Suitability of collagenase treatment for the radioautographic identification of newly synthesized collagen labeled with 3H-glycine or 3H-proline.

Abstract

Sections of Carnoy-fixed tissues from young rats and adult mice were incubated for 5 hr at 37°C in a 0.1% solution of Clostridium histolyticum collagenase purified by ion exchange chromatography. This treatment causes collagenous fibers as well as dentinal and bone matrices to lose their ability to stain red with the van Gieson technique and pink with eosin. In view of the high specificity of the enzyme, it is assumed that the stained component of these tissues is collagen. Immediately after injection of 3H-glycine or 3H-proline, radioautography shows the label in the cytoplasm of several cell types: osteoblasts of femur and alveolar bone, odontoblasts of growing molar and incisor teeth. and fibroblasts of the periodontal membrane. Later, the label appears outside the cells, that is, in bone matrix, dentinal matrix and intercellular spaces of periodontal membrane, respectively. Collagenase treatment extracts about half of the label which is present in the cells soon after injection, all or nearly all the label which appears later in bone and dentinal matrix, but only a fraction of that which appears in the intercellular spaces of the periodontal membrane. It is concluded that the radioactivity which occurs in osteoblasts, odontoblasts and some fibroblasts soon after injection of 3H-glycine and 3H-proline is composed in about equal parts of collagen and of other protein(s). In bone and dentinal matrix collagen alone seems to be deposited, whereas in the periodontal membrane collagen is laid down in association with other protein(s).