Preparation And Multifunctional Modification Of Three-layer Small Diameter Electrospun Artificial Blood Vessels

In recent years,the development of small diameter(<6 mm)artificial blood vessels that can maintain long-term patency has gained clinical importance.The ideal small diameter blood vessels should have excellent biocompatibility and degradability and be able to provide support through matching mechanical properties to promote the regeneration of tissue in vivo.When the vascular scaffold degrades,the regenerated vascular tissue can provide the same function as that of the autologous vessel.Currently,these synthetic scaffolds still face many challenges that need to be overcome,such as thrombosis,intimal hyperplasia and calcification.Therefore,it is an urgent need to design tissue-engineered vascular scaffolds with sufficiently excellent biocompatibility,mechanical strength and anticoagulant properties.This thesis focuses on the construction of three different small diameter electrospun artificial vascular scaffolds using electrostatic spinning technology to mimic the three-layer structure of natural blood vessels and to investigate their performance:(1)A PCL-COL@PCL@PCL-GEL three-layer small-diameter artificial vascular scaffold was constructed by layer-by-layer electrostatic spinning method.To improve the biocompatibility and cell adhesion of the inner layer of the artificial blood vessel,polycaprolactone(PCL)and collagen(COL)were used as the inner layer raw materials,PCL was used in the middle layer to improve the mechanical properties,gelatin(GEL)and PCL were used to construct the outer layer to further improve the compatibility of vascular grafts in the human environment.The performance of the vascular scaffold was also regulated by changing the raw material ratio of PCL and COL in the inner layer.The artificial vessels obtained by electrospinning had good mechanical properties with a maximum longitudinal stress of 2.63 ± 0.12 MPa,which exceeded the maximum stress that many natural vessels can withstand.The addition of COL to PCL significantly enhanced the hydrophilicity of the vascular grafts,and human umbilical vein endothelial cells(HUVECs)adhered more easily to the vascular grafts.In particular,the cytocompatibility and proliferation of HUVECs in the PCL:COL=2:1 scaffold was superior to other ratios of artificial vessels.And the prepared artificial vascular scaffolds did not cause erythrocyte hemolysis.Therefore,the electrospun resulting PCLCOL@PCL@PCL-GEL artificial blood vessels were a promising material for vascular tissue engineering.(2)The crosslinking modification technology of EDC/NHS was used to further improve the performance of small caliber artificial blood vessels.Firstly,three layers of small caliber artificial blood vessels were prepared by electrospinning technology,the inner layer was made of polylactic acid(PLA)and collagen,the middle layer was made of polylactic acid(PLA)and polycaprolactone,and the outer layer was made of polycaprolactone and gelatin.Collagen and gelatin added to artificial blood vessels can be cross-linked by EDC/NHS.The mechanical properties and hydrophilicity of the crosslinked artificial blood vessels have been greatly enhanced.After cross-linking,the maximum axial stress of the artificial blood vessel with inner layer composition proportion PLA:COL=4:1 reached 9.56 MPa,and the radial maximum stress reached 9.31 MPa,which was higher than that of many natural blood vessels.The improvement of hydrophilicity of vascular inner layer before cross-linking was due to the addition of COL.After cross-linking,the inner layer of artificial blood vessel produced hydrophilic urea derivatives,which greatly improved the hydrophilicity of vascular inner layer after crosslinking.The improvement of hydrophilicity would also make it easier for cells to adhere to the inner layer of blood vessels,especially the artificial blood vessels of PLA:COL=2:1.The addition and cross-linking of COL made the cells on this kind of scaffold have high proliferation rate and adhesion.The prepared three-layer vascular stent would not cause hemolysis.Therefore,the three-layer vascular stent crosslinked by EDC/NHS was a kind of artificial vascular material with excellent properties in all aspects.(3)The anti-coagulant properties of the artificial vessels were improved by depositing chitosan followed by cross-linking and grafting heparin on the artificial vessels prepared above.The deposited chitosan provided more amino active sites,and the crosslinked and grafted heparin had excellent hydrophilicity,and the endothelial cells adhered and proliferated more easily on the inner layer of the artificial vessels.On the 5th day of cell proliferation,the PC21-CHep vascular scaffold was almost completely covered by endothelial cells.And the maximum tensile stress in the axial direction of PC41-CHep vascular scaffold reached 8.47 MPa,which has far exceeded the tensile strength of natural blood vessels.In terms of anticoagulation,the artificial vascular scaffold grafted with more heparin by depositing chitosan showed a much higher anticoagulant effect than the artificial vessel without functionalized modification of heparin.The prepared artificial vascular scaffold was a tissue-engineered vascular scaffold with excellent anticoagulant effect.