Figure 3

Sphingomyelinase increases de novo sGAG and collagen synthesis in articular chondrocytes. Bovine articular chondrocytes were cultured for 7 days with 20 μCi/ml [³H]-proline and 10 μCi/ml [³⁵S]-sulphate in the presence or absence of SMase (0.1 U/ml). At the end of the culture period, unincorporated label was removed and (a) [³⁵S] counts (cpm) were measured in cell associated material and media as a measure of de novo sGAG. De novo collagen synthesis was determined by digesting labelled protein in media and cell extracts with 8U bacterial collagenase overnight at 37°C. Digested collagen fragments were removed using Ultrafree^®-MC filter units and remaining [³H] counts taken as a measure of noncollagenous protein. (b) Collagenous protein was calculated using the following equation: collagen (cpm) = total protein ([³H] counts before collagenase digestion) – noncollagenous protein (counts remaining after collagenase digestion). Data are normalized to cell number and presented as mean ± standard error. * P < 0.05 versus control. cpm, counts/min; sGAG, sulphated glycosaminoglycan.