Insights of the Nanoscale Compositional Variations in Dental Enamel Revealed by Statistical Atom Probe Tomography

Abstract

Dental enamel is a critical tissue in the body, acting as the primary surface for mastication as well as serving an aesthetic role in a smile. Enamel is also an inspirational material from a materials science perspective, as it endures decades in a challenging and complex oral environment whilst being subjected to cyclic loads. For these reasons, it is worthwhile to develop a thorough understanding of the microstructural and compositional features that provide this tissue its longevity, and how those features can be altered as a function of location within the tooth, age, or external factors such as disease or lifestyle choices. This dissertation is concerned with exploring the composition of the hydroxyapatite nanocrystals that constitute enamel at the smallest scale, elucidating where changes take place at a nanometric level and providing a perspective as to how those changes occur. Atom probe tomography (APT) is a powerful tool that is well suited to this investigation. Over the past decade, APT has had a significant impact on the study of enamel structure, however small sample sizes and inconsistent use of parameters have limited its ability to make rigorous comparisons between samples from distinct locations, age groups, or conditions. To address these issues, we have developed a routine for atom probe data analysis which enables more granular and robust statistical tests to provide a measure of confidence when comparing multiple groups. The routine is strengthened by complimentary analysis with other techniques such as TEM and Raman spectroscopy. Applied to human enamel from young and senior age groups, this routine reveals an enrichment of fluorine with age in the outer shell of nanocrystals, but not in the nanocrystal core or the intergranular space between nanocrystals. This reflects the culmination of decades of cyclical de- and re-mineralization during which fluorine is incorporated into the nanocrystal shells, though the nanocrystals themselves become slightly smaller and the separation between them slightly larger. The routine described above was also applied to “aprismatic” crocodilian enamel (i.e. without rods), complimenting a broader investigation of the sharp gradient in mechanical properties, composition, and nanocrystal morphology that arises at the outermost enamel surface. Parameter selection during atom probe experimentation and choices made during analysis can have a substantial impact on the reported results. In pursuit of improved accuracy, precision, and repeatability for these and future experiments, this dissertation also includes a systematic exploration of the parameter space for atom probe experiments on synthetic hydroxyapatite. As part of this, we present a facile and automated approach to ranging of the mass-to-charge spectrum, from which the composition is derived. The significance of the wavelength of the laser pulse used during APT is also studied, with evidence indicating that hydroxyapatite is photoionized by the deep ultraviolet wavelength laser which comes equipped on the newest generation of commercial laser pulsed atom probe systems. Finally, this dissertation incorporates the developments described above to compare inner enamel from primary, young, and senior age groups, with a particular focus on the presence of nanoscale organic precipitates that reside between some nanocrystals. Statistical analysis of these precipitates allows for the confident differentiation between organic (e.g., CO) and inorganic (e.g., CO2H) carbon-containing signals in atom probe reconstructions, which can then be applied to better understand the variations in both mineral and organic components between age groups. Additionally, retention of some precipitates after exposure of the enamel to a prolonged bleaching solution indicates that these organic features can be occluded by mineral and thus are resistant to change under attack by external threats. Altogether, this research advances both our understanding of dental enamel as well as the measurement science required to explore nanoscale features in this and other material systems. We envision that future investigations will be able to delve deeper into specific conditions of global and clinical concern, such as aging populations, environmental exposures, caries, molar-incisal hypomineralization, and amelogenesis imperfecta.