Establishment and characterization of a culture system for enzymatically released rat dental pulp cells

To establish a cell culture system that reflects the dentin formation in dental pulp tissue, we used dental pulp cells enzymatically isolated from rat incisor teeth. During the 20-day culture period, the cells exhibited various phenotypes of the odontoblast differentiation process, from the immature stage to the terminal mineralization stage. The cells began to form the mineralized nodules from day 10, and the nodules became larger by day 20. Alkaline phosphatase (ALP)-positive cells surrounded the mineralized nodules. The ALP activity in the cell layers was maximal on day 5, and gradually decreased to day 20. The calcium content in the cell layers was very low by day 10, and significantly increased from day 15. Sulfated glycosamino-glycans (GAGs) contained in the cell layers increased by day 15, but the content then decreased by day 20. The dental pulp cells produced a small amount of osteocalcin that was released into the culture medium by day 10, and the amount secreted increased markedly from day 15. The expression of osteocalcin and parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor mRNA was evident as early as day 5, and the expression of each gradually increased with the number of days in culture. Dentin matrix protein (Dmp1) mRNA gene transcripts were identified by use of the reverse transcription polymerase chain reaction (RT-PCR) in the cells throughout the culture period. The present results demonstrate that this culture system is useful for studying the differentiation process of the odontoblast-like cells.