Stratification of fluoride uptake among enamel crystals with age elucidated by atom probe tomography

Abstract

Dental enamel is subjected to a lifetime of de- and re-mineralization cycles in the oral environment, the cumulative effects of which cause embrittlement with age. However, the understanding of atomic scale mechanisms of dental enamel aging is still at its infancy, particularly regarding where compositional differences occur in the hydroxyapatite nanocrystals and what underlying mechanisms might be responsible. Here, we use atom probe tomography to compare enamel from a young (22 years old) and a senior (56 years old) adult donor tooth. Findings reveal that the concentration of fluorine is elevated in the shells of senior nanocrystals relative to young, with less significant differences between the cores or intergranular phases. It is proposed that the embrittlement of enamel is driven, at least in part, by the infusion of fluorine into the nanocrystals and that the principal mechanism is de- and re-mineralization cycles that preferentially erode and rebuild the nanocrystals shells.

Keywords: Biomineralization; Imaging techniques.

Fig. 1. Preparation of samples for atom probe tomography.

a Optical micrograph of polished cross section of young molar. b Scanning electron micrograph of cervical enamel from young molar, with enamel (EN) and dentin (DE) noted for clarity. The sample was taken from a location approximately 850 µm from the dentin-enamel junction, and (c) approximately 10 µm from the outer enamel surface. The cutting plane line indicates the sectional view of the cantilever presented in (d). d A cantilever with triangular cross section was produced in situ using focused ion beam milling to be lifted out. e A segment of the liftout was attached to a Si micropost then milled with progressively smaller annular ion beam patterns to shape the needle before a final low-kV polishing step was used to achieve a tip diameter <100 nm (f). The perspective of the micrographs in (d–f) is fixed and at a stage tilt of 52°, (d, e) are at the same scale.

Fig. 2. Labeled segment of representative mass-to charge spectra from young and senior enamel.

A full list of the ranges used is provided in the supplementary information.

Fig. 3. Representative APT reconstructions for young and senior enamel.

Only the Mg ions are visible. a, c Properly aligned reconstructions show the Mg-enriched core of each nanocrystal surrounded by a Mg-depleted shell. The intergranular space between nanocrystals also has a relatively high Mg concentration. The boundaries of each nanocrystal are outlined. b, d Rectangular ROIs are drawn and assigned to the intergranular, shell, or core phase.

Fig. 4. Comparison of minor element concentrations by nanocrystal phase.

a Schematic of an APT reconstruction that has captured several hydroxyapatite nanocrystals, with phases labeled. b–e Average composition in atomic% of Mg, F, Na, and C, respectively. For each phase, data from the young sample is on the left, senior is on the right. Error bars represent one standard deviation, each dataset is represented as a point. These concentrations are measured after molecular ions (e.g., CaF) have been decomposed into their constituent elements.

Fig. 5. Comparison of nanocrystal dimensions and morphology by STEM high-angle annular dark field (HAADF).

a Young enamel. The yellow pointers highlight several regions where the mineral domain appears equiaxed, globular, or fragmented. b Senior enamel. c–e Box-and-whisker plots summarizing the distribution of (c) nanocrystal area, (d) fit ellipse major axis length, and (e) fit ellipse minor axis length for individual nanocrystals measured by STEM. Asterisks in the upper right corner denote the p value from an unequal variance t-test. In each plot, the box encloses the second and third quartile, the line represents the median, the notch is the 95% confidence interval for the median, the x marks the mean, and the whiskers stretch beyond the box by 1.5 times the inter-quartile range. Outliers are marked by a hollow circle.