Differentiation of Human Dental Pulp Stem Cells Into Odontoblast-like Cells

Objectives: Odontoblasts play a key role in dental tissue engineering. The aim of this study is to establish a reproducible and simple method for differentiation of human dental pulp stem cells into odontoblasts in vitro and to investigate the mechanisms of odontoblast differentiation through changes in morphology and gene expression.
Methods: The differentiation of human dental pulp stem cells (hDPSCs) was assessed by in vitro morphologic appearance, gene and protein expression of odontoblast phenotype. hDPSCs at passage 4 (P4) were cultured in differentiated medium containing β – glycerophosphate, dexamethasone, acid ascorbic, vitamin D3 and KH₂PO₄ for 4 weeks. After each period of one week, inverted microscope was used to investigate morphological changes and RT-PCR to assess the expression patterns of odontoblast-specific genes: DMP1, DSPP, Nestin, RunX2. Immunocytochemistry was also performed to analyze the expression of specific proteins (DMP1 and DSPP).
Results: The results showed that hDPSC had a fibroblast-shaped morphology. After 7 days of differentiation, the cells displayed a weak-polarized shape and a short cellular extension. RT-PCR analysis showed expression of RunX2 only. From day 14, the well-polarized cells appeared with eccentric nuclei and their typical cellular process; furthermore, all odontoblast-specific genes that are DMP1, DSPP, Nestin, RunX2 were also expressed.
Conclusions: The study defined an experimental protocol for differentiating hDPSC into pre-odontoblast and odontoblast-like cells. This could have an important impact for dental tissue engineering.