The Study Of Intima Surface Modified By Drug Delivery System Promotes Endothelialization Of Tissue Engineered Blood Vessels

Cardiovascular disease has become a major cause of disability and death in current society,and the number of people dying from cardiovascular disease is still high and increasing year by year.The main cause of the disease is ischemia and anoxia of target organs caused by vascular obstruction,stenosis,or inflammation,which leads to cardiovascular disease and its complications.Autograft is usually chosen for the treatment of this disease in clinical medicine,but the quality of autograft also decreased due to various diseases,so the autograft is no longer suitable for transplantation.TEBVs,a substitute of artificial blood vessel,can effectively alleviate the problem of the limited source of ideal blood vessel donor,showing a broad application prospect.Among them,small-caliber tissue engineered blood vessels(Φ≤6 mm)meet the diameter requirement of patients’ own vessels and are suitable for clinical vascular transplantation,but a series of pathological problems such as thrombosis and intimal hyperplasia often occur after transplantation,and cause a decrease in the patency of the blood vessels,which leads to the failure of the operation.Endothelium plays an important role in post of repairing transplanted TEBVs,and intact endothelium layers inhibits thrombosis and intimal hyperplasia.Speeding up the process of endothelialization of TEBVs,that is,promoting the proliferation and migration of endothelial cells,can effectively reduce various kinds of postoperative complications,and is also an important measure to improve the rate of vascular patency and the rate of successful operation.Ginsenoside Rg1 is an active component extracted from the root and stem of Panax Ginseng.It has high content and wide pharmacological action,such as protecting nerve,anti-depression,improving cognition,lowering blood glucose,protecting liver and anti-oxidation.In addition,ginsenoside Rg1 can promote the proliferation and migration of endothelium in angiogenesis and myocardial protection,which shows good therapeutic and medical value.The classical drug carrier cyclodextrin has the chemical structure that can carry the drug molecules and keep the drug releasing function for a long time and the biocompatibility is well.It has been widely used in medical industry.There are amino groups in chitosan molecule,which can dehydrate and condense with the carboxyl group of collagen molecule to form covalent bond.Based on the above,the purpose of this study was to investigate the effects and mechanisms of ginsenoside Rg1 on the proliferation and migration of RAVECs,and prepared drug delivery system Rg1-HGC/CS NPs and modified them into the lumen of blood vessels,and characterized the blood vessels.SD Rats were used as experimental animal models,to evaluate the effect of endothelialization-promoting TEBVs by transplanting into the common carotid artery of SD rats,the patency and endothelialization were observed.Methods1.Effect and mechanism of ginsenoside Rg1 on RAVECs in vitro: Expanding cultivation of RAVECs;CCK-8 was used to detect the effects of different concentrations of Rg1 on cell viability and proliferation,to determine the appropriate working concentration of the drug,and to explore its related mechanisms;The effects of drugs of working concentration on cell migration and its mechanism were examined by cell scratch test;Study on the effect of drug concentration on apoptosis behavior by flow cytometry.2.Characterization and construction of endothelialization-promoting TEBVs and drug delivery system: Rg1-HGC/CS NPs were prepared and modified to the inner surface of TEBVs;Study on the synthesis of drug delivery system by infrared spectroscopy;Particle size distribution of nanoparticles detected by DLS;SEM scanning of NPs and micrograph of inner surface of blood vessel;Mechanical properties measured the maximum mechanical load of blood vessels;Drug release tested for release characteristics of endothelialization-promoting TEBVs;Co-culture test for biocompatibility of TEBVs.3.Animal experimental study on endothelialization-promoting TEBVs: Common carotid artery transplant operation in SD rats,divided into collagen group,blank CS group and endothelialization-promoting TEBVs group,that is,collagen treated vessels,blood vessels modified by CS blank particles and endothelialization-promoting TEBVs were transplanted respectively;The vascular patency of the 30 th day and 90 th day of rats was detected by ultrasound and Micro-CT;HE and Masson sections were stained to observe the structural integrity and the condition of collagen hyperplasia in blood vessels;CD31 and VWF immunofluorescence staining were used to observe the endothelialization of blood vessels on the 90 th day after transplantation.Results1.Rg1 has shown the effect of promoting the proliferation and migration of RAVECs in a certain concentration range,the optimum concentration is 15.6 μmol/L,and the drug could produce the above effect via VEGFR2 on RAVECS possibly;At this concentration,no drug-induced apoptosis of cells were observed.2.The results of FTIR showed that Rg1-HGC/CS was prepared;DLS suggested that the particle diameter was about 130 nm and the size was uniform(PDI < 0.3);The results of SEM showed that NPs had been successfully modified on the luminal surface of blood vessels;The mechanical properties of the prepared TEBVs showed they were similar to the maximum mechanical load of natural blood vessels;Drug release experiments showed that the endothelialization-promoting TEBVs could release drug continuously and smoothly;The results of cell co-culture showed that the blood vessels had a good biocompatibility and was suitable for cell adhesion and growth.3.The results of small animal ultrasound(30 d)and Micro-CT(90 d)showed that the vascular patency of the transplanted endothelialization-promoting TEBVs group was better than that of the other groups;The results of HE and Masson staining showed that the collagen group and the blank CS group lost their normal structures and became blocked due to thrombosis and intimal hyperplasia,while endothelialization-promoting TEBVs group had complete structure and unobstructed lumen;CD31 and vWF immunofluorescence showed that endothelialization of endothelialization-promoting TEBVs group was better than that of other groups,and the lumen was covered with endothelial cells.ConclusionIn this study,the endothelialization-promoting TEBVs can promote the process of endothelialization in transplanted blood vessels,correcting the local vascular disorder,and finally achieve long-term vascular patency and improve the success rate of the transplantation sugery.