The damage to
cartilage collagen is a central event in the pathogenesis of cartilage aging and
osteoarthritis (OA). We have previously developed an in vitro model of cartilage degradation which shows that chondrocyte-dependent lipid peroxidation mediates
cartilage collagen degradation. The goal of our study was to investigate the role of
vitamin C in this degradation model and to investigate effect of chondrocyte-dependent lipid peroxidation in the oxidation of
cartilage collagen.
METHODS: We studied primary articular chondrocytes. Effect of
vitamin C was investigated in the previously described model. Serum-free stimulated and unstimulated chondrocyte-matrix extracts were subjected to SDS-PAGE and immunoblot analysis.
Malondialdehyde (MDA)-
protein oxidation of cartilage
proteins was demonstrated by the reactivity of chondrocyte extracts to a
monoclonal antibody, MDA2, which detects MDA-
lysine adducts.
RESULTS:
Vitamin C treatment of chondrocyte cultures resulted in significant enhanced incorporation of 3H-proline label in cell-matrix. Cells treated with
vitamin C, as compared to control untreated cells showed decreased spontaneous release of labeled matrix.
Vitamin C treated or not treated chondrocytes responded comparably to stimulation with the agonist
calcium ionophore A23187. The serum-free in vitro culture of chondrocytes resulted in MDA-
protein oxidation. The treatment of chondrocytes with
A23187 resulted in the enhancement of MDA-
protein oxidation. The immunoblot reactivity pattern of extracts to MDA2 antibody and to polyclonal anti-
type II collagen antibody was somewhat similar, which suggests that these two different types of
antisera exhibit a crossreaction to chondrocyte
proteins. Chondrocyte extracts were pretreated both with and without pure
collagenase, and then subjected to immunoblot analysis. Only
collagenase treated extracts showed a disappearance, or significant reduction, of larger than 60 kDa size MDA2 immunoreactive
proteins. This suggests that the
proteins that disappeared after the
enzyme treatment were
collagen proteins and which had also been modified by MDA oxidation.
CONCLUSIONS: These observations suggest that
collagen hydroxylation of matrix by
vitamin C does not play a role in this model of chondrocyte-dependent
collagen degradation. Also, this study demonstrates that chondrocyte-derived lipid peroxidation product MDA mediates oxidation of cartilage
collagens. Oxidative modification of
cartilage collagen in vivo could result in alteration of biochemical and biophysical properties of
cartilage collagen fibrils, making them prone to degradation, thus initiating the changes observed in aging and OA.