Proteomic evaluation of saliva pellicle on restorative material surfaces

Abstract

The salivary conditioning film, namely salivary pellicle, is a thin proteinaceous film that forms on both natural and artificial dental hard surfaces when exposed to saliva. Among all the biological functions that salivary pellicle possesses, the ability to assist the attachment of bacteria has aroused much attention in recent decades, as biofilm‐associated oral bacterial infections have created great challenges to oral health professions. This study focused on characterizing and profiling salivary pellicle proteins on different restorative material surfaces, as well as to explore their possible relationship with the bacterial infection process.

In order to explore the pellicle characteristics on the natural tooth surface, in the first part of the study, saliva samples were collected from five healthy donors, (three female/two male, aged 24-33), filter sterilized, from which the two-hour salivary pellicle protein adsorption on dentine surfaces was characterized. For in vitro pellicle formation, dentine specimens were polished and characterized regarding surface roughness, surface morphology, surface free energy, and surface potential. After pellicle formation, the adsorbed protein quantity and content were examined using bicinchoninic acid (BCA) assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Three hundred and ninety-eight proteins were identified; the protein quantity on each specimen (approx.70mm2) was around 2.74 ± 0.35 μg. Within the surface protein detect, proteins that previously identified responsible for lubrication and calcium ion homeostasis were detected in the in vitro formed tooth pellicle. Also, both independent antibacterial proteins/peptides and proteins that may involve in immune processes cascades were detected.

To acquire better insight into the salivary protein adsorption on direct restorative material surfaces, in the second study, the salivary pellicle proteome was further profiled on commercially available dental restorative materials [resin composite (RC), resin-modified glass ionomer cement (R-GIC), conventional glass ionomer cement (GIC) and GIC containing casein phosphopeptide - amorphous calcium phosphate (CPP-ACP)], using saliva samples collected from the same five donors. A total of 553 proteins were identified from this study. There were variations among acquired pellicle protein quantity and in a subtle fashion quality on various material surfaces, with RC surfaces possessing the highest quantity (5.74 ± 1.21μg /block, p<0.001) of proteins. Apparently, more protein varieties (n=441) were observed on R-GIC surfaces than the other restorative materials (GIC, n =360; CPP-ACP GIC, n=349; RC, n=425). According to protein classification, nucleic acid binding proteins (PC00171) and enzyme modulator proteins (PC00095) were most frequently encountered among the proteins detected (no. of proteins > 50).

The last part of this dissertation detailed a preliminary study of salivary pellicle formation on two common implant/indirect restorative materials, namely titanium (Ti) and fine-particle feldspathic ceramic, using saliva samples collected from the same five healthy donors. Titanium and ceramic exhibited similar surface microroughness but distinct surface chemistry after standardized polishing. Three hundred and sixty-nine and 298 proteins were identified respectively. Within them, 18 proteins (C3, C4A, C4B, HNRNPC, IGHA1, IGHA2, IGHG1, IGHG2, IGHG3, IGHG4, IGHM, IGKV1-5, IGKV4-1, IGLC1, IGLC2, IGLC3, IGLL5, and SERPING1) were detected on both material surfaces tested, and one (C1QBP) detected on Ti surfaces were identified as key proteins based on the ontological analysis. Fourteen out of 19 hub proteins were found possible existence in the extracellular region, and all of which are likely related to immune response regulations.

Restoration related infection is a complicated, multiphasic process that involves interactions between cells, proteins, bacteria and/or other biological components. The results outlined in this study displayed the compositions and the possible lubrication, demineralization/remineralization and protective abilities of in vitro salivary conditioning films on tooth and various restorative material surfaces, which may facilitate a better understanding of the underlying pathogenesis of oral microbial infection initiations at the restorative material surfaces.