Arthritis Research & Therapy

ObjectiveThis study investigates joint injury-induced angiogenesis and the effects of genetic deficiency of thrombospondin-2 (TSP2), an anti-angiogenic factor, in joint homeostasis and post-traumatic osteoarthritis (PTOA). MethodWe utilized a murine non-invasive anterior cruciate ligament rupture (ACLR) model of PTOA and mined published synovial transcriptomics datasets to investigate injury-induced synovial angiogenesis. Spatial transcriptomics and flow cytometry of TSP2-GFP reporter mice were used to assess injury-induced thrombospondin-2 and its cellular origins in synovium. Global TSP2 knockout mice (TSP2-KO) were used to assess the effect of TSP2 deficiency on early and late stages of PTOA development via molecular imaging of inflammation and angiogenesis, histopathology, micro-computed tomography, Raman spectroscopy, and synovium bulk RNA-sequencing. ResultsIntra-articular angiogenesis peaked at 7d post-ACLR and declined but remained elevated above baseline at 28d post-ACLR. We identified synovial crosstalk between endothelial cells and sublining fibroblasts as a key driver of angiogenesis and source of thrombospondin-2 signaling, with TSP2 primarily upregulated in sublining fibroblasts. TSP2-KO mice exhibited increased peri-articular inflammation at 7d post-ACLR and inferior bone quality. Histopathology revealed greater PTOA severity but paradoxically lower synovitis in TSP2-KOs. Additionally, aberrant structural remodeling of the entire knee joint was observed in uninjured and ACLR TSP2-KO limbs. The uninjured TSP2-KO synovial transcriptome demonstrated elevated immune, fibrotic, and angiogenic activation; however, TSP2-KO and WT synovial transcriptomes partially converged upon injury. ConclusionTSP2 is essential for joint homeostasis and trauma response. Global TSP2 deficiency causes premature OA and worsened PTOA, suggesting that therapeutic targeting with TSP2 mimetic could be used to prevent OA.

ObjectivePain in osteoarthritis (OA) is often discordant with structural joint damage, particularly in obesity-associated OA, where adipose-derived signals may drive nociception independently of cartilage pathology. Leptin has been demonstrated to be necessary, but not sufficient, to drive obesity-associated OA. Here, we tested the hypothesis that leptin mediates OA-associated pain through sensory neuron reprogramming rather than chondrocyte-intrinsic signaling, suggesting a fat-sensory nerve axis. DesignMale and female constitutive leptin-deficient (Ob/Ob), heterozygous (Ob/+), and wild-type (WT) mice, as well as chondrocyte-specific leptin receptor knockout mice (Aggrecan-CreERT2;LepRfl/fl), were challenged with destabilization of the medial meniscus (DMM) surgery to induce OA. Pain-related behaviors, joint pathology, serum cytokines, and lumbar dorsal root ganglia (DRG) transcriptomes were assessed. Human DRG cultures treated with leptin underwent transcriptomic profiling. Secondary analyses of human infrapatellar fat pad and synovium single-cell datasets evaluated leptin and leptin receptor expression patterns. ResultsChondrocyte-specific deletion of the leptin receptor did not mitigate OA pathology or pain. Global leptin-deficient (Ob/Ob) mice exhibited worse structural joint outcomes than WT and Ob/+ animals following DMM yet were robustly protected from OA-associated hyperalgesia - directly dissociating pain from structural pathology and demonstrating that leptin is involved in nociceptive sensitization. Serum cytokine profiles were sex-dependent and did not align with pain outcomes, separating systemic inflammation from nociceptive differences. Transcriptomic analysis of DRGs revealed that leptin drives enrichment of lipid metabolism, eicosanoid, and inflammatory programs, whereas leptin deficiency shifts sensory neurons toward a cytoskeletal remodeling state that does not sustain pain signaling. In human DRG cultures, leptin treatment produced a transcriptomic shift to enrich for neuronal excitability while vehicle treated cells were enriched for inflammatory signaling. Human infrapatellar fat pad and synovium transcriptomic data demonstrated adipocyte-enriched leptin expression and broad distribution of the leptin receptor across stromal, vascular, immune, and adipocyte populations. ConclusionsLeptin contributes to OA pain through neuro-immune crosstalk between adipose tissue and sensory neurons rather than through direct cartilage signaling. These findings identify leptin-associated neuronal programs linked to nociceptor sensitization and support targeting leptin-modulated neuro-immune pathways as a strategy to alleviate OA pain independently of structural disease progression.

Background and rationale: Knee osteoarthritis (KOA) is a leading cause of lower limb disability worldwide, characterized by functional limitations, stiffness and pain. The incidence of KOA is especially tied to age and obesity. It is a disabling disease that often makes patients less physically active, thus increasing the risk of other diseases and mortality1. The clinical diagnosis of KOA is based on the symptoms and functional limitations of the joint. The diagnosis is usually supported with a radiograph (X-ray) of the weight-bearing knee. Radiographic features, such as Kellgren-Lawrence grade, are used as eligibility criteria for clinical studies while other features, such as joint space width (JSW), are used as endpoints for structural KOA progression2,3. While the use of these radiographic features is standard in academia, the use of JSW as a structural biomarker has received criticism. Critics point out that JSW is an indirect and projection dependent measure of cartilage deterioration which is sensitive to technical factors such as the angulation of the X-ray beam and the positioning of the knee. Small differences in these factors can alter the measured joint space and may not reflect true disease progression4,5. Despite limitations, minimum joint space width (mJSW) remains as one of the most widely used structural biomarkers in KOA trials and is currently one of the only structural imaging accepted in regulatory guidance as evidence of disease modification in OA drug development3. For JSW to be reliable and consistent in determining the advancement of KOA, the use of fixed-flexion devices is crucial to reduce the risk of unwanted narrowing or widening of the radiographic joint space width6,7. The LOSEIT trial, which the present study is based on, acknowledges the angulation problem and uses a standard clinical fixed-flexion device in weight-bearing PA views to get reliable JSW results8. Historically, a radiologist would draw on and grade radiographs of the knee-joint to extract the features. However, manual reading and annotation is time consuming with notable interobserver variance9. With increasing computational power and the use of deep neural networks, off-the-shelf artificial intelligence (AI) tools have become available for automatic extraction of radiograph features. Automation would free up time from radiologists and provide more consistent measurements due to the reproducible nature of the models10. These tools have received regulatory approval for commercial use, however, regulatory approval does not guarantee uniform or bias free performance when used on real-world data11. Furthermore, in a large multi-hospital chest X-ray study, Zech et al., showed that convolutional neural networks achieved worse results on data from other hospitals than on the original hospitals in which it was tested12. This highlights the risk of overestimating the accuracy of AI tools when only internally validated. It is therefore apparent that external validation is required when testing these AI models. Objectives: The aim of this analysis is to evaluate the agreement of a commercially available AI tool for measuring JSW with the best practice radiologist annotation in the tibiofemoral joint of the knee in radiographs stabilized with a fixed-flexion device and acquired as part of a clinical trial. Methods: This study is a secondary analysis of the data from the LOSEIT trial, a randomized, double-blind, placebo-controlled, single-center trial, where patients were randomized to either liraglutide or identically appearing placebo after an initial weight-loss period to investigate the effects on KOA. Radiographs of the tibiofemoral joint were acquired at enrollment (week -8) and at end-of-trial (week 52) for a total acquisition-to-acquisition time of 60 weeks13. The primary analysis will assess agreement between AI-derived and reference-derived change in JSW from enrolment to follow-up. Change will be calculated as follow-up minus enrolment separately for the AI tool and the reference measurement. The main measure of interest will be the change in medial minimal JSW (mmJSW), with change in lateral minimal JSW (lmJSW), medial fixed JSW (mfJSW) and lateral fixed JSW (lfJSW) as secondary measures. This study will follow an equivalence framework using the two one-sided tests (TOST) approach with a Bland-Altman analysis as the main outcome. The equivalence margin will be set at {delta} = 0.5 mm. Agreement consistent with equivalence will be considered established if the upper limit of the 95% confidence interval (95% CI) for the upper limit of agreement (LoA) and the lower limit of the 95% CI for the lower LoA are within the established margins. The reference JSW will be the average measurement of two independent resident radiologists. If there is a mismatch in the measurements of more than 0.40 mm between the two radiologists, the radiologists will re-annotate the case independently. If the difference remains greater than 0.40 mm, a musculoskeletal radiology consultant will review the radiograph and establish the reference JSW. The index test will be the measurements output by the AI tool. Populations: Patients aged 18 to 74 with symptomatic knee osteoarthritis, radiographically confirmed KL grade 1-3, with a BMI [≥]27, motivated for weight loss and in accordance with the LOSEIT trial inclusion criteria Further statistical details Sample size: Not applicable as this is a secondary analysis. Framework: This is an agreement study assessing the equivalence of a commercially available AI tool for radiographic evaluation of knee osteoarthritis with best practice radiologist measurements. Confidence intervals and P values: All 95% confidence intervals and P-values will be two-sided. Statistical software: SAS Studio and/or R version 4.2.2 (or newer).

Osteoarthritis (OA), a degenerative joint disease, is associated with increased systemic inflammation, chronic pain, and cardiovascular dysfunction. Epidemiological evidence establishes that OA increases the risk of cardiovascular disease (CVD) threefold, yet the causal role of OAs contributions remains underexamined. We assessed cardiac function longitudinally following destabilization of the medial meniscus (DMM) surgery to induce osteoarthritis in mice. DMM-mice exhibited significant, sexually dimorphic alterations in echocardiographic parameters. Female DMM mice developed impaired relaxation with altered E/A ratios, increased E/e ratios, and prolonged intraventricular relaxation time with no change in ejection fraction, while male DMM mice showed progressive systolic dysfunction with decreasing ejection fraction, increased E/e ratio, and prolonged intraventricular contraction time. Transcriptomic profiles and biochemical analyses demonstrated divergent cellular responses involving fibrosis and oxidative stress in female mice, whereas autophagic and apoptotic responses were observed in male mice. Using a tumor necrosis factor 2 (TNFR2) agonist shown to reduce systemic inflammation, we investigated its potential therapeutic role in the context of OA-induced cardiovascular dysfunction. TNFR2 agonism proved to be effective both prophylactically and therapeutically for female diastolic dysfunction. While prophylactic and therapeutic administration delayed male systolic dysfunction, the efficacy declined over time. Our findings demonstrate evidence of a novel sexually dimorphic model of OA-induced CVD that recapitulates the sexually dimorphic pattern of patient phenotypes and a promising new therapeutic approach to CVD. Translational RelevanceOsteoarthritis patients have higher, often unrecognized, cardiovascular risk, yet preclinical models linking joint disease to cardiac dysfunction remain unexplored. Using a murine preclinical model of OA reveals the key findings. First, OA alone drives sex-specific cardiac phenotypes - females develop diastolic dysfunction, whereas males develop progressive systolic impairment. Second, selective TNFR2 agonism prevents and reverses OA-induced diastolic dysfunction in female mice and delays systolic decline in males. These findings suggest sex-dependent cardiac monitoring in OA patients and indicate that TNFR2-targeted therapy will likely be a sex-informed intervention to provide cardioprotective benefit. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/736778v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1f41661org.highwire.dtl.DTLVardef@1e94cb7org.highwire.dtl.DTLVardef@1abbac0org.highwire.dtl.DTLVardef@171cc86_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background Pain frequently persists in rheumatoid arthritis (RA), despite effective control of inflammation. The mechanisms driving this residual pain remain poorly characterised in individual patients. Methods In 172 patients with established RA and clinically relevant pain (mean NRS 6.5/10) and 80 pain free controls, we combined indicators of inflammatory disease (CRP, joint counts, power Doppler ultrasound), centrally mediated pain (Widespread Pain Index, painDETECT), psychological distress (PHQ ADS) and quantitative sensory testing (QST). Latent profile analysis was applied without predefined thresholds. Results Four phenotypes were identified: a peripheral, low-inflammation/low-central phenotype (38%); a predominantly inflammatory phenotype (7%); and moderate (43%) and severe (12%) centrally mediated phenotypes. Centrally mediated phenotypes reported the highest pain (NRS 8.2), worst disease impact and lowest employment. DAS28 CRP was similar in both the inflammatory and severe centrally mediated phenotypes but for different reasons, swollen joints and CRP versus tender joints , and did not distinguish them. Conditioned pain modulation was impaired relative to controls (p<0.001) and most reduced in the severe centrally mediated phenotype. Psychological distress was the strongest independent predictor of pain severity (model R squared=0.33), whereas inflammatory markers were not. Principal components analysis identified swollen joint count (loading 0.63) and the tender swollen joint difference (loading 0.60) as accessible clinical markers of the inflammatory and centrally mediated phenotypes respectively. Conclusions A data driven approach identified four mechanism-based pain phenotypes in RA. This framework moves pain assessment beyond inflammation alone and provides a basis for testing analgesic strategies to target the predominant pain mechanism in individual patients.

ObjectiveEnhanced glycolysis is a metabolic hallmark of chondrocytes in osteoarthritis (OA); however, the roles of the glycolytic rate-limiting enzyme hexokinase 2 (HK2) in cartilage remain poorly understood. MethodsPharmacological approach (3-bromopyruvate (3-BrPA) treatment) and mice model involving HK2 knockout in Col2a1-expressing chondrocytes are utilized to access the impact of HK2 blockage on cartilage ex vivo and in vivo. The in vivo effects of HK2 inhibition on OA progression were evaluated using a destabilization of the medial meniscus (DMM)-induced OA mouse model, through both intra-articular 3-BrPA administration and chondrocyte HK2 deletion. Additionally, we analyzed published single-cell RNA sequencing (scRNA-seq) datasets from human articular cartilage and integrated these with bulk RNA-seq data from HK2-deficient chondrocytes to characterize HK2 expression features across conditions. ResultsBoth pharmacological inhibition and genetic deletion of HK2 impair cartilage formation ex vivo. Bulk RNA-seq analysis and ex vivo studies demonstrated a promoted ossification-like process due to HK2 ablation in chondrocytes. Through pseudotime analysis of published single-cell RNA sequencing (scRNA-seq) datasets from human articular cartilages, we further identified that HK2 is differentially expressed across conditions, with a feature of a relatively high expression level at terminal stages of chondrocyte differentiation in the context of OA. We next confirmed HK2 deficiency in chondrocytes significantly exacerbated OA progression but having no impact on skeletal development in mice. ConclusionsHK2 plays a critical role in maintaining cartilage health, likely through the regulation of calcification, thereby highlighting the potential risks associated with targeting glycolytic enzymes as a therapeutic strategy for OA.

Osteoarthritis (OA) is a major cause of chronic pain and disability worldwide, characterized by progressive degeneration of cartilage and subchondral bone. Post-traumatic OA (PTOA) develops in as many as 25-50% of individuals following major joint injury, making it a leading cause of OA in younger and otherwise healthy populations.1,2 Connexin 43 (Cx43), a gap junction protein involved in intercellular communication and cellular stress responses, has been linked to OA; however, its role in the progression of PTOA remains unclear. Here, we examined how cartilage-specific loss of Cx43 influences PTOA and chondrocyte metabolic function. Using a murine model of conditional Cx43 deletion in cartilage, we demonstrate that male knockout mice exhibited severe cartilage surface damage and matrix loss, whereas female knockout mice showed cartilage thinning accompanied by reduced chondrocyte hypertrophy, decreased subchondral bone density, and increased osteophyte formation. Thus, loss of Cx43 disrupts cartilage integrity and osteochondral remodeling in a sex-specific manner, predisposing joints to maladaptive bone changes and cartilage degeneration. Complementary mechanistic studies in human articular chondrocytes revealed that Cx43 deficiency impairs mitochondrial respiration, reduces spare respiratory capacity, and lowers ATP production, consistent with compromised cellular bioenergetics. Together, these findings identify Cx43 as an important coordinator of metabolic and structural responses to joint injury. These results position Cx43 as a context-dependent regulator of joint homeostasis and suggest that maintenance of Cx43 expression may support cartilage resilience following injury.

ObjectiveRecently, alternatives to animal testing, such as new approach methodologies, are being developed in the orthopedic research field; animal models still provide valuable insights into the pathogenesis of knee osteoarthritis (OA). However, commonly used models develop OA much more rapidly and severely than those observed in human patients. We aimed to develop a novel murine model that closely mimics the slow progression of human OA with posterior Cruciate ligament (PCL) rupture. Design12-week-old C57BL/6 mice were induced to PCL-rupture (PCL-R) by manually applying an external tibial posterior translation force. We analyzed joint kinematics, histological observations, and bone structure to confirm the absence of concurrent injury on day 0. Then, joint stability and the pathophysiological progression of knee OA were analyzed at 8, 16, and 34 weeks post-PCL-R. The destabilized medial meniscus (DMM) model was also analyzed to compare the OA progression. ResultsNon-invasive PCL-R intervention induced the complete rupture in the central region of PCL without concurrent injury. The PCL-R group showed larger posterior tibial deviation than the INTACT (P=0.008). Regarding the range of motion in the PCL-R group, there was no limitation in range of motion on day 0, but extension limitations occurred at weeks 16 and 34 weeks. Histologically, articular cartilage degeneration in PCL-R was milder than DMM. In the subchondral bone, micro-CT reconstruction images indicated that, compared with the INTACT group, the DMM group observed progressive subchondral bone formation from 16 weeks post-surgery. In contrast, the PCLR group maintained the subchondral bone structure even at 34 weeks. ConclusionsPCL-R model induced mild abnormal mechanical stress depending on posterior instability, and cartilage degeneration occurred more slowly in this model than in DMM models.

Background Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease which can lead to progressive disability and damage to multiple organs. Obesity is associated with higher disease activity in RA and inadequate long-term outcomes, so better understanding of mechanisms linking adiposity to immune dysregulation might help to refine optimal treatments. Monocytes are important contributors to immune activation in RA through antigen presentation and costimulatory signaling. We hypothesized that adiposity enhances monocyte costimulatory programming in RA, thereby promoting adaptive immune activation. Methods Single-cell RNA sequencing was performed using the 10x Genomics Flex platform on purified circulating monocytes from 31 donors (16 RA participants fulfilling 2010 ACR/EULAR classification criteria and 15 non-RA controls) generating transcriptomic profiles for approximately 135,599 monocytes. Donor-level pathway enrichment scores were calculated for predefined immune activation pathways including antigen processing and presentation, interferon signaling, and regulation of T-cell costimulation. Analyses were performed at the donor level to avoid cell-level pseudoreplication. Associations with disease status and body mass index were evaluated using factorial linear models and Spearman correlation analyses. Results Single-cell transcriptomic profiling identified classical, intermediate-like, non-classical, and interferon-responsive monocyte populations. RA was associated with enrichment of antigen processing and presentation programs in circulating monocytes (p=0.0106), indicating a primed antigen-presenting state. In contrast, regulation of T-cell costimulation pathway enrichment did not differ by RA status alone. However, within RA participants, higher BMI was associated with increased enrichment of monocyte T-cell costimulatory pathways (Spearman {rho}=0.56, p=0.0248), unlike in non-RA controls. Gene-level analyses demonstrated strong baseline expression of CD86, while ICOSLG and TNFSF4 transcripts were expressed at low levels overall, consistent with inducible costimulatory signaling programs. Conclusions These findings support a model in which metabolic dysregulation amplifies monocyte-mediated immune activation and may contribute to worsened disease outcomes in RA.

Osteoarthritis (OA) drug development remains constrained by preclinical models that fail to recapitulate the multicellular interactions that regulate human joint inflammation and extracellular matrix degeneration in response to investigational drugs. Tanezumab, a humanized anti-nerve growth factor monoclonal antibody developed for non-opioid pain relief, advanced to late-stage clinical trials but was discontinued due to unresolved joint-localized safety concerns, including rapidly progressive OA. This study evaluated whether a human microfluidic joint-on-a-chip co-culture system could detect early biomarker responses to tanezumab exposure that were not apparent in conventional chondrocyte monoculture. Tanezumab was first tested in human chondrocyte monoculture under untreated and disease-like (i.e., IL-1{beta}-treated) conditions. Across a 20-analyte panel of inflammatory and matrix-remodeling biomarkers, statistically significant monoculture responses to tanezumab were limited to decreased IL-1{beta} from 335 to 132 pg/mL ([~]0.39-fold) and increased IL-8 from 575 to 675 pg/mL ([~]1.17-fold). Major OA-associated matrix-remodeling markers, including MMP-1, MMP-3, and MMP-13, remained largely unchanged, indicating that monoculture conditions are insufficiently sensitive to detect clinically predictive drug-related molecular changes. Tanezumab was then evaluated in co-cultures containing chondrocytes, osteoblasts, fibroblast-like cells, and macrophages under low-inflammation (i.e., M0 macrophage-based) and high-inflammation (i.e., M1 macrophage-based) conditions. In the M0-based co-culture, tanezumab increased MMP-1 from [~]4.20 x 104 to [~]6.20 x 104 pg/mL ([~]1.48-fold), MMP-3 from [~]8.00 x 104 to [~]1.20 x 105 pg/mL ([~]1.50-fold), and MCP-1 from 2.85 x 103 to 4.31 x 103 pg/mL ([~]1.51-fold). In contrast, the M1-based co-culture showed decreases in MMP-13 from [~]1.66 x 104 to [~]1.17 x 104 pg/mL ([~]0.70-fold) and IFN-{gamma} from [~]1.95 x 104 to [~]1.56 x 104 pg/mL ([~]0.80-fold), changes that may appear beneficial despite the drugs known clinical risks. Collectively, these findings show that low-inflammation multicellular co-culture revealed coordinated matrix remodeling and inflammatory responses to NGF blockade that were missed in monoculture and were partly obscured in highly stimulated disease-like conditions. This platform may provide a useful, human-relevant approach for safety signal assessment and early evaluation of OA therapeutics within a defined context of use focused on joint-specific, tissue-level drug-response testing.

Rheumatoid arthritis is a chronic inflammatory joint disease in which progressive destruction of cartilage and bone drives long-term disability. Current disease-modifying therapies target the immune and cytokine networks that sustain synovial inflammation, but none is directed at the chondrocyte, the resident cell responsible for maintaining cartilage matrix. Chondrocyte survival and matrix homeostasis depend on {beta}1-integrin-mediated adhesion to the extracellular matrix, and dysregulated integrin signalling has been implicated in cartilage injury. Here we test the hypothesis that allosteric modulation of {beta}1 integrin, rather than simple adhesion blockade, is chondroprotective. Using the monoclonal antibody JB1a, which binds an epitope in the hybrid domain of {beta}1 integrin and stabilises the receptor in a low-affinity conformation, we show that intra-articular administration produces both functional and structural amelioration of Freunds complete adjuvant (FCA)-induced arthritis in mice. JB1a abolished the FCA-induced increase in joint diameter and hyperalgesia and markedly reduced synovial inflammation, pannus formation and cartilage erosion, with no effect on the contralateral joint and no observed adverse effects. These changes were accompanied by a reduction in chondrocyte apoptosis in vivo. In primary human articular chondrocytes, JB1a abolished interleukin-1{beta} (IL-1{beta})-induced caspase 3/7 activation, reduced IL-8 secretion, and restored the sinusoidal oscillation of intracellular ATP that was otherwise abrogated by IL-1{beta}. In contrast, the adhesion-blocking, integrin-clustering antibody 6S6 activated caspase 3/7 and amplified IL-1{beta}-induced IL-8 secretion, indicating that the therapeutic effect is a property of the specific mode of receptor engagement rather than of adhesion blockade per se. These findings identify {beta}1-integrin conformational state as a determinant of chondrocyte energy homeostasis and survival, and nominate allosteric {beta}1-integrin modulation as a mechanistically distinct, chondrocyte-directed therapeutic strategy in inflammatory arthritis.

Osteoarthritis is increasingly recognised as a disease of failed integration across the whole joint unit; however, the mechanisms that co-ordinate tissue integrity from development through to adult homeostasis remain largely unresolved. The LIM-homeodomain transcription factor LMX1B is a key determinant of embryonic skeletal patterning, but how it functions to regulate skeletal integrity in the mature skeleton is unknown. Recently, LMX1B was identified as a key driver of osteoarthritis. Here we show that loss of lmx1ba in zebrafish causes premature and progressive severe osteoarthritic pathology in adult spines and jaws, despite largely normal early skeletal patterning, revealing a conserved and continuous requirement for lmx1ba in joint maintenance beyond development. At a cellular level, loss of lmx1ba decouples osteoblast and osteoclast-mediated remodelling leading to bone overgrowth, heterogeneity of bone properties causing increased incidence of spontaneous fractures, and progressive abnormalities in spine morphology. In parallel, we observe degeneration of the intervertebral disc and dysregulation of the proteome and glycosaminoglycans indicative of disrupted extracellular matrix and a breakdown of the coordinated regulation of hard and soft tissue interfaces, which at the organismal level leads to altered joint performance. Notably, degeneration is restricted to mobile joints, and is not observed in cranial sutures, demonstrating a selective requirement for lmx1ba in mechanically active tissues. These changes are consistent with a model of spatially disrupted matrix properties that, under cyclic loading, promote progressive tissue damage. Our findings support a model in which continued expression of LMX1B in adulthood is required to maintain joint structures throughout life.

BackgroundIn osteoarthritis (OA) TLR4 signaling leads to downstream activation of the phosphoinositide 3-kinases/ protein kinase B/ mammalian target of Rapamycin (PIK3/AKT/mTOR) pathway, a known modulator of autophagic mechanisms in chondrocytes. This paper focuses on creating a realistic ex vivo OA model that mimics elements of the pathophysiology of OA, allowing for further hypotheses-based investigations, and for use as a bench test for new therapeutic targets. ObjectiveTo study the downstream inflammatory and matrix changes in cartilage due to TLR4 signaling and the recovery achieved by a commonly used immunosuppressive drug, Rapamycin. MethodsIn an ex vivo 3D model based on healthy porcine cartilage explants, we mimicked the OA environment by LPS stimulation activating TLR4 signaling. Furthermore, we inhibited mTOR signaling via Rapamycin, which is accepted to attenuate the cartilage response to LPS-TLR4 activation. Histology and immunohistochemistry were used to evaluate the structural and biomolecular modifications driven by LPS and Rapamycin. ResultsThe explant model captured key features of OA, such as extracellular matrix degeneration and altered autophagy. The OA-like changes in the model were driven by TLR4 activation and mTOR signaling, well-known OA-related molecular pathways, and reversed by Rapamycin. ConclusionWe demonstrate that our explant model is responsive to LPS stimulation, leading to activation of OA-related biomolecular pathways, closely mimicking the native physiological processes. This evidence supports the potential of our model to act as a platform for OA studies, in particular related to the gut-joint axis in age-related OA, and for the screening of new disease-modifying molecules.

Post-traumatic osteoarthritis (PTOA) is a common long-term consequence of joint injury and a major cause of chronic pain and disability, yet no disease-modifying therapies are currently available. A central barrier to effective intervention is the persistence of maladaptive synovial inflammation, driven in part by macrophage-mediated signaling that sustains tissue degeneration and pain. Here, we developed a scalable, chemically defined platform to generate human induced pluripotent stem cell (iPSC)-derived anti-inflammatory macrophages (iMac-M2) as an off-the-shelf cell therapy designed to restore joint immune homeostasis after injury. These cells maintained a stable anti-inflammatory phenotype and function under osteoarthritis-relevant inflammatory conditions and suppressed inflammatory and catabolic responses in human joint cell co-culture systems. In a preclinical model of PTOA, intra-articular delivery of iMac-M2 after injury improved functional and structural outcomes while modulating synovial inflammatory and pain-associated transcriptional programs. Treatment was well tolerated, with no evidence of systemic immune activation or ectopic tissue formation. Together, these findings support iPSC-derived macrophage therapy as a clinically translatable immunomodulatory strategy to interrupt early inflammatory drivers of PTOA and preserve joint health following injury. One Sentence SummaryAn iPSC-derived macrophage therapy restores joint balance, protects cartilage, and relieves pain after traumatic joint injury.

Background: Rheumatoid arthritis (RA) has a preclinical period characterised by elevations in serum autoantibodies. Identifying the timing and magnitude of autoantibody trajectory changes may inform screening strategies and preventative interventions. Methods: Using a Bayesian multivariate segmented regression, we jointly modelled longitudinal autoantibody trajectories from two Department of Defense Serum Repository cohorts (Sample A: 209 matched case-control pairs, 1566 samples, six biomarkers; Sample B: 309 cases with two matched controls each, 2758 samples, eight biomarkers). Change-points and magnitudes of change were estimated simultaneously under a multivariate likelihood with an unstructured residual correlation matrix. Results: In Sample A, five of six biomarkers exhibited pre-diagnostic trajectory shifts with 95% highest posterior density intervals excluding zero. RF-IgM demonstrated the earliest change-point at 8.10 years before diagnosis (95% HPDI: -10.47, -5.73), followed by ACPA-IgG at 7.43 years (95% HPDI: -9.33, -5.76). In Sample B, only the four IgG isotypes showed pre-diagnostic shifts, with anti-CCP3 (IgG) earliest at 7.00 years (95% HPDI: -8.48, -5.29). A composite metric integrating timing and magnitude reordered rankings. Conclusions: This Bayesian framework enables simultaneous estimation of change-points and magnitudes across correlated autoantibodies while fully characterising uncertainty, offering a complementary approach to prior divergence-based methods for understanding preclinical RA autoimmunity.

Introduction Knee pain is a highly prevalent condition in the general population and is more common than knee osteoarthritis. Population-based evidence linking metabolic dysfunction to knee pain remains limited, and data on sex-specific effects are scarce. Therefore, we examined sex-specific associations between metabolic dysregulation and knee pain in a population-based cohort. Method We analyzed data from a population-based cohort of 1,512 adults (mean age 37.2 years at baseline), of whom 250 completed follow-up after a mean of 9.4 years. Metabolic dysfunction was assessed using a continuous MetS severity score (cMetS) derived from waist circumference, triglycerides, HDL cholesterol, fasting glucose, and systolic blood pressure. Knee pain at follow-up was defined using a combined measure based on a standardized question and a body manikin. Logistic regression models were used to examine associations between baseline cMetS and knee pain, including interaction analyses by sex. Results At follow-up, 28.5% of participants reported knee pain. Higher baseline cMetS was associated with increased odds of knee pain in males (odds ratio [OR] 1.41, 95% confidence interval [CI] 1.17-1.69) but not in females (OR 0.94, 95% CI 0.84-1.07), with evidence of interaction by sex (interaction P < 0.001). Findings were consistent across sensitivity analyses. Conclusions These results indicate that metabolic dysfunction is associated with knee pain in males but not in females, suggesting sex-specific mechanisms linking metabolic dysfunction and knee pain.

Postoperative resilience varies widely among older adults, yet the biological drivers of recovery remain unclear. We evaluated whether preoperative immune profiles, measured in plasma and through ex vivo whole blood stimulation, predict resilience to the acute stress of total knee arthroplasty. A total of 152 adults (greater or equal to 60 years) in the PRIME KNEE cohort underwent elective total knee arthroplasty and had available blood samples for measurement of 45 immune biomarkers, quantified in plasma and in whole blood stimulated ex vivo for 24 hours with lipopolysaccharide (LPS) or influenza antigen (FLU). Resilience was assessed using Expected Recovery Differential (ERD) and Resilience Trajectory (RT) across pain severity, pain interference, lower extremity physical activities of daily living (LE PADLs), and step counts. An exploratory stability selection framework using LASSO identified biomarker predictors of postoperative outcomes. Plasma and stimulated biomarkers showed broadly similar predictive performance. A shared set of biomarkers, including LBP, leptin, TNFR1, CD30, and LIF, was consistently selected across models. Immune predictors explained ~12-24% of the variance in resilience outcomes. Distinct immune signatures emerged for pain versus functional recovery: pain related predictors mapped to local inflammatory and neuroimmune pathways, whereas function related predictors reflected systemic inflammatory load and cytokine signaling. Preoperative immune biomarkers, whether measured in plasma or after ex vivo stimulation, capture meaningful variance in postoperative resilience. The divergence between pain related and function related immune signatures highlights biologically distinct pathways underlying different dimensions of recovery and supports further development of immune based perioperative risk assessment.

Objective We developed a patient- and joint-specific explant co-culture system to model active psoriatic arthritis (PsA) and capture donor-specific tissue responses to therapeutic interventions. Methods Based on convergent joint pathology between end-stage osteoarthritis (OA) and PsA, OA cartilage-bone and synovium tissues from arthroplasty patients were exposed to synovial fluid (SF) obtained from PsA and OA patients. Histological outcomes (synovitis, proteoglycan distribution), curated gene expression, soluble mediators, and proteinase activity were assessed over 7-21-days. Model responses to dexamethasone (DEX) and the anti-tumor necrosis factor antibody adalimumab (ADA) were evaluated. Results PsA SF induced distinct inflammatory and tissue remodeling responses compared to OA SF and control conditions, including altered cartilage proteoglycan distribution, increased synovitis, and tissue-specific transcriptional changes. Multivariate analyses identified distinct osteochondral and synovial transcriptional responses to PsA SF, characterized by reduced osteochondral COL2A1 expression and increased synovial expression of inflammatory and matrix-remodeling genes, including MMP1 and CXCL8. DEX and ADA elicited donor-specific responses across histological, transcriptional, and protein readouts. Among multivariable model outputs, histologic synovitis scores emerged as the most clinically aligned parameter, demonstrating associations with baseline PsA donor disease activity, active joint counts, pain, high-sensitivity C-reactive protein (hsCRP), and radiographic scores. Synovitis score changes to DEX and ADA treatments also aligned with corresponding PsA SF donor clinical improvements to corticosteroid and TNF-modifying therapies. Conclusion This osteochondral-synovial explant co-culture model captured donor-specific inflammatory and treatment-responsive features of PsA SF-induced pathology, thereby providing a clinically relevant ex vivo platform for studying patient-specific therapeutic responses in PsA.

ObjectiveTo develop and apply a preclinical functional imaging assay for visualizing and analyzing activated synovial fibroblasts (SFs) at single-cell resolution using high-content imaging. MethodsA multiparametric functional imaging assay was developed to simultaneously interrogate six key cellular processes in cultured SFs from non-inflammatory control (NIC), osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Two complementary fluorescent panels -- comprising LipidTOX, MitoSOX, TMRM, EdU Click-iT, CYTO-ID, and Sir-Lysosome -- collectively captured autophagy dynamics, mitochondrial health, lipid metabolism, and cellular proliferation within a single imaging workflow. Automated image acquisition and quantitative feature extraction via CellProfiler yielded approximately 1,200 morphological and intensity-based features per cell, enabling high-dimensional, unbiased phenotypic profiling at the individual cell level. ResultsApplication of this assay revealed marked heterogeneity in basal cellular functions among SFs stratified by disease state, and robustly differentiated between NIC, OA and RA SFs. Stimulation with inflammatory cytokines (TNF-, IL-1{beta}, IFN{gamma}) and toll-like receptor ligands (LPS, poly I:C) elicited distinct, stimulus-dependent phenotypic responses across disease groups. Multiparametric analysis and feature importance ranking identified IL-1{beta} as a key driver of enhanced autophagic activity, accompanied by significant remodeling of lipid metabolic profiles. ConclusionWe developed a scalable, sensitive approach for dissecting functional heterogeneity in primary SF cultures, revealing previously unappreciated complexity in SF biology across disease states. Our approach provides a robust framework for high-throughput drug screening and identification of candidate therapeutics selectively targeting pathogenic fibroblast functions in inflammatory arthritis.

Background: Psoriatic arthritis (PsA) is an inflammatory condition involving joints, tendon-bone entheses and synovium that can develop in individuals with psoriasis. Early, accurate clinical diagnosis remains difficult. Extracellular vesicles (EVs) carry proteins and miRNAs that Methods: PubMed and Embase were searched from inception through May 21st, 2026, and human studies examining EV-associated protein or miRNA biomarkers in PsA and related psoriatic or inflammatory diseases were included, with risk of bias assessed using a modified Newcastle-Ottawa Scale and diagnostic accuracy summarized using HSROC/BRMA models when data were sufficient. Results: Seven studies met the inclusion criteria, including 119 individuals with PsA (weighted mean age: 49.8 years; 43.7% female), 205 individuals with non-PsA psoriasis (weighted mean age: 46.4 years; female %: NA), 55 controls (weighted mean age: 44.5 years; 38.2% female), and 50 individuals with other inflammatory joint disorders (weighted mean age: 58.0 years; 58.0% female). EV-associated protein markers demonstrated heterogeneous findings related to immune, vascular, inflammatory, and osteoimmunological signaling. Only 4.2% (4/95) of miRNAs were consistently identified across studies comparing PsA with non-PsA psoriasis, with lower overlap (1.5%, 1/67) in studies comparing PsA with controls. ROC meta-analysis suggested preliminary diagnostic potential, particularly for distinguishing PsA from non-PsA psoriasis, although evidence was constrained by small study numbers. Conclusions: EV-associated proteins and miRNAs are potential biomarker candidates for PsA, reflecting inflammatory, vascular, and osteoimmunological processes underlying disease pathophysiology. However, current evidence remains preliminary and limited by small cohorts, methodological heterogeneity, and inconsistent reporting across studies.