateral healthy knee joints were subsequently collected to assess the impact of the treatment on disease progression. Micro-computed tomography analysis revealed a preservation of bone volume in the arthritic joints that received the AP2 inhibitor peptide treatment, in contrast to the scrambled peptide group. Conclusion: These findings demonstrate that the inhibition of nociceptor endocytosis by a small lipidated peptide presents a promising approach to provide sustained relief from joint pain in individuals with arthritis.

Skeletal muscle dysfunction is a major contributor to disability and incomplete functional recovery in patients with end-stage knee osteoarthritis (KOA), yet the spatial components of disease-associated molecular remodeling remain poorly understood. Here, we applied spatial transcriptomics to paired skeletal muscle biopsies obtained from the surgical (Sx) and contralateral (Ct) limbs of individuals undergoing total knee arthroplasty (TKA) to define the cellular architecture of periarticular muscle and determine how muscle inflammation susceptibility (MuIS) shapes local transcriptional programs. Integrated analysis of 27,087 spots obtained from 22 muscle histological cross-sections (11 Sx-Ct pairs) revealed seven spatially resolved transcriptional domains corresponding to slow and fast myofiber states, an extracellular matrix/fibroadipogenic-enriched domain, a pericyte/smooth muscle domain, and a satellite cell/myogenic-enriched domain. Despite advanced unilateral disease, the major annotated cellular compartments were similarly represented between Sx and Ct limbs. KOA-associated remodeling was reflected primarily by within-cluster transcriptional changes, with the most informative differences observed in fibroadipogenic, pericyte/smooth muscle, and satellite/myogenic domains. Within Sx, MuIS stratification identified a coordinated transcriptional program characterized by denervation- and regeneration-associated genes and altered contractile and metabolic features. Neighborhood analysis localized denervation-associated signals primarily to fast-myofiber-rich regions, while local adjacency patterns among the examined myofiber, fibroadipogenic, and pericyte/smooth muscle domains were broadly preserved. Our findings provide the first spatial transcriptomic analysis of periarticular skeletal muscle in end-stage KOA and identify MuIS as a distinct local transcriptional phenotype in diseased muscle.

Many microRNAs (miRNAs) regulate tissue remodeling, cellular plasticity, and repair across evolutionarily distant vertebrate lineages that are capable of regenerating appendages such as amputated limbs, fins, and antlers, as well as in human articular cartilage responding to injury. These miRNAs often belong to the same families and exert conserved, though occasionally inverted, regulatory effects. This strong cross-species overlap motivates the present literature-derived analysis. Osteoarthritis (OA), the most prevalent joint disease, still lacks disease-modifying therapies, in part because of the longstanding assumption that adult mammalian cartilage demonstrates no intrinsic reparative capacity. Yet human cartilage retains a latent repair program activated by mechanical and inflammatory stress. Some injured or degenerating joints may never be clinically recognized as osteoarthritic because their intrinsic repair capacity is sufficient to restore tissue integrity; in others, where repair capacity is diminished or damage exceeds it, the repair program is insufficient and OA becomes clinically manifest. MiRNAs are established post-transcriptional regulators of cartilage homeostasis, degeneration, and appendage regeneration, yet because the OA and regeneration research fields have advanced largely independently, the insights available at their intersection have gone unrecognized. To close this gap, we systematically mined both literatures to construct an auto-updating, cross-referenced atlas of OA- and appendage regeneration-associated miRNAs. Integrating these datasets identified a core set of shared miRNA families, delineated miRNAs unique to each field, and mapped convergent families onto common pathways governing matrix remodeling, dedifferentiation, senescence, and inflammation. We propose that regeneration-competent species can inform the identification of therapeutic miRNAs, such as miR-133, miR-21, and let-7, capable of activating endogenous cartilage repair. Collectively, this synthesis and its accompanying web-based miRNA atlas (https://mirnaatlas.shinyapps.io/mirnaatlas/) establish a comparative framework for regenerative miRNA biology and provide a continually updated resource to accelerate discovery of disease-modifying, RNA-based therapies for OA.

Cartilage thickness on MRI is a structural biomarker of osteoarthritis (OA), yet anatomical variation and aging can mask early disease, including changes occurring 12-36 months before incident OA. We propose longitudinal local centile maps that use normative growth charts as prior information and integrate a knee's earlier scans to identify cartilage that is thinner or thicker than expected. We built the reference charts from 5,045 scans of 957 persistently radiographically normal knees in the Osteoarthritis Initiative. Under participant-separated nested cross-validation, longitudinal centile profiles achieved higher 12- and 24-month AUCs for predicting incident radiographic OA than regional PCA, radiomics, cross-sectional centile features, and raw-map deep features across most classifiers. These conventional descriptors added little when combined with centile profiles, and cross-sectional centiles added little beyond longitudinal centiles. Deep centile features achieved the best AUCs (0.954 [95% CI 0.931-0.974] at 12 months and 0.969 [0.949-0.985] at 24 months; balanced accuracy 0.917 and 0.931) and outperformed raw-map deep features in all 12 classifiers after correction for multiple comparisons. No representation consistently improved 36-month prediction. Longitudinal centiles transform cartilage thickness maps into reference-based deviation maps: deep longitudinal centile features provide the highest predictive accuracy, while regional longitudinal centile profiles provide interpretable prognosis with the second-highest accuracy.

Osteoarthritis is the most common joint disease for which disease-modifying therapies remain unavailable. Intra-articular gene delivery of interleukin-1 receptor antagonist (IL-1Ra) using high-capacity adenovirus (HCAd) has shown therapeutic promise; however, the duration of therapeutic benefit and the feasibility of repeat dosing under anti-adenoviral immunity remain unresolved. Using the murine anterior cruciate ligament transection model of osteoarthritis, we show that a single intraarticular injection of HCAd5-NF{kappa}B-IL-1Ra provides structural preservation and functional improvement in early-stage osteoarthritis but fails to sustain cartilage protection as disease progresses. However, HCAd5 transduction following repeated treatment is limited due to pre-existing immunity against this serotype. Notably, exchanging serotypes for repeated treatments effectively restores vector transduction and transgene expression in both healthy and osteoarthritic joints. Leveraging this strategy, we demonstrate that sequential intra-articular HCAd-NF{kappa}B-IL-1Ra administration does not further improve pain or motor function compared to the initial treatment, but preserves cartilage as assessed by histopathology and phase-contrast CT, irrespective of serotype. These findings establish HCAd serotype switching as a feasible approach to overcome immune barriers to repeat intra-articular gene therapy. Importantly, sequential HCAd-NF{kappa}B-IL-1Ra administration enhances therapeutic durability in post-traumatic osteoarthritis, providing a translational framework for repeat intra-articular gene delivery strategies aimed at long-term disease modification in osteoarthritis.

Objectives. Observational studies report large, conflicting associations between common drugs and hip or knee replacement. We tested them in the serious adverse event (SAE) tables of randomized trials on ClinicalTrials.gov. Methods. We included randomized drug trials with at least 500 participants. A language model classified arms from summaries without outcome counts (masked by instruction only). We pooled osteoarthritis-related SAEs by drug class as Mantel-Haenszel risk ratios (RRs). Because the plan followed a preliminary analysis of 72 trials, we report estimates rather than tests, checked by positive and negative controls and calibration. Results. The 881 placebo- or usual-care-controlled trials included 1.75 million participants and 9,408 osteoarthritis-related SAEs. The tables reproduced CANTOS's own canakinumab effect (RR 0.57, 95% CI 0.39 to 0.82), the anti-nerve growth factor excess (2.31, 1.82 to 2.92) and two of three modest effects specified before extraction: gallbladder disease with glucagon-like peptide-1 receptor agonists (GLP-1RAs; 1.36, 1.22 to 1.51) and ketoacidosis with sodium-glucose cotransporter-2 (SGLT2) inhibitors (1.96, 1.48 to 2.60). In the same trials, osteoarthritis-related SAEs did not differ detectably with GLP-1RAs (0.97, 0.86 to 1.10; calibrated 0.87, 0.54 to 1.41) or SGLT2 inhibitors (0.96, 0.82 to 1.12; calibrated 0.86, 0.53 to 1.40). Other proposed drugs gave imprecise estimates. Post hoc, 8 of 21 published observational estimates differed from randomized ones, in both directions (3 to 6 after calibration). Conclusions. Over about two years, GLP-1RAs and SGLT2 inhibitors had no detectable effect on osteoarthritis-related SAEs in trials that detected their known effects. Slow or modest structural effects remain possible.

Objective: Osteoarthritis (OA) is a degenerative joint disease associated with metabolic dysregulation. This study aims to identify mitochondrial-lipid metabolism-related hub genes and explore their mechanisms in OA. Methods: Training (GSE55235) and validation (GSE55457 and GSE82107) cohorts were retrieved from the Gene Expression Omnibus (GEO) database. Candidate genes were derived by overlapping differentially expressed genes, mitochondrial-lipid metabolism-related genes, and weighted gene co-expression network analysis module genes. Machine learning algorithms were applied to screen hub genes. The diagnostic performance, functional pathways, immune infiltration, regulatory networks, drug interactions, and molecular docking of the hub genes were systematically evaluated. An interleukin-1{beta}-stimulated chondrocyte OA model was assayed for cell viability, interleukin-6 (IL-6), apoptosis, and hub-gene expression via Cell Counting Kit-8, enzyme-linked immunosorbent assay, flow cytometry, and reverse transcription-polymerase chain reaction. Results: Twenty-five candidate genes were screened, from which six hub genes (alpha-methylacyl-CoA racemase [AMACR], uncoupling protein 2 [UCP2], acyl-CoA thioesterase 7 [ACOT7], aldehyde dehydrogenase 3 family member A2 [ALDH3A2], armadillo repeat containing X-linked 2 [ARMCX2], and monoamine oxidase A [MAOA]) were further identified. The six hub genes exhibited robust diagnostic ability in the training cohort, correlated with immune-cell infiltration, and participated in lipid-metabolism pathways. In the OA model, chondrocyte viability was reduced, while IL-6 levels and apoptosis rate were elevated; five hub genes were markedly upregulated, whereas MAOA was significantly downregulated. Conclusion: Mitochondrial-lipid metabolism-related hub genes may serve as potential diagnostic biomarkers for OA, providing new insights into OA pathogenesis and potential therapeutic targets.