Development Of Prevascularized Tissue Engineered Grafts Using Cell Sheet Technology For Tissue Regeneration

The formation of vascular network of tissue engineering grafts is still a difficult problem.The objective of this experiment is to explore a new way of developing pre-vascularized tissue engineered grafts using cell sheet technology for tissue regeneration,and detect whether the engineered grafts can form functional vascular system in vivo.Methods: 1)Isolation and culture of rabbit bone marrow stem cells(rBMSCs): rBMSCs were isolated by the way of whole bone marrow adherence.The proliferation of rBMSCs were observed by microscope.2)Differentiation of rBMSCs into endothelial like cells: The second passage rBMSCs were cultured in the culture medium which contained inducing factor: vascular endothelial growth factor(VEGF,10 g/L)and basic fibroblast growth factor(b-FGF,10 g/L)for 2 weeks.The morphological changes,proliferation and growth characteristics of the induced cells were observed under inverted microscope.And the induced endothelial cells were identified by the flow cytometry.3)Development of pre-vascularized cell sheet: The second passage rBMSCs were seeded on the culture plate at a density of 9 × 104 / cm2 and cultured for 2 weeks to form undifferentiated cell sheet.The induced endothelial cells from rBMSCs were seeded on the surface of undifferentiated cell sheet at a density of 5 × 104 / cm2 and cultured for 72 hours in the ECs culture medium.The distribution of endothelial cells and angiogenesis were observed under microscope.4)Construction of three dimensional tissue engineering pre-vascularized graft: The ECs/BMSCs cell sheet were cultured for 7 days in the ECs culture medium,and a cell scraper was used to gently scratch the edge of the thick cell sheet and detach it.The harvested cell sheet was clipped into approximately a square.The manicured ECs/BMSCs cell sheet was then folded 3 times to form 8layers EC/BMSCs constructs with a point forceps,which was defined as experimental group.At the same time,the composite construct of the undifferentiated cell sheet was defined as control group.The folded cell sheet was incubated in the ECs culture medium for 24 hours.5)Transplantation experiment in vivo: After culturing for 24 hours,the experimental group andcontrol group cell sheets were implanted into subcutaneous of nude mice.At the time of 1,2weeks after implantation,gross,histological and immunohistochemistry staining were performed.Results: 1)With the whole bone marrow adherence method,we successfully obtained the rBMSCs,which was spindle and polygonal in shape under the microscope.2)BMSCs could be induced into ECs,and the induced ECs showed a "cobblestone like" cell morphology under the microscope.And the rate of expression for CD-31 positive reached to 49.4% by Flow cytometry detection.3)When ECs were seeded onto undifferentiated cell sheet,the ECs migrated and rearranged.CD31 staining showed cavity structure on surface after 3 days.4)After 1,2 weeks,HE staining and CD-31 immunohistochemistry staining showed that the number of blood vessels of control group was significantly lower than that of the experimental group at two time points.However,there was no significant difference between the two time points in the experimental group.The results of CD-31 immunohistochemistry staining showed that the blood vessels produced by the pre-vascularized cell sheets could be functional anastomosis with the host vessels.Conclusion: A pre-vascularized cell sheet could be constructed in vitro entirely based on bone marrow mesenchymal stem cells.After implantation,the pre-fabricated capillary network on the cell sheet could integrate with the host blood vessel to form functional blood vessels.And this provided a new strategy for vascularization of engineered tissues.