Synovial Explants Research Articles

Attachment of cartilage wear particles to the synovium negatively impacts friction properties

Cartilage wear particles are released into the synovial fluid by mechanical and chemical degradation of the articular surfaces during osteoarthritis and attach to the synovial membrane. Accumulation of wear particles could alter key tissue-level mechanical properties of the synovium, hindering its characteristically low-friction interactions with underlying articular surfaces in the synovial joint. The present study employs a custom loading device to further the characterization of native synovium friction properties, while investigating the hypothesis that attachment of cartilage wear particles increases friction coefficient. Juvenile bovine synovium demonstrated characteristically low friction coefficients in sliding contact with glass, in agreement with historical measurements. Friction coefficient increased with higher normal load in saline, while lubrication with native synovial fluid maintained low friction coefficients at higher loads. Cartilage wear particles generated from juvenile bovine cartilage attached directly to synovium explants in static culture, with incorporation onto the tissue denoted by cell migration onto the particle surface. In dilute synovial fluid mimicking the decreased lubricating properties during osteoarthritis, wear particle attachment significantly increased friction coefficient against glass, and native cartilage and synovium. In addition to providing a novel characterization of synovial joint tribology this work highlights a potential mechanism for cartilage wear particles to perpetuate the degradative environment of osteoarthritis by modulating tissue-level properties of the synovium that could impact macroscopic wear as well as mechanical stimuli transmitted to resident cells.

Interleukin 4 promotes anti-inflammatory macrophages that clear cartilage debris and inhibits osteoclast development to protect against osteoarthritis

ObjectiveOsteoarthritis (OA), the leading cause of joint failure, is characterized by breakdown of articular cartilage and remodeling of subchondral bone in synovial joints. Despite the high prevalence and debilitating effects of OA, no disease-modifying drugs exist. Increasing evidence, including genetic variants of the interleukin 4 (IL-4) and IL-4 receptor genes, implicates a role for IL-4 in OA, however, the mechanism underlying IL-4 function in OA remains unknown. Here, we investigated the role of IL-4 in OA pathogenesis. MethodsIl4-, myeloid-specific-Il4ra-, and Stat6-deficient and control mice were subjected to destabilization of the medial meniscus to induce OA. Macrophages, osteoclasts, and synovial explants were stimulated with IL-4 in vitro, and their function and expression profiles characterized. ResultsMice lacking IL-4, IL-4Ra in myeloid cells, or STAT6 developed exacerbated cartilage damage and osteophyte formation relative to WT controls. In vitro analyses revealed that IL-4 downregulates osteoarthritis-associated genes, enhances macrophage phagocytosis of cartilage debris, and inhibits osteoclast differentiation and activation via the type I receptor. ConclusionOur findings demonstrate that IL-4 protects against osteoarthritis in a myeloid and STAT6-dependent manner. Further, IL-4 can promote an immunomodulatory microenvironment in which joint-resident macrophages polarize towards an M2 phenotype and efficiently clear pro-inflammatory debris, and osteoclasts maintain a homeostatic level of activity in subchondral bone. These findings support a role for IL-4 modulation of myeloid cell types in maintenance of joint health and identify a pathway that could provide therapeutic benefit for osteoarthritis.

15 The effect of nutraceuticals on markers of equine joint health

Acute injury and degradation of joint function can limit the performance of horses. To evaluate the ability of a combination of nutraceuticals to impact joint health, an in vitro trial was designed utilizing cartilage (CART) and synovium (SYN) explants harvested aseptically from the carpal joint of 2 horses euthanized for unrelated reasons. CART and SYN explants (n = 3/horse/group) were created using a 6 mm dermal biopsy punch and cocultured in appropriate media with the CART explant in the well and the SYN explant in a trans well insert with a membrane allowing for the flow of media between the insert and the well. Tissues were randomly assigned to one of 5 culture groups: 1) Negative control; 2) Positive control (10ng/ml recombinant equine (re)IL-1β); 3) Low (10ng/ml reIL-1β, 0.2 ng/mL green tea (GT), 100 ng/mL hydrolyzed collagen (HC), 25 ng/mL cranberry powder (CP)); 4) Mid (10ng/ml reIL-1β, 2 ng/mL GT, 1 µg/mL HC, 250 ng/mL CP); or 5) High (10ng/ml reIL-1β, 20 ng/mL GT, 10 µg/mL HC, 2.5 µg/mL CP). Dosages were determined via a previous study. Tissues were cultured for 21 d, and media collected on d 3, 9, 15, and 21. On d 21 viable chondrocyte density (VCD) was determined. Tissue digests were tested for proteoglycan and collagen content. Culture media was analyzed for matrix metalloproteinase (MMP), prostaglandin e 2 (PGE2), nitric oxide (NO), proteoglycan content, interleukin-6 and 8 (IL-6, 8), and growth-related oncogene (GRO). Data were analyzed utilizing a one-way ANOVA in SAS 9.4. Data are presented in the table below. There were no treatment differences for any parameter in any of the culture groups with the exception of the positive and negative control groups. Differences by time existed with increases in inflammation over time in the positive control group and no changes in the negative control group. Significant time x treatment interactions existed only in the positive control group. No combination of nutraceuticals was able to mitigate the inflammatory challenge. At the high level, the combination appeared to have cytotoxic effects. Taken together, these data shed light on the challenges associated with developing an efficacious joint supplement for horses.

Evaluation of Villus Synovium From Unaffected Metacarpophalangeal Joints of Adult and Juvenile Horses

Horses are a widely accepted model for osteoarthritis (OA) research. Synovial tissue sampling is commonly used in studies to evaluate and grade the progress of OA or to assess treatment effects. Synovial explants play an important role in ex-vivo studies, increasingly replacing the use of living animals. To understand histomorphological changes in the process of joint-related diseases such as OA, detailed information about histomorphometric parameters of unaffected synovial villi is necessary.The objective of the present study was to evaluate the mean width of the intimal synovial lining and its cellularity as well as the vascularization of the subintimal layer in juvenile and adult horses not affected by a joint-related disease.One hundred synovial samples from both metacarpophalangeal joints from 25 horses (one day to 24 years old) were collected to evaluate the following parameters on digitalized hematoxylin-eosin stained samples: Width of intimal synovial lining measured by the distance from the inner joint surface to the subintimal layer; density of the cells making up the intimal synovial lining by counting cell nuclei; vascularization of the subintimal layer measured by the number and size of vessels in relation to the subintimal area.The median width of the intimal lining did not differ among juvenile (22.34 µm) and adult (23.34 µm) horses. The cellularity of the intimal lining was significantly lower in juvenile (one cell/143.8 µm2) than in adult (one cell /188.7µm2), (P < .001) horses as well as the density of blood vessels per mm2 within the subintimal layer (juveniles 1/mm2 vs. adults 0.05/mm2), (P < .001).This study provides morphometric data regarding synovial intimal width, intimal cellularity, and vascularization of equine synovial villi of unaffected horses. For future studies, age-related characteristics should be taken into consideration when synovial tissue samples are used for in-vivo and in-vitro studies.

Size-Dependent Effective Diffusivity in Healthy Human and Porcine Joint Synovium.

Intra-articular drug delivery can be effective in targeting a diseased joint butis hampered by rapid clearance times from the diarthrodial joint. The synovium is a multi-layered tissue that surrounds the diarthrodial joint and governs molecular transport into and out of the joint. No models of drug clearance through synovium exist to quantify diffusivity across solutes, tissue type and disease pathology. We previously have developed a finite element model of synovium as a porous, permeable, fluid-filled tissue and used an inverse method to determine urea's effective diffusivity (Deff) in de-vitalized synovium explants.22 Here we apply this method to determineDefffrom unsteady diffusive transport of model solutes and confirm the role of molecular weight in solute transport. As molecular weight increased, Deff decreased in both human and porcine tissues, with similar behavior across the two species. Unsteady transport was well-described by a single exponential transient decay in concentration, yielding solute half-lives (t1/2) that compared favorably with theDeff determined from the finite element model fit. Determined values forDeffparallel prior observations of size-dependentin vivodrug clearance and provide an intrinsic parameter with greater ability to resolve size-dependencein vitro. Thus, this work forms the basis for understanding the influence of size on drug transport in synovium and can guide future studies to elucidate the role of charge and tissue pathology on the transport of therapeutics in healthy and pathological human synovium.

Cartilage Oligomeric Matrix Protein-Derived Peptides Secreted by Cartilage Do Not Induce Responses Commonly Observed during Osteoarthritis.

ObjectiveTo evaluate if 3 peptides derived from the cartilage oligomeric matrix protein (COMP), which wounded zones of cartilage secrete into synovial fluid, possess biological activity and might therefore be involved in the regulation of specific aspects of joint regeneration.MethodsThe 3 peptides were produced by chemical synthesis and then tested in vitro for known functions of the COMP C-terminal domain from which they derive, and which are involved in osteoarthritis: transforming growth factor-β (TGF-β) signaling, vascular homeostasis, and inflammation. Results. None of the peptides affected the gene expression of COMP in osteochondral progenitor cells (P > 0.05). We observed no effects on the vascularization potential of endothelial cells (P > 0.05). In cultured synovium explants, no differences on the expression of catabolic enzymes or proinflammatory cytokines were found when peptides were added (P > 0.05).Discussion and ConclusionsThe 3 peptides tested do not regulate TGF-β signaling, angiogenesis and vascular tube formation, or synovial inflammation in vitro and therefore most likely do not play a major role in the disease process.

Effects of All-&lt;i&gt;Trans&lt;/i&gt; Retinoic Acid on the Optimization of Synovial Explant Induced by Tumor Necrosis Factor Alpha

Current studies focused on the effects of all-trans-retinoic acid (ATRA) on synovial explants from rats with rheumatoid arthritis (RA) induced by lipopolysaccharides (LPS). In our study, synovial membranes were extracted aseptically from the quadriceps femoris of the knee joint of rats, and then incubated in medium containing 10% neonate bovine serum for 24 h adaptive culture. We first measured variations of correlation factors in synovium at 24, 48, 72, 96 and 120 h in control medium or in medium containing 20 ng/mL tumor necrosis factor alpha (TNF-α) (TNF-α-experiment). Then, we investigated the synovium exposed to three ATRA concentrations after 48 h incubation (ATRA-experiment). The effects of ATRA on synovitis were evaluated by observing the expression of inflammatory cytokines, angiogenic factors and the production of proteases in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and apoptosis and autophagy. In TNF-α-experiment, the secretion of nitric oxide (NO), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9) increased significantly after TNF-α stimulation without pathological damage to the synovium. Hence, we successfully obtained the synovial explants model, which had longer inflammatory response time. In the ATRA-experiment, ATRA suppressed the secretion of IL-6 and NO, downregulated the NF-κB P65 and Bcl-2, increased levels of autophagy marker protein LC3, but different doses of ATRA showed inconsistent regulatory effects on VEGF and MMP-9. In short, ATRA inhibited TNF-α induced synovitis by the regulation of inflammatory cytokines and inhibiting NF-κB signal transduction and potentially promoting autophagy, apoptosis and angiogenesis, displaying its role in alleviating synovial inflammation in patients with RA.

Effects of Autologous Conditioned Serum, Autologous Protein Solution, and Triamcinolone on Inflammatory and Catabolic Gene Expression in Equine Cartilage and Synovial Explants Treated With IL-1β in Co-culture.

Autologous conditioned serum (ACS) and autologous protein solution (APS) are newer therapeutic options for osteoarthritis (OA). Co-culture of cartilage and synovium stimulated with IL-1β produces a similar physiologic response to tissues from naturally-ocurring OA. The study objective was to investigate the effects of ACS, APS, and triamcinolone (TA) on inflammatory and catabolic gene expression of inflamed joint tissues in co-culture. Blood was collected and processed for ACS and APS from six horses. Cartilage and synovial explants were harvested from the stifle, placed in co-culture, and treated as: (1) unstimulated control (2) stimulated control (3) ACS at 25% v/v (4) ACS at 50% v/v (5) APS at 25% v/v (6) APS at 50% v/v, (7) TA (10−6 M). Treatment groups 2–7 were stimulated with IL-1β (10 ng/ml). Cultures were maintained for 96 hours, and then both media and explants were harvested for measurement of gene expression and protein. IL-1β stimulation significantly increased IL-1β (p = 0.029), IL-8 (p = 0.011) and MMP-3 (p = 0.043) expression in synovium and IL-1β (p = 0.003) and TNF-α (p = 0.001) expression in cartilage. Treatment with 50% ACS and APS v/v downregulated IL-1β expression in cartilage more than TA treatment (p = 0.001 and p = 0.0004) and APS downregulated MMP-1 expression in synovial membrane (p = 0.025). Treatment with ACS and APS caused a trend in upregulation of IL-10 expression in synovium and type II collagen and aggrecan expression in cartilage. PGE2 media concentrations were significantly reduced following treatment with APS (13.7-fold decrease, p = 0.0001) and ACS (4.13-fold decrease, p = 0.024); while TA did not reduce PGE2 significantly (2.3-fold decreased p = 0.406). As disease-modifying therapies, ACS and APS modified the cellular response from synovial membrane and articular cartilage. ACS and APS may offer an improved strategy to improve clinical signs of horses with naturally occurring OA, compared to TA treatment.

Insulin‐Resistant Pathways Are Associated With Disease Activity in Rheumatoid Arthritis and Are Subject to Disease Modification Through Metabolic Reprogramming: A Potential Novel Therapeutic Approach

To investigate a role for insulin-resistant pathways in inflammation and therapeutic targeting for disease modification in rheumatoid arthritis (RA). RA disease activity and cardiovascular risk factors, including insulin resistance and body mass index (BMI), were assessed in an Irish RA cohort. Glucose transporter 1 (GLUT-1) and GLUT-4 activity in RA and osteoarthritis (OA) synovial tissue was examined using immunohistochemistry. Spontaneous release of proinflammatory mediators from ex vivo RA synovial explants and primary synovial fibroblast (SF) cell culture supernatants was quantified by enzyme-linked immunosorbent assay. Phosphorylated AMP-activated protein kinase (p-AMPK) and GLUT-1 protein expression was analyzed by Western blotting. Cellular glycolytic and oxidative phosphorylation was assessed using extracellular flux analysis. Insulin resistance was independently associated with both BMI (unstandardized coefficient B 0.113 [95% confidence interval (95% CI) 0.059-0.167]; P < 0.001) (n = 61) and swollen joint count in 28 joints (SJC28) (B 0.114 [95% CI 0.032-0.197]; P = 0.008) (n = 61). Increased GLUT-1 expression in RA synovium (n = 26) versus OA synovium (n = 16) was demonstrated (P = 0.0003), with increased expression in the lining, sublining, and vascular regions. In contrast, decreased GLUT-4 expression in the RA lining layer (n = 21) versus the OA lining layer (n = 8) was observed (P = 0.0358). Decreased GLUT-1 protein expression was observed in parallel with increased p-AMPK protein expression in SFs in the presence of metformin (n = 4). Metformin increased glycolytic activity and decreased oxidative phosphorylation in RASFs (n = 7) (P < 0.05 for both). Metformin or aminoimidazole carboxamide ribonucleotide presence decreased spontaneous production of interleukin-6 (IL-6), IL-8, and monocyte chemotactic protein 1 in RA synovial explants and SFs (n = 5-7). Insulin resistance is significantly associated with BMI and synovitis in RA, suggesting distinct interplay between glucose availability and inflammation in RA. Furthermore, the effect of metformin on proinflammatory mechanisms suggests a role for AMPK-modifying compounds in the treatment of RA.

Innovative translational models to study human synovial pathology: Target validation and preclinical imaging

Purpose: Many experiments to study inflammation, hyperplasia, and fibrosis in the synovium have been performed in animal models of RA and OA. However, the predictive value of these models for the screening of potential drugs in RA is variable and for OA, none were sufficiently effective in clinical trials. Translational arthritis research with human cells is often performed in monolayer culture where the absence of extracellular matrix and other cell types results in alterations of cell functions and loss of phenotype. To improve the predictive value of preclinical arthritis research by developing and optimizing innovative translational models to study human synovial pathology in vitro and in vivo. Methods: Synovial biopsies from arthritis patients were obtained during joint replacement surgery and processed for either (1) explant cultures, (2) 3D-synovial micromasses, (3) RA-SCID transplantation studies, and/or (4) a biobank for corresponding mRNA and IHC profiling. For explant culture, 3mm biopsies were cultured for 24 hours w/o various inhibitors, and cytokine production was analyzed by Luminex. 3D-micromasses were generated from primary RA FLS and CD14+ PBMCs, stimulated for 3 weeks with 10 ng/ml TNFα or TGFβ and analyzed by histology, IHC and QPCR. For target validation and preclinical imaging, 6mm biopsies were engrafted into SCID mice. Radio- and fluorescently-labelled anti-FcγRI antibodies were injected intravenously, and targeting was determined by biodistribution, μSPECT/CT and IVIS imaging analysis. Results: Our first assay with human arthritis synovium explants demonstrated to be highly suitable to test the therapeutic efficacy of inhibitors for TNFα, TLR4, p38 and the JAK-pathway, resulting in significantly reduced production of proinflammatory cytokines and chemokines at 24 hours of culture (1) . In contrast to the explant cultures, our 3D synovial micromasses could be followed for weeks. In this second translational model, lining formation was observed at day 7 and the micromasses could be stimulated to mimic RA- or OA-like features of synovial hyperplasia or fibrosis respectively. Long-term exposure to the RA-related cytokine TNFα lead to hyperplasia of the lining and an altered macrophage phenotype characterized by reduced CD163 expression. Conversely, the repair-related growth factor TGFβ induced fibrosis-like changes in the micromass lining, a hallmark of OA. This was accompanied by an increased expression of PLOD2, alpha-SMA, and collagen type 1 (2) Our third preclinical model, the human arthritis synovium SCID mouse, was previously validated using adalimumab, secukinumab, and rituximab (3), and was now used to study FcγRI as a potential marker to image synovitis. Gene expression of FCGRI in synovial explants from RA patients was shown to correlate positively and significantly to gene expression of pro-inflammatory factors IL1B, TNFA, IL8, S100A8 and S100A9 while gene expression of FCGRI in synovial explants from OA patients significantly correlated with S100A9. Interestingly, dual-labelled [111In]In-DTPA-IrDye800CW-anti-FcγRI antibody showed high uptake in the synovial transplants of the engrafted SCID mice, and specifically visualized the subcutaneous synovial grafts by both uSPECT/CT and IVIS imaging. Conclusions: The development of these translational models allows us to bridge the gap between preclinical and clinical drug development research. Whereas our synovial explant assay is ideal for short-term interventions, and the RA-SCID mouse a valuable translational model for in vivo preclinical studies, donor variability and access to sufficient tissue may be challenging. In such cases, the 3D synovial micromass model may be an excellent alternative. Especially in combination, these 3 translational approaches will improve target validation, lead optimization and preclinical development of novel anti-rheumatic drugs. (1) Abdollahi S., J Clin Invest. 2008; 118(1): 205-216. (2) Broeren M. et al., ALTEX. 2019; 36(1):18-28 (3) Koenders et al., Arthritis Rheum. 2012 Jun;64(6):1762-70

ADAMTS-5 degraded cartilage increases tissue turnover in synovial membrane explants and stimulation is inhibited by the anti ADAMTS-5 nanobody, M6495

Purpose: Toll-like receptors are important for activating the innate immune system in response to foreign substances. In addition, it has been proposed that cartilage fragments in the joint may act TLR ligands in the joint, effectively driving inflammation and pain in osteoarthritis. The breakdown of cartilage is mediated by key enzymes: ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) and MMPs (matrix metalloproteinases). The aim of this study was to investigate the effect of ADAMTS-5 degraded cartilage fragments on tissue turnover in a synovial membrane explant model (SME).

Combined Experimental Approach and Finite Element Modeling of Small Molecule Transport Through Joint Synovium to Measure Effective Diffusivity.

Trans-synovial solute transport plays a critical role in the clearance of intra-articularly (IA) delivered drugs. In this study, we present a computational finite element model (FEM) of solute transport through the synovium validated by experiments on synovial explants. Unsteady diffusion of urea, a small uncharged molecule, was measured through devitalized porcine and human synovium using custom-built diffusion chambers. A multiphasic computational model was constructed and optimized with the experimental data to extract effective diffusivity for urea within the synovium. A monotonic decrease in urea concentration was observed in the donor bath over time, with an effective diffusivity found to be an order of magnitude lower in synovium versus that measured in free solution. Parametric studies incorporating an intimal cell layer with varying thickness and varying effective diffusivities were performed, revealing a dependence of drug clearance kinetics on both parameters. The findings of this study indicate that the synovial matrix impedes urea solute transport out of the joint with little retention of the solute in the matrix.

Inflammation and joint destruction may be linked to the generation of cartilage metabolites of ADAMTS-5 through activation of toll-like receptors

Links between pain and joint degradation are poorly understood. We investigated the role of activation of Toll-like receptors (TLR) by cartilage metabolites in initiating and maintaining the inflammatory loop in OA causing joint destruction. Synovial membrane explants (SMEs) were prepared from OA patients' synovial biopsies. SMEs were cultured for 10 days under following conditions: culture medium alone, OSM+TNFα, TLR2 agonist - Pam2CSK4, Pam3CSK4 or synthetic aggrecan 32-mer, TLR4 agonist - Lipid A. Release of pro-inflammatory and degradation biomarkers (acMMP3 and C3M) were measured by ELISA in conditioned media along with IL-6. Additionally, human cartilage was digested with ADAMTS-5, with or without the ADAMTS-5 inhibiting nanobody - M6495. Digested cartilage solution (DCS) and synthetic 32-mer were tested for TLR activation in SEAP based TLR reporter assay. Western blotting confirmed TLR2 and TLR4 in untreated OA synovial biopsies. TLR agonists showed an increase in release of biomarkers - acMMP3 and C3M in SME. Synthetic 32-mer showed no activation in the TLR reporter assay. ADAMTS-5 degraded cartilage fragments activated TLR2 invitro. Adding M6495 - an anti-ADAMTS-5 inhibiting nanobody®, blocked ADAMTS-5-mediated DCS TLR2 activation. TLR2 is expressed in synovium of OA patients and their activation by synthetic ligands causes increased tissue turnover. ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments which activates the TLR2 receptor invitro. M6495 suppressed cartilage degradation by ADAMTS-5, limiting the activation of TLR2. In conclusion, pain and joint destruction may be linked to generation of ADAMTS-5 cartilage metabolites.

Systemic Resolvin E1 (RvE1) Treatment Does Not Ameliorate the Severity of Collagen-Induced Arthritis (CIA) in Mice: A Randomized, Prospective, and Controlled Proof of Concept Study.

Specialized proresolving mediators (SPRM), which arise from n-3 long-chain polyunsaturated fatty acids (n-3FA), promote resolution of inflammation and may help to prevent progression of an acute inflammatory response into chronic inflammation in patients with arthritis. Thus, this study is aimed at determining whether systemic RvE1 treatment reduces arthritis onset and severity in murine collagen-induced arthritis (CIA) and spontaneous cytokine production by human rheumatoid arthritis (RA) synovial explants. 10-week-old DBA1/J male mice were subjected to CIA and treated systemically with 0.1 μg RvE1, 1 μg RvE1, 5 mg/kg anti-TNF (positive control group), PBS (negative control group), or with a combination of 1 μg of RvE1 plus 5 mg/kg anti-TNF using prophylactic or therapeutic strategies. After CIA immunization, mice were treated twice a week by RvE1 or anti-TNF for 10 days. Arthritis development was assessed by visual scoring of paw swelling and histology of ankle joints. Moreover, human RA synovial explants were incubated with 1 nM, 10 nM, or 100 nM of RvE1, and cytokine levels (IL-1β, IL-6, IL-8, IL-10, INF-γ, and TNF-α) were measured using Luminex bead array. CIA triggered significant inflammation in the synovial cavity, proteoglycan loss, and cartilage and bone destruction in the ankle joints of mice. Prophylactic and therapeutic RvE1 regimens did not ameliorate CIA incidence and severity. Anti-TNF treatment significantly abrogated signs of joint inflammation, bone erosion, and proteoglycan depletion, but additional RvE1 treatment did not further reduce the anti-TNF-mediated suppression of the disease. Treatment with different concentrations of RvE1 did not decrease the expression of proinflammatory cytokines in human RA synovial explants in the studied conditions. Collectively, our findings demonstrated that RvE1 treatment was not an effective approach to treat CIA in DBA1/J mice in both prophylactic and therapeutic strategies. Furthermore, no effects were noticed when human synovial explants were incubated with different concentrations of RvE1.

Intra-articular injection of magnesium chloride attenuates osteoarthritis progression in rats

To explore the effects of Mg2+ on the expression of osteoarthritic markers in human cartilage and synovium tissue explants. To investigate the therapeutic effect of intra-articular injection of Mg2+ in an established rat OA (Osteoarthritis) model of anterior cruciate ligament transection with partial medial meniscectomy (ACLT+PMM). Human cartilage and synovium explants were collected from total knee replacement surgeries and incubated with MgCl2 (20mmol/L) invitro. A rat OA model was established by ACLT+PMM surgery in 450-500g male Sprague Dawley (SD) rats. To select the optimal dose, intra-articular injections of MgCl2 (0.05, 0.5, 5mol/L) were performed at 4 weeks after the surgery every 3days for 2weeks. The effect of optimized MgCl2 was further determined by histology, immunohistochemistry, and quantitative real-time polymerase chain reaction. The expressions of osteoarthritic markers in human cartilage and synovium explants were inhibited by Mg2+invitro. Immunohistochemical analysis further suggested the inhibitory effects of Mg2+ on the expression of MMP-13 and IL-6 in the human tissue explants. Cartilage degeneration and synovitis in ACLT+PMM rats were significantly improved by intra-articular injections of Mg2+ (0.5mol/L). Immunohistochemical analysis also showed the regulatory effects of Mg2+ on osteoarthritic markers in both cartilage and synovium in rats, consistent with invitro results. Intra-articular injections of Mg2+ at 0.5mol/L attenuatethe progression of OA in the ACLT+PMM rat model. Such effect was at least in part explained by the promotion of cartilage matrix synthesis and the suppression of synovial inflammation.

Promotion of macrophage activation by Tie2 in the context of the inflamed synovia of rheumatoid arthritis and psoriatic arthritis patients.

ObjectiveTo examine the role of Tie2 signalling in macrophage activation within the context of the inflammatory synovial microenvironment present in patients with RA and PsA.MethodsClinical responses and macrophage function were examined in wild-type and Tie2-overexpressing (Tie2-TG) mice in the K/BxN serum transfer model of arthritis. Macrophages derived from peripheral blood monocytes from healthy donors, RA and PsA patients, and RA and PsA synovial tissue explants were stimulated with TNF (10 ng/ml), angiopoietin (Ang)-1 or Ang-2 (200 ng/ml), or incubated with an anti-Ang2 neutralizing antibody. mRNA and protein expression of inflammatory mediators was analysed by quantitative PCR, ELISA and Luminex.ResultsTie2-TG mice displayed more clinically severe arthritis than wild-type mice, accompanied by enhanced joint expression of IL6, IL12B, NOS2, CCL2 and CXCL10, and activation of bone marrow-derived macrophages in response to Ang-2 stimulation. Ang-1 and Ang-2 significantly enhanced TNF-induced expression of pro-inflammatory cytokines and chemokines in macrophages from healthy donors differentiated with RA and PsA SF and peripheral blood-derived macrophages from RA and PsA patients. Both Ang-1 and Ang-2 induced the production of IL-6, IL-12p40, IL-8 and CCL-3 in synovial tissue explants of RA and PsA patients, and Ang-2 neutralization suppressed the production of IL-6 and IL-8 in the synovial tissue of RA patients.ConclusionTie2 signalling enhances TNF-dependent activation of macrophages within the context of ongoing synovial inflammation in RA and PsA, and neutralization of Tie2 ligands might be a promising therapeutic target in the treatment of these diseases.

Co-culture of osteochondral explants and synovial membrane as in vitro model for osteoarthritis.

The purpose of the current study was to establish an in vitro model for osteoarthritis (OA) by co-culture of osteochondral and synovial membrane explants. Osteochondral explants were cultured alone (control-1) or in co-culture with synovial membrane explants (control-2) in standard culture medium or with interleukin-1β (IL1β) and tumor necrosis factor (TNFα) added to the culture medium (OA-model-1 = osteochondral explant; OA-model-2 = osteochondroal-synovial explant). In addition, in OA-model groups a 2-mm partial-thickness defect was created in the centre of the cartilage explant. Changes in the expression of extracellular matrix (ECM) genes (collagen type-1 (Col1), Col2, Col10 and aggrecan) as well as presence and quantity of inflammatory marker genes (IL6, matrix metalloproteinase-1 (MMP1), MMP3, MMP13, a disintegrin and metalloproteinase with-thrombospondin-motif-5 (ADAMTS5) were analysed by immunohistochemistry, qPCR and ELISA. To monitor the activity of classically-activated pro-inflammatory (M1) versus alternatively-activated anti-inflammatory/repair (M2) synovial macrophages, the nitric oxide/urea ratio in the supernatant of osteochondral-synovial explant co-cultures was determined. In both OA-model groups immunohistochemistry and qPCR showed a significantly increased expression of MMPs and IL6 compared to their respective control group. ELISA results confirmed a statistically significant increase in MMP1and MMP3 production over the culturing period. In the osteochondral-synovial explant co-culture OA-model the nitric oxide/urea ratio was increased compared to the control group, indicating a shift toward M1 synovial macrophages. In summary, chemical damage (TNFα, IL1β) in combination with a partial-thickness cartilage defect elicits an inflammatory response similar to naturally occurring OA in osteochondral explants with and without osteochondral-synovial explant co-cultures and OA-model-2 showing a closer approximation of OA due to the additional shift of synovial macrophages toward the pro-inflammatory M1 phenotype.

The relation between the inflammatory status of human end stage OA synovium and levels of low density lipoprotein

Purpose: High systemic levels of low density lipoprotein (LDL) is a shared metabolic risk factor for osteoarthritis (OA) and cardiovascular disease (CVD), which may point to common biochemical pathways. One of the key events in atherosclerosis is formation and uptake of oxidized LDL (oxLDL) as a consequence of macrophage-mediated inflammation. Studies performed in our lab have previously shown that in mice, high LDL levels enhance synovial activation and ectopic bone formation during development of collagenase-induced OA. Furthermore, injection of oxLDL in naïve macrophage depleted joints led to infiltration of myeloid progenitor cells. Although these results strongly indicate towards (ox)LDL-mediated events that enhance OA severity, these processes have not yet been shown to occur in human osteoarthritic synovium. In the present study, we investigated whether the inflammatory status of OA synovium is associated to systemic and local levels of (ox)LDL. Methods: Blood was collected from patients planned to undergo total knee replacement and LDL serum levels were determined (n=16). After surgery, synovial explants from the same patients (2-8 per patient) were collected and incubated at 37°C for 24 hours in presence of LDL (50 μg ApoB/mL), oxLDL (50 μg ApoB/mL) or control medium. Levels of IL-1β, TNF-α, IL-10 and MCP-1 secreted in the conditioned medium were measured using Luminex. Levels of S100A8/A9 were measured using ELISA. Concentrations of secreted factors were normalized for tissue sample weight. Synovial explants were further processed for histological analysis. Immunohistochemical staining of ApoB was performed to assess presence and uptake of (ox)LDL in the synovium. Hematoxylin & eosin-stained sections were used to arbitrarily score cellular infiltration and synovial hyperplasia. Results: Around 50% of the patients in our cohort showed synovial activation exemplified by high secretion levels of IL-1β, TNF-α and IL-10. Cellular infiltration and synovial hyperplasia was observed in the majority of synovial tissue samples, their grades did however not correlate with cytokine secretion. Secretion levels of IL-1β positively correlated with serum levels of LDL and total cholesterol and did not correlate with BMI, suggesting a link that runs via systemic metabolic processes rather than increased weight-mediated joint loading. Considering that around 50% of OA patients suffers from synovitis characterized by increased macrophage presence and activity, high LDL in these patients may trigger oxLDL formation and uptake in the synovium. Staining of LDL-associated protein ApoB in histological sections of synovia showed distinct speck-like staining within macrophages and endothelial cells especially, suggesting oxLDL accumulation in these cells. Unexpectedly, presence of high concentrations of LDL or oxLDL during incubation of synovial explants did not significantly affect secretion of IL-1β, TNF-α, IL-10 and MCP-1, whereas presence of LDL but not oxLDL negatively affected secretion of S100A8/A9. Conclusions: IL-1β secretion by end stage OA synovium is associated to systemic levels of LDL. Intervention with high concentrations of LDL or oxLDL for 24-hours after synovium extraction did however not alter cytokine secretion, suggesting that LDL and synovitis are linked through more long-term local interactions, or alternatively via systemic inflammatory factors.

Human cartilage-bone-synovium microphysiological system to study ptoa pathogenesis and treatment on earth and in space

Purpose: Acute joint injuries from sports, exercise or accident-related trauma often progress to post-traumatic Osteoarthritis (PTOA), comprising ∼12% of all OA cases. Such joint injuries can cause mechanical damage to cartilage combined with an inflammatory response associated with the synovial lining of the joint capsule. Together, these events lead to cartilage and subchondral bone pathologies including cell death, tissue degradation, neoangiogenesis, osteophyte and cyst formation and synovial fibrosis. Astronauts on International Space Station-National Laboratory (ISS-NL) sustain a higher rate of exercise-related injuries, including those compromising joints, during their training and mission period. Acknowledging the urgent need to identify disease-modifying therapeutics to ameliorate degenerative evolution of OA/PTOA, we carried out this study to (1) simulate aspects of acute joint injury biology on earth and in space using a human Cartilage-Bone-Synovium coculture model (CBS-MPS), (2) to investigate the potential of dexamethasone (Dex) in reducing cell death and ECM degradation, and (3) to utilize a Multi-Use Variable-g Platform (MVP) to study PTOA-related pathogenesis and management on the ISS. Methods: Osteochondral plugs (3.5mm diameter and 5mm thick, including 1.5-2mm thick cartilage) harvested from condyles and trochlear grooves of cadaveric human distal femurs (n=8 knees, Collins grade 1/2, 64-82 yr) were co-cultured with 4-5mm diameter synovium explants harvested from synovial joint capsule of the same knees. All procedures were approved by the Rush University Medical Center Institutional Review Board and the Committee on the use of Humans as Experimental Subjects at MIT. PTOA-like mechanical trauma was simulated by subjecting the cartilage surface of the osteochondral plugs to a single injurious unconfined compression of 60% strain rate to a peak stress of 5MPa using an incubator-housed loading apparatus. The study design included the following groups: (1) Mechanical injury (INJ) - injured osteochondral plugs were cultured in presence of pro-inflammatory cytokines (including TNF-α, IL-6 and IL-8) that were released by synovial explants and quantified by multiplex-readout in situ, (2) Treatment group (INJ + DEX): 100nM Dex was added to the cocultures of injured osteochondral plugs and synovial tissues immediately after injury. A preliminary science validation test was performed to identify a suitable mechanism to adapt CBS cocultures to the custom experiment module and the MVP facility. Engineering validation testing was then carried out to build logistical protocols for the spaceflight including transport of cultures and simulation of tasks preceding launch operations. Results: The levels of pro-inflammatory cytokines such as IL-6, IL-8 and TNF-α were upregulated in CBS cocultures. The combination of mechanical injury and increased release of inflammatory cytokines from synovial tissues caused extensive cell death and biochemical changes in both bone and cartilage. In the INJ group, cartilage tissue showed widespread cell death [Fig1 a], and subchondral bone showed increased cell death as well, indicated by numerous empty lacunae in the matrix. Biochemical analysis showed aggravated release of matrix component (including GAGs) into the medium, associated with cartilage degradation [Fig 1c]. Addition of Dex significantly reduced the injury-induced cell death [Fig 1b] and GAG loss by week 2 [Fig 1c]. For spaceflight preparation, tissue cards [Fig 1d] were optimized for long-term holding and maintenance of CBS co-cultures, which were placed into custom culture chambers [Fig 1e]. Multiple tests using this instrumentation were used to guide the design and operation of automated MVP facility [Fig 1f]. Investigation of disease biology and acute injury management is currently being tested in preparation for eventual spaceflight experiments in microgravity aboard ISS-NL. Conclusions: By providing a platform to study cartilage, bone and synovium together, our CBS MPS can potentially enable a more physiologically relevant understanding of tissue interactions that may lead to PTOA pathogenesis. This system can also be used to test the efficacy of therapeutics. In Space, this MPS will be used to explore the role of therapeutic agents to ameliorate cartilage degradation in microgravity to support astronaut health during long space missions.

Effects of All-Trans Retinoic Acid on Lipopolysaccharide-Induced Synovial Explant.

The present study was conducted to assess the effect of all-trans retinoic acid (ATRA) on synovial explants from rats with rheumatoid arthritis (RA) induced by lipopolysaccharides (LPS). In our study, synovial membranes were excised from the knees of healthy adult Wistar female rats under sterile conditions. We first investigated the synoviums incubated in a control medium or in a medium containing 10 μg/mL LPS, each for 24, 48, and 72 h (LPS-experiment). The changes in inflammatory response from the synoviums were observed at different culture times. Then, we assessed the synoviums exposed to different ATRA concentrations for 24 h (ATRA-experiment). The controls (blank, model group, and solvent groups) were set up. The effects of ATRA on synovitis were evaluated by measuring the production of cytokines, and nitric oxide (NO) and the expression of cartilage damage related proteases. In the LPS-experiment, LPS contributed to the release of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and matrix metalloproteinase-9 (MMP-9) in synovial explants. Importantly, LPS did not cause a significant pathological damage. The inflammatory response observed in this model was significant for 24 h, suggesting that LPS-induced synovial explants were successfully established. In the ATRA-experiment, ATRA suppressed the expression of IL-6, TNF-α, NO, a disintegrin and metalloprotease with thrombospondin motifs-4 (ADAMTS-4), MMP-3, and MMP-9. Taken together, ATRA exhibited inhibitory effects on LPS-induced synovial immune inflammatory response stimulated by the regulation of inflammatory mediators and cartilage damage related proteases in synovial explants, demonstrating a potential protective effect on synovitis and joint destruction in the patients with RA.