Construction Of Functional Tissue Engineered Blood Vessels With Endothelial Cells Derived From Embryonic Stem Cells And MCC950

At the beginning of the 21 st century,about 17 million people worldwide died of cardiovascular and cerebrovascular diseases,and it is estimated that the number of deaths will reach 30 million by 2025.About 600,000 people worldwide need vascular surgery every year.Vascular transplantation is the main method for clinical treatment of vascular diseases(such as atherosclerosis,aneurysms,congenital vascular malformations)and traumatic vascular injuries.Autologous arteries and veins are ideal arterial grafts,but the source,length,and diameter of autologous blood vessels available for transplantation are extremely limited.The problems of immune rejection and other postoperat ive complications in allogeneic vascular transplantation have not been resolved.Therefore,small-caliber tissue-engineered vessels(TEBV)have a wide range of clinical needs.As the main functional cells of blood vessels,endothelial cells can synthesize and secrete a variety of vasoactive factors,regulate coagulation and anticoagulation,change the permeability of blood vessels,promote nutrient exchange and metabolism,and can regulate the function of smooth muscle cells.Previous studies have shown that forming an endothelial cell layer on the inner surface of TEBVs and constructing a vascular wall barrier can effectively prevent the occurrence of thrombosis and intimal hyperplasia after implantation.The human endothelial cells have perfect functions and no immunogenicity,but the problems of primary cell extraction,culture,and expansion are difficult to solve.Embryonic stem cells have the potential for infinite proliferation and multi-directional differentiation.In theory,they can generate a sufficient number of vascular cells(vascular endothelial cells,smooth muscle cells)to meet the needs of clinical and scientific research.Therefore,embryonic stem cells have broad application prospects as a source of endothelial cells.Another important cause of thrombosis and intimal hyperplasia is the inflammatory response.After the TEBV is implanted in the body,it triggers an acute inflammatory response and accelerates the occurrence and development of thrombosis.In addition,inflammation stimulates abnormal migration and proliferation of smooth muscle cells,and the cascade reaction recruits more inflammatory cells,leading to intimal hyperplasia in TEBV.Inflammation is a double-edged sword,proper inflammation can promote the reconstruction of TEBVs in vivo,but uncontrollable inflammation can greatly affect the long-term patency of TEBV.The activation of NLRP3 inflammatory bodies is a key stage of the inflammatory response.Therefore,we want to use small molecule inhibitor MCC950 to inhibit inflammatory bodies,regulate the inflammatory response,improve the local microenvironment,and achieve in-vivo reconstruction and long-term patency of small-caliber TEBVs.We induced embryonic stem cells to differentiate into vascular endothelial cells,and achieved endothelialization of TEBVs by in vitro implantation technology.Next,the small molecule inhibitor MCC950 was modified on the outer membrane of blood vessels through layer-by-layer self-assembly technology.After transplantation in vivo,endothelial cells secrete active factors,inhibiting thrombosis and intimal hyperplasia.Small molecule inhibitors regulate the inflammatory response,improve the local microenvironment,and ultimately achieve reconstruction and long-term patency of TEBVs.Methods:1.Targeted delivery of small molecule inhibitor MCC950First,we used acute myocardial infarction as the animal model,which is the most common and severe cardiovascular disease,to study the inflammation regulation function of the small molecule inhibitor MCC950,and to observe whether it has an effect on the recovery of cardiovascular function.To avoid secondary damage from local injections,we use targeted delivery method.We design a targeted delivery system with MCC950.Particle size analysis and transmission electron microscopy were used to characterize the materials.Sustained release and enzyme response experiments were performed to determine the efficiency of the delivery system.In vivo fluorescence imaging was used to observe the targeting ability of the drug delivery system.Ultrasound,Masson staining,HE staining were used to observe the recovery of cardiac function after myocardial infarction treatment.2.Induced differentiation of embryonic stem cells into vascular endothelial cellsEmbryonic stem cells are cultured and stably expanded.The expression of ESC pluripotency factor was observed by immunofluorescence staining.ESCs were differentiated into vascular endothelial cells using a variety of induction differentiation protocols.Immunofluorescence staining,Weston blot and RT-qPCR were used to detect the expression of endothelial-specific markers.Flow cytometry was used to detect the efficiency of induced differentiation.Tubule formation experiments were performed to verify the biological function of induced endothelial cells.3.Construction of MCC950 modified endothelialized TEBVs and transplantation in vivoMCC950 was modified on the outer membrane of acellular vascular scaffold by layer-by-layer self-assembly technology and characterized by scanning electron microscopy.After endothelial cells were implanted in the lumen of blood vessels,the implantation was observed by scanning electron microscopy and HE staining.After functionalized small-caliber TEBV transplantation in vivo,the patency of vascular grafts,inflammatory cell infiltration,and vascular remodeling were observed by ultrasound,CTA,HE staining,masson staining,and immunofluorescence staining.Results:1.TEM and particle size analysis showed that the drug delivery system was successfully constructed with uniform size and consistent morphology.The sustained release and enzyme response experiments showed that MCC950 was stable in loading and the enzyme response was fine.In vivo fluorescence imaging showed that MCC950 was efficiently delivered to the MI area.Ultrasound,Masson staining and HE staining showed that MCC950 can inhibit the function of inflammatory bodies,protect myocardial cells from further damage,and restore heart function.2.Embryonic stem cells can efficiently differentiate into vascular endothelial cells.Immunofluorescence,immunoblotting and qPCR results showed that induced endothelial cells express CD31 / CD144 / vWF and other endothelial cell-specific markers.Flow cytometry analysis showed that CD31/CD144 double positive cells accounted for 90.5% Induced endothelial cells have the ability to form tubules.3.TEM and HE tests showed that endothelialization of tissue engineered blood vessels was successfully achieved in vitro,and MCC950 was modified to the surface of the adventitia of blood vessels.One day after transplantation in vivo,the MCC950-modified endothelialized TEBVs were unobstructed,and the control group developed stenosis and blockage.Immunofluorescence staining and HE staining showed that there were less infiltrated inflammatory cells in the experimental group and no intimal hyperplasia occurred.Autologous vascular cell invasion had begun to occur,and the reconstruction was good.Conclusion:The small molecule inhibitor MCC950 acts on inflammatory bodies and can effectively suppress the immune inflammatory response and promote the recovery of cardiovascular system function.Embryonic stem cells can efficiently differentiate into vascular endothelial cells and perform corresponding biological functions.By modifying the small molecule inhibitor MCC950 on the outer surface of endothelialized TEBVs,it inhibits the infiltration of inflammatory cells,further prevents thrombosis and intimal hyperplasia and promotes the in vivo reconstruction of TEBVs,and finally achieves long-term patency.