Osteoarthritis and Cartilage

ObjectiveTo validate SERPINB2 and SERPINA9 as chondrogenic biomarker candidates across independent transcriptomic platforms and cell sources, to characterise the complete SERPIN expression landscape during kartogenin (KGN)-induced chondrogenic differentiation of human mesenchymal stem cells (hMSCs), and to identify novel SERPIN biomarker candidates and their signalling context during cartilage lineage commitment. DesignMulti-platform transcriptomic analysis across three independent datasets: (i) Affymetrix HGU133+2 microarray of KGN-induced chondrocytes versus undifferentiated hMSCs (ATCC source); (ii) Affymetrix Clariom D whole-transcriptome array of KGN-treated versus control hMSCs from an independent Mexican source (SINREG Laboratories); and (iii) previously published qPCR validation. Differential expression was computed using limma with Benjamini,Hochberg correction. SERPIN-focused cross-platform correlation and targeted pathway analysis were performed. ResultsThe Clariom D dataset yielded 1,869 differentially expressed genes (925 upregulated, 944 downregulated; FDR < 0.05) from 29,124 transcripts tested. SERPINB2 was concordantly upregulated across all three platforms (Clariom D: fold-change [FC] +3.54, FDR = 0.006; HGU133+2: log2FC = +3.29, nominal P = 0.027; qPCR confirmed), establishing it as one of the most reproducible transcriptomic signals in chondrogenic differentiation. In the direct Bone versus Cart comparison, SERPINB2 showed [~]45-fold chondrogenic enrichment (log2FC = -5.45, adjusted P < 0.0001). Cross-platform SERPIN correlation was significant (Pearson r = 0.54, P = 0.0025; n = 29 shared genes). Four additional SERPINs reached genome-wide significance on Clariom D: SERPINE2 (FC +2.57), SERPING1, SERPIND1, and SERPINE1. SERPINA9 was not replicated in the independent SINREG source, identifying it as a context-dependent marker. ConclusionsSERPINB2 is a robust, cross-platform chondrogenic biomarker with translational potential for osteoarthritis (OA) monitoring. The coordinated SERPIN programme activates a multi-layered proteolytic and signalling network during cartilage lineage commitment, positioning SERPINB2 as a functional regulator of the chondro-osteogenic lineage decision.

BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease characterized by the progressive deterioration of articular cartilage, significantly impacting the quality of life in middle-aged and elderly populations. The Ras and Hippo signaling pathways play critical roles in regulating cell proliferation, differentiation, and stress responses; however, their interactive mechanisms in OA remain unclear. This study aimed to identify key genes associated with these two pathways using bioinformatic approaches and to elucidate their potential mechanisms in OA. MethodsTranscriptomic data of OA along with Ras signaling pathway-related genes (RSPRGs) and Hippo signaling pathway-related genes (HSPRGs) were obtained from public databases. Differentially expressed genes (DEGs) were identified, and key genes were screened through machine learning, expression validation, and receiver operating characteristic (ROC) curve analysis. Functional insights were further explored via gene set enrichment analysis (GSEA), subcellular localization, immune infiltration analysis, regulatory network construction, and drug prediction. Finally, the expression of key genes was validated in clinical samples. ResultsKIT and CSF1R were identified as key genes. GSEA indicated their involvement in pathways such as the lysosome pathway. Subcellular localization predicted that KIT and CSF1R are distributed in the nucleus, extracellular region, and plasma membrane. Immune infiltration analysis revealed that KIT showed a positive correlation with eosinophils and a negative correlation with immature dendritic cells, while CSF1R was positively correlated with macrophages and negatively correlated with CD56 natural killer cells. Drug prediction suggested interactions between the key genes and several therapeutic agents, including avapritinib and IMC-CS4. Subsequently, we validated our findings in cartilage tissue samples and discovered that compared to the control group, both CSF1R mRNA and protein expression was significantly upregulated in OA tissue, while KIT expression was significantly downregulated.The same results were also validated in immunofluorescence staining of chondrocytes. ConclusionThis study identified KIT and CSF1R as key genes in OA, providing new theoretical insights and potential targets for mechanistic research and targeted therapy.

ObjectiveDuring osteoarthritis (OA) progression, the synovial membrane undergoes profound structural and compositional remodeling and fibrosis. We sought to elucidate how evolving synovial microenvironmental mechanics during fibrotic remodeling influence cell behavior and drive the progression of synovial pathology. MethodsSkeletally-mature male C57BL/6J mice were subjected to destabilization of the medial meniscus (DMM). To control for surgical confounders, both sham-operated and unoperated mice were included, with evaluation at 4- and 8-weeks. Synovial micromechanics were quantified via atomic force microscopy (AFM). Single-cell RNA sequencing (scRNA-seq), RNA fluorescence in situ hybridization (FISH), and flow cytometry were employed to investigate cellular heterogeneity, spatial organization, and crosstalk within fibrotic and non-fibrotic synovial niches. ResultsProgressive fibrotic remodeling and marked matrix stiffening were observed in DMM-operated synovium but absent in sham- and un-operated controls. While both sham and DMM joints mounted an acute stromal and immune response to surgery, these changes resolved over time in sham conditions but persisted in DMM synovium. During disease progression, distinct functional subsets of synovial fibroblasts and immune cells emerged, with mechanosignalling pathways and distinct immune cell-fibroblast crosstalk robustly activated within DMM-induced fibrotic microenvironments. ConclusionThis study demonstrates the complex cellular dynamics and crosstalk that differentiate the evolution of the pathological synovial response in the fibrotic DMM condition relative to surgical sham controls. Our findings highlight mechanotransduction as a central mechanism driving OA synovial pathogenesis and underscore the utility of the DMM model as a platform to dissect the molecular underpinnings of synovial fibrosis.

Traumatic joint injuries both disrupt chondrocyte metabolism and increase the risk for post-traumatic osteoarthritis. Yet the relationships between trauma, altered metabolism, and cartilage degradation remains unclear. This study compares the metabolic responses of bovine (normal) and osteoarthritic (OA) chondrocytes to physiological and injurious mechanical stimuli under normoxic (20% O2) and hypoxic (5% O2) conditions. Using primary chondrocytes encapsulated in agarose, physiological and injurious mechanical stimulation, targeted metabolomic profiling of central carbon metabolites, and O2 saturation measurements, we find that healthy bovine chondrocytes exhibit robust, time-dependent adaptation to mechanical stimuli, whereas OA chondrocytes display a blunted response, particularly under injury conditions. Injurious mechanical stimuli led to altered O2 consumption and glutamine accumulation, suggesting disrupted respiration and reduced protein synthesis hypothesized to be a result of altered mitochondrial metabolism in OA cells. These findings underscore the role of mechanical cues in chondrocyte metabolism and inform future studies aimed at identifying metabolic targets relevant to post-traumatic osteoarthritis progression.

BackgroundPeople with hip or knee joint arthroplasties are commonly advised to avoid high-impact physical activities, despite increasing demand to return to sport and vigorous exercise. Current implant testing standards do not reflect real-world loading during high-impact tasks, and few studies have quantified implant loads in high-functioning individuals who have returned to such activities. MethodsHigh-functioning adults with a total hip arthroplasty (THA, n = 11), total knee arthroplasty (TKA, n = 4), or unicompartmental knee arthroplasty (UKA, n = 3) performed a range of low-to high-impact activities, including walking, running, hopping, countermovement jumps, landings, and change-of-direction tasks. Three-dimensional trunk and lower-limb kinematics and ground reaction forces were collected. Musculoskeletal modelling was used to quantify three-dimensional hip and knee joint contact forces. Linear mixed-effects models were used to rank implant loads across activities and to compare peak resultant joint loads with healthy controls from a prior study. ResultsFor people with THR, relative to walking, a 45{degrees} change of direction generated the highest predicted hip contact force (8.38 BW, 95% CI 7.70-9.06), followed by running and unilateral hopping (all >1.5x walking, p < 0.05). Unilateral hopping and running produced the highest predicted knee contact force in TKA and UKA participants (8.0-9.1 BW), and both significantly greater than walking (p < 0.05). Compared with healthy controls, THA participants exhibited a lower predicted HCF during walking (-1.58 BW, 95% CI -2.46 to -0.69), but no group differences were observed for running, hopping, or jumping. ConclusionHigh-impact activities vary widely in model-estimated hip and knee contact forces. Several tasks were not substantially higher than walking. These data provide a biomechanical basis for evidence-informed activity prescription, regulatory implant testing, and future computational simulation of implant performance under realistic loading conditions.

ObjectivePhysical activity is a first-line therapeutic intervention for managing osteoarthritis-related pain and functional impairment. However, the growing literature questions the long-term relevance of exercise-induced improvements in patients, while pre-clinical research evidence base is limited by reliance on stressful, forced exercise paradigms which do not reflect voluntary engagement. Here, we aimed to investigate the effects of voluntary wheel running on the pain experience in mice with joint pain. DesignWe investigated the impact of free access to a running wheel on sensory, functional and affective outcomes following unilateral intra-articular injection of monoiodoacetate in single-housed male and female C57Bl/6J mice. ResultsMonoiodoacetate injection transiently reduced running activity in both sexes; however, females rapidly resumed and sustained high activity levels over a two-month period, while males showed a progressive decline in running distance. Active males and females showed improvements in the monoiodoacetate-induced hindpaw secondary mechanical hypersensitivity. Moreover, mechanical thresholds positively correlated with the distance ran after injury, suggesting a functional relationship between exercise and secondary pain relief. However, access to a wheel temporarily exacerbated several monoiodoacetate-induced gait impairments in both sexes. Finally, while there were no obvious effects of running on anxio-depressive-like behaviours or cognitive functioning, exercise significantly impacted stress-induced faecal output and phenotypic regulation of body weight. ConclusionsOur findings suggest that persistent loading of an injured knee joint may compromise functional outcomes independently of pain relief away from the joint, underscoring a critical consideration for exercise-based therapeutic strategies in osteoarthritis.

Aberrant bone remodeling is a hallmark of osteoarthritis, the most common arthritis affecting over 27 million US adults. Subchondral bone sclerosis, one sign of aberrant bone remodeling observable by routine x-rays, occurs as the trabeculae thicken, leading to increased bone volume. Toll-like receptors, pattern-recognition receptors of the innate immune system, have been implicated in OA pathogenesis, with TLR ligands, receptors, and co-receptors shown to mediate the severity and progression of OA. We have previously shown that CD14-deficiency protects mice against post-traumatic OA, and specifically reduces subchondral sclerosis post-injury. We hypothesized that depletion of CD14 protects against TLR4-dependent inhibition of osteoclastogenesis and therefore increases OC density in the SCB after injury, mitigating aberrant bone deposition in a murine model of OA. To determine how cellular changes correlate with bone structure derangements post-DMM, we performed MicroCT, Tartrate-resistant acid phosphatase staining, and alkaline phosphatase staining. To establish mechanistic changes in cellular signaling, we isolated WT and CD14-deficient osteoclast precursors and subjected them to LPS, an osteoarthritis-relevant TLR ligand, during differentiation. CD14-deficient mice, as well as WT mice treated with an anti-CD14 monoclonal antibody, show protection from post-injury increases in both bone volume fraction and bone mineral density. CD14-deficient mice had an increased osteoclast presence in the SCB two weeks post-injury, potentially protecting them from increases in bone volume and density. In vitro, CD14-deficient OCPs differentiated faster than WT OCPs, due to reduced Type I Interferon (IFN-I) signaling. In the presence of an LPS challenge, CD14-deficient OCPs were protected against LPS and TLR4-mediated inhibition, likely due to decreased MyD88-dependent TLR4 signaling. This work opens up new potential pathways to therapeutically target aberrant bone remodeling in the setting of joint injury and PTOA. Lay SummaryOsteoarthritis is one of the leading causes of disability worldwide. One of the hallmarks is subchondral sclerosis, or thickening of the bone in and around the joint. In this work, we used a mouse model of osteoarthritis to show that decreasing inflammatory signaling, through removal of CD14, protects against subchondral sclerosis, due to an increased presence of osteoclasts, cells that combat bone thickening. Osteoclasts without CD14 differentiate faster than osteoclasts with CD14, due to decreased Type I Interferon, an inflammatory cytokine. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/705094v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@176bdd5org.highwire.dtl.DTLVardef@a914bborg.highwire.dtl.DTLVardef@902748org.highwire.dtl.DTLVardef@2f9b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ex vivo models bridge in vitro and in vivo systems by preserving native extracellular matrix architecture and multicellular interactions. In articular joint research, osteochondral-synovial co-cultures are particularly valuable for studying bone-cartilage crosstalk and synovial inflammatory regulation. However, a lack of standardized culture conditions regarding glucose and oxygen, two key regulators of cellular metabolism, limits reproducibility and translational relevance. This study aims to define how glucose and oxygen conditions influence joint tissues maintenance in an ex vivo model. Bovine osteochondral explants and synovium are harvested from the stifle joint and co-cultured using either high glucose DMEM (HG, 4.5 g/L) or low glucose DMEM (LG, 1 g/L) under hyperoxic (21% O2) or physioxic (5% O2) conditions. Cell viability, gene expression, and metabolomic profiles are evaluated across tissues. LG conditions increase cell death in the deep zone of cartilage and in subchondral bone. Gene expression and metabolomic analyses reveal tissue-specific effects of glucose and oxygen. In cartilage and bone, glucose-dependent gene regulation and metabolic changes occur under hyperoxia but are largely absent under physioxia, indicating buffering of glucose responses. Gene-specific sensitivity to glucose and oxygen is observed in bone and synovium; however, glucose-induced metabolic responses persist under physioxia only in synovium. Overall, these findings identify oxygen and glucose as critical modulators of joint tissue physiology and support the use of HG, physioxic culture conditions to improve cell viability and stabilize molecular outcomes in ex vivo joint models. This optimized ex vivo model provides platforms for investigating mechanisms relevant to joint-related diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/704322v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1393324org.highwire.dtl.DTLVardef@4c9393org.highwire.dtl.DTLVardef@16cc00eorg.highwire.dtl.DTLVardef@b4d9ea_HPS_FORMAT_FIGEXP M_FIG C_FIG This study evaluates the effects of glucose concentration and oxygen tension in an ex vivo joint co-culture system to define optimal culture conditions. High glucose medium and physioxic conditions support tissue viability, preserve homeostasis, and enhance the physiological relevance of the ex vivo model.

OBJECTIVESOsteoarthritis is a heterogeneous disease, with diverse structural patterns likely reflecting distinct genetic drivers. Robust, data-driven methods to identify and characterise such phenotypes are lacking. This study leveraged the UK Biobank to define machine learning-derived structural osteoarthritis phenotypes and evaluate their clinical and genetic profiles. METHODSMachine learning models were applied to knee and hip DXA scans to derive osteophyte area, minimum joint space width, and B-scores (a combined shape vector predictive of osteoarthritis). Imaging and demographic features were clustered using k-means to classify individuals with at least one osteoarthritis feature. Phenotypes were compared with healthy controls for associations with joint pain and total joint replacement (TJR). Genetic correlations, osteoarthritis risk loci, and polygenic risk scores were analysed to define shared and distinct genetic mechanisms between phenotypes. RESULTSAmong 59,539 participants (mean age 65 years; 53% female), nine reproducible phenotypes were identified, spanning joint-specific and multi-joint patterns. Hypertrophic and end-stage knee phenotypes showed the highest odds of pain (OR 7.8 [95% CI 7.1,8.7], 13.4 [9.5,19.0]) and TJR (66.0 [46.6,93.5], 127.6 [72.6,224.1]). A novel increased-cartilage phenotype was associated with greater odds of hip (3.5 [2.4,5.2]) and knee replacement (4.1 [2.6,6.6]). Distinct genetic architectures were observed; increased- and atrophic-cartilage phenotypes were inversely genetically correlated (rg -0.46 [-0.9,-0.2]) with opposing effects at DOT1L and COL27A1. CONCLUSIONSMachine learning revealed nine reproducible osteoarthritis structural phenotypes with divergent clinical and genetic signatures. These findings demonstrate that simple imaging and demographic data can stratify patients into biologically distinct phenotypes likely to require tailored treatments. Key messagesWhat is already known on this topic? O_LIDifferent osteoarthritis phenotypes have been proposed, which could guide patient stratification for drug trials and pharmacotherapy. However, these proposals have mainly been based on analysis of small numbers of patients that are focused on the knee joint alone. C_LIO_LITo our knowledge, no systematic, hypothesis-free approach has been applied to classify different osteoarthritis phenotypes using structural features derived from large numbers of individuals. C_LI What this study adds? O_LIThis study identifies and characterises nine reproducible structural phenotypes of osteoarthritis across both the hip and knee using high-resolution DXA imaging in UK Biobank. C_LIO_LIIt demonstrates that these phenotypes have distinct clinical profiles, with widely varying risks of joint pain and subsequent joint replacement. C_LIO_LIIt provides robust evidence that the phenotypes differ in their genetic architecture, supporting the existence of genetically determined endotypes within osteoarthritis. C_LI How this study might affect research, practice or policy? O_LIThe findings advance understanding of the structural heterogeneity of osteoarthritis and highlight that distinct phenotypes represent different biological pathways guiding research into future disease modifying therapeutics. C_LIO_LIThe automated, scalable methods used here could support patient stratification in clinical trials, enabling targeted evaluation of treatments in phenotypes most likely to benefit, an essential step towards a precision medicine approach in osteoarthritis. C_LI

Objective To evaluate the Cartilage Thickness Score (CTh-Score) as a quantitative measure of cartilage damage severity by assessing its association with three osteoarthritis (OA) milestones and comparing its performance with conventional morphometric measures (radiographic minimum joint space width (JSW) and regional average cartilage thickness). Methods Data were obtained from the Osteoarthritis Initiative (OAI) and the publicly available OAI CTh-Maps and CTh-Score dataset. Three matched case-control designs were used to represent major OA milestones: (i) incident radiographic OA onset, (ii) combined pain and structural progression, and (iii) knee replacement (KR) in the coming 2 years. Progression subjects were extracted from the FNIH Biomarkers Consortium cohort. Cases and controls were compared at 4 years (T-4Y), 2 years (T-2Y), and 0 years (T0) before the milestone. MRI-based CTh-Score and regional average cartilage thickness, as well as JSW, were analyzed cross-sectionally and longitudinally. Associations with case status were assessed using adjusted logistic regression models, and responsiveness was evaluated using longitudinal change and standardized response means. Results The onset cohort included 307 matched case-control pairs, the progression cohort 164 cases and 369 controls, and the KR cohort 81 cases and 324 controls. Across all three study designs, the CTh-Score significantly differentiated cases from controls at all timepoints. In the onset cohort, the CTh-Score was higher in future cases than controls at T-4Y (16.2 vs 12.6, p=0.007), T-2Y (23.5 vs 16.7, p<0.001), and T0 (39.8 vs 18.6, p<0.001), whereas JSW and regional thickness measures showed limited or later discrimination. Similar findings were observed for progression (43.2 vs 33.0 at T-4Y; p<0.001) and KR (55.4 vs 46.1 at T-4Y; p=0.02) cohorts. Longitudinally, CTh-Score changes differentiated cases from controls earlier and more consistently than JSW or regional average thickness, and its responsiveness was consistently the highest across OA milestones and time intervals. In adjusted models, the CTh-Score was independently associated with all outcomes at T-4Y and T-2Y, with odds ratios per standard deviation increase ranging from 1.3 to 2.2. Conclusion The CTh-Score captures high-resolution cartilage thickness patterns associated with OA onset, progression, and future knee replacement, outperforming conventional morphometric measures in early discrimination, responsiveness, and predictive association. These findings support CTh-Score as a sensitive quantitative marker of cartilage damage severity across the OA continuum.

Purpose To determine whether clinically significant weight loss (>5% of body weight) is associated with slower 2-year knee cartilage degeneration in individuals with and without radiographic osteoarthritis. This study used a cartilage structural assessment score derived from the spatial distribution of cartilage thickness, referred to as the Cartilage Thickness Score (CTh-Score). It is based on cartilage thickness patterns and scores the cartilage between 0 and 100, with higher scores indicating greater severity. Methods We conducted a retrospective matched cohort study within the Osteoarthritis Initiative. High-resolution cartilage thickness maps (CTh-Maps), along with their corresponding CTh-Score, were extracted from a public repository. Participants with complete radiographic and MRI data at baseline and 24 months were stratified by baseline Kellgren-Lawrence (KL) grade into non-radiographic OA (non-ROA; KL<2) and radiographic OA (ROA; KL>=2). Within strata, cases (>5% 2-year weight loss) were propensity score-matched 1:2 to weight-stable controls on age, sex, height, weight, KL grade, joint space width (JSW), KOOS Pain, baseline CTh-Score, and mean cartilage thickness in the medial and lateral femoral and tibial compartments. The primary outcome was 2-year change (delta) in CTh-Score, where higher values indicate worsening. Secondary outcomes were delta JSW, delta regional mean cartilage thickness, and delta KOOS Pain. Non-parametric tests were used. Results We included 164 cases and 328 controls in non-ROA, and 266 cases and 532 controls in ROA. Median (interquartile range) weight loss was -6.10 kg (-8.90, -4.70) versus +0.30 kg (-1.30, 2.20) in non-ROA and -6.80 kg (-9.10, -5.02) versus +0.40 kg (-1.40, 2.82) in ROA (both p<0.001). Weight loss was associated with significantly smaller 2-year increases in CTh-Score: in non-ROA, median 1.58 (0.61, 6.53) vs 3.14 (0.44, 7.12) (p=0.005); in ROA, median 1.69 (0.97, 6.71) vs 2.90 (0.19, 7.38) (p=0.004). No between-group differences were detected for delta JSW or delta regional mean cartilage thickness in any of the 4 ROIs. A trend toward greater KOOS Pain improvement with weight loss was observed in ROA: 2.75 (-3.35, 13.40) vs 0.00 (-5.60, 8.40) (p=0.06). Conclusions Achieving >5% weight loss over 2 years is associated with approximately 50% lower progression in median cartilage degeneration, as assessed by CTh-Score, in both non-ROA and ROA. No change was observed with conventional structural metrics. These findings support weight management as a structural disease-modifying strategy and highlight CTh-Score as a sensitive endpoint.

The osteochondral junction is a specialized region ensuring the biomechanical and biological integration of the unmineralized articular cartilage with the subchondral bone through an intermediate layer of mineralized cartilage. This location is of clinical relevance, being the target of osteoarthritis. While aging is considered a risk factor for osteoarthritis, the interplay between microstructural and material changes during aging and predisposing to joint degeneration is not fully clear. This is especially true for mineralized cartilage, which remains understudied despite its critical role in load transfer from unmineralized articular cartilage to bone. We investigate age-related alterations of mineralized cartilage and subchondral bone in rat tibiae of adult and aged animals using a multimodal, high-resolution, correlative analysis. Our approach includes micro-computed tomography to measure microstructural features, second harmonic generation imaging to visualize collagen organization, quantitative backscattered electron imaging to map local mineral content, and nanoindentation to obtain mechanical properties. Mineralized cartilage and subchondral bone exhibited distinct age-related modifications. At the architectural level, the subchondral plate thickened and the trabecular network became coarser, those changes being different from those observed in the metaphysis. At the tissue level, mineralized cartilage was less mineralized than bone but exhibits a greater relative increase of mineral content with age, underlying differences in mineralization. A central observation is that aging led to an abrupt transition in mineral content and mechanical properties across the interface between unmineralized and mineralized cartilage, with a conceivable impact on stress localization. Overall, these changes may alter load transfer and contribute to age-related joint degeneration.

BackgroundOsteoarthritis (OA) is a leading cause of disability worldwide, yet no licensed therapies can prevent or slow its progression. We aimed to identify potential targets for disease-modifying OA drugs (DMOADs) by integrating genetic and differential protein expression (DPE) evidence. MethodsWe evaluated genetically predicted perturbations of plasma protein levels using cis-protein quantitative trait loci (cis-pQTLs) across three large European cohorts (UK Biobank Pharma Proteomics Project, deCODE, and Fenland) and outcome data from the Genetics of Osteoarthritis Consortium, covering 11 OA phenotypes. DPE analyses were performed in 44,789 UKB participants, comparing 2,920 protein measurements between OA cases and controls, supported by sensitivity analyses. Proteins identified through genetic and/or DPE approaches were further assessed in downstream analyses. FindingsIn total, 305 proteins showed evidence of association with OA through genetically predicted perturbations, with 81 supported by colocalisation across datasets. DPE analyses identified 605 proteins associated with at least one OA phenotype, of which 450 (74{middle dot}4%) remained robust after sensitivity testing. Several novel targets were identified, including PPP1R9B, PCSK7, and ITIH4. Integration of both approaches prioritised 5 proteins, 4 of which demonstrated druggable potential, including 3 high-confidence candidates DLK1, TNFRSF9, and OGN. Downstream analyses highlighted key biological pathways and candidate compounds with potential for repurposing. InterpretationThis large-scale study combines genetic and DPE evidence to prioritise candidate DMOAD targets. Findings reinforce established biology while revealing novel proteins and pathways, providing a foundation for therapeutic development in OA. FundingWL is supported by the Guangzhou Elite Project (project no. JY202314). SSZ is supported by The University of Manchester Deans Prize, Arthritis UK Career Development Fellowship (grant no. 23258). This work is supported by the NIHR Manchester Biomedical Research Centre (NIHR203308). Research in contextO_ST_ABSEvidence before this studyC_ST_ABSCirculating proteins have been linked to osteoarthritis (OA) in observational studies, supporting their potential as biomarkers and drug targets. However, differential protein expression analyses are vulnerable to confounding and reverse causation. Mendelian randomisation (MR) studies using proteomic GWAS instruments have suggested causal roles for several circulating proteins in OA-related traits and highlighted druggable candidates. However, many analyses relied on earlier OA GWAS data (e.g., Genetics of Osteoarthritis Consortium 1{middle dot}0) and smaller proteomic GWAS datasets, and typically did not integrate MR findings with large-scale differential protein expression. As a result, it remains unclear how well genetically predicted protein effects align with observed protein expression in OA, and how robust prioritised targets are when replicated across proteomic data from multiple cohorts. Added value of this studyThis study integrates large-scale proteomic MR and differential protein expression (DPE) analyses across multiple OA phenotypes using the largest datasets to date. By combining genetic evidence with observed protein dysregulation in population-based cohorts, we strengthen causal inference and improve robustness of target prioritisation. This approach allows us to distinguish proteins that are likely to play a causal role in OA from those that reflect downstream disease processes, and to highlight targets with greater translational relevance than identified by either method alone. Implications of all the available evidenceTaken together, our findings support a causal role for a subset of circulating proteins in OA and demonstrates the value of integrating genetic and observational proteomic data for target prioritisation. Proteins supported by both MR and DPE are more likely to represent biologically relevant drivers of disease and actionable therapeutic targets. This integrated framework reduces false positives arising from confounding or reverse causation and provides a more reliable basis for drug development, biomarker discovery, and patient stratification in OA.

Objectives: Knee osteoarthritis (KOA) is a leading cause of disability, yet which patients will experience structural decline remains unclear. Body mass index (BMI) and lower limb alignment are established risk factors for KOA, but their independent and interactive effects on compartment-specific cartilage loss and total knee replacement (TKR) have not been characterized at scale. Methods: We analyzed 5,832 limbs from 3,016 participants in the Osteoarthritis Initiative followed over 7 years. Cartilage thickness in the weight-bearing medial and lateral femur and tibia was quantified, and lower limb alignment was measured using hip-knee-ankle (HKA) angle obtained from full-limb radiographs. Linear mixed-effects models estimated the independent and interactive effects of BMI and lower limb alignment on longitudinal cartilage thinning, and mixed-effects logistic regression modeled TKR risk. Results: In the medial compartment, BMI and varus alignment interacted multiplicatively, with their combined effect exceeding the sum of independent contributions (femur: p = 0.011; tibia: p < 0.001). At +10 kg/m2 BMI and +10 degrees varus, the rate of medial femur cartilage thinning was 243.5% faster than the reference rate. In the lateral compartment, BMI and valgus alignment were independently associated with faster cartilage thinning, with no significant interaction. TKR risk increased exponentially with HKA deviation (odds ratio [OR] = 1.38 per 1 degree; ~five-fold at 5 degrees malalignment) but was not associated with BMI. Conclusion: BMI and lower limb alignment influence structural KOA progression through compartment-specific pathways. The multiplicative interaction in the medial compartment identifies high BMI combined with varus malalignment as a discrete high-risk phenotype, with implications for clinical risk stratification and disease-modifying intervention design.

BackgroundKnee osteoarthritis (OA) is a prevalent painful degenerative disease without effective disease-modifying drugs. The rising prevalence of comorbid obesity and knee OA underscores the urgent need for effective management to delay or prevent disease progression. In a recently completed randomized, placebo-controlled trial in adults with comorbid obesity (BMI >30 kg/m{superscript 2}) and unilateral or bilateral knee OA (Kellgren-Lawrence grade II-III), we were the first to demonstrate that a 6-month prebiotic intervention (16 g/day oligofructose-enriched inulin) significantly improved physical function and metabolic health. MethodsTo elucidate the underlying mechanisms, we incorporated metagenomics, metabolomics, and machine-learning-based multi-omics integration in 30 participants who completed baseline and at least one follow-up assessment and sample collection at months 3 and 6. ResultsPrebiotic supplementation reshaped gut microbial composition and function, increasing diet-derived carbohydrate availability, mitigating excessive host-glycan degradation and mucosal barrier disruption, reducing systemic inflammation and metabolic dysregulation, and ultimately improved physical performance and metabolic health. In a diet-induced obese rat model, prebiotic treatment reduced tibial cartilage degeneration and synovial membrane thickening, providing protection against OA onset and progression through a shared inflammatory pathway. ConclusionsOur findings provide mechanistic evidence supporting the therapeutic potential of prebiotic supplementation as a conservative management in humans and as a preventive approach for obesity-related knee OA in a preclinical rat model, mediated through the gut-joint axis. Trial registrationClinicaltrials.govNCT04172688

Osteoarthritis (OA) is a leading cause of disability worldwide, with chronic pain representing its most debilitating symptom and frequently accompanied by affective and cognitive comorbidities. Increasing evidence implicates maladaptive supraspinal plasticity within cortical regions involved in pain affect, including the anterior cingulate cortex (ACC) and anterior insular cortex (AIC), however, the relationship between these behavioral impairments and neuronal alterations, as well as potential sex-specific vulnerability, remains poorly documented. Using a monosodium iodoacetate (MIA) model of knee OA in adult male and female mice, we examined the temporal progression of sensory, affective, and cognitive alterations at early (day 7) and advanced (day 28) stages of disease. Pain sensitivity, locomotor and gait changes, anxiety- and depression-like behaviors, and working-memory performance were assessed using established behavioral paradigms, followed by analyses of apoptosis-associated neuronal signaling in the ACC and AIC. MIA induced robust mechanical and thermal hypersensitivity and gait impairment in both sexes, while early emotional and cognitive alterations were not observed. In contrast, advanced OA was associated with pronounced anxiety- and depression-like behaviors and impaired working memory. Notably, analysis at day 28 post-MIA revealed a significant increase in apoptotic signaling and neuronal loss in both cortical regions, with females exhibiting greater vulnerability, particularly within the AIC, paralleling their more severe affective phenotypes. Together, these findings indicate that chronic OA pain is associated with progressive, sex-dependent neuronal loss within key cortical pain-affective circuits and highlight supraspinal remodeling as a potential substrate underlying the emotional and cognitive burden of OA pain.

Osteoarthritis (OA) is a multifactorial joint disease driven by complex interactions among chondrocytes, osteoblasts, fibroblasts, and immune cells across cartilage, bone, and synovial tissues. Conventional monoculture systems are unable to capture this crosstalk, limiting their physiological relevance. Building on our previously established joint-on-a-chip platform, this study evaluated multicellular communication and assessed whether a microfluidic co-culture provides a more realistic representation of joint inflammation compared with monoculture models. Two configurations were established: a healthy, low-inflammation model containing M0 macrophages and an OA-like, high-inflammation model with M1 macrophages. In healthy models, co-culture significantly increased MMP-1 ([~]4-fold), MMP-3 ([~]15-fold), TIMP-2 ([~]5-fold), IL-6 ([~]6-fold), and IL-8 ([~]5-fold) relative to monoculture, indicating that endogenous signaling initiates basal matrix remodeling and inflammatory pathways. In disease models, M1-driven co-culture elevated MMP-10 ([~]300-fold) and MMP-13 ([~]60-fold), along with TIMP-2 ([~]5-fold), compared with monoculture, reflecting amplified catabolic activation. Direct comparison of disease versus healthy co-culture revealed additional increases in MMP-10 ([~]55-fold), MMP-13 ([~]95-fold), MCP-1 ([~]1.6-fold), MMP-1 ([~]1.6-fold), MMP-3 ([~]1.8-fold), TIMP-1 ([~]1.4-fold), and TIMP-2 ([~]1.5-fold), representing a macrophage-mediated shift from homeostasis to OA-like pathology. However, neither IL-1 nor TNF, each a key inflammatory mediator of OA, differed measurably between healthy and disease models under either monoculture or co-culture conditions. Thus, the microfluidic joint inflammation-on-a-chip model presented here more faithfully recapitulates the pathogenic MMP profile of OA than monoculture systems, but it does not yet fully recapitulate the pathogenic inflammatory environment of OA.

ObjectivesPatients with osteoarthritis (OA) affecting multiple joints have poorer health outcomes than those without, yet most research examines isolated joints, leaving a gap in multi-joint disease. This study aimed to describe radiographically defined hip (rHOA) and knee OA (rKOA) within UK Biobank (UKB), exploring interrelationships across joints, and associations with joint pain, obesity, race and deprivation. MethodsAutomated machine learning was applied to left and right hip and knee dual-energy X-ray absorptiometry scans. Radiographic OA (rOA) was defined as custom grades [&ge;]2. Joint pain was assessed through self-reported questionnaires. Descriptive statistics summarised the population characteristics. Logistic regression models examined bilateral and cross-joint associations, as well as associations with joint pain. Adjustments were made for age, sex, race, height, weight and deprivation. Other models examined the associations between body size and OA. ResultsAmong 59,475 individuals (mean age 65 years; 52.8% female), rHOA prevalence was 4,098 (6.9%)) and 4,841 (8.1%) for the right and left joints, respectively. The corresponding estimates for rKOA were 3,750 (6.3%) and 4,220 (7.1%). Overall, increasing grades of rOA and number of joints affected were more strongly associated with joint pain. Regarding joint-interrelationships, bilateral associations were stronger at the knee, whereas cross-joint associations (hip-knee) were weaker. Associations with BMI and height differed between the hip and knee. ConclusionsRadiographic hip and knee OA exhibit distinct patterns of interrelationship, associations with symptoms and risk factors, suggesting heterogeneity in disease process and the need for joint-specific treatment. Key MessagesO_ST_ABSWhat is already known on this topic?C_ST_ABSO_LIOsteoarthritis (OA) commonly affects the hip and knee and is associated with pain and disability, with recognised risk factors such as age, obesity and deprivation. C_LIO_LIIncreasing interest in multi-joint OA challenges the traditional concept of lower-limb OA as a monoarthritis, but most research examines joints in isolation. C_LIO_LIGenetic evidence suggests that hip and knee OA may differ in underlying mechanisms, yet population-scale comparisons are limited. C_LI What this study adds?O_LIAmong 59,574 individuals, this study identifies that radiographic OA captures structurally and clinically relevant disease with increasing severity and greater number of joints affected, positively associated with chronic joint pain. C_LIO_LIRadiographic hip and knee OA demonstrated strong bilateral but weaker cross-joint associations, indicating preferential within-joint symmetry. C_LIO_LIRisk factors differed by anatomical site with BMI and weight strongly associated with knee OA and weakly associated with hip OA. Height showed the opposite associations. C_LI How this study might affect research, practice or policy?O_LIThese findings support that hip and knee OA may partially represent different disease processes rather than a single condition. C_LIO_LIClinical practice should consider cumulative joint involvement and joint-specific risk factors. C_LIO_LIFuture research should consider the development of more targeted treatment to prevent multi-joint progression. C_LI

BackgroundAnterior knee pain (AKP) is common in adolescent athletes and encompasses heterogeneous osseous and soft tissue pathologies, yet its developmental mechanisms remain poorly integrated. HypothesisPain-generating tissues within the knee extensor mechanism are redistributed from osseous to soft tissue structures with skeletal maturation. Study DesignRetrospective observational cohort study. Level of EvidenceLevel 3. MethodsA total of 1,595 patients with sports-related knee injuries (2017-2025) were included. Skeletal maturity was determined by proximal tibial physeal status on radiographs, classifying participants into open-physes (n = 707) and closed-physes (n = 888) groups. AKP was classified into bony and non-bony subtypes based on maximal tenderness. Prevalence was compared using odds ratios (ORs) with 95% confidence intervals (CIs). ResultsOverall, 575 patients (36.1%) had AKP. AKP was more prevalent in the open-physes group than in the closed-physes group (60.1% vs 16.9%; OR, 7.4; 95% CI, 5.9-9.3; p < 0.001). Bony AKP showed a marked difference (42.4% vs 3.7%; OR, 19.1; 95% CI, 12.8-28.6; p < 0.001), whereas non-bony AKP showed only a modest difference (17.7% vs 13.2%; OR, 1.4; 95% CI, 1.1-1.9; p = 0.013). ConclusionThe association between AKP and skeletal maturity was primarily driven by bony AKP, supporting structural redistribution of pain-generating tissues during growth. Clinical RelevanceTenderness-based classification may aid identification of tissue-specific vulnerability and inform growth-stage-specific load management.

Intervertebral disc degeneration (IVDD) compromises disc structures and mechanics, yet systematic evaluations of the mechanical responses and their relationship to morphological changes in preclinical models remain limited. This systematic review and meta-analysis synthesized mechanical and morphological alterations following experimental disc injury in in vivo animal models. Searches of MEDLINE, EMBASE and Web of Science databases were conducted in accordance with PRISMA guidelines. Study quality and risk of bias were assessed using modified CAMARADES and SYRCLE tools. Twenty-eight studies were included. Pooled analyses showed significant reductions in stiffness, Youngs modulus, and disc height, and significant increases in range of motion and degeneration grade, indicating both mechanical and structural deterioration. Youngs modulus appeared to be the most sensitive marker of functional degeneration. By contrast, creep and other viscoelastic responses showed non-significant changes. High heterogeneity was evident across studies, reflecting variability in injury models, species, timepoints, and testing methods. Evidence of publication bias was detected in several domains, and moderate methodological quality was noted with overall insufficient blinding and lack of sample size calculations. In vivo animal models of IVDD demonstrate robust and consistent mechanical and morphological degeneration after injury. Youngs modulus is a sensitive mechanical indicator, supporting its use in future preclinical research. Standardization of outcome definitions, methodology, and reporting is essential to improve comparability and enhance translation of preclinical findings to clinical research.