| BackgroundWith the improvement of people's life and change in diets, more and more people are risking cardiovascular disease. Usually, vascular bypass graftings are needed to improve their living quality and even save lives. The patients' own arteries or veins remain the conduit of choice, but unfortunately, most of the patients do not have suitable artery or vein for grafting, especially for the redo patients. Also, the procedure of grafting onto pathological vessel from another part would create new trauma to the patient, so scientists begin their finding of substitute materials for blood vessel prosthesis. Development of tissue engineering provides theory and method for construction of tissue engineered blood vessels(TEBVs).Construction of decellularized xenogarfts(DXs) is one of the hotspots in blood vessel tissue engineering. Compared with other types of tissue engineered blood vessel, the DXs have several advantages. To synthetic prosthesis, the decellularized grafts have better mechanical properties and biocompatibility; to scaffolds made from fixed extracellular matrix proteins(ECM), they have more delicate three-dimensional structure; to cells self-assembly TEBVs, their fabrication are easier and need less time.Although their advantages over other type of TEBVs, which had been proved by many animal trials, whether the decellularized grafts can also be successfully used in human being is waiting for clinic tests, because some experiments showed that animals have inherent ability to self-cellularize their vascular grafts. Actually, replacements of dysfunctional vessels or valves with decellularized xenografts didn't meet encouraging results due to inflammation, calcification and degradation of these grafts. Many problems should be addressed before the popular use of decellularized vessels or valves.Firstly, How to control the degradation speed of the DXs? Fixation of their collagen fibres can not only slow their degradation speed, but also elimilate risks of inflammation and calcification. Secondly, how to recellularize the DXs in vitro? There are enough scientific evidences showed endothelial cells(EC) can improve the patency rates of TEBVs because they can effectively prevent the formation of thrombus and stenosis. While smooth muscle cells(SMC), another dominating cell component in natural vessels do not arouse enough attentions, moreover, researches on constructing a TEBV involving both SMC and EC were rare. The interaction between EC and SMC are considered to be involved in the control of growth and function of natural blood vessel, and many researches on relationship between SMC and EC had showed that the interaction between SMC and EC can occur both in morphology and molecular levels. So, seeding the TEBVs with both SMC and EC will favour the normal proliferation, differentiation and function of both cells, thereby improve the patency rate of the TEBVs.In this study, canine aortas were decellularized, then their mechanical and biological properties were evaluated, and finally the decellularized canine aortas were recellularized in vitro, including the seeding of SMC and EC separately and unitedly, for constructing a novel TEBV based on decellularized xenografts with higher integrity in both structure and function. Hereinafter is the detail. Methods1. Decellularization of the canine aortasPrevious studies of our team showed ethylene glycol diglycidyl ether(EX-810) was effective not only in fixing the collagen fibres but also in removing the cell components of porcine arteries. At the same time, the structure of their extracellular matrices(ECM) was maintained and not any cellular toxicity was detected. While, EX-810 could just disrupt the cells and cell debris are left, which would result in the formation of calcification. So, in this study, ion-free water and sonication were added to enhance the removal of cell components in canine aortas. Hematoxylin-eosin(HE) staining and fluorescence staining(6-Diamidino-2-phenylindole dihydrochloride hydrate, DAPI) were used to test the efficiency of decellularization, and Masson staining was used to test the preservation of ECM structure.2. Mechanical properties evaluation of the decellularized canine aortaFor a decellularized blood vessel scaffold, complete removal of cell components is not enough, the mechanical properties of the scaffold should be retained. In INSTRON8874 biomechanical testing system was used to test the mechanical properties of the canine aortas before and after decellularization, including max load, energy at max load, tensile stress at max load, tensile extention at max load, and tensile strain at max load.3. Biocompatibility testing for the decellularized canine aortas and its recellularization in vitroSMC and EC were separately seeded on the decellularized canine aortas in different densities to test the biocompatibility of these scaffolds on the one hand, and to make sure the suitable seeding density needed for rapid recellularization on the other. How to obtain confluent EC layer on SMC was studied by seeding high density EC on different density SMC. Scanning electron microscope(SEM), methabenzthiazuron(MTT) and immunohistochemical staining were used. SEM graphs were analyzed by software of Image-Pro Plus, then covering rates of EC on the decellularized aortas and on SMC layer were calculated.4. Retention of cells on the decelluiarized canine aorta under flow shear stress.Large diameter TEBVs based on synthetic materials have been successfully used in clinic, so in this study we will focus on the fabrication of small diameter ones. To test the retention of cells on the decellularized canine aortas, 5 dyne/cm~2 flow shear stress was chosen to treat the recellularized TEBV scaffolds for 2 h and 4 h respectively. High flow shear stress doesn't used in this study because of limited time and materials.Results1. HE and DAPI staining results showed that EX-810 could partly remove the cell components of canine aortas, but not completely. Both ion-free water and sonication could help removing the cell debris. Under the co-action of these three treatments, almost all the cell components were removed and the structure of the ECM was maintained according to Masson staining results.2. There was no significant change in mechanical properties of the canine aortas after decellularization.3. SMC and EC grew well on the decellularized canine aortas. With the increase of seeding density, cells arranged in a more regular manner on the TEBVs and showed higher covering rate. The recellularization process could be accelerated by increasing the seeding density. In the dual seeding system, EC could grow well directly on the layer of SMC with different seeding density, while the morphology and covering rate of EC were influenced by the density of their co-culture SMC. EC were cobblestone-like when seeded on low density SMC, but shuttle-like when seeded on high density SMC. Highest covering rate could be obtained when EC were seeded on medium density SMC. 4. A confluent EC layer were kept after treatment with 5 dyne/cm~2 flow shear stress for either 2 h or 4 h not only in EC single seeding model, but also in the dual seeding model with EC on SMC with different seeding density, which indicated the recellularized TEBVs we had constructed could perform their normal function when planted in vivo.Conclusions1. Under the co-action of EX-810, ion-free water and sonication, cell components in canine aortas can be removed completely, resulting in sharp decrease in immunogenicity.2. After decellularization, the ECM structure and mechanical properties of the canine aortas do not change significantly.3. The decellularized canine aortas show good biocompatibility. Both SMC and EC can grow well on their surface. EC can grow well on SMC layer with different seeding density. A confluent EC layer can be achieved either on the decellularized graft or on their coculture SMC layer.4. EC can retain on the TEBV scaffolds after treatment with 5dyn/cm~2 flow shear stress both in single seeding model and dual seeding model.
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