Angiogenesis Of Stem Cells From Human Exfoliated Deciduous Teeth Stimulated By Catechol-chitosan Loaded With RhVEGF₁₆₅

The pulp disease of young permanent teeth caused by infection and trauma is one of the common diseases in oral medicine,The traditional treatment for pulp and periapical disease of young permanent teeth are apexification.Although apexification can promote the development of the root,the lost circulation of the teeth will become brittle,more likely to break or split than the normal teeth,or even premature missing.In recent years,with the development of cell and molecular biology,pulp tissue regeneration has become a biological therapy which can not only regenerate damaged pulp tissue but also restore its physiological function.However,the key to the success of pulp tissue regeneration is to induce the formation of blood vessels quickly and effectively,and to provide a nutritional basis for tissue regeneration.As one of the key factors of tissue engineering,scaffolds are the focus of research in recent years.Chitosan is widely used in biomedical field,although it has good biological properties,However,the structural instability limits its application in biomedical field,and catechol compounds have good adhesion properties.Therefore,we envisage whether chitosan can be combined with catechol to prepare a new scaffold material with vascular endothelial growth factor(VEGF),which can promote angiogenesis,as a carrier of dental pulp stem cells,and to promote the regeneration of blood vessels.To improve the success rate of pulp regeneration,to provide a new treatment for pulp tissue repair.In our research group,we have successfully grafted catechol groups into chitosan molecules by chemical method,and crosslinked with natural cross-linking agent Genipin to prepare catechol-chitosan scaffolds with good biological properties and three-dimensional reticular structure,In this experiment,catechol-chitosan was used as carrier of stem cells from human exfoliated deciduous teeth to observe its effect on adhesion and proliferation of dental pulp stem cells on the surface of deciduous teeth,rhVEGF165 and catechol-chitosan were combined to observe their ability to promote the differentiation of stem cells from human exfoliated deciduous teeth into vascular endothelial cells in order to provide a new scaffold material for pulp regeneration of young permanent teeth.In this study,stem cells from human exfoliated deciduous teeth were extracted by enzyme digestion method and inverted attachment method,and identified by cell morphology,alkaline phosphatase and alizarin red staining;The pore diameter structure of chitosan and catechol-chitosan after freeze-drying was observed by scanning electron microscope,After the disinfection,the material was pretreated with High glucose medium containing 10%FBS and 1%double antibody,and the pH of the culture solution was monitored;The biocompatibility of chitosan and catechol-chitosan was detected by MTT;The stem cells from human exfoliated deciduous teeth were co-cultured with chitosan and catechol-chitosan respectively,The proliferation and adhesion of the cells on the surface of the materials were observed by scanning electron microscope(SEM)and HE staining,ten 400-fold fields of view were randomly selected in each group to calculate the average cell density to evaluate whether catechol-chitosan is more favorable to the proliferation of cells on the surface and the extension of pore size;The effect of rhVEGF165 loaded catechol-chitosan scaffolds on the angiogenesis ability of stem cells from human exfoliated deciduous teeth was detected by Real timePCR to detect the expressionof CD31,VEGFR2,VE-cadherin on the surface of endothelial cells.The experimental results show that we have successfully extracted stem cells from human exfoliated deciduous teeth,and the stem cells have typical fibroblast-like morphology,Alkaline phosphatase and alizarin red staining showed that the extracted dental pulp stem cells had good osteogenic differentiation ability;Scanning electron microscopy(SEM)showed that chitosan and catechol-chitosan were both three-dimensional pore-like structure,but the pore size of catechol-chitosan was more uniform than that of chitosan;The results of MTT test showed that the biocompatibility of catechol-chitosan was better than that of chitosan;Scanning electron microscopy(SEM)and HE staining showed that catechol-chitosan was more favorable to the adhesion of stem cells on the surface and to the extension of stem cells in the pore size;The results of ReaL-Time PCR showed that the combination of catechol-chitosan and rhVEGF165 was more beneficial to the differentiation of stem cells from human exfoliated deciduous teeth into vascular endothelial cell.In conclusion,the prepared catechol-chitosan has good biocompatibility and can be used as a scaffold material for dental pulp regeneration.