Assessing Enamel Remineralization at the Nanoscale - an in vitro Platform

Objectives: Remineralization of caries lesions in enamel is accomplished through flow of salivary mineral components, facilitated by usage of oral care products. Fluoride addition in such products has demonstrated to enhance remineralization and reduce demineralization, both in vitro and in vivo. However, little is known about fluoride incorporation in the redeposited mineral locally at the nanoscale, and how varying concentrations of fluoride alters the pathway of the (onset of) remineralization. We attempt to address this by establishing an in vitro platform under controlled conditions.
Methods: We exposed artificially demineralized enamel sections to a solution containing calcium and phosphate concentrations similar to those found in saliva, and fluoride (1.5 - 20 mM). Lamellae of the remineralized layers and underlying enamel were prepared by focused ion beam (FIB) and imaged by high-resolution (scanning)TEM. (Nano)diffraction was used to probe the local crystal structure. Furthermore, atom probe tomography (APT) was employed on FIB-prepared sharpened tips to locally resolve fluoride concentrations.
Results: At low concentrations of fluoride, (S)TEM imaging revealed a ~100 nm layer consisting of small apatite crystallites, also present in the precipitate. Using an intermediate concentration of fluoride (5 mM), a much thicker apatitic remineralized layer (3-4 μm) was grown, while fluorite was precipitated. Interestingly, at 20 mM fluoride present in the solution, no remineralization was observed. Preliminary APT analysis of the layer resulting from the in vitro experiment containing 1.5 mM fluoride indicated a rather homogeneous fluoride content of ~1 atom %, higher than expected for untreated enamel fluoride content.
Conclusions: This platform shows that solely changing fluoride concentration alters the remineralized enamel layer thickness and morphology significantly, and that the layers' varying fluoride content can be determined locally. The absence of remineralization at higher concentrations suggests a threshold concentration at which remineralization proceeds advantageously. Transformation of fluorite into apatite appears a key factor guiding these changes.