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

The development of a representative human, in vitro OA model could deepen understanding of disease mechanisms. Our research aimed to reprogram healthy and OA-derived synoviocytes to induced pluripotent stem cells (iPSCs), thereby generating a novel OA in vitro model. Comparison between the two models shall enable research into underlying processes with potential for clinical translation. A meta-analysis of OA synovial biomarkers was conducted, identifying up to thirteen relevant pathophysiology-related factors, including, amongst others, IL-13, IL-10, IL-6, PIICP, and HA, with PIICP demonstrating the largest effect (SMD 6.11 [3.50, 8.72], p <0.00001). With these findings in mind, human fibroblast-like synoviocytes (HFLS) from healthy and OA patients were transduced using Sendai viral reprogramming. Two clones for each of the resulting iPSC lines were expanded and preliminarily analysed in triplicate by ICC and RT-qPCR for pluripotency characteristics. Healthy HFLS-derived and OA-HFLS-derived iPSC (UoS-B and UoS-C lines, respectively) were generated, indicating successful reprogramming. Morphological observations demonstrated typical iPSC appearance, and ICC confirmed presence of pluripotency markers Tra-1-60, Oct3/4 and Nanog. Expression of Oct3/4, Nanog and Sox2 were confirmed by RT-qPCR with OA-iPSC lines expressing higher levels of all markers compared to non-OA iPSC. In particular, expression of Oct3/4 and Sox2 was 3.5 fold and 4.6 fold higher (p <0.001) in OA-iPSCs (UoS-C) vs. non-OA iPSCs (UoS-B), respectively. Sendai virus clearance was confirmed by passage 4. The successfully obtained OA and non-OA iPSCs can be differentiated towards mesenchymal lineages, including chondrocyte and bone progenitor cells, enabling phenotypic comparison and biomarker analysis as identified in meta-analysis. Cell bank dissemination of these cell lines could deepen further in vitro OA research, with potential impact for clinical translation via the identification of novel cellular and molecular targets

Osteoarthritis (OA) is the most common degenerative joint disease causing joint immobility and chronic pain. Treatment is mainly based on alleviating pain and reducing disease progression. During OA progression the chondrocyte undergoes a hypertrophic switch in which extracellular matrix (ECM) -degrading enzymes are released, actively degrading the ECM. However, cell biological based therapies to slow down or reverse this katabolic phenotype are still to be developed. Bone morphogenetic protein 7 (BMP-7) has been shown to have OA disease-modifying properties. BMP-7 suppresses the chondrocyte hypertrophic and katabolic phenotype and may be the first biological treatment to target the chondrocyte phenotype in OA. However, intra-articular use of BMP-7 is at risk in the proteolytic and hydrolytic joint-environment. Weekly intra-articular injections are necessary to maintain biological activity, a frequency unacceptable for clinical use. Additionally, production of GMP-grade BMP-7 is challenging and expensive. To enable its clinical use, we sought for BMP-7 mimicking peptides better compatible with the joint-environment while still biologically active and which potentially can be incorporated in a drug-delivery system. We hypothesized that human BMP-7 derived peptides are able to mimic the disease modifying properties of the full-length human BMP-7 protein on the OA chondrocyte phenotype. A BMP-7 peptide library was synthesized consisting of overlapping 20-mer peptides with 18 amino-acids overlap between sequential peptides. OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty (n=18 donors). HACs were exposed to BMP-7 (1 nM) or BMP-7 library peptides at different concentrations (1, 10, 100 or 1000 nM). Gene-expression levels of important chondrogenic-, hypertrophic-, cartilage degrading- and inflammatory mediators were determined by RT-qPCR. GAG and ALP activity were determined using a colorimetric assay and PGE levels were measured by EIA. During the BMP-7 peptide library screening human BMP-7 derived peptides were screened for their full-length human BMP-7 mimicking properties at different concentrations (1, 10, 100 or 1000nM) on a pool of human chondrocytes. Gene expression as well as GAG, ALP and PGE2 level analysis revealed two distinct peptide regions in the BMP-7 protein based on their pro-chondrogenic and anti-OA phenotype actions on human OA chondrocytes. The two most promising peptides were further analysed for their OA chondrocyte disease modifying properties in the presence of OA synovial fluid, showing similar OA phenotype suppressive activity. Conclusively, we successfully identified two peptide regions in the BMP-7 protein with in vitro OA suppressive actions. Further biochemical fine-tuning of the peptides, and in vivo evaluation, will potentially result in the first peptide-based experimental OA treatment, addressing the hypertrophic and katabolic chondrocyte phenotype in OA

Introduction. Osteoarthritis (OA) involves pathological change in all joint tissues, including cartilage degradation and synovitis. Synovial inflammation is significantly associated with pain severity and incidence in knee OA. It is becoming evident that synovitis also plays an active role in the initiation and progression of cartilage erosion in OA, through direct secretion of catabolic enzymes as well as factors that stimulate chondrocyte catabolic activity. Therapeutic agents that target both synovitis and cartilage pathology are likely to be maximally beneficial in treating pain and slowing cartilage breakdown in OA. We have previously shown that an amide-derivative of HA (HYMOVIS™) was superior to native HA of the same MW in improving gait, and reducing synovial hyperplasia in a sheep OA model. In the present study the mechanisms whereby the chemically modified HA may be beneficial were examined using chondrocytes and synovial fibroblasts from knees of OA patients. Patients & Methods. Chondrocytes (HAC, n=6) and synovial fibroblasts (HSF, n=6) were isolated from OA patients at the time of knee replacement. HYMOVIS™ (0, 0.5, 1.0 or 1.5mg/mL) was added to simultaneously or 1 hour before interleukin-1β (IL1, 2ng/mL). Cultures were terminated 30 minutes later for Bioplex. ®. quantitation of p-JNK, p-NFκB and p-p38; or 24 hours later for RNA isolation and analysis of gene expression by real time RT-PCR, and measurement of MMP13 activity in the media. Only statistically significant results are reported. Results. In HAC in the absence of IL1, HYMOVIS™ decreased MMP13, ADAMTS5, PTGS2 and IL6 and increased COL2A1 mRNA (2–10fold). In HSF in absence of IL1, HYMOVIS™ decreased TIMP1, TIMP3, CD44, IL6 and increased PTGS2 (2–3fold). In HAC and HSF, IL1 increased expression of MMP1, MMP13, PTGS2, IL6 (>100fold), ADAMTS4 (∼10 fold), all phosphoproteins (3–10fold), and APMA-activated MMP13 activity in media. IL1 increased expression of ADAMTS5 (∼10fold) only in HSF. As expected, IL1 reduced expression of the key matrix proteins in HAC (2–3 fold decrease in COL2A1 and ACAN) and HSF (2 fold decrease in COL1A1). When added simultaneously with IL1, HYMOVIS™ decreased expression of MMP13, ADAMTS5, PTGS2, IL6 expression, and normalised matrix protein expression in both HAC and HAS. Pre-incubation with HYMOVIS™ for 1 hour inhibited IL1-stimulated p-JNK, p-NFκB and p-p38 in both cell types (excluding p-JNK in HSF). In HAC, HYMOVIS™ pre-incubation was superior to simultaneous addition in reducing expression of MMP1, MMP13, ADAMTS4, PTGS2, and IL6 expression. There was a less dramatic effect of HYMOVIS™ pre-incubation on gene expression in HSF compared with HAC. The inhibitory effects of HYMOVIS™ on IL1 stimulated gene expression in HAC and HSF was partially ameliorated by pre-incubation with a CD-44 blocking antibody. Discussion/Conclusions. The present studies have demonstrated several potential key mechanisms whereby the intra-articular injection of a hexadecylamide-derivative of HA (HYMOVIS™) may have both symptom and disease-modifying effects in OA. The previously described increased joint retention of the hexadecylamide-derivative, might act in a similar manner to the pre-incubation studies in our cell culture studies, to reduce the initiation of degradative events with recurrent/cyclic inflammatory episodes that typify OA

Introduction. Osteoarthritis (OA) is a progressively debilitating disease that affects mostly cartilage, with associated changes in the bone. The increasing incidence of OA and an ageing population, coupled with insufficient therapeutic choices, has led to focus on the potential of stem cells as a novel strategy for cartilage repair. Methods. In this study, we used scaffold-free mesenchymal stem cells (MSCs) obtained from bone marrow in an experimental animal model of OA by direct intra-articular injection. MSCs were isolated from 2.8 kg white New Zealand rabbits. There were ten in the study group and ten in the control group. OA was induced by unilateral transection of the anterior cruciate ligament of the knee joint. At 12 weeks post-operatively, a single dose of 1 million cells suspended in 1 ml of medium was delivered to the injured knee by direct intra-articular injection. The control group received 1 ml of medium without cells. The knees were examined at 16 and 20 weeks following surgery. Repair was investigated radiologically, grossly and histologically using haematoxylin and eosin, Safranin-O and toluidine blue staining. Results. Radiological assessment confirmed development of OA changes after 12 weeks. Rabbits receiving MSCs showed a lower degree of cartilage degeneration, osteophyte formation, and subchondral sclerosis than the control group at 20 weeks post-operatively. The quality of cartilage was significantly better in the cell-treated group compared with the control group after 20 weeks. Conclusions. Bone marrow-derived MSCs could be promising cell sources for the treatment of OA. Neither stem cell culture nor scaffolds are absolutely necessary for a favourable outcome. Cite this article: Bone Joint Res 2014;3:32–7

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

TGF-β/Smad2 signaling is considered to be one of the important pathways involved in osteoarthritis (OA) and protein phosphatase magnesium-dependent 1A (PPM1A) functions as an exclusive phosphatase of Smad2 and regulates TGF-β signaling, here, we investigated the functional role of PPM1A in OA pathogenesis. PPM1A expressions in both human OA cartilage and experimental OA mice chondrocytes were analyzed immunohistochemically. Besides, the mRNA and protein expression of PPM1A induced by IL-1β treatment were also detected by q-PCR and immunofluorescence in vitro. OA was induced in PPM1A knockout (KO) mice by destabilization of the medial meniscus (DMM), and histopathological examination was performed. OA was also induced in wild-type (WT) mice, which were then treated with an intra-articular injection of a selective PPM1A inhibitor for 8 weeks. PPM1A protein expressions were increased in both human OA cartilage and experimental OA mice chondrocytes. We also found that treatment with IL-1β in mouse primary chondrocytes significantly increased both mRNA and protein expression of PPM1A in vitro. Importantly, our data showed that PPM1A deletion could substantially protect against surgically induced OA. Concretely, the average OARSI score and quantification of BV/TV of subchondral bone in KO mice were significantly lower than that in WT mice 8 weeks after DMM surgery. Besides, TUNEL staining revealed a significant decrease in apoptotic chondrocytes in PPM1A-KO mice with DMM operation. With OA induction, the rates of chondrocytes positive for Mmp-13 and Adamts-5 in KO mice were also significantly lower than those in WT mice. Moreover, compared with WT mice, the phosphorylation of Smad2 in chondrocytes was increased in KO mice underwent DMM surgery. However, articular-injection with SD-208, a selective inhibitor of TGF-β/Smad2 signaling could significantly abolish the chondroprotective phenotypes in PPM1A-KO mice. Additionally, both cartilage degeneration and subchondral bone subchondral bone sclerosis in DMM model were blunted following intra-articular injection with BC-21, a small-molecule inhibitor for PPM1A. Our study demonstrated that PPM1A inhibition attenuates OA by regulating TGF-β/Smad2 signaling. Furthermore, PPM1A is a potential target for OA treatment and BC-21 may be employed as alternative therapeutic agents for the management of OA

Establishing disease biomarkers has been a long-sought after goal to improve Osteoarthritis (OA) diagnosis, prognosis, clinical and pharmaceutical interventions. Given the role of the synovium in contributing to OA, a meta-analysis was performed to determine significant synovial biomarkers in human OA tissue, compared to non-OA patients. Outcomes will direct future research on marker panels for OA disease modelling in vitro/in vivo, aiding clinical research into OA disease targets. A PRISMA compliant search of databases was performed to identify potential biomarker studies analysing human, OA, synovial samples compared to non-OA/healthy participants. The Risk of Bias In Non-Randomised Studies of Interventions (ROBINS-I) tool assessed methodological quality, with outcome analysed by Grading of Recommendations Assessment, Development and Evaluation (GRADE). Meta-analyses were conducted for individual biomarkers using fixed or random effect models, as appropriate. Where three or more studies included a specific biomarker, Forest Plot comparisons were generated. 3230 studies were screened, resulting in 34 studies encompassing 25 potential biomarkers (1581 OA patients and 695 controls). Significant outcomes were identified for thirteen comparisons. Eleven favoured OA (IL-6, IL-10, IL-13, IP-10, IL-8, CCL4, CCL5, PIICP, TIMP1, Leptin and VEGF), two favoured non-OA controls (BMP-2 and HA). Notably, PIICP showed the largest effect (SMD 6.11 [3.50, 8.72], p <0.00001, I. 2. 99%), and TIMP1 resulted critically important (0.95 [0.65, 1.25], p <0.00001, I. 2. 82%). Leptin and CCL4 showed lower effects (SMD 0.81 [0.33, 1.28], p =0.0009; 0.59 [0.32, 0.86], p <0.0001, respectively). Thirteen significant synovial biomarkers showed links with OA bioprocesses including collagen turnover, inflammatory mediators and ECM components. Limitations arose due to bias risk from incomplete or missing data, publication bias of inconclusive results, and confounding factors from patient criteria. These findings suggest markers of potential clinical viability for OA diagnosis and prognosis that could be correlated with specific disease stages

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 common degenerative joint disorder. Its multifactorial etiology includes age, sex, joint overloading, genetic or nervous influences. In particular, the autonomic nervous system is increasingly gaining in importance. Its two branches, the sympathetic (SNS) and parasympathetic nervous system, are well-balanced under healthy conditions. OA patients seem to be prone to an autonomic imbalance and therefore, we analyzed their autonomic status. More than 200 participants including patients with early and late stage knee OA (before and 1 year after knee replacement surgery) and healthy probands (age-matched) were analyzed. Heart rate variability was measured via electrocardiogram to assess long-term sympathetic (low-frequency=LF) and parasympathetic (high-frequency=HF, pRR50) activities or general variability (RMSSD, SDRR). Serum cortisol concentrations were measured by ELISA. Perceived chronic stress (PSQ) was assessed via questionnaire. Multivariant regression was performed for data analysis. LF/HF value of early OA was slightly increased compared to healthy controls but significantly higher compared to late OA patients before (p>0.05) and after TKR (p>0.01). HF in late OA patients before TKR was significantly decreased compared to patients after TKR (p>0.001) or healthy controls (p>0.05). Healthy probands exhibited the highest SDRR values, early OA patients had slightly lower levels and late OA patients before TKR displayed significantly reduced SDRR (p>0.001). The same differences were observed in pRR50 and RMSSD. Serum cortisol concentrations and PSQ scores increased in late OA patients before TKR. At the time point of TKR, women with beta blocker medication had significantly higher age (71 ± 9 years) than those without (63 ± 12 years)(p>0.01). An autonomic dysfunction with sympathetic dominance occurs in OA patients. The fact that beta blocker medication in women delayed the need of TKR indicates that SNS inhibition might counteract OA. Future therapeutic interventions for OA should consider a systemic approach with special regard on the ANS

Osteoarthritis (OA) is the most common disorder of the Sternoclavicular Joint (SCJ). In our case-control study, we evaluated the relationship between clavicular length and OA at the SCJ. CT scans of adults presenting to the Emergency Department of our hospital were examined to look for OA, defined as the presence of osteophytes, subchondral cysts, or cortical sclerosis at the SCJ. Medial-most and lateral-most points of the clavicle were marked on the slices passing through the SC and AC joints respectively. Using x, y, and z-axis coordinates from the DICOM metadata, clavicular length was calculated as the distance between these two points with 3D geometry. Preliminary data of 334 SCJs from 167 patients (64% males, 36% females) with a mean age of 48.5 ± 20.5 years were analysed. Multivariate regression models revealed that age and clavicular length were independent risk factors for OA while gender did not reach statistical significance. A 1mm increase in length was associated with 9% and 7% reduction in the odds of developing OA on the left and the right respectively. Comparing the mean clavicular length using t-test showed a significantly shorter clavicle in the group with OA (145.8 vs 152.7, p=0.0001, left and 144.2 vs 150.3, p=0.0007, right). Our data suggest that the risk of developing OA at the SCJ is higher for shorter clavicles. This could be of clinical relevance in cases of clavicular fracture where clavicular shortening might lead to a higher risk of developing OA. Biomechanical studies are needed to find out the mechanism of this effect

Osteoarthritis (OA), the most prevalent chronic joint disease, represents a relevant social and economic burden worldwide. Human umbilical cord mesenchymal stem cells (HUCMSCs) have been used for injection into the joint cavity to treat OA. The aim of this article is to clarify whether Huc-MSCs derived exosomes could inhibit the progression of OA and the mechanism in this process. A rabbit OA model was established by the transection of the anterior cruciate ligament. The effects of HUCMSCs or exosomes derived from HUCMSCs on repairing articular cartilage of knee osteoarthritis was examined by micro-CT. Immunohistochemical experiments were used to confirm the expression of relevant inflammatory molecules in OA. In vitro experiments, Transwell assay was used to assess the migration of macrophages induced by TNF-a. Results showed that a large number of macrophages migrated in arthcular cavity in OA model in vivo, while local injection of HUCMSCs and exosomes did repair the articular cartilage. Immunohistochemical results suggested that the expression of CCL2 and CD68 in the OA rabbit model increased significantly, but was significantly reduced by HUCMSCs or exosomes. Transwell assay showed that both HUCMSCs and exosomes can effectively inhibit the migration of macrophage. In conclusion, the exosomes derived by HUCMSCs might might rescue cartilage defects in rabbit through its anti-inflammatory effects through inhibiting CCL2

Osteoarthritis (OA) of the equine distal interphalangeal joint (DIPJ) is a common cause of lameness. MicroRNAs (miRNAs) from biofluids such as plasma and synovial fluid make promising biomarker and therapeutic candidates. The objectives of this study are (1) Identify differentially expressed (DE) miRNAs in mild and severe equine DIPJ OA synovial fluid samples and (2) Determine the effects of DE miRNAs on equine chondrocytes in monolayer culture. Synovial fluid samples from five horses with mild and twelve horses with severe DIPJ OA were submitted for RNA-sequencing; OA diagnosis was made from MRI T2 mapping, macroscopic and histological evaluation. Transfection of equine chondrocytes (n=3) was performed using the Lipofectamine® RNAiMAX system with a negative control and a miR-92a mimic and inhibitor. qPCR was used to quantify target mRNA genes. RNA-seq showed two miRNAs (miR-16 and miR-92a) were significantly DE (p<0.05). Ingenuity Pathway Analysis (IPA) identified important downstream targets of miR-92a involved in the pathogenesis of osteoarthritis and so this miRNA was used to transfect equine chondrocytes from three donor horses diagnosed with OA. Transfection was successfully demonstrated by a 1000-20000 fold increase in miR-92a expression in the equine chondrocytes. There was a significant (p<0.05) increase in COMP, COL3A1 and Sox9 in the miR-92a mimic treatment and there was no difference in ADAMTS-5 expression between the miR-92 mimic and inhibitor treatment. RNA-seq demonstrated miR-92a was downregulated in severe OA synovial fluid samples which has not previously been reported in horses, however miR-92a is known to play a role in the pathogenesis of OA in other species. Over expression of miR-92a in equine chondrocytes led to significantly increased COMP and Sox9 expression, consistent with a chondrogenic phenotype which has been identified in human and murine chondrocytes

Osteoarthritis (OA) is a degenerative disease that lacks regenerative treatment options. Current research focuses on mesenchymal stem cells (MSCs) and Platelet-Rich Plasma (PRP) as regenerative therapies, but extracellular vesicles (EVs) have shown to be more advantageous. This study compares the regenerative potential of human umbilical cord MSC-derived EVs (cEVs) and platelet-derived EVs (pEVs) in ex vivo and in vivo OA models. In the ex vivo study, OA conditions were induced in human cartilage explants, which were then treated either with pEVs or cEVs. Results showed a higher content of DNA and collagen in the pEVs group compared to control and cEVs groups, suggesting that pEVs could be a potential alternative to cEVs. In the in vivo study, an OA model was established in the knee joints of rats through MIA (monoiodoacetate) injection and then treated either with pEVs or cEVs. Results showed that pEVs-treated knee joints had better subchondral bone integrity and greater OA reversion, particularly in female rats, indicating that pEVs are a viable regeneration treatment for OA and outperform cEVs in terms of efficacy. Overall, the study demonstrates the potential of EVs as a regenerative treatment for OA, with pEVs showing promising results in both ex vivo and in vivo models. The use of pEVs in clinical practice could provide a faster path to translation due to the established use of platelet concentrates in therapeutics. However, further studies are needed to fully evaluate the potential of pEVs for OA treatment and to elucidate the mechanisms behind their regenerative effects. Acknowledgments: The authors thank Dr Fernando Hierro (UIB) for their technical contribution with TEM, Mª Trinidad García (UIB) for the access to radioactivity facilities, Aina Arbós (IUNICS) for her contribution in the histology staining, María Tortosa (IdISBa) for her assistance with the animal care and ADEMA School of Dentistry for the access to the cone beam computed tomography (CBCT). Funding: This research was funded by Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (MS16/00124; CP16/00124), PROGRAMA JUNIOR del proyecto TALENT PLUS, construyendo SALUD, generando VALOR (JUNIOR01/18), financed by the sustainable tourism tax of the Balearic Islands; the Direcció General d'Investigació and Conselleria d'Investigació, Govern Balear (FPI/2046/2017); the Mecanisme de Recuperació i Resiliència, intended to execute research projects of «Noves polítiques públiques per a un mercat de treball dinàmic, resilient i inclusiu», collected in Pla de Recuperació, Transformació i Resiliència, financed by European Union-Next Generation EU and driven by SOIB and Conselleria de Fons Europeus, Universitat i Cultura i la Conselleria de Model Econòmic, Turisme i Treball (NG0421) and the grant SYN20/03 from IdISBa

Osteoarthritis (OA) affects the whole joint and leads to chronic pain. The sympathetic nervous system (SNS) seems to be involved in OA pathogenesis, as indicated by in vitro studies as well as by our latest work demonstrating that sympathectomy in mice results in increased subchondral bone volume in the OA knee joint. We assume that chronic stress may lead to opposite effects, such as an increased bone loss in OA due to an elevated sympathetic tone. Therefore, we analyzed experimental OA progression in mice exposed to chronic stress. OA was induced in male C57BL/6J mice by surgical destabilization of the medial meniscus (DMM) and Sham as well as non-operated mice served as controls. Half of these groups were exposed to chronic unpredictable mild stress (CUMS). After 12 weeks, chronic stress efficiency was assessed using behavioral tests. In addition to measuring body weight and length, changes in subchondral bone were analyzed by μCT. Dynamic Weight Bearing system was used to monitor OA-related pain. Histological scoring will be conducted to investigate the severity cartilage degeneration and synovial inflammation. CUMS resulted in increased anxiety and significant decrease in body weight gain in all CUMS groups compared to non-CUMS groups. CUMS also increased serum corticosterone in healthy mice, with even higher levels in CUMS mice after DMM surgery. CUMS had no significant effect on subchondral bone, but subarticular bone mineral density and trabecular thickness were increased. Moreover, CUMS resulted in significant potentiation of DMM-associated pain. Our results suggest that the autonomic imbalance with increased sympathetic nervous activity induced by chronic stress exacerbates the severity of OA pain perception. We expect significantly increased cartilage degeneration as well as more severe synovial inflammation in CUMS DMM mice compared to DMM mice

Joint tissues release extracellular vesicles (EVs) that potentially sustain joint homeostasis and contribute to osteoarthritis (OA) pathogenesis. EVs are putative novel therapeutics for OA, and transport biologically active molecules (including small non-coding RNAs (SNCRNAs)) between cells. This study identified altering SNCRNA cargo in EVs in OA which may act as early diagnostic markers and treatment targets. OA was surgically induced in four skeletally mature Standardbred horses using an osteochondral fragment model in the left middle carpal joint. The right joint underwent sham surgery. Synovial fluid (SF) and plasma were obtained weekly throughout the 70-day study. EVs were isolated using size exclusion chromatography and characterised using nanoparticle tracking (Nanosight), and exosome fluorescence detection and tetraspanin phenotyping (Exoview). RNA was extracted from EVs derived from SF (sham and OA joints) and plasma collected at days 10, 35, 42, 49, 56, 63, and subjected to small RNA sequencing on a NovaSeq SP100 flow cell (Illumina). Nanosight-derived EV characteristics of size and concentration were not significantly different following disease induction. The diameter of the temporal population of plasma and SF-derived exosomes changed significantly for CD9 and CD81 following OA induction with significant temporal, and disease-related changes in CD63 and CD81 protein expressin in plasma and SF. In SF and plasma-derived EVs snoRNAs, snRNAs, tRNAs, lncRNA, y-RNA, piRNAs and scRNA were found. Following pairwise analysis of all-time points we identified 27 miRs DE in plasma and 45 DE miRs in SF. Seven were DE in plasma and SF; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a. In plasma and SF 35 and 21 snoRNAs were DE with four DE in plasma and SF; U3, snord15, snord46, snord58. This work has identified alterations to OA EV sncRNAs in plasma and SF providing a greater understanding of the role of EVs in early OA

Abstract. Cranial cruciate ligament (CrCL) disease/rupture is a highly prevalent orthopaedic disease in dogs and common cause of pain, lameness, and secondary joint osteoarthritis (OA). Previous experiments investigating the role of glutamate receptors (GluR) in arthritic degeneration and pain revealed that OA biomarkers assessing early bone turnover and inflammation, including osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa-B ligand (RANKL) are more likely to be influenced by glutamate signalling. Moreover, interleukin-6 (IL-6) has a complex and potentially bi directional (beneficial and detrimental) effect, and it is a critical mediator of arthritic pain, OA progression and joint destruction. Objectives. 1) to recruit dogs undergoing CrCL disease/rupture surgery and obtain discarded synovial fluid (SF) and serum/plasma (ethics approval, RCVS:2017/14/Alves); 2) to quantify the biomarkers listed above in the SF and serum/plasma by enzyme linked immunosorbent assay (ELISA); 3) to assess radiographic OA at the time of surgery and correlate it with the biomarkers and clinical findings. Methods. Abnova, Abcam and AMSBIO ELISA kits were tested using a validation protocol relating the standard curve to a dilution series of SF and serum/plasma (1× to 1/50×), with and without SF hyaluronidase treatment to evaluate linearity, specificity and optimal dilutions. Validated ELISA kits were used to measure [IL-6], glutamate [glu], [RANKL] and [OPG] in SF and serum/plasma. For each dog, CrCL disease pre-operative lameness scores were graded as: (1) mild, (2) moderate (easily visible), (3) marked (encumbered), (4) non-weightbearing lameness. Blinded OA scoring was performed on radiographs [15–60, normal-severe OA]. Results. canine population (n=14) was of various breeds, aged between 2–10 years and weighing 17.1–45.5Kg; 42.86% male; 57.14% female; 83.33% males and 62.5% females were neutered. Lameness scores varied from 1 and 4 (average 2.07±1.12) and radiographic OA scores from 18 and 36 (average 27.86±5.11). Individual correlations in concentrations with respect to age, weight, lameness score (1–4) and OA scores (15–60) were tested. SF [glu] and lameness score were inversely correlated with higher levels of lameness corresponding to lower SF [glu] (P=0.0141). SF [RANKL] inversely correlated with weight (P=0.0045) and lameness score (P=0.0135), and serum [RANKL] inversely correlated with weight (P=0.0437). There was also a negative correlation between SF and serum [OPG] and weight (P=0.0165 and P=0.0208, respectively). No other significant correlations were detected. Overall, [glu] and [IL-6] are increased in SF compared to serum/plasma, by 12.84 and 1.28, respectively, whereas all the remaining biomarkers are higher (2–3 times) in the serum/plasma compared to SF. Principal component analysis (PCA) and Pearson correlation coefficient matrix [IL-6/glu/RANKL/OPG] (n=7) showed SF [IL-6] correlates with SF [glu] (rs=0.64) and strong positive correlations between SF/serum [RANKL] and SF/serum [OPG] (rs 0.68–0.96). Conclusions. Dogs with CrCL disease show an association between the bone remodelling markers RANKL and OPG, and the inflammatory cytokine IL-6, and to a lesser extent SF [glu]. Therapeutics targeting bone remodelling, IL-6 or GluR/[glu] may be of interest for the management of OA in dogs. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Cartilage diseases have a significant impact on the patient's quality of life and are a heavy burden for the healthcare system. Better understanding, early detection and proper follow-up could improve quality of life and reduce healthcare related costs. Therefore, the aim of this study was to evaluate if difference between osteoarthritic (OA) and non-osteoarthritic (non-OA) knees can be detected quantitatively on cartilage and subchondral bone levels with advanced but clinical available imaging techniques. Two OA (mean age = 88.3 years) and three non-OA (mean age = 51.0 years) human cadaveric knees were scanned two times. A high-resolution peripheral quantitative computed tomography (HR-pQCT) scan (XtremeCT, Scanco Medical AG, Switzerland) was performed to quantify the bone microstructure. A contrast-enhanced clinical CT scan (GE Revolution Evo, GE Medical Systems AG, Switzerland) was acquired with the contrast agent Visipaque 320 (60 ml) to measure cartilage. Subregions dividing the condyle in four parts were identified semi-automatically and the images were segmented using adaptive thresholding. Microstructural parameters of subchondral bone and cartilage thickness were quantified. The overall cartilage thickness was reduced by 0.27 mm between the OA and non-OA knees and the subchondral bone quality decreased accordingly (reduction of 33.52 % in BV/TV in the layer from 3 to 8 mm below the cartilage) for the femoral medial condyle. The largest differences were observed at the medial part of the femoral medial condyle both for cartilage and for bone parameters, corresponding to clinical observations. Subchondral bone microstructural parameters and cartilage thickness were quantified using in vivo available imaging and apparent differences between the OA and non-OA knees were detected. Those results may improve OA follow-up and diagnosis and could lead to a better understanding of OA. However, further in vivo studies are needed to validate these methods in clinical practice

Osteoarthritis (OA) is an inflammatory disease affecting the complete synovial joint including the cartilage layer and the subchondral bone plate. Due to the multifactorial causes and the not yet completely resolved molecular mechanisms, it lacks a gold standard treatment to mitigate OA. Hence, biomaterials capable of delaying or preventing OA are a promising alternative or supplement to antiphlogistic and surgical interventions. Magnesium (Mg) and its alloys are among the promising biomaterials with osteoinductive effects. This work investigated the impact of Mg micro cylinders (length ≈of 1.0 mm and width of 0.5 mm) in vitro, in favoring joint regeneration together with preventing OA progression. Therefore, a mesenchymal stem cell line (SCP-1) was applied in order to assess the compatibility of the degradable material. Furthermore, an in vitro OA model utilizing SCP-1 cells based on the supplementation of the cytokines; IL-1β, TNF-α was established and disclosed the capability of Mg microparticles in differentiating SCP-1 cells into chondrogenic and osteogenic lineages proven through extracellular matrix staining and gene marker analysis. A concentration above 10 mM revealed a reduction in the cell viability by 50 %. An increase in the expression of collagens especially and proteoglycans (COL2A1, Aggrecan) as extracellular matrix proteins as well as an increase in osteogenic marker (ALP, BMP2) favoring the mineralization process were observed. The inflammatory condition reduced the viability and productivity of the applied stem cell line. However, the application of Mg microparticles induced a cell recovery and reduction of inflammation marker such as MMP1 and IL6. The cytocompatible and the ability of Mg microparticles in supporting bone and cartilage repair mechanisms in vitro even under inflammatory conditions make biodegradable Mg microparticles a suitable implant material to treat OA therapy. Acknowledgements: This project OAMag was funded by the German Research Foundation (project number 404534760). The author thank Dr. Björn Wiese (hereon) for the production of Mg based material and Prof. Böcker (MUM Musculoskeletal University Center Munich) for the provision of SCP-1 cell line

Senescent chondrocyte and subchondral osteoclast overburden aggravate inflammatory cytokine and pro-catabolic proteinase overproduction, accelerating extracellular matrix degradation and pain during osteoarthritis (OA). Fibronectin type III domain containing 5 (FNDC5) is found to promote tissue homeostasis and alleviate inflammation. This study aimed to characterize what role Fndc5 may play in chondrocyte aging and OA development. Serum and macroscopically healthy and osteoarthritic cartilage were biopsied from patients with knee OA who received total knee replacement. Murine chondrocytes were transfected with Fndc5 RNAi or cDNA. Mice overexpressing Fndc5 (Fndc5Tg) were operated to have destabilized medial meniscus mediated (DMM) joint injury as an experimental OA model. Cellular senescence was characterized using RT-PCR analysis of p16INK4A, p21CIP1, and p53 expression together with ß-galactosidase activity staining. Articular cartilage damage and synovitis were graded using OARSI scores. Osteophyte formation and mechanical allodynia were quantified using microCT imaging and von Frey filament, respectively. Osteoclast formation was examined using tartrate-resistant acid phosphatase staining. Senescent chondrocyte and subchondral osteoclast overburden together with decreased serum FNDC5 levels were present in human osteoarthritic cartilage. Fndc5 knockdown upregulated senescence program together with increased IL-6, MMP9 and Adamts5 expression, whereas Alcian blue-stained glycosaminoglycan production were inhibited. Forced Fndc5 expression repressed senescence, apoptosis and IL-6 expression, reversing proliferation and extracellular matrix production in inflamed chondrocytes. Fndc5Tg mice showed few OA signs, including articular cartilage erosion, synovitis, osteophyte formation, subchondral plate sclerosis and mechanical allodynia together with decreased IL-6 production and few senescent chondrocytes and subchondral osteoclast formation during DMM-induced joint injury. Mechanistically, Fndc5 reversed histone H3K27me3-mediated IL-6 transcription repression to reduce reactive oxygen species production. Fndc5 loss correlated with OA development. It was indispensable in chondrocyte growth and anabolism. This study sheds light onto the anti-ageing and anti-inflammatory actions of Fndc5 to chondrocytes; and highlights the chondroprotective function of Fndc5 to compromise OA