Objective: To determine the contribution of certain matrix metalloproteinases (MMPs) to the degradation of proteoglycan and type II collagen in cartilage.
Methods: Bovine nasal and articular cartilage explants were cultured with recombinant human interleukin-1 alpha (IL-1 alpha) for up to 4 weeks. Release of proteoglycan and type II collagen into the medium was determined by colorimetric assay and immunoassay, respectively. The activity of MMPs in the medium was assayed using a quenched fluorescent substrate, as well as with a collagen fibril assay, by zymography, and in Western immunoblots. In some experiments, the effects of specific MMP inhibitors on type II collagen degradation were studied.
Results: In cultures of nasal cartilage with IL-1 alpha, almost all the proteoglycan was released within the first week, whereas there was no detectable release of type II collagen for the first 2 weeks of culture. A rapid period of almost complete dissolution of the collagen occurred in the third or fourth week. MMP activity measured using a quenched fluorescent substrate was negligible during the first 2 weeks of culture but was substantially increased in the third week of culture, at the time of collagen degradation. Similarly, there was a large increase in collagenolytic activity (by collagen fibril assay) and gelatinolytic activity (by zymography) during the third week of culture. Articular cartilage cultured with IL-1 alpha lost proteoglycan progressively during the 4-week period; however, there was no loss of type II collagen from the matrix in that time and no significant increase in MMP activity. The loss of type II collagen from nasal cartilage stimulated with IL-1 alpha was inhibited by BB87, an inhibitor of both collagenases and gelatinases, and by BB3003, a selective inhibitor of gelatinase A. In Western immunoblots, procollagenase and active interstitial collagenase could be readily detected in nasal cartilage cultures. Some procollagenase 3 and active collagenase 3 was also shown to be present.
Conclusion: MMP activity correlates with degradation of type II collagen, but not proteoglycan, in cartilage cultures. Interstitial collagenase, collagenase 3, and gelatinases are all likely to contribute to cleavage and removal of collagen from the cartilage matrix. The proteinase(s) responsible for aggrecan breakdown remains unclear.