Kinetics of collagen crosslinking in adult bovine articular cartilage

SummaryObjectiveDetermine the kinetics of collagen crosslinking in adult bovine articular cartilage explants using radiolabel pulse-chase studies.MethodsExplant 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, β-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 (ΔDHLNL), and hydroxylysyl pyridinoline (HP).ResultsExplant cultures of adult bovine articular cartilage metabolized lysine into hydroxylysine and the collagen crosslinks, ΔDHLNL 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 ΔDHLNL 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.ConclusionCollagen crosslinks form in cartilage explants in vitro according to the classical lysyl oxidase-mediated pathway