Abstract: BACKGROUND: The autologous bypass grafting is the best surgical scheme. However, the source of autologous blood vessels is limited, which is far from meeting the huge clinical needs. Therefore, the research and development of small-diameter artificial blood vessels are of great significance. However, small-diameter artificial blood vessels have not been truly applied in clinical practice due to the problems of easy thrombosis and intimal hyperplasia. 
OBJECTIVE: To explore the growth of human umbilical vein endothelial cells and vascular smooth muscle cells on poly(L-lactic caprolactone)/silk fibroin electrospun membrane, the patency, histocompatibility and degradability of poly(L-lactic caprolactone)/silk fibroin small-diameter artificial blood vessels in rats.
METHODS: Electrospinning technology was used to prepare poly(L-lactic caprolactone)/silk fibroin small-caliber artificial blood vessels. Vascular endothelial growth factors were loaded on the artificial blood vessel, and heparin was finally loaded on the artificial blood vessel. Human umbilical vein endothelial cells and vascular smooth muscle cells were respectively seeded on artificial blood vessels loaded and unloaded with vascular endothelial growth factor. The growth of cells on artificial blood vessels was analyzed by confocal laser scanning microscope, scanning electron microscope, and MTT assay. Transwell membrane in cell chamber was replaced with poly(L-lactic caprolactone)/silk fibroin electrospun membrane. Human umbilical vein endothelial cells and vascular smooth muscle cells were inoculated on poly(L-lactic caprolactone)/silk fibroin material for cell co-culture. The growth of cells on poly(L-lactic caprolactone)/silk fibroin materials was analyzed by confocal laser scanning microscope, scanning electron microscope, and MTT assay. The artificial blood vessel loaded with vascular endothelial growth factor and heparin was implanted into the rat carotid artery. The patency, histocompatibility, and degradability of the artificial blood vessel were detected.
RESULTS AND CONCLUSION: (1) Cells separately seeded on artificial blood vessels: Confocal laser scanning microscope, scanning electron microscope, and MTT assay showed that the number of human umbilical vein endothelial cells seeded on the artificial blood vessel loaded with vascular endothelial growth factor was more than that of the artificial blood vessel without vascular endothelial growth factor. The number of vascular smooth muscle cells loaded with and without vascular endothelial growth factor was basically the same. (2) Two kinds of cells seeded together on the artificial blood vessel: Confocal laser scanning microscope, scanning electron microscope, and MTT assay showed the number of cells in co-culture was larger than that of single cells in non-co-culture environment, but less than the sum of the number of two single cells. (3) Implantation experiments in animals: The laser Doppler perfusion imaging showed that the blood flow velocity decreased at 1 day after implantation of the artificial blood vessel, and then gradually decreased, until the third week it had not been surveyed. Histological and immunohistochemical staining exhibited inflammatory cells appeared on the third day after implantation of artificial blood vessel, and disappeared one week later. At the third week, blood flow was observed in the sample, and a uniform endodermis and new angiogenesis were formed on the inner wall of the sample. Scanning electron microscopy showed that the fibers were basically broken at the 6th week of implantation of the artificial blood vessel, and the fibers became thinner. At the 4th week, the retention strength of artificial blood vessel suture decreased to 42.2% of the original data. (4) These findings suggest that poly(L-lactic caprolactone)/silk fibroin small-diameter artificial blood vessels have good cytocompatibility and histocompatibility, but the patency and degradability need to be improved. 

Key words: small-diameter artificial blood vessel, poly(l-lactide-co-ε-caprolactone), silk fibroin, cell co-culture, in vivo experiment