Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats aged 0-21 days: II. Two proteinases, gelatinase B and collagenase-3, are implicated in the lysis of collagen fibrils

In the transformation of the cartilaginous epiphysis into bone, the first i ndication of change in the surfaces destined for resorption is the cleavage of aggrecan core protein by unidentified matrix metalloproteinases (MMPs) (Lee et al., this issue). In cartilage areas undergoing resorption, the cle avage leaves as superficial, 6-mum-thick band of matrix, referred to as "pr e-resorptive layer." This layer harbors G1-fragments of the aggrecan core p rotein within a framework of collagen-rich fibrils exhibiting various stage s of degeneration. Investigation of this layer in every resorption area by gelatin histozymography and TIMP-2 histochemistry demonstrates the presence of an MMP whose histozymographic activity is inhibited by such a low dose of the inhibitor CT1746 as to identify it as gelatinase A or B. Attempts at blocking the histozymographic reactions with neutralizing antibodies capab le of inhibiting either gelatinase A or B reveals that only those against g elatinase B do so. Immunostaining of sections with anti-gelatinase B IgG co nfirms the presence of gelatinase B in every pre-resorptive layer, that is, at the blind end of excavated canals (stage I; 6-day-old rats), at sites a long the walls of the forming marrow space (stage II; 7 days), at sites wit hin the walls of this space as it becomes the ossification center (stage II I; 9 days) and along the wall of the maturing center (stage IV, 10-21 days) . We also report the presence of collagenase-3 in precisely the same sites, possibly as active enzyme, but this remains to be proven. Because the resu lts reveal that collagenase-3 is present beside gelatinase B in every pre-r esorptive layer and, because these sites exhibit various signs of degradati on including fibrillar debris, reduction in fibril number, or overt loss, w e propose that gelatinase B and collagenase-3 mediate the lysis of this pre -resorptive layer-most likely through a cooperative attack leading to the d isintegration of the collagen fibril framework. (C) 2001 Wiley-Liss, Inc.