Transmission and scanning electron microscopic observations of epithelial–mesenchymal interface of rat tooth germs using dithiothreitol separation

Abstract

Aperiodic fibrils (AF) project from the interstitial side of the lamina densa of the basement membrane (BM) of the inner enamel epithelium (IE), and show remarkable changes in their morphology during development. The three-dimensional morphology of aperiodic fibrils during development has not been observed, because of the difficulty of exposing the interstitial surface of the BM of the inner enamel epithelium. In the present study, the dithiothreitol separation method was applied to expose the interstitial side of the inner enamel epithelial BM of rat tooth germs for the purpose of observing the exposed aperiodic fibrils by transmission and scanning electron microscopy (TEM and SEM, respectively). After dithiothreitol treatment, the enamel organ (EO) was mechanically separated from the dental papilla (DP). In the region with poorly-developed aperiodic fibrils, the separation occurred at the junction between the inner enamel epithelial BM and the dental papilla, and the aperiodic fibrils were exposed, showing the typical picture of dithiothreitol separation. SEM observation of this region revealed that the aperiodic fibrils were connected to each other and they formed networks. These networks resembled those formed by the anchoring fibrils of epidermal and mucosal epithelial BMs. TEM and SEM observations revealed that there were sidechain-like structures on the surface of the aperiodic fibrils. In the region with well-developed aperiodic fibrils, dithiothreitol treatment was not entirely effective, and some mesenchymal tissues remained on the BM. In this region, TEM observation revealed that the aperiodic fibrils were arranged in parallel with each other, and were connected by the sidechains. Several thin collagen fibrils, which were thought to be immature collagen fibrils (CF) of the predentine, were also connected to the aperiodic fibrils with these sidechains and arranged in parallel with them. Based on SEM and TEM observations, the aperiodic fibrils may be regarded as a kind of anchoring fibrils and they may play a role in connecting the BM with the mesenchymal tissue below. They are also thought to guide the arrangement of collagen fibrils in the surface layer of the predentin.