Reproduced Chondrons Maintain Chondrocyte Phenotype and Promote Inflammatory Response in a 3d Osteoarthritic Cartilage Model

Chondrocytes are surrounded by a distinct thin layer of pericellular matrix (PCM), which differs from the extracellular matrix (ECM) in both content and properties. The combination of chondrocytes and the pericellular matrix is known as a chondron, which is the smallest functional unit of cartilage tissue. Changes in the PCM are often observed before those in the ECM during osteoarthritis (OA). However, there has been limited research on the role of chondrons and PCM in regulating chondrocyte phenotype and inflammatory response in OA. To develop a 3D osteoarthritic cartilage model by 3D culture of reproduced chondrons. Human OA chondrocytes were isolated from OA femoral head and expanded in monolayer. To obtain the reproduced chondrons, P1 chondrocytes were seed into alginate beads (1.2%) and treated with chondrogenic media (10 ng/mL TGFβ) for promoting the production of pericellular matrix. After two weeks of chondrogenesis, the alginate beads were dissolved and the reproduced chondrons were obtained. The chondrons were characterised by immunofluorescence of collagen VI under confocal microscopy. Then the P1 chondrocytes and the reproduced chondrons were seeded into fibrin gel /polyurethane (PU) scaffolds separately and followed by treatment with/without 10ng/mL IL1β for 7 days. Chondrogenic markers (COL2, ACAN) and inflammatory markers (IL6, IL8, COX2) were tested by RT-qPCR and cell morphology was evaluated by histology. Immunofluorescence of collagen VI showed that a thin layer of pericellular matrix was produced after 2 weeks of chondrogenic stimulation. Different from the fibroblast-like chondrocytes in PU scaffolds, the chondrons in PU scaffolds better reflected the physical chondrocyte morphology, indicated by typical double cell clusters surrounded by pericellular matrix forming chondrons under Safranin-O staining (Fig. 1A). The reproduced chondrons in PU scaffolds had significantly elevated COL2 and ACAN expression compared to chondrocytes in PU scaffolds. The expression of inflammatory markers, COX2 and IL8, increased significantly in reproduced chondron scaffolds and chondrocytes seeded scaffolds upon treatment of IL1β, and COX2 showed a higher expression level in chondrons compared with chondrocytes under IL1β treatment. Compared to single cells, reproduced chondrons cultured in 3D provided superior physiological relevance in terms of chondrocyte cell morphology and response to inflammatory OA-like conditions. Our model suggests a promising tool for studying the role of the pericellular matrix in cartilage degradation and inflammation during osteoarthritis progression, offering insights into potential therapeutic targets for OA management. For any figures or tables, please contact the authors directly.