Studies Of Small-diameter Artificial Vascular Graft With High Anticoagulant Property And Ability To Induce Regeneration

Objective:Treatment of obstructive atherosclerotic disease, which is a major cause of mortality and morbidity in the world, may involve replacement and bypassing of affected arteries using autologous, allogenic and synthetic vascular grafts. Although vascular grafts have been used successfully to replace large-diameter blood vessels, the long term patency of small-diameter (<6mm) vascular grafts is still disappointing, primarily due to stenosis and thrombus formation. Vascular tissue engineering has provided new approaches for implantation, especially small-diameter vascular grafts. Vascular grafts with a confluent lining of endothelial cells can be obtained after expansion of cell numbers by cell culture in vitro. Drawbacks of this approach are the time interval between the need of an endothelialized vascular graft and its availability, as well as the increased risk of bacterial infection, limiting its use to non-emergency situations. In this study, decellularized fresh specimens of bovine pericardiums in vitro and pig small-diameter blood vessels in vivo were studied with respect to crosslinking, heparinization and vascular endothelial cell growth factor (VEGF) binding and release, in order to produce high-performance small-diameter artificial blood vessels with highly anticoagulant property and ability to induce regeneration in short time.Methods:1 Matrix preparation:Fresh specimens of bovine pericardiums were treated by repeatedly freezing and thawing+TritonX-100. Tissue samples were then observed by HE staining to confirm the removal of cells. Acellular tissue samples were crosslinked by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS),and the content of crosslink was detected by mechanical properties, in vitro degradation by collagenase and the shrinkage temperature indicating the resistance against thermal denaturation.2 Heparin immobilization,3 methods:(1) Heparinized EDC treated acellular tissue samples; (2) Heparinized poly(ethyleneimine) (PEI, MW=1800D, 10kD,20kD) treated acellular tissue samples; (3) Heparinized EDC-PEI treated acellular tissue samples. Determination of immobilized heparin was tested by ICP-MS and FTIR; the surface wettability was tested by contact angle; the moisture uptake was analyzed; cell and blood compatibility were evaluated by cytotoxic test, hemolysis test, platelet attachment test, PT/APTT and recalcification time.3 VEGF165 labeling and binding:VEGF165 was labeled with 125I in comparison of EDC treated group, PEI treated group and EDC-PEI treated group in order to observe the binding and release of VEGF165. Proliferation of human umbilical vein endothelial cells was tested.4 Small-diameter vascular xenografts were modified by the best heparinization way and implanted in canine left carotid artery to evaluate the feasibility.Results:1 Histological analysis revealed that repeatedly freezing and thawing +TritonX-100 resulted into a complete loss of cellular structures and no loss or disruption of extracellular matrix. This suggested that it was an effective, economic, fast decellularization method. Mechanical properties and heat shrinkage temperature were greater in matrix crosslinked by EDC than that in fresh specimens of bovine pericardiums, and had the perfect anti-degradeation function.2 Amount of heparin with PEI was increasing by multilayers. Different multilayers would produce different amount of heparin. Heparin immobilized by EDC equaled to heparin produced by 18 multilayers. Heparin was treated by 15 multilayers with 3 MW of PEI, moisture uptake and contact angle tests revealed that wettability and hydrophilia abiltity were greater in PEI1800D treatment group than that in PEI10kD and PEI20kD group. In hemolysis test, hemolysis ratio was 2.66% in PEI1800D treatment group,5.02% in PEIlOkD treatment group,5.58% in PEI20kD treatment group. These revealed that only PEI1800D treatment group was qualification. Hemolysis test, platelet attachment, PT/APTT and recalcification time were tested in control group, EDC-Hep group, PEI1800D-Hep group and EDC-PEI1800D-Hep group, confirmed that anticoagulant properties were the best in EDC-PEI1800D-Hep group during immersed in saline for 15 days. In cytotoxic test, PEI1800D-Hep group was 0.5 degree, EDC-PEI1800D-Hep group was 1.0 degree, EDC-Hep group was 1.3 degree. These suggested that PEI1800D-Hep group and EDC-PEI1800D-Hep group were better than EDC-Hep group. 3 Through the results of binding and release of VEGF165 from heparized materials, we could make conclusion that controlled release of VEGF165 from EDC-Hep group was better than that of PEI-Hep group, which confirmed the stability of heparin was better in crosslinking. Therefore, the stability of heparin in EDC-PEI1800D-Hep group was satisfactory too. Proliferation of human umbilical vein endothelial cells in EDC-PEI1800D-Hep group was the best.4 EDC-PEI1800D-Hep grafts binding VEGF165 were implanted in canine left carotid artery. All grafts were not occluded in short time. We should observe continuously.Conclusions:Fresh exogenic tissue, binding heparin and VEGF165 with EDC-PEI1800D, had better anticoagulant properties, proliferation of vascular endothelial cells, mechanical properties and anti-degradeation function. Period of preparation was short, and we could make conservation after freezing and sterilization for a long time. These results demonstrated that these small-diameter artificial blood vessels with highly anticoagulant property and ability to induce regeneration would be applied in clinical settings in future.