Construction Of Antithrombotic Tissue-engineered Blood Vessels With Biomimetic Enzymatic Reaction

With the increasing morbidity and mortality of cardiovascular disease,a large number of blood vessels are needed to operate vascular grafting.At present,the blood vessels of clinical transplantation mainly include autologous blood vessels and artificial blood vessels.There are some problems in the process of autologous blood vessel transplantation: on one hand,the quantity is limited,and the quality of autologous blood vessels is not high under the condition of disease;on the other hand,it will cause local injury when taking the blood vessels.Therefore,there is an urgent need for artificial blood vessels of good biocompatibility.But only large-diamater artificial blood vessels are available for the treatment of aortic diseases currently,small-diamater blood vessels have low patency rate due to acute thrombosis,which leads to the failure of transplantation.Preventing acute thrombosis is the key to ensure the patency of small-diameter tissue engineered blood vessels(TEBVs).As xenobiotic,artificial blood vessel will quickly activate platelets after implantation,activated platelets will release adenosine diphosphate(ADP)in high concentration,which is the most important substance that promotes platelet aggregation.The aggregation of platelets will further activate the coagulation reaction and form a stable thrombus at last.Inhibition of platelet aggregation induced by transplantation is an effective way to preve nt acute thrombosis.Reduced graphene oxide(RGO)is a kind of two-dimensional nano materials with unique physical and chemical properties;it has high specific surface area,high absorption,good stability and excellent biocompatibility and is widely used in the field of biomedical research.In physiological conditions,endothelial cells play an antithrombotic role through continually synthesizing antiplatelet and anticoagulant substances.Inspired by this,we designed a novel biomimetic antithrombotic TEBVs modified by RGO-Enzyme complex.We take RGO as the carrier,the Apyrase and 5'-nucleotidase(5'-NT)immobilized on the surface covalently,which not only enhance the stability of the enzyme protein against blood flow,but also prevent the aggregation of enzyme protein and improve its dispersibility.These two kinds of enzymes can synergistically catalyze ADP continuously into the adenine nucleotide(AMP),which can inhibit platelet aggregation and adenosine under physiological conditions.Thus inhibit platelet aggregation,promote endothelial cell proliferation and ensure the patency of TEBVs.Methods1.Fabrication of RGO-Enzyme and its characterizationRGO dispersion was obtained by ultrasonic dispersion of RGO with the assistance of two amphiphilic macromolecules DSPE-PEG2000-COOH.Under the activation of EDC and NHS,Apyrase and 5'-NT was covalently bonded to the carboxyl terminus of RGO,and the RGO-Enzyme complex was obtained.Using the collagen coated slide as a model,the RGO-Enzyme complex was crosslinked on the slide surface to conduct a flow chamber experiment to verify the ability of RGO-Enzyme composite to resist the blood flow and shear stress.The dispersion of RGO was observed by transmission electron microscopy(TEM),and the binding of Enzyme on RGO surface was characterized by Raman spectroscopy.2.Determination of RGO-Enzyme activityDetermination of RGO-Enzyme activity in vitro was induced by molybdate malachite green spectrophotometry.High performance liquid chromatography(HPLC)was used to determine the changes of ADP,AMP and adenosine in plasma under the action of RGO-Enzyme.Blood of rats was obtained to verify the anti platelet aggregation performance of RGO-Enzyme in vitro.The degree of platelet activation was detected by flow cytometry.The effect of RGO-Enzyme on the proliferation of endothelial cells was examined in vitro.3.Animal experimentDecellularized vascular matrix material was prepared and RGO-Enzyme was modified on the surface.7 days after implantation to rat carotid artery,rats were examined by CT.Grafts were taken out and observe using scanning electron microscope(SEM)and HE staining.Results1.The TEM results showed that RGO was fully dispersed,which increased the specific surface area and adsorption capacity.Raman spectra showed that the enzyme molecules were successfully crosslinked to the RGO surface.The plate flow chamber experiment results show that the immobilized enzyme on RGO have stronger resistance to blood flow shear force,and are not easy to agglomerate.2.The results of enzyme activity assay and HPLC showed that RGO-Enzyme had good enzyme activity,which could catalyze the conversion of ADP to AMP and adenosine.The results of in-vitro experiments showed that RGO-Enzyme could enhance the proliferation of endothelial cells and reduce the activation of platelets.3.The results of animal experiments showed that RGO-Enzyme modified TEBVs were kept open for 7 days,and there was no thrombus formation.Compared with the control group,the degree of endothelialization was better.ConclusionUsing RGO as a carrier,a RGO-Enzyme catalytic system with two synergetic enzymes was constructed and was modified on the surface of TEBVs.This system enhance enzyme stability,and will catalyze the conversion of ADP to AMP and adenosine,which could regulate the local micro-environment and inhibite platelet aggregation,thereby inhibiting the platelet aggregation induced by vascular transplantation and thrombosis,promote endothelialization and guarantee the patency of TEBVs.