Experimental Studies Of Cytobiological Characteristics Of HPMSCs By Co-culture With Multilayer Microspheres Loading VEGF And Vancomycin

Purpose:1.Human placenta derived mesenchymal stem cells(HPMSCs) has been an important type of mesenchymal stem cells as seed cells for bone tissue engineering.This research tried to isolate and culture HPMSCs from placenta, and explore the biological characteristic of HPMSCs.2.We have successfully developed multilayer alginate chitosan microspheres loading VEGF and vancomycin before. The mail goal of this research was to develop the microspheres in a sterile way and to study proliferation and osteogenic differentiation of HPMSCs by co-culture with multilayer microspheres.Methods:1.HPMSCs isolated from sterile minced amnion tissues were cultured in vitro.The morphology was observed under inverted microscope. Cell growth curve was detected with MTT. Then under specific induction conditions, HPMSCs were induced to osteoblast and lipoblast, and specific staining methods were adopted to determine their differentiation potential.2.The multilayer alginate chitosan microspheres loading VEGF and vancomycin was explored in a sterile environment. The third generation was cultured with multilayer microspheres. In the experiment, the ability of proliferation of HPMSCs was identified by cck-8 kit and osteogenic differentiation was identified by Alkaline phosphatase kit and alizarin red staining.Results:1.The cells isolated and cultured from human placenta were showed static adherence and typical fibroblastic like morphology by light microscope, the cell growth curve was present by “S” shape. Cells exposed to osteogenic inductive medium resulted in secretion of extracellular calcium crystals, identified by alizarin red staining, indicating osteogenic differentiation. When cells were cultured in adipogenic inductive medium, intracytoplasmic lipid vacuoles were observed, and confirmed by oil red O staining.2.After improved, the size of the multilayer sustained-release microspheres was uniform, and the surface is smooth and round. Bacteriological detection results showed that the microspheres were satisfied with sterile standard.3.The proliferation capacity of HPMSCs was not significantly changed after co-cultured with microspheres loading VEGF and vancomycin(P>0.05). The three groups exposed to osteogenic inductive medium were all positive in alizarin red staining, but the calcium nodes of the cells co-cultured with microspheres loading VEGF and vancomycin were much more than the other two groups, and the plasma of the cells were obviously blue. The other two groups showed significant difference between each other too. The content of alkaline phosphatase detected with ALP kit showed the same results as alizarin red staining, and all of the differences were statistically significant(P<0.05).Conclusion:1.Human placenta was proved to be rich in HPMSCs, which could be obtained from the amnion by specific separation method. The results of differentiation induction indicated that the cultured HPMSCs maintained the cytobiological properties and multi-directional differentiation potential as MSCs.2.Multilayer alginate chitosan sustained-release microspheres loading VEGF and vancomycin was developed in sterile environment successfully.3.There was no significant effect on the proliferation activity of HPMSCs co-cultured with microspheres loading VEGF and vancomycin. But it could significantly improve the osteogenic differentiation activity of HPMSCs.