In this study, we characterized human dental pulp cells (HDPCs) obtained by different culture methods to establish the most suitable methodology for dental tissue engineering and regenerative endodontic applications.

HDPCs were isolated by the outgrowth method (HDPCs-OG), the enzymatic digestion method (collagenase/dispase/trypsin, HDPCs-ED), or the combination of both methods (HDPCs-Combined). The expression of mesenchymal stem cell markers (CD105, CD90, and CD73) was investigated. In vitro differentiation capacities of HDPCs into adipogenic, osteogenic, and chondrogenic lineages were compared. Differentiation markers were analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and western blotting.

Our data indicated that whole HDPCs-ED, HPDCs-OG, and HDPCs-Combined could be differentiated into adipogenic, chrondrogenic, and osteogenic cell types. However, we found that the methods for isolating and culturing HDPCs influence the differentiation capacities of cells. HDPCs-OG and HDPCs-ED were preferably differentiated into adipogenic and osteogenic cells, respectively. Differentiation markers shown by RT-PCR and western blotting analysis were mostly upregulated in the treated groups compared with the control groups.

Our findings confirmed that cell populations formed by two different culture methods and the combined culture method exhibited different properties. The results of this study could provide an insight into regenerative endodontic treatment using HDPCs.

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.