Stage-Specific Analysis of Enamel Formation by Micro-Computed Tomography

The formation of enamel as the hardest biomineral of the body is a gradual multi-stage process. Objectives: The goal was to determine the mineral density of enamel from early to late developmental stages. Methods: Micro-computed tomography (microCT) was performed to analyze the mineral density in mouse mandibular incisors during enamel formation. The continuously erupting rodent incisor displays all developmental stages of enamel formation. Mouse hemimandibles at age 7 weeks were dissected from wildtype, Ambn mutant (missing exons 5 and 6; Ambn^mutant), and Ambn mutant reconstituted with transgenic Ambn by mating (Ambn^mutant/Tg). The Ambn transgene was expressed at a level similar to wildtype. Samples were scanned (Sky Scan 1172 microCT scanner) at a resolution of 5 µm. Hydroxyapatite-based phantoms, ranging in density from 0.25 to 2.9 mg HA/cm³ were used to calibrate mineral density. The samples were reconstructed with correction of beam hardening and ring artifacts. The mineral density of the enamel was analyzed at 0.5 mm intervals from the apical to incisal edge. The start points for analyses were anatomically determined and scaled to ramus size. Results: In the sagittal and cross-sectioned views of microCT images, mineral density of enamel increased steadily during the secretory and maturation stages in wildtype controls. In contrast, the formation of an enamel layer failed completely in Ambn^mutant mice. Ectopic, nodular mineralizations were present within the enamel organ cells. In Ambn^mutant/Tg animals, a secretory stage was present but compared to control, the density of mineral started increasing earlier. The final mineral density of enamel was similar in Ambn^mutant/Tg animals and controls with 2.8 to 2.9 mg HA/cm³. Conclusions: The final mineral density in enamel in wildtype and Ambn^mutant/Tg animals was similar. MicroCT is suitable for determining the mineral density of enamel stage-specifically. This study was funded by the University Research Council, UTHSCSA.