Effects Of Microspheric Deformable Scaffolds With Different Concentrations Of Vascular Endothelial Growth Factor On Osteogenic Activity Of Human Placenta-derived Mesenchymal Stem Cells

Purpose:1.This study attempted to prepare sodium alginate/chitosan microsphere scaffolds with different concentrations of vascular endothelial growth factor xunder aseptic conditions.2.Human placenta-derived mesenchymal stem cells(HPMSCs)are isolated and cultured in vitro.3.To investigate the effects of algirnate/chitosan microsphere scaffolds loaded with different VEGF concentrations on the proliferation and osteogenic activity of HPMSCs,and to explore the optimal VEGF concentration for promoting the proliferation and osteogenic activity of HPMSCs,and at this concentration,the adhesion rate of the cells to the scaffold material is measured.Methods:1.Using the principle of layer self-assembly and the principle of electrostatic and complexartion of anion and cation,prepare multi-layer sustained-release microsphere scaffolds loaded with VEGF and vancomycin,and set the concentration gradient of VEGF as follows:Negative control;Ong/ml;10 ng./_ml;25 ng/ml;50 ng/ml.2.Primary HPMSCs were isolated and cultured from the placenta by tissue block culture method,and the cells after three generations were used for osteogenic differentiation and adipogenic differentiation and subsequent experiments.3.The effect of microsphere-type scaffolds with different concentrations of VEGF on the proliferative activity of HPMSCs was detected by MTT assay.The effects of different concentrations of VEGF microsphere scaffolds on the osteogenic activity of HPMSCs were evaluated by osteogenic induced alizarin red staining,alkaline phosphatase staining,alkaline phosphatase activity assay,and western blot analysis of ALP and BMP-2 genes and expression.Results:1.The multi-layer sustained-release microsphere scaffolds with different concentrations of VEGF prepared under aseptic conditions have both sustained release and scaffold functions,and the morphology is good.:The long spindle-shaped human placenta-derived mesenchymal stem cells were isolated and cultured.After the osteogenic induction,the calcium nodules were observed,and lipid droplets were observed after adipogenic induction.3.The high concentration of VEGF loaded in the scaffold material can promote the proliferation of HPMSCs(P<0.01).The VEGF loaded in the scaffold can promote the osteogenic differentiation of HPMSCs(P<0.01)in a dose-dependent manner.When VEGF is 50 ng/ml,the cells show the best proliferation and osteogenic activity.Conclusions:1.Successfully prepared multi-layered sustained-release microsphere scaffolds loaded with different concentrations of VEGF and verified their sterility.2.Successfully extracted HPMSCs from placental tissue,which was characterized by osteogenic induction and adipogenic induction.3.Through the co-culture of scaffold materials and HPMSCs,the optimal VEGF concentration for promoting the proliferation and osteogenic activity of HPMSCs was 50 ng/ml.4.In this experiment,tissue engineered bone was constructed,in which HPMSCs was used as seed cell,sodium alginate/chitosan microsphere sca-ffold was used as scaffold material,VEGF was used as cytokine,and vancomycin was used as anti-inflammatory drug.This provides a theoretical basis for subsequent experiments in vivo.