Objective: Determine the kinetics of collagen crosslinking in adult bovine articular cartilage explants using radiolabel pulse-chase studies.
Methods: Explant cultures of adult bovine articular cartilage were radiolabeled with [14C]lysine in medium including fetal bovine serum and ascorbate, and then maintained for chase periods up to 28 days. In some samples, beta-aminopropionitrile (BAPN) was included during chase to inhibit lysyl oxidase-mediated collagen crosslinking. Tissue was hydrolyzed and analyzed for [14C]metabolites in the forms of lysine, hydroxylysine, dehydrodihydroxylysinonorleucine (DeltaDHLNL), and hydroxylysyl pyridinoline (HP).
Results: Explant cultures of adult bovine articular cartilage metabolized lysine into hydroxylysine and the collagen crosslinks, DeltaDHLNL and HP. During chase, [14C]hydroxylysine maintained steady-state levels, [14C]DHLNL rose to a plateau, and [14C]HP increased gradually. Addition of BAPN inhibited formation of [14C]DHLNL. Analysis of raw data and that normalized to [14C]hydroxylysine gave characteristic time constants for formation of DeltaDHLNL and HP crosslinks of 1-2 and 7-30 days, respectively. The distribution of [14C]lysine metabolites in collagen crosslinks was described by peak values in [14C]DHLNL/[14C]hydroxylysine of 0.047-0.064 and in [14C]HP/[14C]hydroxylysine of 0.03.
Conclusion: Collagen crosslinks form in cartilage explants in vitro according to the classical lysyl oxidase-mediated pathway.
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