Effect of angiopoietin 4 on odontogenic differentiation of dental pulp stem cells

Abstract

Objective To investigate the effects of angiopoietin 4 (ANGPT4) on the odontogenic differentiation of human dental pulp stem cells. Methods This study has been reviewed and approved by the Ethics Committee, and informed consent has been obtained from patients. Human premolars were fixed, decalcified, dehydrated, embedded, and sectioned. Immunofluorescence staining was used to observe the expression and localization of ANGPT4. Human dental pulp stem cells (hDPSCs) were isolated and cultured in vitro. The growth state and morphology of hDPSCs were observed under an inverted phase contrast microscope. The expression of cell surface-related molecular markers was detected by flow cytometry. Alkaline phosphatase and alizarin red S staining were used to detect the odontogenic differentiation potential of hDPSCs. Oil-red O staining was used to detect the adipogenic differentiation potential of hDPSCs. RNA was extracted from hDPSCs at different time points after odontogenic induction, and RT-qPCR was used to analyze the mRNA expression of ANGPT4 and odontogenic-related genes during the odontogenic differentiation of hDPSCs in vitro. siRNA gene silencing technology was used to silence the expression of ANGPT4 in hDPSCs, and the silencing efficiency was detected by RT-qPCR and Western Blot. After silencing ANGPT4 in hDPSCs for 24 h, odontogenic induction was performed. Alkaline phosphatase and alizarin red S staining were performed on the 7th and 14th of induction to detect the odontogenic differentiation ability of hDPSCs after silencing ANGPT4. Results Immunofluorescence staining of human premolars showed that ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone. hDPSCs were in good condition after 14 days of isolation and culture. Under the microscope, multiple cell colonies were observed, and the cell morphology was uniform and long spindle-shaped. The results of flow cytometry showed that hDPSCs expressed mesenchymal stem cell markers CD105 (90.42%) and CD90 (97.15%), but did not express hematopoietic cell markers CD45 (0.01%) and CD34 (0.08%). After odontogenic and adipogenic induction of hDPSCs, alkaline phosphatase staining, alizarin red S staining and oil red O staining were positive. The results of RT-qPCR after the odontogenic induction of hDPSCs showed that ANGPT4 was highly expressed on the 5th, 7th, 11th and 14th days of differentiation of hDPSCs (P<0.05), with the highest expression level on the 5th day. After hDPSCs were transfected with si-ANGPT4, the expression of ANGPT4 mRNA and protein was significantly down-regulated (P<0.05). The results of alkaline phosphatase staining showed that ALP staining intensity and area in the si-ANGPT4 group were significantly lower than those in the negative control. Alizarin red S staining showed that the formation of calcium nodules in the si-ANGPT4 group was significantly lower than that in the negative control. Conclusion ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone of human premolars. ANGPT4 may be a factor to promote the odontogenic differentiation of hDPSCs.

Keywords

• angiopoietin 4
• dental pulp stem cells
• odontoblast
• gene silencing
• odontogenic differentiation
• growth factor
• tissue regeneration