Objectives: Dental papilla mesenchymal cells differentiate into odontoblasts through epithelial-mesenchymal interactions. However, how enamel epithelial cells facilitate the differentiation of dental mesenchymal cells remains unknown. Alkaline phosphatase (ALPase) is a marker for odontoblast-like differentiation because odontoblasts show much higher ALPase activity than dental undifferentiated mesenchymal cells. The continuously growing rabbit incisor is a good model for analyzing epithelial-mesenchymal interactions in odontogenesis. In the present study, we developed isolation and culture methods for rabbit incisor-derived epithelial cells (E cells) and rabbit incisor-derived fibroblasts (F cells), then examined the effects of E cells on ALPase activity in F cells. Methods: E cells were obtained by enzymatic digestion from incisors dissected carefully from the lower jaws of 4-week-old male Japanese White rabbits. F cells were obtained by explant cultures of the incisor pulp. E cells, F cells and a mixture of both were cultured in MD medium [1:1 (vol/vol) MCDB153:DMEM] for 7 days. ALPase mRNA expression was examined by RT-PCR. ALPase activity was measured by the method of Bessey et al. (1946). ALPase activity was also examined histochemically by an azo dye coupling method. Results: mRNA levels and ALPase activity were very low in separate cultures of E cells or F cells but high in the mixed co-cultures on day 7. ALPase activity was prominent in F cells near E cells. Conclusions: These findings suggest that epithelial cells play an important role in the induction of ALPase expression in dental mesenchymal cells. The co-culture system developed here will be useful for examing the role of epithelial-mesenchymal interaction during odontoblast differentiation.

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