Watching Dentin and Enamel Form Using Focused Ion Beam Microscopy

Objectives: Enamel formation is critically dependent upon events that precede ameloblast differentiation, degradation of the epithelial basement membrane (BM), and the onset of mineralization. On continuously growing mouse incisors all developmental stages are arrayed chronologically from basal to incisal, as the cells that make dentin and enamel move progressively and linearly in the incisal direction. Our objective was to visualize at high magnification mandibular incisor formation along the mesenchymal-epithelial interface from the basal loop to the onset of enamel mineralization.
Methods: Seven week C56BL/6 mice were perfused with 2.5% glutaraldehyde, and the incisors imaged by focused ion beam scanning electron microscopy (FIB-SEM).
Results: Thin “aperiodic” fibrils in predentin extending perpendicular to the epithelial basement membrane (BM) are first observed in the ascending portion of the basal loop. These fibrils appear to terminate on the basement membrane or pass through the membrane to contact the epithelial cell membrane, and concentrate in predentin along the BM. Thicker, distinctly banded collagen fibers are first observed on the horizontal part of the basal loop. These collagen fibers often run parallel to the thin fibrils, but are less likely to reach the BM. Collagen-like banding is observed in some of the smaller fibrils. Finger-like ameloblast processes extend through gaps in the BM. Thin fibrils and fragmented BM collect along the epithelial membrane between the epithelial processes and appear to be resorbed into the ameloblasts, making way for the ameloblast membrane to intimately contact the ends of highly-organized collagen fibers. Once this occurs, predentin starts to mineralize.
Conclusions: FIB-SEM imaged in high resolution how the epithelial membrane and basement membrane organized collagen fibers in predentin resulting in a specific arrangement of collagen fibers in mantle dentin that mineralize and form the platform for the deposition of initial enamel ribbons. This study was supported by NIDCR grant DE015846.