Objective. Excessive mechanical stress on synovial joints causes osteoarthritis (OA) and results in the production of prostaglandin E2 (PGE2), a key molecule in arthritis, by synovial fibroblasts. However, the relationship between arthritis-related molecules and mechanical stress is still unclear. The purpose of this study was to examine the synovial fibroblast response to cyclic mechanical stress using an in vitro osteoarthritis model. Method. Human synovial fibroblasts were cultured on collagen scaffolds to produce three-dimensional constructs. A cyclic compressive loading of 40 kPa at 0.5 Hz was applied to the constructs, with or without the administration of a cyclooxygenase-2 (COX-2) selective inhibitor or dexamethasone, and then the concentrations of PGE2, interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-6, IL-8 and COX-2 were measured. Results. The concentrations of PGE2, IL-6 and IL-8 in the loaded samples were significantly higher than those of unloaded samples; however, the concentrations of IL-1β and TNF-α were the same as the unloaded samples. After the administration of a COX-2 selective inhibitor, the increased concentration of PGE2 by cyclic compressive loading was impeded, but the concentrations of IL-6 and IL-8 remained high. With dexamethasone, upregulation of PGE2, IL-6 and IL-8 was suppressed. Conclusion. These results could be useful in revealing the molecular mechanism of mechanical stress in vivo for a better understanding of the pathology and therapy of OA. Cite this article: Bone Joint Res 2014;3:280–8

Summary Statement. Increased Dkk-1 signaling is associated with OA occurrence and joint microenvironment damage. Interruption of Dkk1 action is beneficial to improve OA knees. Introduction. Osteoarthritis (OA) is a leading cause of disability and healthcare financial burden for total knee arthroplasty, rehabilitation, and disability. Inappropriate mechanical stress, immunological, or biochemical regulation reportedly disturbs homeostasis among cartilage, synovium and subchondral bone microstructure that contributes to OA pathogenesis. Control of joint-deleterious factor action is an emerging strategy to ameliorate OA-induced joint deterioration. Dickkopf-1 (Dkk-1) is a potent inhibitor for Wnt/β-catenin signaling regulation of tissue development and remodeling in physiological or pathological contexts. Dkk-1 also acts as a master deleterious factor that represses osteoblast differentiation capacity and bone repair. Associations among Dkk-1 expression, chondrocyte fate, synovial fibroblast behavior or OA incidence are merit of characterization. Patients & Methods. Cartilage, synovial tissue and fluid were harvested from informed consent OA patients underwent arthroplasty and patient with knee injuries without OA changes as controls. Primary chondrocyte cultures and synovial fibroblasts were treated with inflammatory cytokines or Dkk-1 antisense oligonucleotide or monoclonal antibodies. Knees in experimental animals were subjected to anterior cruciate ligament transection- or intra-articular collagenase injection to induce OA. Joint inflammation, integrity and subchondral bone microstructure in knees as well gait profiles were quantified using 2-deoxyglucose-probed near-infrared in vivo image, µCT, catwalk and histomorphometric analyses. Results. In clinical vignettes, patients with end-stage OA knee had higher abundances of Dkk-1 in cartilage, synovial tissue, and synovial fluid compared to control patients. Disruption of DKk-1 signaling ameliorated the promoting effects of inflammatory cytokines on the survival and cartilage matrix synthesis in primary cartilage chondrocyte cultures. Of interest, Dkk-1 neutralization attenuated the excessive angiogenic activities and matrix metalloproteinase secretion in primary synovial fibroblasts of OA knees. Dkk-1 modulation of survival or metabolic activities in chondrocytes and synovial fibroblasts were through β-catenin-dependent and -independent signaling pathways. Moreover, increased Dkk-1 expression in lesion sites and sera was associated with the incidence of femoral head osteonecrosis. Loss of Dkk-1 action alleviated bone cell apoptosis in osteonecrotic bone microenvironments. In experimental OA knee models, knockdown of Dkk-1 alleviated articular cartilage damage as evidenced by improved Mankin score in OA knees. Dkk-1 disruption also alleviated the adverse effects of OA on subchondral bone exposure and loss of trabecular bone volume and mineral acquisition in injured joints. Loss of Dkk-1 function reduced joint inflammation, vessel number, leukocyte infiltration in synovium compartment of OA joint and improved gait profiles of affected limbs. Conclusion. Dkk-1 signaling is associated with the OA knee occurrence and accelerates apoptosis, matrix degradation and angiogenic activities in chondrocytes and synovial fibroblasts of OA joint. Dkk-1 interference alleviates the promoting effects of OA on cartilage, synovial and subchondral bone remodeling. Blocking the deleterious actions of Dkk-1 in joint microenvironment will be a prospective molecular regime beneficial for retarding excessive joint deterioration in OA knees

Intra-articular injection is a common way to deliver biologics to joints, but their effectiveness is limited by rapid clearance from the joint space. This barrier can be overcome by genetically modifying cells within the joint such that they produce anti-arthritic gene products endogenously, thereby achieving sustained, therapeutic, intra-articular concentrations of the transgene products without re-dosing. A variety of non-viral and viral vectors have been subjected to preclinical testing to evaluate their suitability for delivering genes to joints. The first transfer of a gene to a human joint used an ex vivo protocol involving retrovirally transduced, autologous, synovial fibroblasts. Recent advances in vector technology allow in vivo delivery using adeno-associated virus (AAV). We have developed an AAV vector encoding the interleukin-1 receptor antagonist (AAV.IL-1Ra) for injection into joints with osteoarthritis (OA). It showed efficacy and safety in equine and rat models of OA, leading to a recently-completed, investigator-initiated, Phase I, dose-escalation clinical trial in 9 subjects with mid-stage OA of the knee (. ClinicalTrials.gov. Identifier: NCT02790723). Three cohorts of three subjects with mild to moderate OA in the index knee were injected intra-articularly under ultrasound guidance with a low (10e11 viral genomes) medium (10e12 viral genomes) or high (10e13 viral genomes) dose of AAV.IL-1Ra and followed for one year. The data confirm safety, with evidence of sustained intra-articular expression of IL-1Ra and a clinical response in certain subjects. Funding for a subsequent Phase Ib trial involving 50 subjects (. ClinicalTrials.gov. Identifier: NCT05835895), expected to start later this year, has been acquired. Progress in this area has stimulated commercial activity and there are now at least seven different companies developing gene therapies for OA and a number of clinical trials are in progress. Acknowledgement: Clinical trial funded by US Department of Defense Clinical Trial Award W81XWH-16-1-0540

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

Synovitis has been shown to play a role in pathophysiology of OA promoting cartilage destruction and pain. Synovium is mainly composed of synovial fibroblast (SF) and macrophage (SM) that guide synovial inflammation. Adipose stromal cells (ASC) promising candidate for cell therapy in OA are able to counteract inflammation. Two different subsets of macrophages have been described showing a pro-inflammatory (M1) and an anti-inflammatory (M2) phenotype. Macrophage markers: CD68, CD80 (M1-like) and CD206 (M2-like) were evaluated in osteoarthritic synovial tissue. GMP-clinical grade ASC were isolated from subcutaneous adipose tissue and M1-macrophages were differentiated from CD14+ obtained from peripheral blood of healthy donors. ASC were co-cultured in direct and indirect contact with activated (GM-CSF+IFNγ)-M1 macrophages for 48h. At the end of this co-culture we analyzed IL1β, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9, IL10, CD163 and CD206 by qRT-PCR or immunoassay. PGE2 blocking experiments were performed. In moderate grade OA synovium we found similar percentages of CD80 and CD206. M1-activated macrophage factors IL1β, TNFα, IL6, MIP1α/CCL3, S100A8 and S100A9 were down-modulated both co-culture conditions. Moreover, ASC induced the typical M2 macrophage markers IL10, CD163 and CD206. Blocking experiments showed that TNFα, IL6, IL10, CD163 and CD206 were significantly modulated by PGE2. We confirmed the involvement of PGE2/COX2 also in CD14+ OA synovial macrophages. In conclusion we demonstrated that ASC are responsible for the switching of activated-M1-like to a M2-like anti-inflammatory phenotype, mainly through PGE2. This suggested a specific role of ASC as important determinants in therapeutic dampening of synovial inflammation in OA

Background. Epigenetic regulation of gene transcription affects metabolism of chondrocytes and synovial fibroblasts and is associated with the prevalence of osteoarthritis (OA) of knees. Histone lysine demethylase (KDMs) reportedly modulates tissue homeostasis and deterioration. This study investigated whether KMD6a inhibitor treatment affected the joint injuries in the progression of OA. Methods. Collagenase-induced OA knees in mice were intra-articular administered with KDM6a inhibitor GSK-J4. Walking patterns and footprints of affected animals were detected by Catwalk. Articular cartilage injury was quantified by OARSI scoring; and subchondral bone microstructure was analysed by μCT imaging. Histopathology and mRNA expression of cartilage, fibrosis and bone matrices in joint micro-compartments were detected by histomorphometry and quantitative RT-PCR. Methylation states of chondrogenic transcription factor SOX9 promoter was detected by methylation-specific PCR and chromatin immuno-precipitation. Results. Declined KDM6a expression and SOX9 gene transcription was associated with the pathogenesis of collagenase-induced joint injures. GSK-J4 administration dose-dependently improved gait profiles and footprint characteristics of affected feet and alleviated histopathology of severe cartilage degradation, synovial inflammation, fibrotic matrix accumulation and subchondral bone microarchitecture deterioration in injured joints. Treatment with GSK-J4 decreased expression of fibrogenic factor (TGF-β1, PLOD2 and TIMP) and restored expression of cartilage and bone matrices (collagen II, I, aggrecan, and osteocalcin). KDM6a inhibitor curtailed the hypomethylation of SOX9 promoter and lysine 27 of histone H3 (H3K27) and restored SOX9 mRNA and protein levels in joint tissues. Conclusions. KDM6a enhanced SOX9 promoter and H3K27 hypomethylation that accelerated the progression of OA. KDM6a inhibitor had mitigated effects on SOX9 promoter demethylation thereby restored SOX9 signaling and stabilised homeostasis of cartilage, synovium and subchondral bone compartments in affected joints. This study sheds a new light on the KDM6a-mediated epigenetic dysfunction in OA joints and has a perspective that pharmaceutical KDM6a inhibitor has therapeutic potential for OA knee pathogenesis. Level of evidence. II

We examined the cellular responses to various particles injected into the knees and the intramedullary femoral cavities of rats in the presence of polymethyl-methacrylate (PMMA) plugs. The intra-articular particles were mainly ingested by synovial fibroblasts. Increased numbers of macrophages were not detected and there was only a slight increase in synovial thickness. Cellular responses in the intramedullary space were similarly mild and bone resorption around the PMMA plug did not occur. Bone formation was inhibited only by polyethylene particles. In contrast to current views, our study shows that wear particles per se do not initiate bone resorption

Summary. Hyaluronan suppressed lipopolysaccharide-stimulated prostaglandin E. 2. production via intercellular adhesion molecule-1 through down-regulation of nuclear factor-κB. Administration of hyaluronan into rheumatoid joints may decrease prostaglandin E. 2. production by activated macrophages, which could result in improvement of arthritic pain. Introduction. Prostaglandin E. 2. (PGE. 2. ) is one of the key mediators of inflammation in rheumatoid arthritis (RA) joints. Intra-articular injection of high molecular weight hyaluronan (HA) into RA knee joints relieves arthritic pain. Although HA has been shown to inhibit PGE. 2. production in cytokine-stimulated synovial fibroblasts, it remains unclear how HA suppresses PGE. 2. production in catabolically activated cells. Furthermore, HA effect on macrophages has rarely been investigated in spite of their contribution to RA joint pathology. Objectives. This study was aimed to investigate the inhibitory mechanism of HA on lipopolysaccharide (LPS)-stimulated PGE. 2. in U937 human macrophage culture system. Methods. With or without pretreatment with one of HA, NS-398, and BAY11-7085, differentiated U937 macrophages were stimulated with LPS. In another set of experiments, the cells were incubated with anti-ICAM-1 antibody or non-specific IgG before pretreatment with HA. PGE. 2. concentrations of the cell-free supernatants were determined using an enzyme-linked immunosorbent assay. The cell lysates and nuclear extracts were prepared for immunoblot analysis. HA binding to ICAM-1 was evaluated by fluorescence microscopic analysis. Results. Stimulation of U937 macrophages with LPS enhanced PGE. 2. production in association with increased protein levels of cyclooxygenase-2 (COX-2). Pretreatment with HA of 2,700 kDa resulted in suppression of LPS-induced COX-2, leading to a decrease in PGE. 2. production. While LPS activated NF-κB pathway, inhibition studies using BAY11-7085 revealed the requirement of NF-κB for LPS-stimulated PGE. 2. production. HA down-regulated the phosphorylation and nuclear translocation of NF-κB by LPS. Fluorescence cytochemistry demonstrated that HA bound to ICAM-1 on U937 macrophages. Anti-ICAM-1 antibody reversed the inhibitory effects of HA on LPS-activated PGE. 2. , COX-2, and NF-κB. Conclusion. These results clearly demonstrated that HA suppressed LPS-stimulated PGE2 production via ICAM-1 through down-regulation of NF-κB. Clinical administration of high molecular weight HA into RA joints may decrease PGE2 production by activated macrophages, which could result in improvement of arthritic pain

Objectives

The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known.

Objectives

Given the function of adiponectin (ADIPOQ) on the inflammatory condition of obesity and osteoarthritis (OA), we hypothesized that the ADIPOQ gene might be a candidate gene for a marker of susceptibility to OA.

The patellofemoral joint is an important source of symptoms in osteoarthritis of the knee. We have used a newly designed surgical model of patellar strengthening to induce osteoarthritis in BALB/c mice and to establish markers by investigating the relationship between osteoarthritis and synovial levels of matrix metalloproteinases (MMPs). Osteoarthritis was induced by using this microsurgical technique under direct vision without involving the cavity of the knee. Degeneration of cartilage was assessed by the Mankin score and synovial tissue was used to determine the mRNA expression levels of MMPs. Irrigation fluid from the knee was used to measure the concentrations of MMP-3 and MMP-9. Analysis of cartilage degeneration was correlated with the levels of expression of MMP.

After operation the patellofemoral joint showed evidence of mild osteoarthritis at eight weeks and further degenerative changes by 12 weeks. The level of synovial MMP-9 mRNA correlated with the Mankin score at eight weeks, but not at 12 weeks. The levels of MMP-2, MMP-3 and MMP-14 mRNA correlated with the Mankin score at 12 weeks. An increase in MMP-3 was observed from four weeks up to 16 weeks. MMP-9 was notably increased at eight weeks, but the concentration at 16 weeks had decreased to the level observed at four weeks.

Our observations suggest that MMP-2, MMP-3 and MMP-14 could be used as markers of the progression of osteoarthritic change.

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff.

We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis).

The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears.

These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment.

In order to clarify the role of cytokines in the remodelling of the grafted tendon for ligament reconstruction we compared the responses to interleukin (IL)-1β, platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-β1 of extrinsic fibroblasts infiltrating the frozen-thawed patellar tendon in rats with that of the normal tendon fibroblasts, in regard to the gene expression of matrix metalloproteinase (MMP)-13, using Northern blot analysis. We also examined, immunohistologically, the local expression of IL-1β, PDGF-BB, and TGF-β1 in fibroblasts infiltrating the frozen-thawed patellar tendon.

Northern blot analysis showed that fibroblasts derived from the patellar tendon six weeks after the freeze-thaw procedure in situ showed less response to IL-1β than normal tendon fibroblasts with respect to MMP-13 mRNA gene expression. The immunohistological findings revealed that IL-1β was over-expressed in extrinsic fibroblasts which infiltrated the patellar tendon two and six weeks after the freeze-thaw procedure in situ, but neither PDGF-BB nor TGF-β1 was over-expressed in these extrinsic fibroblasts. Our findings indicated that IL-1β had a close relationship to matrix remodelling of the grafted tendon for ligament reconstruction, in addition to the commencement of inflammation during the tissue-healing process.