We previously identified diabetes-induced alterations in collagen metabolism and in the gingival response to inflammation. We now propose to focus on the alterations in collagen degradation by carrying out three groups of studies: (I) To determine whether diabetes in rats (and, in some experiments, guinea pigs) stimulates the breakdown of collagen precursors as well as extracellular collagen fibrils (the latter effect was identified by us previously). Tissues from control (C) and diabetic (D) rats will be incubated in vitro with 14C-proline to assess the intracellular breakdown of newly synthesized radiolabeled collagen; procollagen degradation will be assessed using the techniques of SDS-polyacrylamide gel electrophoresis and fluorography. Relevant to the effect on degradation, we will also determine (a) whether diabetes affects the hydroxylation and the helical stability of collagen synthesized in vitro, and (b) whether increasing the abnormally low tissue ascorbic acid levels in the diabetic animal normalizes the enhanced degradation of newly synthesized collagen in vivo; (II) To determine whether the enhanced gingival collagenase and impaired crevicular PMNL chemotactic activities in the diabetic rat (identified by us previously) is mediated, in part, by an overgrowth of Gram-negative organisms in the gingival crevice. These abnormalities will be studied in germfree C or D rats and germfree C or D rats mono-infected with various Gram-positive or Gram-negative organisms (including several periodontopathogens). Gingival tissue from these animals will be assayed for collagenase activity by measuring the breakdown of 14C-labeled collagen fibrils in tissue culture, and by extracting the enzyme and measuring 14C-collagen breakdown in vitro. The chemotactic responsiveness of the PMNLs in the gingival crevice of the different groups of rats will be assayed using our previously published in vivo techniques. (III) To determine whether treatment with minocycline, and antibiotic effective against oral Gram-negative organisms, decreases the abnormally elevated collagenase activity in gingiva of diabetic rats and humans (tissues from C's will also be studied). A preliminary study suggests this possibility and that the drug's effectiveness may not be solely dependent on its antibacterial efficacy. Our long term objectives are to continue identifying pathologic mechanisms involvedin the interaction between diabetes and periodontal disease and to identify therapeutic approaches targeted specifically at these abnormalities.
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