Effect of dentin treatment on proliferation and differentiation of human dental pulp stem cells

Abstract

Objectives: Sodium hypochlorite (NaOCl) is an excellent bactericidal agent, but it is detrimental to stem cell survival, whereas intracanal medicaments such as calcium hydroxide (Ca[OH]2) promote the survival and proliferation of stem cells. This study evaluated the effect of sequential NaOCl and Ca[OH]2 application on the attachment and differentiation of dental pulp stem cells (DPSCs).

Materials and methods: DPSCs were obtained from human third molars. All dentin specimens were treated with 5.25% NaOCl for 30 min. DPSCs were seeded on the dentin specimens and processed with additional 1 mg/mL Ca[OH]2, 17% ethylenediaminetetraacetic acid (EDTA) treatment, file instrumentation, or a combination of these methods. After 7 day of culture, we examined DPSC morphology using scanning electron microscopy and determined the cell survival rate with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We measured cell adhesion gene expression levels after 4 day of culture and odontogenic differentiation gene expression levels after 4 wk using quantitative real-time polymerase chain reaction.

Results: DPSCs did not attach to the dentin in the NaOCl-treated group. The gene expression levels of fibronectin-1 and secreted phosphoprotein-1 gene in both the Ca[OH]2- and the EDTA-treated groups were significantly higher than those in the other groups. All Ca[OH]2-treated groups showed higher expression levels of dentin matrix protein-1 than that of the control. The dentin sialophosphoprotein level was significantly higher in the groups treated with both Ca[OH]2 and EDTA.

Conclusions: The application of Ca[OH]2 and additional treatment such as EDTA or instrumentation promoted the attachment and differentiation of DPSCs after NaOCl treatment.

Keywords: Calcium hydroxide; Cell attachment; Cell differentiation; Dental pulp stem cells; Regenerative endodontics; Sodium hypochlorite.

Figure 1. Experimental groups for the attachment and differentiation of dental pulp stem cells. Cell attachment analysis with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (tetrazolium-based colorimetric assay).

Figure 2. Cell viability analysis with MTT assay. Cells in the control group were grown on plates. Data were obtained from 3 separate experiments. MTT assay, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide assay.

Figure 3. Expression levels of (a) fibronectin-1 (FN-1), and (b) secreted phosphoprotein-1 (SPP-1). Data were obtained from 3 separate experiments. ^*Mann-Whitney U test, level of statistical significance was set at p < 0.05.

Figure 4. Scanning electron microscopic views of dental pulp stem cell morphology on dentin surfaces after 7 days of culture. (a) group 2; (b) group 3; (c) group 4; (d) group 5 (magnification, ×1,000).

Figure 5. Expression levels of (a) dentin matrix protein-1 (DMP-1), and (b) dentin sialophosphoprotein (DSPP). Data were obtained from 3 separate experiments. ^*Mann-Whitney U test, level of statistical significance was set at p < 0.05.