Snapshot of the Seconds-Pellicle: Ultrastructure and Proteomic Changes

The dental pellicle is a continuously forming layer present at the interface between oral surfaces and saliva. It protects the dental surfaces by shielding against chemical and mechanical damages. The pellicle represents the basis for further biofilm development, and its formation starts after oral hygiene through the adsorption of mostly salivary proteins to all exposed oral surfaces. In spite of its important physiologic role, its formation process and composition are not yet revealed in all details. The objective of the current study was 1) to visualize and elucidate the individual proteomic composition of the very early (only a few seconds) formed in situ pellicle, named seconds-pellicle, and 2) to analyze changes in its ultrastructure and proteomic composition over time. Transmission electron microscopic analyses demonstrated the seconds-pellicle to consist of an already continuous electron-dense layer without a detectable increase in thickness within the first minutes of formation. Proteomic analyses showed it to consist of up to 841 proteins on an individual level, without elevating protein content or diversity over the first minutes. Analyses of several pellicle formation times enabled the detection of changes over time and the first direct verification of protein desorptions occurring during pellicle formation. Comparisons between the salivary and pellicle proteomes at the different formation times revealed insights in the amount of substance of single pellicle proteins over time. The present study provides first evidence that the pellicle forms instantaneously and dynamically on solid surfaces in the oral cavity. Furthermore, the data suggest these first proteins to be replaced by proteins adsorbing with higher selectivity to stabilize the pellicle within the first minutes of its formation. These insights represent the basis for selective modifications of the pellicle layer to control the originating biofilm and develop preventive strategies for oral biofilm management.