Effect Of Extracellular Matrix Microenvironment On Endothelial Network Formation Based On Cell Sheet Technology

This study aims to fabricate pre-vascularized cell sheets using bone marrow mesenchymal stem cells(BMSCs)in vitro,study the effect of cell sheet extracellular matrix(ECM)micro-environment on vascular network formation,which would offer further theoretical guidance for engineering optimal three-dimensional vascularized tissue.Methods:(1)Fabrication of BMSCs Sheet:The hBMSCs were seeded on a cell culture dish at high density and cultured under two different conditions to obtain undifferentiated and osteogenic differentiated hBMSCs sheets respectively.General observation,microscopes and histological methods were used to evaluate the harvested sheets.(2)The effect of different seeding density of endothelial cells on vascular network formation:The hBMSCs were seeded at high density and cultured for 2 weeks to form a undifferentiated hBMSCs sheet,then HUVECs were seeded onto the sheet at different densities.The hBMSCs sheet morphology,formation of vascular networks under a microscope and CD31 immunofluorescent staining were used to evaluate the vascularized cell sheet.(3)The effect of different cell sheet extracellular matrix(ECM)micro-environment on vascular network formation:The hBMSCs were cultured for 2 weeks to obtain the osteogenic differentiated sheet(OS)and undifferentiated hBMSCs sheet(US)respectively,human umbilical vein endothelial cells(HUVECs)were seeded on the two different sheets at a cell density of 5 X 104cell/cm2.At 3 and 7days,immunofluorescent staining for collagen I(Col I),fibronectin(FN)and CD31 were performed to evaluate the ECM and the vascularized networks formation on the two sheets.Results:(1)The hBMSCs proliferated fast and formed an intact sheet,and the sheet cultured in osteogenic-induced medium showed mineralized nodules,staining of alkaline phosphatase and alizarin red were positive.(2)The HUVECs migrated and rearranged on the hBMSCs sheet.CD31 staining revealed a progressive process of lumen formation.With an increase in the HUVECs density on the sheet,there were more vascularized netwoks formed.Once the HUVECs seeding density was over 5 × 104cell/cm2,there have no any increased in the number of networks.(3)The immunofluorescent staining revealed the presence of Col I and FN,and the amount of Col I in the OS is higher than that of in the US.During tubulogenesis,the Col I remodelled and rearranged,quantitative analysis showed that the amount of Col I significantly decreased and changed more obviously in the OS than in the US.Furthermore,quantitative analysis showed that the number of networks on the OS was higher than that of on the US.Conclusion:(1)hBMSCs sheet can be formed by continuously culturing hBMSCs at high density,and the sheet induced by osteogenic medium have the osteogenic characteristics.(2)It is feasible for HUVECs to assemble into prevascular networks on hBMSCs sheet in vitro and the optimal seeding density of HUVECs was 5×04cell/cm2 in terms of the number of formed network on the sheet.(3)The ECM in the cell sheet was realigned due to the tubulogenesis process,and the OS can significantly facilitated the vascular network formation.