Adenosine triphosphate induced odontoblastic differentiation of human dental pulp cells in vitro and in vivo

Abstract

Objective: To choose a suitable efficient concentration of adenosine triphosphate (ATP) which can induce human dental pulp cells (HDPC) differentiate into odontoblast successfully, and explore the role of this concentration of ATP in dentin regeneration in vivo. Methods: HDPC were treated with different concentrations (0, 10, 400, 600, 800 μmol/L) of ATP. Then cell counting kit-8 (CCK-8), quantitative real-time PCR, and Western blotting were used to detect the cell proliferation and the expressions of odontoblastic differentiation related markers, dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP). Alizarin red S staining experiment was used to analyze the effect of ATP on the mineralization ability of HDPC. By the above experiments, the suitable effective concentration of ATP was chosen to pretreat the HDPC for 7 days, then cells were seeded on gelfoam, inserted into the root canal fragment, and subsequently transplanted into the subcutaneous space on the back of immunodeficient mice, after three months, the samples were stained with HE for histological analysis. Results: The CCK-8 results in 5 d showed that 10 μmol/L ATP obviously promoted the proliferation of HDPC, while the 600 and 800 μmol/L ATP apparently inhibited the HDPC proliferation, however, the proliferation in 800 μmol/L ATP group was lower than that of 600 μmol/L ATP group (P<0.05). qPCR and Western blotting results showed that the 600 and 800 μmol/L ATP significantly up-regulated the DMP1 and DSPP expressions (P<0.05), furthermore, there was no significant difference in the two groups, but no changes were found in other groups (P>0.05). After 21 days of culturing, there were obvious mineralization nodules in 600 and 800 μmol/L ATP groups, but no mineralization nodules in other groups. Quantitative analysis of the staining results showed the A value in 0, 10, 400, 600, and 800 μmol/L ATP groups were respectively 1.05±0.15, 1.11±0.23, 1.15±0.17, 3.65±0.30, and 3.40±0.43, and the A value in 600 and 800 μmol/L ATP groups were higher than those of other groups; however, there was no difference in 600 and 800 μmol/L ATP groups. The histological analysis showed that 600 μmol/L ATP could induce the HDPC differentiate into dentin-like structure in the root canal fragment. Conclusions: Therefore, the suitable effective concentration of ATP is 600 μmol/L, which could induce HDPC differentiate into odontoblast-like cells, and form the dentin-like structure in vivo.