A new ex vivo human model of osteoarthritis cartilage calcification

Abstract

Objective: Cartilage pathologic calcification is a hallmark of osteoarthritis (OA). Here, we aimed to describe a new ex vivo human model to study the progression of cartilage calcification. Method: Cartilage explants (n ¼ 11), as well as primary chondrocytes (n ¼ 3), were obtained from OA patients undergoing knee replacement. Explants and chondrocytes were cultured in control (NT) or calcification (CM) medium (supplemented with ascorbic acid and b-glycerophos-phate). Calcification was evaluated by micro-CT scan at day 0 and 21 in explants, and by Alizarin red staining in chondrocyte monolayers. Raman spectrometry allowed characterization of the crystal type. Interleukin-6 (IL-6) secretion in explant and cell supernatants was measured by ELISA. Finally, matrix degradation was evaluated by Safranin-O staining of explant sections and by glycosaminoglycans (GAG) released in supernatants. Results: Micro-CT scan showed calcifications in all explants at baseline (day 0), which in the CM group increased significantly in number and size after 21 days compared with the NT group. Raman spectrometry revealed that crystals were exclusively basic calcium phosphate crystals (carbonated hydroxyapatite) both in NT and CM. IL-6 secretion was significantly increased in calcifying conditions. Finally, CM significantly increased cartilage catabolism as assessed by decreased Safranin-O staining of tissue explants and increased GAG release in supernatants. CM effects (enhanced calcification, IL-6 secretion and proteoglycans turnover) were recapitulated in vitro in OA chondrocytes. Conclusions: We have described a new ex vivo human model of cartilage calcification that can summarize the triad of events seen during oste-oarthritis progression, i.e. calcification, inflammation and cartilage degradation. This model will allow the identification of new anti-calcification compounds.