The goal of this research is to characterize pellicle-bacterial interactions on the cementum surface in vitro. There are three main stages in dental plaque formation: (1) specific adsorption of salivary macromolecules on the tooth surface; (2) initial adhesion of bacteria; (3) proliferation and accumulation of bacteria. These tooth-pellicle-bacterial interactions play a pivotal role in determining the ultimate microflora of plaque and consequently its potential to induce dental diseases. Some of the molecular mechanisms governing microbial colonization of the enamel surface have been described. In sharp contrast, very little is known concerning the process by which dental plaque forms on the cementum surface. Our initial experiments were designed to demonstrate the feasibility of studying the first two stages of this process in vitro: formation of the cementum pellicle, and subsequently bacterial adherence to that pellicle. First, we have found that unique salivary components are adsorbed to the cementum surface. Second, the adhesion characteristics of clinical isolates of several oral bacteria to the saliva-coated cemental slabs were described. The goal of the proposed experiments is to investigate the composition of the cementum pellicle and the mechanism of attachment of various bacteria to that pellicle, comparing and contrasting these events to those which occur on the enamel surface. Specifically, we will: 1) Investigate various methods of production of cementum pellicle in vitro by varying the following parameters: a) bovine cementum slabs prepared by mechanical cleaning or by chemical extraction; b) incubation of cleaned slabs with various saliva and/or plasma preparations; c) removal of resulting pellicle by mechanical or by chemical means. 2) Characterize the composition of the in vitro cementum pellicle by: a) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of externally-labeled pellicle glycoproteins; b) identifying certain pellicle components by their reaction with specific antisera; c) determining a structural profile of pellicle oligosaccharides using gas-liquid chromatography (g.l.c.) and combined g.l.c.-mass spectrometry; d) determining the structural classes of major lipids. 3) Investigate bacterial adherence to root surfaces in vitro by: a) determining the binding characteristics of various labeled bacteria to the cementum surface; b) determining the structural specificity of the adhesive process using either chemically or enzymatically modified pellicle or competition experiments with known pellicle constituents. Further knowledge of the molecular mechanisms of host-parasite adherent interactions may allow for the development of agents that control bacterial infections either by interfering with bacterial attachment or by promoting bacterial desorption.
