Specific Collagenase From Germ-Free Rat Gingivae

A collagenase from a vertebrate source was first described from cultured tadpole tails by Gross and Lapiere (1), and Lapiere and Gross (2). Soon thereafter, collagenase was detected in culture fluids of gingivae from man (3–8), and from the guinea pig (8). The specific collagenase was subsequently detected in culture fluids of human epithelial and connective tissue gingival fragments cultured separately (9). The present investigation discloses the presence of a specific collagenase in culture fluids of gingivae from germ-free as well as conventional rats. This study provides additional proof of a tissue source of collagenase.

Materials and Methods. Free and attached gingivae surrounding molar and incisor teeth of 30-day-old Sprague-Dawley rats (six germ-free and six conventional) were excised, diced into 2- to 3-mm² pieces, cleansed in several changes of Tyrode's solution with antibiotics and cultured in either (1) Tyrode's solution with antibiotics on the surface of reconstituted collagen gels, or (2) Tyrode's solution with antibiotics as described previously (3, 9). All were cultured in 5% CO₂ in air at 37° for 7 days. Cultures on collagen gels were not disturbed for the 7-day period. Culture fluids from the others were withdrawn and replenished daily.

Reconstituted collagen was prepared from rat skins according to the method of Kang et al. (10). Purity and native helicity of the collagen was determined by optical rotation and resistance to tryptic digestion. Reaction mixtures containing 0.5 μ trypsin per microgram collagen never exceeded 5% digestion.