Calcium hydroxide increases the attachment and the mineralization gene expression of dental pulp stem cells on the dentin treated by sodium hypochlorite

Abstract

Sodium hypochlorite (NaOCl) is an excellent bactericidal agent, but it has a profoundly detrimental effect on stem cell survival, attachment and differentiation. Intracanal medicaments can also affect the survival and differentiation of the dental pulp stem cells (DPSCs). Recent studies found that Ca(OH)2 promoted greater survival and proliferation of stem cells from apical papilla (SCAP). The purpose of this study is to evaluate the effect of the sequential use of NaOCl and Ca(OH)2 on the attachment and differentiation of DPSCs. Addtionally, to further investigate the optimal protocols to reduce the cytotoxicity of NaOCl on DPSCs.Human DPSCs were obtained from human third molars. Dentin specimens were sterilized by ethylene oxide gas sterilization and an EDTA treatment to produce specimens without a smear layer produced during the preparation of the dentin specimen. Group 1 was treated by NaOCl. Groups 2, 3, 4, 5, which underwent other treatment processes after the NaOCl and Ca(OH)2 treatment. Group 2, for which NaOCl and Ca(OH)2 treatments were followed by PBS washing. Group 3, for which NaOCl and Ca(OH)2 treatments were followed by EDTA. Group 4, for which NaOCl and Ca(OH)2 treatments were followed by EDTA and culture media for 24 hours. Group 5, for which NaOCl and Ca(OH)2 treatments were followed by instrumentation and EDTA. DPSCs morphology was observed by SEM after 7 days of culture. A MTT assay was performed to assess the cell survival rate by group after the treatment. After 4 days of culture, gene expression level of cell adhesion was measured. After 4 weeks of culture, gene expression level of odontogenic differentiation by quantitative real-time polymerase chain reaction was investigated.The DPSCs in the Group 1 were not attached, but the cells in the Groups 2, 3, 4, 5 were attached to the dentin surface. The cell viability in the Groups 2, 3, 4, 5

was lower than control group which cells were grown in plates treated with PBS alone. The Fibronectin-1 (FN-1) and Secreted phosphoprotein-1 (SPP-1) gene expression level was significantly higher in Groups 3, 4, 5 than Group 2. The gene expression level of Dentin matrix protein-1 (DMP-1) was significantly higher in the Groups 2, 3, 4, 5 than control. The Dentin sialophosphoprotein (DSPP) level was significantly higher in the Groups 3, 4, 5 than control. But, the DMP-1 and DSPP level was not significantly different between Group 4 and Group 5.In conclusion, application of Ca(OH)2 promoted the attachment and differentiation of DPSCs. After treatment of Ca(OH)2, additional treatment such as EDTA or instrumentation enhanced the attachment and differentiation of DPSCs.