The biological role of Dental integuments has been the focus of interest ever since it was recognized that it represents the site where bacterial attachment initiates Dental plaque formation. The formation of plaque plays a pivotal role in oral pathogenesis since it leads to development of caries and gingivitis/periodontitis, the two major oral diseases. Plaque, or oral biofilm, is formed by a selective attachment and growth of oral microorganisms on a proteinaceous film called the acquired enamel pellicle which is tightly adhering to the mineral surface of teeth. The biological and clinical importance of this structure is emphasized by playing a dual role. The acquired enamel pellicle modulates the mineral homeostasis in de- and de-mineralization processes of tooth surfaces and dictates the initial interactions between the outer pellicle surface and the early bacterial colonizer of the biofilm. The focus of this project is to characterize structurally and functionally the components which constitute the acquired enamel pellicle. The overall goal of the studies proposed is to elucidate the biology of pellicle and to generate results which may lead to therapeutic means for the protection of host tissues and prevention of oral diseases. We will combine nanoscale methods with state-of-the-art electrophoretic and proteomics technologies for protein characterization and will seek to establish for the first time the interplay between pellicle and biofilm in a variety of in vivo conditions including those of health and disease.
The Specific Aims are to: 1) Identify and characterize in vivo formed acquired enamel pellicle components by proteomics approaches using several MudPIT modifications, 2-DE PAGE, and various chromatographic micro-separations to obtain components which will be analyzed prior and after fragmentation with trypsin and other proteolytic enzymes. 2) Investigate the consistency of individual protein/peptide profiles of pellicle and whole saliva samples formed in vivo over time, and to determine differences and similarities in the protein profiles of pellicle and whole saliva of the same subject using 2-DE PAGE and 2-D DICE (Differential In Gel Electrophoresis). 3) Determine the hydroxyapatite binding characteristics of pure pellicle components in binary and multi-component models and to evaluate the effect of pellicle proteins on enamel demineralization. 4) Investigate the relationship between pellicle protein profiles and bacterial colonizers present on tooth surfaces during the early phase of biofilm formation in healthy subjects, gingivitis patients, and caries-free and caries-active children using 2-DE and checkerboard DNA-DNA hybridization.
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