Primary Culture to Study the Differentiation of Ameloblasts

Objective: Dental epithelial cells differentiate to ameloblasts secreting enamel matrix proteins in the process of tooth germ development. However, the molecular mechanism of the ameloblast differentiation has not been well understood. To study the mechanism, we attempted to establish the primary cell culture system to observe the growth and differentiation. Methods: We isolated the cervical loop epithelium from lower incisor tooth germs of seven-day-old Wistar rats. The epithelial cells were dispersed and then cultured with dental papilla cells. As control, only epithelial cells were also cultured in absence of dental papilla cells. Anti nerve growth factor receptor p75 (p75NGFR) and anti ameloblastin antibodies were used as differentiation makers of the inner enamel epithelial cells and the ameloblasts, respectively. Anti cytokeratin 14 (CK14) and anti keratin for wide spectrum (Wide-keratin) antibodies were also used as maker of enamel epithelial cells. We estimated the appearance of the inner enamel epithelial cells and ameloblasts time-dependently by immunostaining of these differentiation markers. Results: At the 2 days co-culture, epithelial cells (wide-keratin-positive cells) expressing p75NGFR were detected, while ameloblastin was not expressed in any cells. At the 4 days, the number of p75NGFR-positive epithelial cells increased subsequently. In this phase, a few of epithelial cells (CK14-positive cells) expressed ameloblastin. At culture day 10, no epithelial cells expressed p75NGFR. In contrast, ameloblastin was expressed in the almost all epithelial cells. In the mono-culture, ameloblastin-positive cells did not appear during the period of culture. Conclusion: We established the useful culture system to observe the ameloblast differentiation from undifferentiated enamel epithelial cells. In this culture the result suggested that the differentiation of dental epithelial cells needs the interaction with dental mesenchymal cells. Supported by Grants from the Ministry of Education, Science, Sports, and Culture (No.13470387, No.13877308) and Kato Memorial Bioscience Foundation in Japan.