Towards a 4-D spatial and temporal model of human incisor enamel biomineralisation using X-ray techniques.

Objectives: Precise timings and spatial progression of human enamel biomineralisation are still largely unknown due to scarcity of developing human enamel specimens for investigation. However, this information is crucial for optimising emerging biomimetic regenerative/reparative dentistry routes. The aim was to characterise and compare the crystallography, microstructure and mineral density (MD) of immature incisal enamel at various development stages.
Methods: Five developing primary upper incisors were obtained from an archaeological source. Two type-matched mature contemporary teeth were used for comparison. X-ray microtomography (XMT) with 15μm³ resolution at 90kV was used to map MD distribution. Linear attenuation coefficient was used to calculate MD (gcm^-3). Specimens were then sectioned into 0.3mm slices to carry out synchrotron X-ray diffraction (S-XRD) on XMaS (BM28) at the European Synchrotron Radiation Facility and B16 at Diamond Light Source in order to map crystallographic structural parameters. 2D diffraction images were collected using 15keV or 18keV X-ray energy, 50μm and 43μm beam size, and a 2048x2048 pixel and 3056x3056 pixel CCD detector on XMaS and B16 respectively.

Results: XMT revealed the average MD of least developed enamel is lower (2.34±0.3gcm^-3) than that of mid (2.63±0.2gcm^-3) and fully developed (2.75±0.1gcm^-3). In fully developed enamel, the MD increased when moving from the enamel-dentine junction (EDJ) to the outermost surface enamel. In contrast, developing enamel MD was relatively higher near the EDJ compared to the surface. The least developed enamel had higher distribution of MD values. S-XRD showed the texture distribution to be more spatially uniform and lower in early developed enamel, becoming more spatially heterogeneous and on average higher in full development. Hydroxyapatite crystallite orientation was perpendicular to EDJ regardless of development stage, indicating initial preferred directions of crystallites persist from early through to full maturation.
Conclusions: These results provide new insight into the fundamental understanding of natural growth and formation of human incisal enamel.