Kinetics of collagen crosslinking in adult bovine articular cartilage.
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.


CONCLUSIONS
Collagen crosslinks form in cartilage explants in vitro according to the classical lysyl oxidase-mediated pathway.
Ahsan
T
T
Department of Bioengineering, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Harwood
F
F
McGowan
K B
KB
Amiel
D
D
Sah
R L
RL
D000596:Amino Acids / Q000378:metabolism; D000629:Aminopropionitrile / Q000378:metabolism; D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism*; D002417:Cattle; D003094:Collagen / Q000493:pharmacokinetics*; D003432:Cross-Linking Reagents / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D006901:Hydroxylysine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism
D000596:Amino Acids; D003432:Cross-Linking Reagents; D004151:Dipeptides; D000629:Aminopropionitrile; D006901:Hydroxylysine; C015484:pyridinoline; D003094:Collagen; D008239:Lysine