S100A8 and S100A9 are increased in synovial fluid and expressed by chondrocytes in patients with osteoarthritis (OA), however their role remains unclear. S100A9^-/- mice are protected from cartilage degradation in more inflammatory arthritis models but not post-traumatic OA. We previously demonstrated that S100A8 and S100A9 “prime” cartilage for degradation by up-regulating catabolic genes; but alone they do not induce activation of these enzymes or cartilage breakdown. We hypothesised that a second signal was required to work in synergy with S100A8 and/or S100A9 to cause OA-like cartilage degradation. The aim of this study was to determine if cartilage degradation was induced by S100A8 or S100A9 in co-culture with a second joint tissue, synovium. Ovine articular cartilage explants were co-cultured with synovial explants for 4 days ± S100A8 or S100A9 (n=6), and analysed for gene expression changes, enzyme activation and release of major cartilage matrix components collagen and aggrecan. There was no change in effect of S100A8 on cartilage ± synovium. In contrast there was an additive effect in co-cultures treated with S100A9 compared to non-treated co-cultures and S100A9-treated cartilage, with up-regulation of MMP-2 (p=0.0163), a key enzyme involved in cartilage breakdown; and down-regulation of TIMP-3 (p=0.025), an inhibitor of MMPs. Addition of S100A9 to co-cultures also increased MMP-9 and -13 activation and aggrecan release from cartilage compared to non-treated co-cultures. The results from this study show that while addition of S100A8 or S100A9 to isolated cartilage is pro-catabolic it is not sufficient to induce degradation. However in the presence of synovium, S100A9 induces enzyme activation and aggrecan breakdown, and is therefore a potential target for OA treatment. This study highlights the importance of a global approach to studying OA, as potential therapies may have differing effects in the presence of multiple tissues that contribute to global OA joint pathology.