Abstract: BACKGROUND: It is difficult to solve the problem of vascularization in tissue engineering, which has become the key to restrict its clinical application.
OBJECTIVE: To investigate the feasibility of forming a prevascularized cell sheet by co-culturing of rabbit umbilical vein endothelial cells and stem cell sheet, and to analyze the gene expression of angiogenesis related factors.
METHODS: Rabbit bone marrow mesenchymal stem cells were isolated and cultured in vitro, forming stem cell sheet by ascorbic acid. Prevascularized cell sheet was prepared by co-culture rabbit umbilical vein endothelial cells with stem cell sheet in the experimental group. Rabbit umbilical vein endothelial cells cultured separately were used as control group. At 3, 7, and 14 days, morphological and histological changes were observed by light microscopy and hematoxylin-eosin staining. mRNA expression of angiogenesis related factors was detected by RT-PCR, and the structure of vascularized network was observed by immunohistochemical staining.
RESULTS AND CONCLUSION: (1) The light microscopy showed that the cells of the experimental group migrated and rearranged at 3 days after culture. Rabbit umbilical vein endothelial cells "connected" and formed a network structure at 7 days. The network structure was more obvious at 14 days. The cell density of the control group increased continuously, forming a cell mass. No rearrangement or migration of cells was observed to form a network structure. (2) Hematoxylin-eosin staining showed that the cells in the experimental group were not uniformly arranged at 3 days, and the cells were arranged in cords at 7 days and reticulated at 14 days. The cells in the control group showed the accumulation of paving stones and the density increased continuously, without cords or reticular structures. (3) RT-PCR showed that mRNA expression of vascular endothelial growth factor was higher in the experimental group than in the control group at 7 days (P < 0.05). The mRNA expression of basic fibroblast growth factor was higher in the experimental group than that of the control group at 14 days (P < 0.05). The mRNA expression of Ang1 was higher in the experimental group than that of the control group at 7 and 14 days (P < 0.05). The mRNA expression of Ang2 was higher in the experimental group than that of the control group at 3, 7, and 14 days (P < 0.05). (4) Immunohistochemical staining of CD31 showed that the vascular network was formed in the experimental group, while no network was formed in the control group. (5) The results showed that rabbit umbilical vein endothelial cells co-cultured with stem cell membrane can successfully construct the pre-vascularized cell sheet, significantly expressing angiogenesis-related genes.

Key words: material, stem cell sheet, bone marrow mesenchymal stem cells, umbilical vein endothelial cells, ascorbic acid, prevascularized cell sheets, angiogenesis-related factors, tissue engineering