Background. Resveratrol is a polyphenolic compound commonly found in the skins of red grapes. Sirtuin 1 (SIRT1) is a human gene that is activated by resveratrol and has been shown to promote longevity and boost mitochondrial metabolism. We examined the effect of resveratrol on normal and osteoarthritic (OA) human chondrocytes. Methods. Normal and OA chondrocytes were incubated with various concentrations of resveratrol (1 µM, 10 µM, 25 µM and 50 µM) and cultured for 24, 48 or 72 hours or for six weeks. Cell proliferation, gene expression, and senescence were evaluated. Results. SIRT1 was significantly upregulated in normal chondrocytes with resveratrol concentrations of 25 µM and 50 µM on both two- (2D) (both p = 0.001) and three-dimensional (3D) cultures (p = 0.008 and 0.001, respectively). It was significantly upregulated in OA chondrocytes treated with 10 µM, 25 µM and 50 µM resveratrol on 2D cultures (p = 0.036, 0.002 and 0.001, respectively) and at 50 µM concentration on 3D cultures (p = 0.001). At 72 hours, the expression of collagen (COL)-10, aggrecan (AGG), and runt-related transcription factor 2 (RUNX2) was significantly greater in both 25 µM (p = 0.011, 0.006 and 0.015, respectively) and 50 µM (p = 0.019, 0.004 and 0.002, respectively) resveratrol-treated normal chondrocyte cultures. In OA chondrocytes, expression of COL10 and RUNX2 was significantly greater in 25 µM (p = 0.004 and 0.024) and 50 µM (p = 0.004 and 0.019) cultures at 72 hours on 3D cultures. Conclusions. At concentrations of 25 µM and/or 50 µM, resveratrol treatment significantly upregulates SIRT1 gene expression in normal and osteoarthritic chondrocytes. Resveratrol induces chondrocytes into a hypertrophic state through upregulation of COL1, COL10, and RUNX2. Cite this article: Bone Joint Res 2014;3:51–9

Onset and progression of osteoarthritis (OA) is affected by a plethora of factors, including joint injury, obesity, aging, and heredity. This multi-factorial etiology obstructs our understanding of driving molecular mechanisms, which likely comprise an interplay between systemic and local factors. Next to biomechanical factors and cytokines, the course of OA appears to be altered by microenvironmental oxidative stress: cumulative evidence now suggests a prominent participation of cell signalling mediated by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master regulator of cellular protective processes, in this process. Nrf2 activation through phosphorylation of mitogen-activated protein kinases (MAPKs) regulates Nrf2 target genes, like hemeoxygenase-1 (HO-1), superoxide dismutase 2 (SOD2), or NAD(P)H Quinone Dehydrogenase 1 (NQO1) in OA chondrocytes. Maintaining high levels of HO-1 appears to be beneficial against OA development. Experimental manipulation of putative antioxidant response element (ARE) binding sites alters the in vitro expression of key transcription factors of chondrocyte markers in promoter-reporter assays. Potentially, Nrf2 is involved in autophagy, intermediary metabolism and unfolded protein response. RNAi-mediated depletion of Nrf2 further significantly abrogated anti-inflammatory and chondroprotective effects and epigenetics link transcriptional pathways of ‘N-factors’, Nrf2 and NFATs, to micro-RNA signalling. Current findings thus reveal novel mechanisms regulating extracellular matrix synthesis by chondrocytes. A further understanding of these pathways and their regulation will lead to important novel targets to slow OA progression

Aims. To evaluate the effect of ultrasound-targeted simvastatin-loaded microbubble destruction (UTMDSV) for alleviation of the progression of osteoarthritis (OA) in rabbits through modulation of the peroxisome proliferator-activated receptor (PPARγ). Methods. In vitro, OA chondrocytes were treated with ultrasound (US), US-targeted microbubble destruction (UTMD), simvastatin (SV), and UTMDSV on alternate days for four weeks. Chondrocytes were also treated with PPARγ inhibitor, PPARγ inhibitor+ UTMDSV, and UTMDSV. The cholesterol efflux rate and triglyceride levels were measured using an assay kit and oil red O staining, respectively. In vivo, the OA rabbits were treated with a single intra-articular injection of UTMD, SV, and UTMDSV every seven days for four weeks. Cartilage histopathology was assessed by safranin-O staining and the Mankin score. Total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) in rabbit knee synovial fluid were detected by enzyme-marker assay. Aggrecan, collagen II, and PPARγ expression levels were analyzed by Western blotting (WB). Results. In vitro, UTMDSV significantly increased the cholesterol efflux rate and aggrecan, collagen II, and PPARγ levels in OA chondrocytes; these effects were blocked by the PPARγ inhibitor. In vivo, UTMD. SV. significantly increased aggrecan, collagen II, PPARγ, and HDL-C levels, while TC levels and Mankin scores were decreased compared with the UTMD, SV, OA, and control groups. Conclusion. UTMDSV promotes cartilage extracellular matrix synthesis by modulating the PPARγ-mediated cholesterol efflux pathway in OA rabbits. Cite this article: Bone Joint Res 2021;10(10):693–703

Abstract

Aims. Osteoarthritis (OA) is a prevalent joint disorder with inflammatory response and cartilage deterioration as its main features. Dihydrocaffeic acid (DHCA), a bioactive component extracted from natural plant (gynura bicolor), has demonstrated anti-inflammatory properties in various diseases. We aimed to explore the chondroprotective effect of DHCA on OA and its potential mechanism. Methods. In vitro, interleukin-1 beta (IL-1β) was used to establish the mice OA chondrocytes. Cell counting kit-8 evaluated chondrocyte viability. Western blotting analyzed the expression levels of collagen II, aggrecan, SOX9, inducible nitric oxide synthase (iNOS), IL-6, matrix metalloproteinases (MMPs: MMP1, MMP3, and MMP13), and signalling molecules associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Immunofluorescence analysis assessed the expression of aggrecan, collagen II, MMP13, and p-P65. In vivo, a destabilized medial meniscus (DMM) surgery was used to induce mice OA knee joints. After injection of DHCA or a vehicle into the injured joints, histological staining gauged the severity of cartilage damage. Results. DHCA prevented iNOS and IL-6 from being upregulated by IL-1β. Moreover, the IL-1β-induced upregulation of MMPs could be inhibited by DHCA. Additionally, the administration of DHCA counteracted IL-1β-induced downregulation of aggrecan, collagen II, and SOX9. DHCA protected articular cartilage by blocking the NF-κB and MAPK pathways. Furthermore, DHCA mitigated the destruction of articular cartilage in vivo. Conclusion. We present evidence that DHCA alleviates inflammation and cartilage degradation in OA chondrocytes via suppressing the NF-κB and MAPK pathways, indicating that DHCA may be a potential agent for OA treatment. Cite this article: Bone Joint Res 2023;12(4):259–273

There is an evolving body of evidence that demonstrates the role of epigenetic mechanisms, such as DNA-methylation in the pathogenesis of OA. This systematic review aims to summarize the current evidence of DNA methylation and its influence on the pathogenesis of OA. A pre-defined protocol in alignment with the PRISMA guidelines was employed to systematically review eight bibliographic databases, to identify associations between DNA-methylation of articular chondrocytes and osteoarthritis. A search of Medline (Ovid), Embase, Web-of-Science, Scopus, PubMed, Cinahl (EBSCOhost), Cochrane Central and Google Scholar was performed between 1st January 2015 to 31st January 2021. Data extraction was performed by two independent reviewers. During the observation period, we identified 15 gene specific studies and 24 genome wide methylation analyses. The gene specific studies mostly focused on the expression of pro-inflammatory markers, such as IL8 and MMP13 which are overexpressed in OA chondrocytes. DNA hypomethylation in the promoter region resulted in overexpression, whereas hypermethylation was seen in non-OA chondrocytes. Others reported on the association between OA risk genes and the DNA methylation pattern close to RUNX2, which is an important OA signal. The genome wide methylation studies reported mostly on differentially methylated regions comparing OA chondrocytes and non-OA chondrocytes. Clustering of the regions identified genes that are involved in skeletal morphogenesis and development. Differentially methylated regions were seen in hip OA and knee OA chondrocytes, and even within different regions of an OA affected knee joint, differentially methylated regions were identified depending on the disease stage. This systematic review demonstrates the growing evidence of epigenetic mechanisms, such as DNA methylation, in the pathogenesis of OA. In recent years, there has been a focus on the interplay between OA risk genes and DNA methylation changes which revealed a reactivation of genes responsible for endochondral ossification during development. These are important findings and may help to identify eventual future therapeutic targets. However, the current body of literature is mostly showing the differences in DNA methylation of OA chondrocytes and non-OA chondrocytes, but a true longitudinal analysis demonstrating the DNA methylation changes actually happening is still not available

Osteoarthritis (OA) is the most prevalent degenerative joint disease that is a leading cause of disability worldwide. Existing therapies of OA only address the symptoms. Liraglutide is a well-known anti-diabetic medication that is used to treat type 2 diabetes and obesity. In inflammatory and post-traumatic OA animal models, liraglutide has demonstrated anti-inflammatory, pain-relieving, and cartilage-regenerating effects1 . The objective of this study is to investigate liraglutide's ability to reduce inflammation and promote anabolism in human OA chondrocytes in vitro. Pellets formed with human OA chondrocytes were cultured with a chondrogenic medium for one week to form cartilage tissue. Afterward, pellets were cultured for another 2 weeks with a chondropermissive medium. The OA group was treated with IL-1β to mimic an inflammatory OA condition. The drug group was treated with 0.5 or 10 µM liraglutide. On days 0, 1, and 14, pellets were collected. Conditioned medium was collected over the 2 weeks culture period. The gene and protein expression levels of regenerative and inflammatory biomarkers were evaluated and histological analyzes were performed. Results showed that the nitric oxide release of the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups were lower than the OA group. The DNA content of the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups were higher than the OA group on day 14. The RT-qPCR results showed that the anabolism (ACAN, COMP, and COL2) markers were higher expressed in the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups when compared with the OA group. The inflammation (CCL-2 and IL-8) markers and catabolism markers (MMP-1, MMP-3, ADAMTS4, and ADAMTS5) had lower expression levels in the OA + liraglutide groups compared to the OA group. The histomorphometric analysis (Figure 1) supported the RT-qPCR results. The results indicate that liraglutide has anabolic and anti-inflammatory effects on human OA chondrocyte pellets. Acknowledgments: This project has received funding from the Eurostars-2 joint program with co-funding from the European Union Horizon 2020 research and innovation program. The funding agencies supporting this work are (in alphabetical order of participating countries): France: BPI France; Germany: Project Management Agency (DLR), which acts on behalf of the Federal Ministry of Education and Research (BMBF); The Netherlands: Netherlands Enterprise Agency (RVO); Switzerland: Innosuisse (the Swiss Innovation Agency). For any figures and tables, please contact the authors directly

Mechanical loading regulates the metabolism of chondrocytes in cartilage1. Nowadays, studies exploring the in vitro response of cartilage towards loading often rely on bioreactor experiments applying only compressive loading. This is likely not sufficiently representative for the complex multi-directional loading profile in vivo (i.e. where typical compressive and shear loading are both present). The impact of multi-axial loading is specifically relevant in the context of the onset of osteoarthritis (OA) due to joint destabilization. Here, alterations in the 3D loading profile, and in particular increased shear forces, are suggested to initiate catabolic molecular responses leading to cartilage degeneration3. However, in vitro/ex vivo data confirming this hypothesis are currently lacking. Therefore, we aim to investigate how increased shear loading affects the metabolism and ECM deposition of a healthy chondrogenic cell line and if this response is different in osteoarthritic primary chondrocytes. A murine chondrogenic precursor cell line (ATDC5) and primary human osteoarthritic articular chondrocytes (hOACs) were encapsulated in 2.2% alginate disks and cultured in DMEM medium for three days. Hydrogels seeded with the different cell groups were loaded in the TA ElectroForce BioDynamic Bioreactor and subjected to following loading conditions: (a) 10% compression at 1Hz for 1h, (b) 10% compression and 10° shear loading at 1Hz for 1h. Unloaded constructs were used as control. After loading, hydrogel constructs were stabilized in culture medium for 2 hours, to facilitate adequate gene expression responses, before being dissolved and snap frozen. RNA was isolated and gene expression levels specific for anabolic pathways, characterized by extracellular matrix (ECM) genes (Col2a1, Aggrecan and Perlecan), catabolic processes (MMP-3 and MMP-13) and chondrogenic transcription factor (Sox9) were evaluated using RT-qPCR. The TA ElectroForce BioDynamic Bioreactor was successfully set-up to mimic cartilage loading. In ATDC5 cells, compression elicits an increase in all measured ECM genes (Col2a1, Aggrecan and Perlecan) compared to unloaded controls, suggesting an anabolic response. This upregulation is decreased when adding additional shear strain. In contrast to ATDC5 cells, the anabolic response of proteoglycans Aggrecan and Perlecan to compressive loading was lower in osteoarthritic chondrocytes, and Col2a1 expression appeared decreased. Adding shear strain reversed this effect on Col2a1 expression. Multi-directional loading increased transcription factor Sox9 expression compared to compression in both ATDC5 and OA chondrocytes. In OA chondrocytes, both loading regimens increased MMP-3 and MMP-13 expression. Shear loading reduces the anabolic effect of compressive loading in both cell types. OA cells presented more catabolic response to mechanical loading compared to precursors, given the increase in catabolic enzymes MMP-3 and MMP-13

Introduction and Objective. Osteoarthristis (OA) has been associated with many genes and yet the genetic basis for this disease has never formally been established. Recent realization that epigenetic changes could be the underlying pathological mechanisms has helped to explain many complex multifactorial diseases with no clear genetic cause. We therefore asked whether epigenetics could also play a role in OA. We have previously shown that the DNA epigenetic modification, specifically the hydroxymethylation on cytosine (5hmC), undergoes a fivefold increase on OA-associated genes which are activated at OA onset. In this study, we further uncovered a set of 5hmC-mediated gene targets and their mechanistic link to OA progression. Materials and Methods. We surgically induced OA on 4 to 6 months old Tet1−/− mice (Tet1tm1.1Jae, the Jackson laboratory) and wild-type littermates by performing destabilization of the medial meniscus (DMM) surgery. Joints were collected for histological assessment through blinded grading with the OARSI scoring system. Human articular chondrocytes were harvested from OA cartilage samples obtained during total knee arthroplasty or from grossly normal cartilage pieces obtained during notchplasty or debridement from patients undergoing anterior cruciate ligament (ACL) reconstruction with no history of OA symptoms, under approved Human subjects Institutional Review Board protocols. Bioinformatic analyses of RNA-sequencing and CCGG sequencing (reduced representation 5hmC profiling) were performed to identify TET1 target genes associated with OA progression. Several measurements were used to assess the effect of TET1 ablation on the phenotype of mouse cartilage tissue and human chondrocytes including, histological evaluation, and quantitative bone assessment by micro-CT imaging and multiplex cytokine analyses in the serum of mice in vivo (mouse 39-plex assay) and in the supernatant of human chondrocyte cultures (human 62-plex assay). Results. We used a mouse model with surgically induced OA and found that OA onset was accompanied by a gain of ∼40,000 differentially hydroxymethylated sites prior the notable histological onset of the disease. We additionally revealed that these changes are mediated by the ten-eleven-translocation enzyme 1 (TET1), since Tet1−/− mice lost 98% of 5hmC sites upon OA induction. Remarkably, Tet1−/− mice were protected from OA development including degeneration of the cartilage surface and osteophyte formation. Silencing of TET1 expression in human OA chondrocytes reduced the expression in a set of genes, which may represent the pathological gene targets that exacerbate OA including MMP3 and MMP13 and several inflammatory cytokines. Therefore, our study reveals the unexpected beneficial role of TET1 inhibition in blocking OA progression. In fact, intra-articular injections of a dioxygenases’ inhibitor, 2 hydroxyglutarate, on mice after surgical induction of OA stalled disease progression. Furthermore, treatment of human OA chondrocytes with the same inhibitor also phenocopied TET1 loss, implicating a therapeutic potential of TET inhibition in OA patients. Conclusions. Collectively, our study not only demonstrate the role of TET1 in OA; the 5hmC-mediated gene targets acting on multiple OA pathways were identified and can be modulated as therapeutic intervention to treat OA

Introduction. Osteoarthritis (OA) is a prevalent joint disorder characterized by cartilage degeneration, inflammation, and pain. Current treatments provide only symptomatic relief, necessitating novel molecular targets. The caspase family, known for its roles in apoptosis and inflammation regulation, may additionally influence crucial processes for cartilage homeostasis such as differentiation and proliferation. However, the specific roles of individual caspases in OA pathogenesis remain unclear. This study aims to investigate the involvement of the caspase family in OA and as potential targets for therapy, with a focus on caspase-1 and -8. Method. Chondrocytes from both healthy and OA donors were cultured in 2D and 3D culture models and stimulated with TNF-α or IL-1β. The expression and activation of caspase-1 and -8 was assessed using RT-PCR, ELISA. Transcriptome analysis of OA and healthy cartilage samples, along with Mendelian randomization (MR) analysis were conducted to explore the involvement of caspase family in OA and to assess its potential as therapeutic targets. Result. Higher expression levels of caspase-1, -8 were observed in OA cartilage compared to healthy cartilage. TNF-α stimulation increased their expression in both healthy and OA chondrocytes, while IL-1β had limited impact. Caspase-8 expression was causally associated with knee OA in MR analysis, suggesting a potential therapeutic target. The caspase-1 inhibitor VX-765 mildly reduced chondrocyte viability, with no significant effect in the presence of TNF-α. While the caspase-8 inhibitor Z-IETD-FMK exhibited slight enhancements in cell viability, these improvements were not statistically significant. Nevertheless, its effectiveness significantly increased in the presence of TNF-α. Conclusion. This study highlights the involvement of caspase-1 and caspase-8 in OA pathology, with caspase-8 emerging as a potential therapeutic target for knee OA treatment. Further investigation into the roles of caspase-1 and -8 in OA pathophysiology, including the efficacy and potential side effects of their corresponding inhibitors, is warranted. Acknowledgements. Funding Inter-Action/Inter-Excellence project (BTHA-JC-2022-36/LUABA22019)

Osteoarthritis (OA) is a disabling disease depriving the quality of life of patients. Mesenchymal stem cells (MSCs) are recently used to modify the inflammatory and degenerative cascade of the disease. Source of MSCs could change the progression and symptoms of OA due to their different metabolomic activities. We asked whether MSCs derived from the infrapatellar fat (IPF), synovium (Sy) and subcutaneous (SC) tissues will decrease inflammatory and degenerative markers of normal and OA chondrocytes and improve regeneration in culture. Tissues were obtained from three male patients undergoing arthroscopic knee surgery due to sports injuries after ethical board approval. TNFa concentration decreased in all MSC groups (Sy=156,6±79, SC=42,1±6 and IPF=35,5±3 pg/ml; p=0,036) on day 14 in culture. On day seven (Sy=87,4±43,7, SC=23±8,9 and IPF=14,7±3,3 pg/ml, p=0,043) and 14 (Sy=29,1±11,2, SC=28,3±18,5 and IPF=20,3±16,2 pg/ml, p=0,043), MMP3 concentration decreased in all groups. COMP concentration changes however were not significant. Plot scores of tissues for PC2-13,4% were significantly different. Based on the results of liquid chromatography-mass spectrometry (LC-MS) metabolomics coupled with recent data processing strategies, clinically relevant seven metabolites (L-fructose, a-tocotrienol, coproporphyrin, nicotinamide, bilirubin, tauro-deoxycholic acid and galactose-sphingosine) were found statistically different (p<0.05 and fold change>1.5) ratios in tissue samples. Focusing on these metabolites as potential therapeutics could enhance MSC therapies. Acknowledgment: Hacettepe University, Scientific Research Projects Coordination Unit (#THD-2020-18692) and Turkish Society of Orthopedics and Traumatology (#TOTBID-89) funded this project. Feza Korkusuz MD is a member of the Turkish Academy of Sciences (TÜBA)

Osteoarthritis (OA) leads to articular cartilage degradation, following complex dysregulation of chondrocyte's metabolism towards a catabolic state. Mechanical and biochemical signals are involved and need to be considered to understand the condition. Regulatory network-based models (RNM) successfully simulated the biological activity of the chondrocyte and the transduction of mechanical signals at the molecular and cell levels. However, the knowledge gap between single-cell regulation and intercellular communication in tissue volumes hinders the interpretability of such models at larger scales. Accordingly, a novel tissue-level biochemical model is proposed. We hypothesise that it is possible to simulate interacting network effects through the transport of diluted species in a finite-element model, to grasp relevant dynamics of cell and tissue regulation in OA. Chondrocyte RNM equations were translated into a reaction term of 18 multi-species diffusion model (e.g., 3 anti-inflammatory and 8 pro-inflammatory interleukins, 3 pro-anabolic and 1 pro-catabolic growth factors, 2 nociceptive factors and 2 pro-inflammatory cytokines). Elements with RNM reaction terms represented the chondrocytes and were distributed randomly through the model, according to known cellular density in the knee cartilage, and could both react to and produce diffusive entities through the pericellular matrix, associated with reduced diffusion coefficients. The model was constructed over a 2D square of 0.47 mm sides considered to be in the middle of the cartilage, so boundary conditions were settled as periodic. Different simulations were initialised with initial concentrations of either healthy or pro-OA mediators. Preliminary results showed that, independently of the initial conditions, the chondrocytes successfully evolved into anabolic states, in absence of sustained pro-catabolic external stimulations, in contrast to single-cell RNM [2]. Our intercellular model suggests that paracrine communication may increase robustness towards cartilage maintenance, and future tests shall reveal new OA dynamics. Acknowledgements: Funding was provided by the European Commission (ERC-2021-CoG-O-Health-101044828)

Irisin is a hormone-like myokine released from skeletal muscle during exercise. It has also been reported that irisin levels in serum and synovial fluid of knee osteoarthritis (OA) patients were negatively correlated with OA severity. We hypothesized that irisin might play a role in the cartilage homeostasis mediated by physical activity. Therefore, this study aims to explore the cross talk between skeletal muscle and cartilage tissues in human with OA mediated by the myokine irisin. Human articular OA chondrocytes were isolated, expanded and cultured in micro-mass 3-D culture system. Pellets were cultured with or without r-Irisin, and then activated by protein inhibitors of p38-MAPK signalling pathway. After one week the amount of GAG content was evaluated. Quantitative gene expression of Coll-X and Coll-II was performed. WB was utilized to detect expressions of p38-MAPK signalling pathway and Coll-X and Coll-II. In the current study, chondrocytes cultured in r-Irisin showed a significant higher GAG/DNA content compared to control (p<0.05). Moreover, r-Irisin promoted a significant increase of the expression collagen type II and decrease of collagen type X in (p<0.05). This OA chondrocytes recovery was abrogated by the p38 MAPK and ERK signalling pathways. Our observation suggests that Irisin targets chondrocytes promoting GAG content, increasing Collagen Type II and decreasing Collagen type X gene expressions. The observed OA chondrocyte recovery mediated by irisin is obtained through the inactivation of p38/ERK MAP kinase signalling cascades in vitro. This is the first study that demonstrates a cross-talk between muscle and cartilage mediated by irisin

Knee osteoarthritis (KOA) diagnosis is based on symptoms, assessed through questionnaires such as the WOMAC. However, the inconsistency of pain recording and the discrepancy between joint phenotype and symptoms highlight the need for objective biomarkers in KOA diagnosis. To this end, we study relationships among clinical and molecular data in a cohort of women (n=51) with Kellgren-Lawrence grade 2–3 KOA through Support Vector Machine (SVM) and a regulation network model (RNM). Clinical descriptors (i.e., pain catastrophism (CA); depression (DE); functionality (FU); joint pain (JP); rigidity (RI); sensitization (SE); synovitis (SY)) are used to classify patients. A Youden's test is performed for each classifier to determine optimal binarization thresholds for the descriptors. Thresholds are tested against patient stratification according to baseline WOMAC data from the Osteoarthritis Initiative, and the mean accuracy is 0.97. For our cohort, the data used as SVM inputs are KOA descriptors, synovial fluid (SL) proteomic measurements (n=25), and transcription factors (TF) activation obtained from RNM [2] stimulated with the SL measurements. The relative weights after classification reflect input importance. The performance of each classifier is evaluated through AUC-ROC analysis. The best classifier with clinical data is CA (AUC = 0.9), highly influenced by FU and SE, suggesting that kinesophobia is involved in pain perception. With SL input, leptin strongly influences every classifier, suggesting the importance of low-grade inflammation. When TF are used, the mean AUC is limited to 0.608, which can be related to the pleomorphic behaviour of osteoarthritic chondrocytes. Nevertheless, FU has an AUC of 0.7 with strong importance of FOXO downregulation. Though larger and longitudinal cohorts are needed, this unique combination of SVM and RNM shall help to map objectively KOA descriptors. Acknowledgements: Catalan & Spanish governments 2020FI_b00680; STRATO-PID2021126469ob-C21-2, European Commission (MSCA-TN-ETN-2020-Disc4All-955735, ERC-2021-CoG-O-Health-101044828). ICREA Academia

While cell morphology has been recognized as a fundamental regulator of cell behavior, few studies have measured the complex cell morphological changes of chondrocytes using quantitative cell morphometry descriptors in relation to inflammation and phenotypic outcome. Acute vs. persistent exposure to IL-1β and how IL-1β modulated dynamic changes in cell morphology in relation to the phenotype, donor and OA grade in healthy and osteoarthritis (OA) chondrocytes was investigated. A panel of quantitative cell morphometry descriptors was measured using an automated high-throughput method. Absolute quantification of gene expression was measured by ddPCR followed by correlation analyses. In OA chondrocytes, chronic IL-1β significantly decreased COL2A1, SOX9, and ACAN, increased IL-6 and IL-8 levels and caused chondrocytes to become less wide, smaller, longer, slimmer, less round and more circular, consistent with a de-differentiated phenotype. In healthy chondrocytes, 3 days after acute (72 h) IL-1β exposure, COL1A2 and IL-6 significantly increased but had minor effects on cell morphology. However, in healthy chondrocytes, persistent IL-1β led to more profound effects in all cell morphology descriptors and chondrocytes expressed significantly less COL2A1 and more IL-6 and IL-8 vs. controls and acutely-stimulated chondrocytes. In both OA and healthy chronically-stimulated chondrocytes, area, width and circularity were sensitive to the persistent presence of the IL-1β cytokine. Moreover, there were many significant and strong correlations among the measured parameters, with several indications of an IL-1β-mediated mechanism. Cell morphology combined with gene expression analysis could guide researchers interested in understanding inflammatory effects in the complex domain of cartilage/chondrocyte biology. Use of quantitative cell morphometry could complement classical approaches by providing numerical data on a large number of cells, thereby providing a biological fingerprint for describing chondrocyte phenotype, which could help to understand how changes in cell morphology lead to disease progression

Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), a potential mechanism of MSC therapies has been attributed to paracrine activity, in which extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor compete with adult MSC in their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSC) appear to provide a promising source. In this study, MSCs and iMSC during long term-expansion using a serum free clinical grade condition, were characterized for surface expression pattern, proliferation and differentiation capacity, and senescence rate. Culture media were collected continuously during cell expansion, and EVs were isolated. Nanoparticle tracking analysis (NTA), transmission electron microscopy, western blots, and flow cytometry were used to identify EVs. We evaluated the biological effects of MSC and iMSC-derived EVs on human chondrocytes treated with IL-1α, to mimic the OA environment. In both cell types, from early to late passages, the amount of EVs detected by NTA increased significantly, EVs collected during cells expansion, retained tetraspanins (CD9, CD63 and CD81) expression. The anti-inflammatory activity of MSC-EVs was evaluated in vitro using OA chondrocytes, the expression of IL-6, IL-8 and COX-2 was significantly reduced after the treatment with hMSC-derived EVs isolated at early passage. The miRNA content of EVs was also investigated, we identify miRNA that are involved in specific biological function. At the same time, we defined the best culture conditions to maintain iMSC and define the best time window in which to isolate EVs with highest biological activity. In conclusion, a clinical grade serum-free medium was found to be suitable for the isolation and expansion of MSCs and iMSC with increased EVs production for therapeutic applications. Acknowledgments: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671

The signaling molecule prostaglandin E2 (PGE2), synthesized by cyclooxygenase-2 (COX-2), is immunoregulatory and reported to be essential for skeletal stem cell function. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in osteoarthritis (OA) analgesia, but cohort studies suggested that long-term use may accelerate pathology. Interestingly, OA chondrocytes secrete high amounts of PGE2. Mesenchymal stromal cell (MSC) chondrogenesis is an in vitro OA model that phenocopies PGE2 secretion along with a hypertrophic OA-like cell morphology. Our aim was to investigate cause and effects of PGE2 secretion in MSC-based cartilage neogenesis and hypertrophy and identify molecular mechanisms responsible for adverse effects in OA analgesia. Human bone marrow-derived MSCs were cultured in chondrogenic medium with TGFβ (10ng/mL) and treated with PGE2 (1µM), celecoxib (COX-2 inhibitor; 0.5µM), AH23848/AH6809 (PGE2 receptor antagonists; 10µM), or DMSO as a control (n=3–4). Assessment criteria were proteoglycan deposition (histology), chondrocyte/hypertrophy marker expression (qPCR), and ALP activity. PGE2 secretion was measured (ELISA) after TGFβ withdrawal (from day 21, n=2) or WNT inhibition (2µM IWP-2 from day 14; n=3). Strong decrease in PGE2 secretion upon TGFβ deprivation or WNT inhibition identified both pathways as PGE2 drivers. Homogeneous proteoglycan deposition and COL2A1 expression analysis showed that MSC chondrogenesis was not compromised by any treatment. Importantly, hypertrophy markers (COL10A1, ALPL, SPP1, IBSP) were significantly reduced by PGE2 treatment, but increased by all inhibitors. Additionally, PGE2 significantly decreased ALP activity (2.9-fold), whereas the inhibitors caused a significant increase (1.3-fold, 1.7-fold, 1.8-fold). This identified PGE2 as an important inhibitor of chondrocyte hypertrophy. Although TGFβ and WNT are known pro-arthritic signaling pathways, they appear to induce a PGE2-mediated antihypertrophic effect that can counteract pathological cell changes in chondrocytes. Hampering this rescue mechanism via COX inhibition using NSAIDs thus risks acceleration of OA progression, indicating the need of OA analgesia adjustment

Pericytes are contractile, motile cells that surround the capillary. Recent studies have shown that pericytes promoted joint fibrosis and induced subchondral bone angiogenesis, indicating the role of pericytes in osteoarthritis (OA). However, whether pericytes are involved in regulating inflammatory and catabolic response, as well as fibrotic repair of cartilage is still unclear. Here we used 2D and 3D models to investigate the communication of pericytes and chondrocytes under inflammatory osteoarthritis conditions. CD34-CD146+ pericytes were isolated and sorted from human bone marrow. Human OA chondrocytes were isolated from OA joints. In 2D studies, monolayer cultured chondrocytes were treated +/- pericyte conditioned media, +/- 1ng/ml IL1β for 24h. In 3D studies, pericytes and chondrocytes were cultured within fibrin gel in 3D polyurethane scaffolds, separately or combined for 7 days, followed by treatment of +/- IL1β for another 7 days (Fig 2A). The inflammatory response, catabolic activity and expression of fibrosis markers of chondrocytes and pericytes were measured by ELISA and/or q-rtPCR. Pericytes had weak inflammatory, catabolic and fibrotic response to IL1β (data not shown). The 2D study showed that pericyte conditioned media promoted inflammation, catabolism and fibrosis markers of chondrocytes, in the absence of IL1β treatment (Figure 1). However, study in 3D showed that coculture of chondrocytes and pericytes reduced the inflammatory and catabolic response of chondrocytes to IL1β and induced fibrosis markers in chondrocytes (Figure 2). Pericytes are involved in regulating inflammatory response, catabolic response and fibrosis of chondrocytes. The opposite results from 2D and 3D experiments indicate the variety of the regulatory role of pericytes in the interaction with chondrocytes within different culture models. The underlying mechanism is under evaluation with on-going studies. Acknowledgements. This study was funded by SINPAIN project, from European Union's Horizon Europe research and innovation programme under Grant Agreement NO. 101057778. Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them. For any figures or tables, please contact the authors directly

The aim of the study was to investigate the expression of genes regulating cholesterol efflux in human chondrocytes and to study the effect of an LXR agonist on cholesterol efflux and lipid accumulation in osteoarthritic chondrocytes. Human cartilage was obtained from 24 patients with primary osteoarthritis (OA) undergoing total knee replacement surgery. Normal cartilage was obtained from 8 individuals undergoing fracture repair surgery, with no history of joint disease. ATP-binding-cassette transporter A1(ABCA1), apolipoprotein A1 (ApoA1), and liver X receptors(LXRα and LXRβ) mRNA expression levels were evaluated using real-time PCR. The effect of the synthetic LXR agonist TO-901317 was studied after treatment of osteoarthritic chondrocytes and subsequent investigation of ABCA1 and ApoA1 mRNA expression levels. Cholesterol efflux was evaluated in osteoarthritic chondrocytes radiolabeled with [1,2(n)-3H] cholesterol after LXR treatment, while intracellular lipid accumulation was studied after Oil-red-O staining. Apoptosis was evaluated using flow cytometry. ApoA1, ABCA1, LXRα and LXRβ mRNA expressions were significantly lower in osteoarthritic chondrocytes compared to normal. Treatment of osteoarthritic chondrocytes with the LXR agonist TO-901317 significantly increased ApoA1 and ABCA1 mRNA expression levels as well as cholesterol efflux, while it significantly reduced apoptosis. Additionally, osteoarthritic chondrocytes presented intracellular lipids deposits, while no deposits were found after treatment with TO-901317. Our findings suggest that impaired expression of genes regulating cholesterol efflux may be a critical player in osteoarthritis, while the ability of the LXR agonist to facilitate cholesterol efflux and decrease apoptosis suggests that it may be a target for therapeutic intervention in osteoarthritis

Osteoarthritis is a complex joint disease in which all involved tissues play an important role in its onset and progression. It has been suggested that osteoarthritis is likely to be a systemic disease involving stromal cell differentiation and lipid metabolism while altered lipid metabolism has been implicated as a critical player in its pathogenesis. As excessive accumulation of free cholesterol is toxic for the cells, the accumulation of lipids in chondrocytes may signify a causal relationship to development and/or progression of osteoarthritis; therefore we investigated the expression of genes regulating reverse cholesterol transport, as ABCA1, ApoA1, LXRa, LXR_, in human osteoarthritic chondrocytes. We also investigated the effect of an LXR agonist on ABCA1 and ApoA1 expression and, for the first time, on cholesterol effiux and lipid accumulation in osteoarthritic chondrocytes. Articular cartilage samples were obtained from femoral condyles and tibial plateaus of patients with primary OA undergoing knee replacement surgery while normal cartilage was obtained from eight individuals undergoing fracture repair surgery, with no history of joint disease. Total cellular RNA was extracted from all samples and ABCA1, ApoA1, and LXRα and LXRβ mRNA and protein expression levels were evaluated using real-time PCR and Western blot analysis respectively. The effect of the synthetic LXR agonist TO-901317 was studied after treatment of osteoarthritic chondrocytes and subsequent investigation of ABCA1 and ApoA1 mRNA expression levels. Cholesterol effiux was evaluated in osteoarthritic chondrocytes radiolabeled with [1,2(n)-3H] cholesterol after LXR treatment, while intracellular lipid accumulation was studied after Oil-red-O staining. ApoA1 and ABCA1 mRNA levels were significantly lower in osteoarthritic cartilage compared to normal (p< 0.01 and p< 0.001 respectively). In addition, the two subtypes of the LXR, namely LXRα and LXRβ, mRNA levels were also found to be significantly lower in osteoarthritic cartilage (p< 0.05 and p< 0.01 respectively). The differential expression pattern of the cholesterol effiux genes between normal and osteoarthritic cartilage remained the same at the protein level as well. Treatment of osteoarthritic chondrocytes with the LXR agonist TO-901317 significantly increased ApoA1 and ABCA1 expression levels, as well as cholesterol effiux. Additionally, osteoarthritic chondrocytes presented intracellular lipids deposits, while no deposits were found after treatment with TO-901317. Our findings suggest that impaired expression of genes regulating cholesterol effiux may be a critical player in osteoarthritis, while the ability of the LXR agonist to facilitate cholesterol effiux suggests that it may be a target for therapeutic intervention in osteoarthritis