Immunohistochemical staining and electron microscopy. (a, e, i) Fresh, non-cultured bovine cartilage and (b, f) human osteoarthritis (OA) cartilage, as well as (c1 to c4, g1 to g4, j1 to j4) bovine cartilage from monocultures or (d1 to d4, h1 to h4, k1 to k4) co-cultures with synovial fibroblast (SFB) after 14 days are shown. Immunostaining for cartilage oligomeric matrix protein (COMP) clearly reveals a (c1 to c4) strong correlation between the appearance/detection of COMP within the cartilage matrix and the stimulation with TNF-α, IL-1β and TNF-α/IL-1β, (d1 to d4) which is dramatically augmented by the co-culture with SFB. (a) Fresh, non-cultured bovine cartilage and (c1) non-stimulated cartilage monocultures do not stain for COMP; in contrast, (b) human OA cartilage shows a positive staining for COMP. (g1 to h4) Immunostaining for the collagen cleavage neo-epitope Col2-3/4C-(short) demonstrates the matrix-degrading capacity of SFB and the amplifying impact of TNF-α, IL-1β and TNF-α/IL-1β on this process. (e) Whereas fresh, non-cultured bovine cartilage lacks signs of collagen cleavage, (f) human OA cartilage exhibits positive staining for the neoepitope. (i to k4) Transmission electron microscopy confirmed the immunohistologically detected collagen breakdown by a decreased optical density of collagen fibres (the dotted line indicates the cartilage surface or the interface between the cartilage and the co-cultured SFB). Magnifications in (a to h4) 200×; inserts 630×; (i to k4) 39,000×.