This study aimed to determine whether collagen triple helix repeat containing-1 (CTHRC1), which is involved in vascular remodeling and bone formation, can stimulate odontogenic differentiation and angiogenesis when administered to human dental pulp stem cells (hDPSCs).

The viability of hDPSCs upon exposure to CTHRC1 was assessed with the WST-1 assay. CTHRC1 doses of 5, 10, and 20 µg/mL were administered to hDPSCs. Reverse-transcription polymerase reaction was used to detect dentin sialophosphoprotein, dentin matrix protein 1, vascular endothelial growth factor, and fibroblast growth factor 2. The formation of mineralization nodules was evaluated using Alizarin red. A scratch wound assay was conducted to evaluate the effect of CTHRC1 on cell migration. Data were analyzed using 1-way analysis of variance followed by the Tukey post hoc test. The threshold for statistical significance was set at p < 0.05.

CTHRC1 doses of 5, 10, and 20 µg/mL had no significant effect on the viability of hDPSCs. Mineralized nodules were formed and odontogenic markers were upregulated, indicating that CTHRC1 promoted odontogenic differentiation. Scratch wound assays demonstrated that CTHRC1 significantly enhanced the migration of hDPSCs.

CTHRC1 promoted odontogenic differentiation and mineralization in hDPSCs.

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.

The aim was to confirm the stem cell-like properties of the dental pulp stromal cells and to evaluate the morphologic changes during in vitro chondrogenesis.

Stromal cells were outgrown from the dental pulp tissue of the premolars. Surface markers were investigated and cell proliferation rate was compared to other mesenchymal stem cells. Multipotency of the pulp cells was confirmed by inducing osteogenesis, adipogenesis and chondrogenesis. The morphologic changes in the chondrogenic pellet during the 21 day of induction were evaluated under light microscope and transmission electron microscope. TUNEL assay was used to evaluate apoptosis within the chondrogenic pellets.

Pulp cells were CD90, 105 positive and CD31, 34 negative. They showed similar proliferation rate to other stem cells. Pulp cells differentiated to osteogenic, adipogenic and chondrogenic tissues. During chondrogenesis, 3-dimensional pellet was created with multi-layers, hypertrophic chondrocyte-like cells and cartilage-like extracellular matrix. However, cell morphology became irregular and apoptotic cells were increased after 7 day of chondrogenic induction.

Pulp cells indicated mesenchymal stem cell-like characteristics. During the in vitro chondrogenesis, cellular activity was superior during the earlier phase (within 7 day) of differentiation.