Effect of 5-Aza-2′-deoxycytidine on Odontogenic Differentiation of Human Dental Pulp Cells

Introduction: Human dental pulp cells (hDPCs) comprise a heterogeneous cell population that possesses the capacity to differentiate into osteoblasts and plays an important role in reparative dentinogenesis. 5-Aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, is known to be involved in cell differentiation. However, its role in the differentiation program of hDPCs remains unknown. The purpose of this study was to explore the role of 5-Aza-CdR in the regulation of odontogenic growth and differentiation of hDPCs. Methods: hDPCs were treated with 1 Amol/L 5-Aza-CdR for 24 hours before being incubated in odontogenic medium for 2 weeks. To identify the effect of 5-Aza-CdR on proliferation and the odontogenic differentiation potential of hDPCs, the cell growth was measured using the Cell Counting Kit-8 assay (Dojindo, Kumamoto, Japan). The expression levels of the odontogenic markers, dentin sialophosphoprotein (DSPP), dentin matrix protein 1, (DMP1) and transcription factors, runt-related transcription factor 2 (RUNX2), distal-less homeobox 5 (DLX5), osterix (OSX) were analyzed. The activity of alkaline phosphatase was determined, and the formation of mineralized nodules was assessed using alizarin red S staining. Results: After treatment with 5-Aza-CdR, the proliferation capacity of hDPCs was suppressed (n = 3, P < .05). 5-Aza-CdR up-regulated the expression of DSPP, DMP-1, OSX, RUNX2, and DLX5; increased the level of alkaline phosphatase activity; and accelerated the formation of calcified nodules (n = 3, P < .05). Conclusions: DNA methyltransferase inhibitor 5-Aza-CdR significantly inhibits the proliferation and enhances the capability of odontogenic differentiation of hDPCs, suggesting that DNA methylation may play an important role in reparative dentinogenesis.