Chondrocytes are enveloped within the pericellular matrix (PCM), a structurally intricate network primarily demarcated by the presence of collagen type VI microfibrils and perlecan, resembling a protective cocoon. The PCM serves pivotal functions in facilitating cell mechanoprotection and mechanotransduction. The progression of osteoarthritis (OA) is associated with alterations in the spatial arrangement of chondrocytes, transitioning from single strings to double strings, small clusters, and eventually coalescing into large clusters in advanced OA stages. Changes in cellular patters coincide with structural degradation of the PCM and loss of biomechanical properties. Here, we systematically studied matrix metalloproteinases (MMPs), their distribution, activity, and involvement in PCM destruction, utilizing chondrocyte arrangement as an OA biomarker. Cartilage specimens were obtained from 149 osteoarthritis (OA) patients, and selected based on the predominant spatial pattern of chondrocytes. Immunoassays were employed to screen for the presence of various MMPs (-1, -2, -3, -7, -8, -9, -10, -12, -13). Subsequently, the presence and activity of elevated MMPs were further investigated through immunolabeling, western blots and zymograms. Enzymatic assays were utilized to demonstrate the direct involvement of the targeted MMPs in the PCM destruction.Introduction
Methods
Osteoarthritis (OA) often results from joint misloading, which affects chondrocyte calcium signaling through mechano-sensitive receptors such as Piezo1, -2, and TRPV4. Activation of Piezo1, especially under inflammatory conditions, can trigger premature chondrocyte apoptosis. Intra-articular glucocorticoid therapy, while beneficial against inflammation and pain in osteoarthritis, may induce oxidative stress and chondrotoxicity at higher doses. This study aims to assess the effects of glucocorticoids, particularly triamcinolone, on chondrocyte elasticity and mechanosignaling. Chondrocytes isolated from articular condyles obtained from patients undergoing knee replacement surgery (n= 5) were cultured for 7 days in triamcinolone acetonide (TA) at different concentrations (0.2µM – 2mM). Cytoskeletal changes were assessed by F-actin labeling. Cell elasticity was measured using atomic force microscopy (AFM). Labeling cells (n=6 patients) with the calcium-sensitive dye (Fluo-4) enabled monitoring changes in intracellular calcium fluorescence intensity during guided single-cell mechanical indentation (500 nN) by AFM.Introduction
Method
