In vitro characterization of enamel epithelium and pulp cells in mouse tooth germs.

Mixed cell populations consisting of enamel epithelium and pulp cells obtained from 18-day embryonic mouse tooth germs were cultured in vitro. Epithelial cells and pulp cells were also cultured individually and examined. Isolated cells were cultured in alpha-MEM supplemented with 10% fetal bovine serum for up to four weeks and examined morphologically using histological procedures including immunostaining, light and electron microscopy, and electron probe microanalysis. The pulp cells proliferated and differentiated in the absence of epithelium, but the number of epithelial cells showed a strong dependence on the pulp cells. Pulp cells showing fibroblastic morphology in the mixed culture gradually became elliptical, and eventually transformed into spherical cells surrounded by a calcified extracellular matrix. Alkaline phosphatase (ALPase) activity was expressed on the pulp cells prior to calcification of the extracellular matrix, as shown by von Kossa's and alizarin-red staining. Calcification deposition, which is closely associated with thick banded type-I collagen fibrils, was shown to be composed of calcium and phosphorous using electron probe microanalysis. Type-I collagen immunoreactivity was detected on the extracellular matrix after two weeks of culturing. The present results show that the proliferation and differentiation of pulp cells, and subsequent calcification of the extracellular matrix occur in the presence or absence of epithelial elements, but that the proliferation of epithelial cells depends on the presence of pulp cells.