Original Article
Manal Yaya, MSc, Joseph Bird,P
Abstract
OBJECTIVE: The aim of this research is to characterize the most appropriate concentration of Foetalbovine serum [FBS] for culture maintenance, and determine the apoptosis rate in the culture system, and also to investigate the induction of proteoglycan catabolism and the role of nitric oxide [NO] on proteoglycans [PG] loss. Furthermore it also aims to study the involvement of specific nitric oxide signaling pathways and to determine the involvement of matrix metalloproteinase [MMPs] in the degradation of proteoglycans. METHODS: Proteoglycan degradation was investigated with 1 μmol/L retinoic acid [RA] and NO donor Diethylenetriamine DETA-NONOate in bovine cartilage explant cultures. Signaling pathways were investigated using specific inhibitors. Nitrite, an end product of NO metabolism, was measured in media by the Griess reaction. Matrix metalloproteinase [MMP] activity was investigated by gelatinase zymography and chondrocyte apoptosis in cultures was assessed using active caspase-3 immunohistochemistry. RESULTS: Low rates of apoptosis were identified relative to positive control samples. Retinoic acid at 1μmol/L caused a significant increase in proteoglycan loss and this effect was completely reversed in the presence of NO. The zymography results did not show a difference in MMP activity. It was also found that p38 and p42/44 MAPKs, ROCK and soluble guanylate cyclase activities were involved in RA induced PG loss but not in NO mediated anti-catabolic. CONCLUSION: This study demonstrates that 1% FBS is the most appropriate concentration to study PG loss in bovine culture model. In our culture system there are likely to be low levels of apoptosis due to the tissue culturing procedure and PG loss in RA-induced cultures is mediated by proteinases other than MMPs.