Effects Of ANGPT4 On Odontogenic Differentiation Of Dental Pulp Stem Cells And Angiogenesis

Purposes:Human dental pulp stem cells(hDPSCs)are a kind of mesenchymal stem cells derived from the cranial nerve crest,which have a multi-directional differentiation potential and play an important role in pulp-dentin regeneration.hDPSCs are good candidates for seed cells for tissue-engineered tooth regeneration.Identification of bioactive molecules that regulate dental pulp stem cell differentiation and angiogenesis will contribute to the development of therapeutic strategies for pulp-dentin regeneration.Angiopoietins are a family of growth factors that regulate the development of blood vessels and lymphatic vessels.It is composed of Angiopoietin 1(ANGPT1),Angiopoietin 2(ANGPT2)and Angiopoietin 4(ANGPT4),which are ligands of the endothelial cell-specific receptor tyrosine kinase TIE2.Among them,the recombinant protein of ANGPT1 can promote angiogenesis and stability through the PI3K/Akt signaling pathway mediated by TIE2 and can regulate the differentiation of bone mesenchymal stem cells(BMSCs)into osteoblasts.However,the clinical application of ANGPT1 is limited due to its polymer structure and poor solubility.As a member of the angiopoietin family,ANGPT4 has a similar function to ANGPT1 and can expand the endothelial lumen structure through the PI3K/Akt pathway mediated by Tie2.However,whether ANGPT4 has a dual role in promoting osteogenic differentiation and angiogenesis similar to ANGPT1 remains unclear.So far,the role of angiopoietins in dental pulp angiogenesis has not been studied,and the physiological importance of angiopoietins in dental pulp regeneration is still unknown.This study aims to investigate the expression and localization of ANGPT4 in mouse mandibular incisors and human premolars,as well as its effects on the differentiation of hDPSCs and angiogenesis.It has important theoretical significance to explore the mechanism of hDPSCs in pulp dentin regeneration.This study will provide effective therapeutic strategies for the regeneration of dental-pulp lesions.Methods:human premolars were fixed,decalcified,dehydrated,embedded and sectioned.Immunohistochemical staining was used to observe the expression and localization of ANGPT4.hDPSCs were isolated and cultured from the pulp tissue of healthy teeth.After 7 days of static culture,the growth status and morphology of hDPSCs were observed under an inverted phase contrast microscope,and the expression of surface-related molecular markers was detected by flow cytometry.The odontogenic differentiation potential of hDPSCs was identified by ALP and alizarin red S staining after 7 and 14 days of odontogenic induction,respectively.The adipogenic differentiation potential of hDPSCs was identified by oil red 0 staining.RNA was extracted from hDPSCs at different time points after odontogenic induction,and RT-q PCR was used to analyze the expression of ANGPT4 and odontogenic-related genes during the odontogenic differentiation of hDPSCs in vitro.The expression of ANGPT4 in hDPSCs was silenced by si-RNA gene silencing technology,and the silencing efficiency was detected by RT-q PCR and Western blot.ALP and alizarin red S staining were performed on the 7th and 14 th day of induction to detect the odontogenic differentiation ability of hDPSCs after silencing ANGPT4.After silencing ANGPT4 in hDPSCs,the human umbilical vein endothelial cell fusion cells(EA.hy926)were cultured in conditional medium(CM).Scratch test was used to detect the scratch healing rate of EA.hy926 cells.The angiogenesis ability of EA.hy926 cells was detected by angiogenesis assay in vitro.Results:Immunohistochemical staining of ANGPT4 in human premolars showed that ANGPT4 was expressed in odontoblasts and the sub-odontoblastic rich-cell zone.hDPSCs were in good condition after 14 days of culture.Under the microscope,multiple cell colonies were observed,with uniform cell morphology and long spindle shape.Flow cytometry results showed that hDPSCs expressed mesenchymal stem cell surface markers CD105 and CD90,but did not express hematopoietic cell markers CD45 and CD34.After 7 days of odontogenic induction,ALP staining was positive in hDPSCs.After 14 days of odontogenic induction,calcium nodule formation was observed by alizarin red S staining.Lipid droplet formation was observed by oil red O staining after 30 days of adipogenic induction of hDPSCs.The results of RT-q PCR after the odontogenic induction of hDPSCs showed that ANGPT4 was highly expressed in the differentiation of hDPSCs on the 5th,7th,11 th and 14 th days,with the highest expression level on the 5th day,which was similar to the expression pattern of RUNX2 gene.RT-q PCR and Western Blot results showed that the expression of ANGPT4 in hDPSCs was successfully silenced by si RNA gene silencing technology.The results of ALP staining showed that the intensity and area of ALP staining in si-ANGPT4 group were significantly lower than those in NC group.Alizarin red S staining showed that the formation of calcium nodules in si-ANGPT4 group was significantly lower than that in NC group.The results of scratch test showed that the scratch healing rate of EA.hy926 cells in si-ANGPT4 CM group was lower than that in NC CM group.The results of angiogenesis assay in vitro showed that compared with the NC CM group,the angiogenesis ability of EA.hy926 cells in the si-ANGPT4 CM group was decreased.Conclusions:1.ANGPT4 is highly expressed in odontoblasts and sub-odontoblastic cell-rich zone of human premolars.2.ANGPT4 promotes the odontogenic differentiation of hDPSCs.3.hDPSCs promote EA.hy926 migration and angiogenesis in vitro through ANGPT4,which may contribute to the angiogenesis of dental pulp.