Osteoarthritis and Cartilage

ObjectivesOsteoarthritis (OA) is a debilitating joint disease that affects millions of people worldwide, with the temporomandibular joint (TMJ) and knee joint being prominently affected. Despite its prevalence, TMJ-OA remains understudied. This study aimed to investigate the transcriptional signature of the TMJ compared to that of the knee joint and to explore transcriptional differences in the medial and superficial layers of the TMJ-OA. DesignSix-month-old C57BL/6J mice TMJ and knee samples were collected. Goat TMJ superficial and medial layer cartilage was separated and treated with IL-1{beta}. All samples were subjected to bulk RNA sequencing followed by differential expression and gene set enrichment analysis. ResultsWe identified 4,031 protein-coding genes differentially expressed in the TMJ compared to the knee, with significant enrichment of neuronal system genes and lower enrichment of innate immune system genes. Key osteoarthritis biomarkers such as Mmp13, Postn, and Col1a1 were more highly expressed in the TMJ, indicating a higher vulnerability to OA development. IL-1{beta} treatment in goat TMJ chondrocytes mimicked the natural TMJ-OA-like transcriptional changes and immune responses, which are also observed in the rabbit TMJ-OA model. This validated the in vitro goat TMJ-OA model. The IL-1{beta}-treated goat TMJ medial cartilage layer was enriched in OA-associated transcription factors (TFs), senescence genes, and epigenetic regulators. ConclusionOur study demonstrated the unique transcriptomic signature of the TMJ compared with the knee joint, highlighting its vulnerability to OA and pain. These findings provide valuable insights into the molecular mechanisms of TMJ and offer a resource for potential therapeutic target selection for TMJ-OA treatment. HighlightsO_LISignificant enrichment of neuronal system genes and lower enrichment of innate immune system genes in temporomandibular joint. C_LIO_LIKey osteoarthritis biomarkers such as Mmp13, Postn, and Col1a1 have higher expression in temporomandibular joint, indicating a higher vulnerability to osteoarthritis development. C_LIO_LIInterleukin-1beta treatment in goat temporomandibular joint medial layer cartilage mimics natural temporomandibular joint osteoarthritis-like transcriptional changes and immune responses observed in rabbit temporomandibular joint osteoarthritis model. C_LI

ObjectiveTo evaluate the therapeutic potential of iopanoic acid (IOP), a thyroid hormone pathway inhibitor, in preserving cartilage and bone integrity in osteoarthritis (OA), using in vivo and ex vivo tissue models. DesignIn the DMM mouse model, IOP was administered through intra-articular (i.a.) injection, either alone or combined with a thermosensitive hydrogel to enable sustained release. Histological analyses included Safranin O/Fast Green staining and OARSI scoring. Immunohistochemistry was performed for COL2, MMP13, and CCDC80 to evaluate anabolic, catabolic, and hypertrophic markers. Micro-CT assessed subchondral bone changes. In the ex vivo studies, IOP was applied to lesioned human osteochondral OA explants. Matrix degradation and repair were evaluated by sulfated glycosaminoglycan (sGAG) release, Mankin histology scores, and RT-qPCR for cartilage matrix genes. ResultsAdministration of IOP significantly reduced cartilage degeneration in DMM mice (P [≤] 1.0x10-4), characterized by increased COL2, and decreased MMP13 and CCDC80 expression. Notably, IOP also prevented pathological subchondral bone thickening. In human explants, IOP treatment led to a significant reduction in sGAG release compared to untreated explants on day 6 of the IOP treatment. Moreover, Mankin scores were significantly improved in IOP-treated compared to untreated explants, indicating reduced cartilage degradation. ConclusionIOP demonstrates strong chondroprotective effects, reducing cartilage degradation and promoting repair in OA models. Its combination with a thermosensitive hydrogel amplifies therapeutic potential, offering a promising strategy for OA treatment. Next steps are to optimize delivery and validate early molecular effects.

ObjectivesTo identify synovial transcriptional clusters in human knee osteoarthritis (OA) and determine how these relate to synovial histologic features, cell-type-associated gene expression, and cartilage degeneration severity. MethodsBulk RNA sequencing (RNA-seq) of synovial tissue from n = 135 patients with knee OA was analyzed using consensus clustering. Clusters were compared by clinical and histologic features, including cartilage degeneration severity (OARSI score). Single-cell RNA-seq (n = 18) and spatial transcriptomics were used to relate cartilage degeneration-associated gene expression patterns to synovial cell populations. ResultsFour synovial transcriptional clusters that differed in synovial histologic features and cartilage degeneration severity were identified. Greater cartilage degeneration was associated with enrichment of lining fibroblast- and inflammatory myeloid-associated gene expression, whereas lesser cartilage degeneration was associated with enrichment of sublining fibroblast, endothelial, mural cell, and adipocyte-associated gene expression. ConclusionsHuman knee OA synovium segregates into transcriptional clusters associated with cartilage degeneration severity. Synovial transcriptional heterogeneity corresponds to cell-type-associated gene expression. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LIOsteoarthritis synovium exhibits marked histologic and molecular heterogeneity. C_LIO_LISynovial inflammation detected by MRI correlates with cartilage degeneration and predicts progressive cartilage loss in knee OA. C_LIO_LIPrior transcriptomic studies have identified molecular subsets of OA synovium, but their relationship to cartilage degeneration severity remains unclear. C_LI What this study addsO_LIOA synovium segregates into four transcriptional clusters: Sublining (C1), Lymphomyeloid (C2), Myeloid (C3), and Major trauma (C4). C_LIO_LIGreater cartilage degeneration is associated with enrichment of inflammatory myeloid and lining fibroblast gene expression, whereas lesser degeneration is associated with enrichment of adipocyte, sublining fibroblast, endothelial, and mural cell-associated gene expression. C_LI How this study might affect research, practice or policyO_LIProvides a framework for a clinically relevant biological stratification of OA patients based on synovial molecular features. C_LIO_LIInforms future efforts to link synovial biology with OA prognosis, cartilage degeneration, treatment allocation, and development of targeted therapeutic strategies. C_LI

Proteomic studies have implicated clusterin as a potential biomarker of osteoarthritis (OA). However, there are two isoforms of clusterin with opposing functions, and their roles in OA have not previously been clarified. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone which prevents protein aggregation and enhances cell proliferation and viability, whereas nuclear clusterin (nCLU) acts as a pro-death signal. In this study, we focused on the role of sCLU and used established, pathophysiologically relevant, in vitro culture models to validate this potential biomarker of cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and chondrocytes was analysed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with or without pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}) and tumour necrosis factor- (TNF-). The amount of sulphated glycosaminoglycans (sGAG) released into the medium was determined by dimethylmethylene blue (DMMB) analysis. Clusterin mRNA expression was quantified by real-time PCR. MMP-3, MMP-13, COMP and sGAG released from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. Release of sCLU was attenuated with cytokine treatment in all three in vitro models. Expression of clusterin mRNA in cartilage explants and chondrocytes was down-regulated 7-days post cytokine stimulation. Cytokine stimulation attenuated expression and secretion of sCLU, therefore potentially limiting the cytoprotection which sCLU provides. These observations further implicate sCLU as having a role in OA, and diagnostic value as a potential biomarker for cartilage degradation.

ObjectivesThe inositol phosphatase SHIP2 plays a crucial role in skeletal development and chondrocyte differentiation, and mutations in INPPL1 (encoding SHIP2) cause opsismodysplasia, a chondrodysplasia with marked cartilage abnormalities. We investigated whether SHIP2 contributes to structural joint remodeling in osteoarthritis (OA). MethodsA cartilage-specific conditional knockout of SHIP2 was generated using Ship2fl/fl mice crossed with AggrecanCreERT2 mice. OA was induced at 9 weeks of age via destabilization of the medial meniscus (DMM). Sham surgery served as control. Mice were sacrificed 12 weeks post-surgery. Histological evaluation of articular cartilage, synovium, osteophytes, and subchondral bone was performed. Chondrocyte hypertrophy was assessed by type X collagen (COLX) staining, and SHIP1 was evaluated as a potential compensatory mechanism. ResultsDMM surgery induced OA-like changes in all genotypes, including cartilage damage, synovial inflammation, osteophyte formation, and subchondral bone thickening. However, Ship2cCART-KO mice showed no differences in OA-related parameters compared to control littermates. COLX expression increased following DMM surgery, independent of SHIP2 deletion. SHIP1 protein levels were not elevated in SHIP2-deficient mice. ConclusionThese findings indicate that SHIP2, while essential for cartilage development, does not act as a structural disease modifier in post-traumatic OA, suggesting that within this context, SHIP2 is not required for maintaining adult articular cartilage structure and is unlikely to represent a major therapeutic target for modifying structural disease progression.

ObjectivesAlthough rodent models of traumatically or chemically induced intervertebral facet joint osteoarthritis (FJOA) were previously described, the characteristics of spontaneous FJOA animal models have not been documented. This study aimed to identify the characteristics of a murine model of spontaneous FJOA and its underlying mechanisms. MethodsThe lumbar facet joints of mutant mice carrying a disrupted NFAT1 (nuclear factor of activated T cells 1) allele and of wild-type control mice were examined by histochemistry, quantitative gene expression analysis, immunohistochemistry, and histomorphometry using a novel FJOA scoring system at 2, 6, 12, and 18 months of age. The reproducibility of the FJOA scoring system was analyzed by inter-observer and intra-observer variability tests. Tissue-specific histomorphometric and gene expression changes were statistically analyzed. ResultsNFAT1-mutant facet joints displayed dysfunction of articular chondrocytes and synovial cells with aberrant gene and protein expression in cartilage and synovium as early as 2 months, followed by osteoarthritic structural changes such as articular surface fissuring and chondro-osteophyte formation at 6 months. Deeper cartilage lesions, synovitis, separation of cartilage from thickened subchondral bone, and tissue-specific molecular and cellular alterations in NFAT1-mutant facet joints became evident at 12 and 18 months. Osteoarthritic structural changes were not detected in wild-type facet joints at any ages, though age-related cartilage degeneration was observed at 18 months. ConclusionsUsing NFAT1-mutant mice, this study has identified for the first time an animal model of spontaneous FJOA with age-dependent osteoarthritic characteristics, developed the first FJOA scoring system, and elucidated the molecular mechanisms of NFAT1 mutation-mediated FJOA.

ObjectivesIdentification of osteoarthritis(OA)-specific synovial inflammatory pathways, and when in the clinical course they are active, is critical for their utility as therapeutic targets. We directly compared the mononuclear inflammatory/immune-cell responses following joint injury that does and does-not lead to OA, to define bona-fide OA-associated cellular events. MethodsWe undertook detailed temporal flow-cytometric and mRNA expression analysis in mice after sham or medial-meniscal-destiblization (DMM) surgery. We compared this with patients with meniscal injury and OA, and evaluated the role of synovial monocytes/macrophages versus lymphocytes in catabolic metalloproteinase secrection in vitro. We determined the effect of transient acute or delayed systemic T-cell depletion on DMM-induced OA pathology. ResultsOA-inducing/DMM and non-OA-inducing/Sham surgery had identical synovial monocyte/macrophage number, activation and polarization. The number and activation of synovial (not splenic or peripheral-blood) CD4 and CD8 lymphocytes was increased from 1-day after DMM versus Sham, and showed a persistent cyclical elevation throughout OA onset and progression. There was a temporal imbalance in synovial Th17/Treg and Th1/Th2 lymphocytes during DMM-induced OA initiation and progression. We confirmed early post-injury and late-OA CD3/CD8 T-cell responses in synovial tissues from patients, identified an association between CD8 and early post-injury symptoms, and defined a significant role for CD3+T-cells in synovial metalloproteinase secretion. Anti-CD3 cell-depletion studies in mice confirmed a key role for the earliest post-injury T-cell response in long-term OA pathology. ConclusionsWe identify a hitherto unappreciated pathophysiological role of acute T-cell activation after joint injury in long-term post-traumatic OA risk, providing a novel diagnostic and therapeutic target. Key MessagesO_ST_ABSWhat is already known about this subject?C_ST_ABSThe presence of synovitis/joint-inflammation increases the risk not only of osteoarthritis (OA) progression but incident disease. While numerous inflammatory effectors including macrophages and lymphocytes have been identified in OA, their disease-specificity, temporal regulation, and association with risk of pathology onset and progression is lacking. How does this study add?By directly comparing the mononuclear inflammatory/immune-cell responses following significant joint injury that does (medial-meniscal-destabilization; DMM) and does-not (Sham-surgery) lead to OA in mice, we have defined bona-fide OA-associated cellular events. There was no difference in synovial or systemic monocyte/macrophage cell number, activation or polarization between DMM and Sham, both showing a successful wound-healing response. In contrast, increases in number and activation of synovial Th1- and Th17-CD4, and CD8 T-cells in DMM compared with Sham occurred within the first 3 days, and while recurring cyclically through subsequent disease onset, depletion studies indicated this initial influx was key to long-term ptOA risk. How might this impact on clinical practice of future developments?Acute increases in synovial T-cells following jont injury may be both a novel marker of OA risk, and a target to reduce long term structural damage.

ObjectiveEarly knee osteoarthritis (KOA) presents as minor structural abnormalities in joint tissues, such as cartilage and subchondral bone, that cannot be assessed radiographically. Identification of a sensitive and convenient marker for early disease has the potential to enhance patient outcomes. This study determined 1) whether fat infiltration in muscle (i.e., muscle quality), as measured by ultrasound, is associated with structural abnormalities seen in early KOA and 2) which quadriceps muscles are appropriate as a novel marker for early KOA. MethodsParticipants with early symptomatic KOA (Kellgren Lawrence grade 1-2) underwent ultrasound assessment to measure the echo intensity of the vastus medialis and rectus femoris. The echo intensity corrected for ultrasound wave attenuation caused by subcutaneous fat was then calculated (i.e., corrected echo intensity). Structural abnormalities were assessed using the whole-organ magnetic resonance score (WORMS). A generalized linear mixed model was used to assess the relationship between the corrected echo intensity and WORMS score. ResultsForty-nine participants (ages: 44-78 years, 65.3% women) with 52 knees were included. After adjustment for covariates, increased corrected echo intensity (i.e., poor muscle quality) in the vastus medialis muscle was significantly associated with greater structural abnormalities, including disrupted cartilage integrity in the medial tibiofemoral joint. The association was not significant in the rectus femoris muscle. ConclusionIndividuals with poor muscle quality in the vastus medialis displayed compromised joint integrity. This study suggests that fat infiltration in vastus medialis assessed by ultrasound is an indicator of early symptomatic KOA.

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.

Temporomandibular joint osteoarthritis (TMJ-OA) affects a significant proportion of the population worldwide. However, there has been no substantial progress in the development of FDA-approved drugs for treatment due to a lack of understanding of the specific factors regulating key TMJ-OA molecular mechanisms. Lysyl Oxidase Like-2 (LOXL2) promotes knee joint cartilage protection, and it is downregulated in TMJ-OA animal model. We evaluated the role of LOXL2 in TMJ cartilage, its molecular mechanism and gene networks using in vivo Loxl2 knockout mice (Acan-Cre; Loxl2flox/flox) and ex vivo goat TMJ cartilage. Our results show that Loxl2 knockout in mice cartilage upregulates Il1b, Mmp9, Mmp13, Adamts4, and Adamts5, whereas it reduces the levels of aggrecan and proteoglycan. Loxl2 deleted TMJ cartilage show a higher enrichment of inflammatory response, TNFA signaling via NF-kB, extracellular matrix (ECM), and collagen degradation pathway network. Conversely, LOXL2 treatment reduces interleukin-1 beta (IL-1{beta})-induced expression of Mmp13, protects mitochondrial function and ECM from degeneration. Importantly, LOXL2 attenuates IL-1{beta}-induced chondrocyte apoptosis via phosphorylation of NF-{kappa}B and expression of pain-related gene PTGS2 (encodes COX2). Taken together, Loxl2 knockout mice exacerbate TMJ-OA through cartilage/ECM degradation, mitochondrial dysfunction, chondrocyte apoptosis, and inflammatory gene expression, whereas LOXL2 treatment mitigates these effects. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/653519v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@cb7540org.highwire.dtl.DTLVardef@17ebe2eorg.highwire.dtl.DTLVardef@1f7fd59org.highwire.dtl.DTLVardef@19fdd7_HPS_FORMAT_FIGEXP M_FIG C_FIG

ObjectivesMechanical cues are essential for maintaining cartilage function, yet how they integrate with molecular pathways dysregulated in osteoarthritis (OA) remains poorly defined in human tissue. Canonical Wnt signalling influences cartilage biology and cell-matrix interactions, but its role in integrin-dependent mechanoregulation in human cartilage is not fully understood. This study aimed to determine how Wnt activation affects chondrocyte responses to physiological mechanical loading, with a focus on 5{beta}1integrin and cytoskeletal organisation. MethodsHuman cartilage explants from non-OA and OA donors were subjected to short-term physiological cyclic compression. Canonical Wnt signalling was activated with CHIR99021, and integrin-mediated adhesion was modulated using the 5{beta}1 blocking peptide ATN-161 during loading. Chondrocyte responses were assessed by analysing mechanoresponsive and matrix-related gene expression, 5{beta}1 complex formation via proximity ligation assay and actin cytoskeletal organisation by confocal microscopy. ResultsOA chondrocytes exhibited a distinct integrin profile, characterised by increased ITGA5 and ITGB1 but reduced ITGA10 expression. In non-OA cartilage, canonical Wnt activation increased ITGB1 expression and 5{beta}1 integrin complex formation, while mechanical loading further enhanced ITGA5 and ITGB1 transcription under Wnt-activated conditions. Under control conditions, loading induced mechanoresponsive and anabolic gene expression in non-OA cartilage; these responses were attenuated following Wnt-activation and partially restored by 5{beta}1 blockade. Mechanical loading induced F-actin reorganization toward a more cortical distribution across cartilage zones, irrespective of disease status or treatment. Wnt activation did not result in distinct cytoskeletal phenotypes under load, and load-induced actin remodelling was comparable between groups. ConclusionThese findings identify 5{beta}1integrin as a key mediator linking canonical Wnt signalling to altered chondrocyte mechanoresponsiveness in human cartilage. While mechanical loading consistently induced cortical F-actin reorganization, Wnt-associated changes in load responsiveness arose primarily from integrin-dependent mechanisms rather than major alterations in actin organization. This study highlights the complexity of cartilage mechanoregulation and identifies integrin-mediated signaling as important contributors to canonical Wnt-driven alterations in load responsiveness relevant to OA.

Osteoarthritis (OA) is characterized by progressive cartilage degeneration, yet the initiating molecular events remain incompletely understood. Transmembrane protein 2 (TMEM2), a cell-surface hyaluronidase that degrades hyaluronan (HA), has been implicated in extracellular matrix homeostasis. Here, we delineate the spatiotemporal expression pattern of TMEM2 in mouse knee joints and assess its functional role during OA development. Single-cell RNA sequencing and histological analyses of normal joints revealed predominant Tmem2 expression in non-calcified articular chondrocytes and synovial cells. Following destabilization of the medial meniscus (DMM), Tmem2 was transiently upregulated during early OA, particularly within the non-calcified cartilage zone, coinciding with a pronounced reduction in HA content indicative of accelerated HA turnover. In contrast, Tmem2 expression declined at later stages, suggesting a temporally restricted activation phase. Functionally, Tmem2 deficiency exacerbated DMM-induced OA, leading to more severe structural deterioration, increased chondrocyte apoptosis, reduced proliferation, and elevated type X collagen, consistent with impaired cartilage homeostasis. Collectively, these findings identify TMEM2 as a key regulator of HA metabolism and a context-dependent modulator of OA progression. We propose that early, transient TMEM2 upregulation represents an adaptive remodeling response, whereas dysregulated or prolonged activity may contribute to HA depletion and cartilage breakdown.

Objectivesi) develop and use a new cryogenically-enhanced phase contrast method to visualise hyaline articular cartilage (HAC); ii) to measure HAC, articular calcified cartilage (ACC) and total articular cartilage thicknesses in male STR/Ort (osteoarthritis, OA) and CBA (healthy) mouse tibial epiphyses, reflecting divergent OA predisposition, at three age timepoints chosen to reflect pre-OA, OA onset and late-progression; iii) to compare HAC, trans-zonal and ACC 3D chondrocyte anatomy in tibial epiphyses. MethodsSTR/Ort and CBA mouse knees (n=4 per age and strain group) were synchrotron-CT scanned at high-resolution while fresh frozen, without staining, fixation, dissection or dehydration of the joint capsule. Both cartilage thickness and cellular characteristics (chondrocyte n=420) were manually measured and statistically compared (SPSS). ResultsCryo-enhanced phase contrast allowed cartilage to be seen in full thickness with cellular detail. HAC was thicker in STR/Ort than age-matched CBA mice in 16/24 knee joint compartments and timepoints (all p<0.04). In contrast, HAC was thicker only in the posterior lateral femur of CBA mice at 10weeks (p<0.001, Table 1). ACC and total cartilage were also thicker in STR/Orts. Trans-zonal chondrocytes were smaller than ACC and HAC chondrocytes (p-values<0.001, volumes 878, 1,567m3 and 1,348m3 respectively). O_TBL View this table: org.highwire.dtl.DTLVardef@191d5a9org.highwire.dtl.DTLVardef@162523borg.highwire.dtl.DTLVardef@4b7caorg.highwire.dtl.DTLVardef@1be6005org.highwire.dtl.DTLVardef@192143d_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTABLE 1:C_FLOATNO O_TABLECAPTIONCondylar compartments in which HAC thickness was measured, and the presence or absence of a significant difference between STR/Ort and CBA mouse strains. * indicates a significant difference between strains (p<0.05). Note that the posterior lateral femur at 10 weeks of age is the only region in which STR/Ort mice have significantly thinner HAC than CBA mice. C_TABLECAPTION C_TBL ConclusionsCryogenically-enhanced phase-contrast imaging allowed cellular detail to be seen in 3D as never before in HAC in this (or any other) model. Our findings challenge current understanding by associating STR/Ort OA vulnerability with regions of thick, rather than thinning-with-age, cartilage. Our data affirm an association between excessively hypertrophic chondrocytes and OA is present in STR/Ort mice.

ObjectivesThe etiology of osteoarthritis revolves around the interplay between genetic predisposition and perturbing environmental cues, such as mechanical stress. The pericellular matrix, with its hallmark proteins collagen type VI and fibronectin, surrounds chondrocytes and is critical in transducing the biomechanical cues. The objective is to study the functional effects of an OA disease-risk mutation in COL6A3 in interaction with hyper-physiological mechanical cues in a tailored human induced pluripotent stem cells (hiPSCs) derived cartilage organoid model. MethodTo identify pathogenic OA mutations exome sequencing in symptomatic OA patients was performed. To study functional effects, CRISPR-Cas9 genome engineering was used to introduce the mutation in our established human induced pluripotent stem cell-derived in-vitro neo-cartilage organoid model in interaction with hyper-physiological mechanical loading conditions. ResultsA high-impact mutation in COL6A3 was identified that resulted in significantly lower binding between the PCM proteins COLVI and fibronectin (FN) and provoked an osteoarthritic chondrocyte state. Moreover, aberrant function of the PCM, secondary to the COL6A3 mutation, abolished the initial stress responses marked particularly by upregulation of PTGS2 encoding cyclooxygenase-2 (COX-2), after hyper-physiological mechanical loading conditions. ConclusionThese findings demonstrate that ablating the characteristic transient COX-2 response after injurious mechanical cues may have a direct negative impact on chondrocyte health. What is already knownO_LIThe etiology of osteoarthritis revolves around the interplay between genetic predisposition and perturbing environmental cues, such as mechanical stress. C_LIO_LIThe pericellular matrix, with its hallmark proteins collagen type VI and fibronectin, surrounds the chondrocytes and is critical in transducing biomechanical cues from the extracellular matrix to chondrocytes henceforth it determines the chondrocyte mechanical environment. C_LIO_LIThe mechanical environment of the chondrocytes is a critical factor that influences chondrocyte health as it determines the balance between synthesis and degradation of the articular cartilage extracellular matrix. C_LI What this study addsO_LIA sustainable human induced pluripotent stem cell-derived in-vitro neo-cartilage organoid model that is tailored to study detailed biologic effects of mechanical cues to chondrocytes. C_LIO_LIAn OA disease-risk mutation in COL6A3 reduces the binding between collagen type VI to fibronectin and provoked an osteoarthritic chondrocyte state. C_LIO_LIUpon hyper-physiological mechanical loading, aberrant function of the pericellular matrix, secondary to the COL6A3 mutation, ablates the initial transient inflammatory response, characterized particularly by PTGS2 encoding cyclooxygenase-2 (COX-2). C_LI How this study might affect research practice or policyO_LIInhibiting COX-2, as an important transient inflammatory response after hyper-physiological mechanical cues, could worsen the loss of structural integrity of the cartilage in osteoarthritis patients. Henceforth, prescription of COX-2 inhibitors as pain treatment for OA patients should be reconsidered. C_LI

Background and purposeIn the QUADX-1 trial, we randomized 140 patients with severe knee osteoarthritis (OA) eligible for a knee arthroplasty to home-based exercise for 12 weeks. Seventy-nine (68%) of the 117 patients, who completed the exercise intervention, postponed surgery. Here, we report how many patients, who completed the 12-week exercise intervention, had received a knee arthroplasty at 2 years and describe their initial exercise response. MethodsFrom the QUADX-1 trial, we had the following: isometric knee-extensor strength, Oxford Knee Score (OKS), Knee Osteoarthritis Outcome Score (KOOS), average knee pain last week (0-10 numeric rating scale [NRS]), 6-minute walk test, stair climbing test, and self-reported exercise behaviour. ResultsAt the 2-year follow-up, 50 (43%) of the 117 patients had received a knee arthroplasty (KA group) and 67 (57%) had not (NO-KA group). Compared with the KA group, the NO-KA group had less severe radiographic OA at baseline (KL grade 4: 38% vs 55%) and showed greater-- and often clinically relevant--improvements after the 12-week exercise intervention, including knee pain (-2.1 vs -0.1 NRS points), OKS (+6.9 vs +0.5 points), and KOOS ADL (+13.9 vs +1.3 points). ConclusionTwo years after completing the initial 12-week QUADX-1 exercise intervention, more than half the cohort had not received a knee arthroplasty despite initially being considered eligible. Those who had not received a knee arthroplasty at two years had less severe radiographic OA at baseline and generally responded better to 12-week exercise two years earlier, compared to those who had. ClinicalTrials.gov-IDNCT02931058.

IntroductionThe natural history of Femoroacetabular Impingement (FAI) has been clinically associated with the development of hip osteoarthritis (OA); however, the pathobiological mechanisms underlying the transition from focal impingement to global joint degeneration remain unclear. The goal of the study was to investigate differences in transcriptomic profiles of the cartilage from FAI and hip OA patients using whole-genome RNA sequencing. MethodsThirty-seven patients were included in the study with 20 diagnosed with FAI undergoing arthroscopic treatment and 15 diagnosed with hip OA undergoing total hip arthroplasty (THA). Cartilage samples were obtained intraoperatively over the femoral head-neck junction for both FAI and OA cohorts. Whole-genome RNA sequencing was performed on 10 gender-matched patients in the FAI and OA cohorts with the remaining samples used for histopathologic analysis using the Osteoarthritis Research Society International (OARSI) grading system and qRT-PCR validation. Target validation was further confirmed with immunohistochemical staining for FGF18 on FAI and OA cartilage samples. ResultsWe identified a total of 3,531 Differentially Expressed Genes (DEGs) between the FAI and OA cohorts with multiple targets for genes implicated in canonical OA pathways. qRT-PCR validation confirmed increased expression of FGF18 and WNT16 in the FAI samples, while there was increased expression of MMP13 and ADAMTS4 in the OA samples. Expression levels of FGF18 and WNT16 were also higher in FAI samples with mild cartilage damage (OARSI grades 1-2) compared to FAI samples with severe cartilage damage or OA cartilage (OARSI grade 4-6). Immunohistochemical evaluation identified increased FGF18 staining in OARSI grade 1-2 FAI samples compared to OARSI grade 5-6 FAI and OA samples. ConclusionsRNA sequencing of cartilage of FAI and hip OA patients identified a negative association of FGF18 expression levels with cartilage damage severity, suggesting that FGF18 may be used as a marker for hip OA progression. Future evaluation of FGF18 signaling as well as other markers in early OA may yield further insight into disease prevention and treatment.

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

Knee osteoarthritis (OA) is the most common form of OA which affects knee joints and there is currently no disease-modifying treatment available for OA. Therefore, an ideal strategy to prevent the development of OA is to identify and intervene at the modifiable risk factors for the development and progression of OA. Early-life factors such as obesity and malalignment may affect the mechanical aspect of the knee (i.e. alterations in normal knee kinematics) and could be the risk factor for the development of knee OA in later life. Identifying early-life (gestational factors, congenital defects, childhood, adolescence, early adulthood) factors which affect the development of knee OA in later stages of the life may help to develop targeted prevention programs in early-life itself to prevent the development of knee OA. Hence, this systematic review protocol provides the method to be used to comprehensively summarise the existing evidence on early life modifiable risk factors associated with the development and progression of knee OA.

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 the most prevalent joint disorder occurring with articular cartilage degradation, which includes a switch from an articular to a growth-plate chondrocyte phenotype. Epigenetics serves as a new therapeutic target but histone modification changes in OA remain elusive. Here, we investigated the profiles of four histone modifications in normal and OA chondrocytes. The repressive mark H3K27me3 was significantly lost in OA, associated with up-regulated gene expression. Surprisingly, many of these genes were occupied by both H3K27me3 and H3K4me3 in normal chondrocytes, showing a poised bivalent state. These bivalent genes are deemed to be activated during the hypertrophy of growth plate chondrocytes. Furthermore, inflammation induced the expression of demethylase KDM6B and decreased H3K27me3 level in OA chondrocytes, which was rescued by the KDM6B inhibitor GSK-J4. Altogether, our results suggest an inherited bivalent epigenetic signature on developmental genes that makes articular chondrocytes prone to hypertrophy and contribute to a promising epigenetic therapy for OA. The Paper ExplainedO_ST_ABSProblemC_ST_ABSOsteoarthritis (OA) affects as much as 40% of the elderly population, representing the largest cause of age-related disability. The high susceptibility to OA suggests an intrinsic and systemic characteristic in articular chondrocytes that makes cartilage prone to degeneration. ResultsEpigenetic bivalent genes, which are occupied with both H3K27me3 and H3K4me3, are considered to poise expression of developmental genes. Surprisingly, we reported bivalency for hypertrophy related genes in normal articular chondrocytes. These bivalent genes need to be activated in growth plate chondrocytes for extracellular matrix degradation and ossification, but are left as a "bomb" for degeneration in articular chondrocytes. We further found that inflammation induced KDM6B remove H3K27me3 to activate hypertrophy related genes that promote OA. ImpactOur results suggest an inherited epigenetic signature that makes articular chondrocytes prone to hypertrophy and ossification and contribute to a promising epigenetic therapy for OA.