High-Resolution Characterization of Enamel Remineralization Using Time-of-Flight Secondary Ion Mass Spectrometry and Electron Microscopy

Abstract

<b><i>Introduction:</i></b> The aim of this in vitro study was to assess the suitability of high-resolution time-of-flight secondary ion mass spectrometry (ToF-SIMS) for visualizing cross-sectional changes in human enamel microstructure and chemical composition during treatment and remineralization cycling of artificially generated caries lesions underneath an artificial plaque. <b><i>Methods:</i></b> Treatments consisted of exposure to twice daily toothpaste/water slurries prepared from 0, 1,100, and 5,000 μg/g fluoride (F) NaF/silica toothpastes. In addition, treatments with slurries prepared from 1,100 μg/g F SnF₂/silica toothpastes were done using ⁴⁴Ca in the remineralization solution to allow for differentiation of newly formed mineral and exploration of incorporated metal dopants using ToF-SIMS. Complementary microhardness, scanning electron microscopy, and high-resolution transmission electron microscopy (HR-TEM) investigations were performed on enamel cross sections. <b><i>Results:</i></b> HR-TEM was used for the first time to determine the change in crystallinity during remineralization revealing distinct microstructural zones within one lesion. Chemical mapping using ToF-SIMS demonstrated that the distribution of F, while observed primarily in the new mineral phase, was widespread throughout the lesion with ⁴⁴Ca substantially limited to the remineralizing mineral. Both penetrated the inter-rod spaces of the sound enamel illustrating how acid damage propagates into the native mineral as the caries lesion deepens. HR-TEM examination revealed different regions within the lesion characterized by distinct micro- and ultrastructures. Importantly, HR-TEM revealed a return of crystallinity following remineralization. F dose-response observations verified the ability of these high-resolution techniques to differentiate remineralization efficacy. <b><i>Conclusion:</i></b> The collective results provided new insights such as the visualization of F or calcium penetration pathways, as well as new tools to study the caries process.