In situ localization of gelatinolytic activity during development and resorption of Meckel's cartilage in mice

Degradation of Meckel's cartilage in the middle portion is accompanied by hypertrophy and death of chondrocytes, calcification of the cartilaginous matrix, and chondroclastic resorption. We hypothesize that the gelatinolytic activity of matrix metalloproteinases (MMPs) largely contributes to the degradation of extracellular matrix (ECM) in the process. The activity in Meckel's cartilage of mouse mandibular arches at embryonic days 14-16 (E14-E16) was examined by a combination of in situ zymography (ISZ), using quenched fluorescent dye-labeled gelatin as a substrate, with CTT (a selective inhibitor of MMP-2 and -9) or with EDTA (a general MMP inhibitor). On E14 and E15, ISZ showed fluorescence in the perichondrium, in the intercellular septa between chondrocytes, and in the nucleus of chondrocytes. CTT attenuated fluorescence, and EDTA eliminated it. On E16, calcified cartilaginous matrix showed intense fluorescence, and dot-like fluorescence was observed in as-yet uncalcified intercellular septa, even after CTT treatment. EDTA inhibited fluorescence, but unexpectedly intense fluorescence was found in the cytoplasm of hypertrophic chondrocytes facing the resorption front. MMP-2, -9, and -13 immunoreactivity was detected in the perichondrium and chondrocytes of Meckel's cartilage. These findings suggest that MMPs and other proteinases capable of degrading gelatin play an integral role in the development, calcification, and resorption of Meckel's cartilage through ECM reconstitution.