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).

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.

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.

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