Experimental setup of the in vitro model and histological characterization of the cartilage co-cultured with SFB. Upper panel: Embedding of cartilage and subsequent co-culture with synovial fibroblasts (SFB). (a) Hot 2% agarose was filled in each well of a 48-well plate and (b) a cylinder was created in the agarose by inserting a metal pin plate and removing the plate after polymerisation. (c-d) Subsequently cartilage disc were embedded in the preformed cylinder and pre-cultured for two days. (e) The SFB suspension was then applied and (f) left for three hours for settling and initial adherence of the SFB on the cartilage surface. Finally, (g-h) co-culture medium was carefully added into the upper well compartment. Middle panel: Experimental setup. Cultures were maintained for two weeks, medium was replaced every two to three days, and supernatants were collected and subjected to protein analysis. Cultured constructs were either further processed for histological evaluation and quantification of cartilage oligomeric matrix protein (COMP) content in cartilage or used for gene expression analysis of SFB and cartilage. Lower panel: Histological and immunohistochemical staining of cartilage co-cultures with SFB after 14 days of in vitro culture. (a, b) H&E staining demonstrates the formation of a SFB multilayer on the cartilage surface. (c) Immunostaining for prolyl-4-hydroxylase verifies the viability of SFB and chondrocytes and (d) immunostaining for human Thy-1 proves the fibroblast origin of the co-cultivated cells. Magnification (a) 40×, (b) 630× and (c, d) 400×.