Fabrication Of Pre-vascularized Cell Sheets Based On Rabbit Bone Marrow Mesenchymal Stem Cells

The aim of this study is to is to explore a new method of developing a pre-vascularized cell sheet. based on the rabbit bone marrow mesenchymal stem cells, Mehods:(1):Isolation and culture of rabbit bone marrow mesenchymal stem cells(rBMSCs):rBMSCs were isolated and purified from the rabbit femoral marrow by the method of whole bone marrow adherent, the morphology and characteristic of rBMSCs were observed using inverted microscope and MTT.(2):The rBMSCs were induced to differentiate into endothelial-like cells (ECs):rBMSCs were cultured in DMEM-LG medium that added the10μg/LVEGF and10μg/L b-FGF, at different time points, the morphology and characteristic of rBMSCs were observed using inverted microscope and MTT, immunofluorescent staining for CD31/vWF was used to evaluate the induction efficiency.(3):Fabrication of a pre-vascularized cell sheet:The undifferentiated rBMSCs were seeded on a cell culture dish at a cell density of9×104cells/cm2and cultured for2weeks to form a thick cell sheet, and ECs were then seeded on the rBMSCs sheet at a cell density of5×10cells/cm2to develop vascularized cell sheets. The immunofluorescent staining and histological analysis were performed to evaluate the vascularized cell sheet. Results:(1) Using the method of whole bone marrow adherent,we can get the BMSCs. The BMSCs,displayed morphologically as spindle-like、polygon morphology and the colony-shape,and the "MTT" test showed the growth curve is "S" type.(2)At the designated time points, the morphology of rBMSCs changed from long fusiform shape to cobblestone-like morphology under the effects of VEGF and b-FGF. The expression of platelet endothelial cell adhesion molecule (PECAM-1, CD31) and von Willebrand factor (vWF) were positive in ECs, while the rBMSCs were negative.(3)When the ECs were seeded on the rBMSCs sheet, the ECs migrated and rearranged. The images showed the formation of the intracellular vacuoles and networks. Furthermore, immunofluorescent staining for CD31also revealed a developing process of cord formation after the ECs were seeded on the rBMSCs sheet. The histological evaluations indicated the lumen formed. Conclusion:(1) The rBMSCs can be effectively isolated and purified by the method of whole bone marrow adherent,then the rBMSCs have the Strong ability of proliferation.(2) rBMSCs have ability to differentiate into vascular endothelial like cells under the investigated condition.(3)Endothelial like cells differentiated from mesenchymal stem cells can develop into pre-vascular network constructs when seeded on the rBMSCs sheet, which provides a new method for engineering a pre-vascularized tissue construct.