Transmission electron microscopy comparison of methods for collecting in situ formed enamel pellicle

The in vivo formed salivary pellicle is composed of an outer globular and a densely structured basal layer. This study developed a method for selective recovering of these pellicle layers from the enamel surface. Two-hour in situ pellicles were formed by intraoral exposure of enamel specimens in two adults. Pellicle-covered enamel specimens were treated either mechanically (scraping with scaler, curette or razor blade, or rubbing with a sponge) or chemically (phosphate buffer, NaCl, NaOCl, CaCl2, NaSCN, urea, tetrahydrofurane, guanidine, SDS, HCl, or EDTA with or without additional ultrasonication). Specimens were processed for transmission electron microscopic analysis to detect pellicle residues remaining on the enamel surface after the different treatments. Most of the chemical treatments caused partial, incomplete removal of the globular layer. Complete removal of the globular layer without disruption of the basal layer was obtained by sponge rubbing or by CaCl2 combined with ultrasonication, whereas scraping caused partial disruption of the basal layer. Removal of the basal layer was observed after treatment with HCl, EDTA, or NaOCl combined with ultrasonication. Electrophoretical analysis of recovered pellicle fractions indicate that combination of sponge-rubbing followed by EDTA treatment can be recommended for stepwise removal of the globular and basal pellicle layers.