Periodontitis is a progressive and destructive disease of the connective tissue around teeth that is caused by gram-negative bacteria and the substances they produce. One such substance, endotoxin, has been shown to activate the inflammatory response in gingival tissue and have direct deleterious effects on the growth and morphology of the connective tissue cells of the periodontium. At present, little is known about the effect of endotoxin on the production of collagenous proteins by these cells. This research project will provide information as to whether endotoxin extracted from periodontally diseased human teeth, or that extracted from Bacteriodes gingivalis, an oral pathogen associated with chronic periodontitis, affect collagen synthesis in cultured gingival fibroblasts. The culture of gingival fibroblasts derived from healthy and inflammed tissue will be employed to isolate and study the direct cellular effects of endotoxin on collagen production from those of the inflammatory response of the host. Two experimental approaches will be used: (1) confluent cultures of gingival fibroblasts will be exposed to endotoxin to determine whether endotoxin directly affects collagen production in nondividing cells, and (2) gingival fibroblasts will be repeatedly subcultured in the presence of endotoxin to determine whether prlonged exposure of proliferating cells alters collagen synthesis in subsequent cell generations. Gingival fibroblasts will be exposed to endotoxin and then incubated with tritiated proline and glycine to label the newly synthesized collagenous proteins. The medium and cells will be analyzed for types I and III collagens and the type I collagen trimer by carboxy methyl cellulose chromatography, gel filtration and SDS-polyacrylamide gel electrophoresis. Because gingival tissue exhibits a high turnover of collagenous proteins and because a unique collagen molecule, the type I trimer, is detectable in diseased gingiva, cultured gingival fibroblasts provide an excellent model for studying the effects of endotoxin on the ability of these cells to maintain and repair gingival tissue.
