Toward understanding the role of cartilage particulates in synovial inflammation

Abstract

Objective Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with osteoarthritis (OA). Here, we developed an in vitro model to study the interaction of cartilage wear particles with fibroblast-like synoviocytes (FLS) to better understand the interplay of cartilage particulates with cytokines on cells of the synovium. Method In this study sub-10μm cartilage particles or 1μm latex particles were co-cultured with FLS ± 10 ng/mL interleukin-1α (IL-1 α) or tumor necrosis factor- α (TNF-α). Samples were analyzed for DNA, glycosaminoglycan (GAG), and collagen and media samples were analyzed for media GAG, nitric oxide (NO) and prostaglandin-E2 (PGE2). The nature of the physical interaction between the particles and FLS was determined by microscopy. Results Both latex and cartilage particles could be phagocytosed by FLS. Cartilage particles were internalized and attached to the surface of both dense monolayers and individual cells. Co-culture of FLS with cartilage particulates resulted in a significant increase in cell sheet DNA and collagen content as well as NO and PGE2 synthesis compared to control and latex treated groups. Conclusion The proliferative response of FLS to cartilage wear particles resulted in an overall increase in ECM content, analogous to the thickening of the synovial lining observed in OA patients. Understanding how cartilage particles interface with the synovium may provide insight into how this interaction contributes to OA progression and may guide the role of lavage and synovectomy for degenerative disease.

Cartilage Wear Particles Induce an Inflammatory Response Similar to Cytokines in Human Fibroblast‐Like Synoviocytes

“…In lapine, canine, and equine in vivo studies, injection of cartilage particles into the knee joint led to rapid development of synovitis followed by gradual onset of fibrotic synovium thickening and decreased cartilage thickness, similar to traditional animal models of OA such as ACL transection or meniscal release . Our group has also demonstrated that small CWP (<10 µm diameter) both attach to the cell membrane and are phagocytosed, and stimulate proteinase activity, cellular proliferation, collagen synthesis, and nitric oxide production in bovine FLS monolayer cultures …”

“…Cells were expanded in α‐minimum essential medium (α‐MEM; Life Technologies, Carlsbad, CA) containing 10% fetal bovine serum, 1% antibiotic–antimycotic and 5 ng/ml fibroblast growth factor‐2 (Life Technologies) to obtain a homogenous FLS population of sufficient quantity. To create dense synovial monolayers, FLS at passage 3 were plated at 1.25 × 10 4 cell/cm 2 and cultured as previously described . Flow cytometry was performed as previously described to confirm that isolated cells maintained the characteristic FLS phenotype …”

“…Expired cartilage tissue grafts were obtained from the Musculoskeletal Transplant Foundation (Edison, NJ) and stored at 4°C until use. As described previously, cartilage samples were submerged in sterile phosphate‐buffered saline (PBS) and manually abraded with waterproof 120 grit sandpaper (McMaster‐Carr, Elmhurst, IL). Effort was made to ensure that no bone was abraded during the process, and residual sandpaper grit was removed gravimetrically and via subsequent filtration with 70, 40, and 10 μm porous nylon mesh filters to achieve a pure dispersion of sub‐10 μm diameter CWP.…”

“…Concentrations of up to 6.25 × 10 6 CWP/ml were cultured for 6 days with confluent FLS monolayers isolated from four different OA donors to determine a dosage that would result in a measurable cell response. Using the MTT assay (Sigma‐Aldrich, St. Louis, MO) as previously described, the lowest particle dose that significantly increased metabolic activity was identified for each donor. From these results, 3.125 × 10 6 CWP/ml was selected for future experiments to ensure that as many donors as possible responded to the treatment.…”

Fibroblast-like synoviocyte mechanosensitivity to fluid shear is modulated by interleukin-1α

“…Cells were cultured in αMEM containing 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 5 ng/mL FGF (Life Technologies) for two passages to obtain a pure population of FLS (Sampat, O’Connell et al 2011, Silverstein, Stefani et al 2017). …”