Construction Of Functional Small Caliber Tissue Engineering Blood Vessel With Bionic Structure

Background: vascular occlusion caused by cardiovascular disease remains an important cause of death in patients worldwide.Although balloon angioplasty and other traditional operations have been developed,many patients still need to be treated with vascular graft procedures such as coronary artery bypass surgery.In addition,many diseases need to use vascular substitutes for transplantation,such as arteriovenous shunt in hemodialysis patients,vascular transplantation in patients with vascular injury caused by trauma,etc.At present,autogenous grafts such as internal thoracic artery and saphenous vein are gold standard grafts.However,the long-term performance of vein grafts is not good enough when they are transplanted to the arterial sites that need to bear high pressure.Besides,many patients cannot use autogenous vessels for transplantation because of their poor health.Therefore,a variety of artificial vascular grafts made of polymer materials have become substitutes for autologous blood vessels,such as expanded polytetrafluoroethylene(e-PTFE).However,there are many deficiencies in their biocompatibility,compliance and so on,and they are prone to form thrombus when their caliber is small,which limits their clinical applications.Over the past decade,vascular tissue engineering has become one of the fastest growing research fields.The aim of vascular tissue engineering is to design tissue engineered blood vessel(TEBV)with bioactivity similar to natural blood vessel.However,TEBV at present still has many disadvantages,such as long preparation time,limited cell source,insufficient mechanical properties and thrombus formation after implantation.The reason why natural blood vessels have excellent biological function and mechanical properties is closely related to their composition and structure.Blood vessels are composed of a three layered structure,intima,media,and adventitia.The intima as an endothelial cell layer that is able to regulate coagulation balance and prevent thrombus formation;the media is composed of arranged smooth muscle cells,elastic fibers,and collagen fibers that provide the primary mechanical strength;and the outer layer is the connective tissue that contains fibroblasts and peripheral nerves.From the perspective of bionics,this paper studies the construction of TEBV in the following aspects.Objective and Methoeds: first,thrombosis is the first issue to be solved after TEBV implantation in vivo.First of all,thrombosis is the first problem to be solved after TEBV implantation.As a foreign body,TEBV is easy to cause platelet activation,aggregation and subsequent coagulation reaction after implantation.In this process,the high concentration of adenosine diphosphate(ADP)released by activated platelets plays an important role in promoting coagulation.In physiological state,two extracellular nucleotidases on the surface of endothelial cells,CD39 and CD73,regulate the concentration of ADP.In addition,endothelial cells can synthesize anticoagulants such as prostacyclin and nitric oxide.However,it takes a long time for TEBV to complete endothelialization in vivo.In order to achieve anticoagulant effect before endothelialization,we designed a nanocomposite that produced antithrombotic substances by cascade enzymatic reaction and modified it on the inner surface of TEBV to form anticoagulant coating.We used reduced graphene oxide(r GO)as a carrier,CD39 and CD73 were covalently bonded on the surface of r GO,and then the RGO enzyme complex was modified on the surface of acellular blood vessels.The results show that the purinergic pathway formed by CD39 and CD73 can convert ADP released by platelet into adenosine monophosphate(AMP)and adenosine under physiological conditions,which can regulate microenvironment,prevent thrombosis and promote endothelialization.The sheet structure of r GO can not only ensure the progress of cascade enzyme reaction,but also enhance the dispersion and stability of enzyme protein.The results of animal experiments showed that the modified TEBV was less likely to form acute thrombosis,and had a better degree of endothelialization compared with the unmodified vessels.Secondly,we fabricated high strength hydrogel vessels with biomimetic components and microstructures by using 3D printing technology.Although acellular blood vessels from animals have the advantages of good mechanical properties and wide sources,the immune rejection caused by xenogeneic materials and the inability to freely choose the size and caliber limit their application.Hydrogels are widely used in tissue engineering,but hydrogel engineering structures which can withstand large pressure or tension are still rare.We first designed a double-network(DN)hydrogel containing three components,which enhanced the strength and compliance through the energy dissipation mechanism.The photocurable hydrogel was used as biological ink to print vascular structure through 3D biological printing technology,and then restricted air drying(RAD)was performed.During the process of losing water,the stress generated in the hydrogel makes the polymer gradually form a hierarchical aligned fiber structure,which is similar to the structure of the vascular wall.The test results show that the elastic modulus,fracture strength and ultimate deformation of DN hydrogel were improved.After RAD treatment,the directional fiber alignment structure is formed in axial and circumferential direction,and the toughness and strength of the blood vessel are further enhanced.The pure hydrogel vessels still do not possess the three layer structure of natural arterial vessels.Endovascular elastic membrane,as the boundary between intima and media,is related to material exchange and cell migration.It is very important for the normal function of blood vessels.We use acellular intima as the inner layer of three-layer blood vessels.The inner layer is coated PCL fiber membrane,which is prepared by micro nano 3D printing technology.The micro PCL fiber has a crisscross directional arrangement structure,which is similar to the directional arrangement of fibers and cells in the middle layer of blood vessels,and can provide sufficient mechanical strength.The outer layer was printed with bioink containing human umbilical cord derived mesenchymal stem cells(MSC).Subsequently,induced pluripotent stem cells(i PS cells)were induced to differentiate into vascular endothelial cells,which were planted on the inner surface of blood vessels using a self-made vascular perfusion culture system.In vitro experiments showed that MSC cells grew well in hydrogels.The cells grew along the fiber direction under the guidance of PCL fiber.The endothelial cells planted on the inner surface are aligned under the action of scouring force,which is close to the arrangement of human vascular endothelial cells.The results showed that MSCs could survive for more than 7 days,and vessels seeded with endothelial cells were less likely to form thrombosis,and remained unobstructed 14 days after transplantation.In summary,this paper contributes to the biomimetic design of antithrombotic TEBV through the solution of vascular function and structure at the molecular,cellular,compositional,and structural scales.We also built TEBV that resembles the fibrous microstructure of native blood vessels using hydrogel materials and 3D bioprinting technology,and finally three layered biomimetic blood vessels were prepared.In vivo transplantation experiments were performed.The results demonstrate that these biomimetic methods can achieve antithrombotic effect of blood vessels,and rapidly prepare TEBV of any size with excellent mechanical properties,which provides a new method for the design and preparation of small caliber TEBV in vitro.