| Coronary artery disease(CAD)is one of the diseases with high morbidity and mortality in the world.With the increase of CAD,the demand of artificial vascular grafts continues to increase year by year.In most cases,the surgical replacement of damaged blood vessels may include the use of autologous vessels,allogeneic vessels from organ donors,or even synthetic grafts such as Gore-Tex or Dacron.However,due to age,disease,or other complications,autologous vessels are limited by quantity,although they are the standared of care at present.Synthetic materials have been successfully used as medial and large internal diameter prosthetics(ID>6 mm).Unfortunately,when applied to SDVGs,these artificial grafts exhibited acute thrombogenicity,anastomotic intimal hyperplasia,infection,aneurysm formation,and atherosclerotic disease.Therefore,it is very important to find the vascular materials with good biocompatibility and rich source.Acellular blood vessels has retained the extracellular matrix(ECM),mainly collagen and elastic fibers which have high homology in mammals.And decellularized blood vessels grafts have good compliance and cellular compatibility,which can promote the adhesion,proliferation,differentiation and spread of cells,and complete the re-endothelialization of blood vessels.However,after decellularzation the EC on the inner lumen was removed with the explored collagen,which can easily activate the coagulation system and result in thrombosis.Besides the decellularization process might accelerate the elastin deformation and degradation,damage the vessel structure,increase porosity,and decrease the mechanical properties of the vessel,resulting in vascular graft expansion and even aneurysm formation.So in the current study,we designed a bilayer tissue-engineered vascular graft(BTEV)by electrospunning the PLCL/gelatin fibers outside of the decellularized porcine coronary artery to enhance it's mechanical properties.Heparin was used to functionalize the intima of acellular vessels to improve blood compatibility.Main research contents and results of this paper:(1)Microscopic detection,matrix components Analysis and Mechanics performance test of decellularized porcine coronary arteryThe native porcine coronary artery(NPCA)and DPCA were performed by mechanical property test,histological analysis,SEM and matrix components analysis,respectively.The mechanical property test results showed that decellularization significantly decreased the max load.There is no significant difference in elongation and surface young's modulus.The results of histological analysis and SEM showed the repeated freeze-thaw and trypsin treatment can effectively remove the cells,and remain the majority of collagen.After decellularization,the vessel get loose,the intima histoarchitecture of DPCAs was similar to that of NPCA,and no cellular debris or cell remnants in DPCAs were detectable,while some of the filaments were exposed.The contents of DNA,elastin and GAG of decellularized porcine coronary artery showed a significant decrease compared with those of native porcine coronary artery.There is no significant difference in hydroxyproline content.(2)The preparation and characterization of bilayer tissue-engineered vascular graft(BTEVs)After vacuum freeze drying the DPCAs was gently immobilized on the steel mandrel and keep a good tubular structure,then the steel mandrel was fixed in collector and electrospinninged by customized electrospinning apparatus.SEM images showed that PLCL/gelatin layer and inner DPCA layer were tightly attached to each other without obvious delamination.The PLCL/gelatin layer was the structure of nanofiber layer and showed a homogeneous fibrous morphology.The diameter of PLCL/gelatin nanofibers was approximately 650 nm.The intima was roughly smooth with several small holes.The external PLCL/gelatin layer improved the max load and water penetration pressure of the BTEV graft,even better than that of NPCA.The zebra fish test showed no significant difference was observed between untreated control group and BTEV-treated group.The result of cytotoxicity showed after1,3 and 5 d of cultivation,the cell viability in different concentrations of BTEV extract were still above 75%,meeting the standards of medical equipment.Subcutaneous rat embedding experiments showed that electrospun gelatin-PLCL layer could inhibit the infiltration of inflammatory cells to a certain extent.(3)Assessment of heparin immobilization and platelet adhesionThe results of toluidine blue staining and energy spectrum analysis showed the heparin can be immobilized on the DPCA though covalent bonding.The platelet adhesion test showed that the heparin modification of DPCAs decreased platelet adhesion,activation and aggregation,and extend the recalcification time.Therefore,the heparin modification of DPCAs improved the anticoagulant activity.(4)Implantation testThe Colored doppler ultrasound showed that HBTEVs were able to maintain the graft patency with a constant luminal diameter without significant stenosis,aneurysm or dilatation for the duration of the four weeks study,while HDPCAs presented serious expansion.The histology and immunohistochemistry revealed that four weeks after implantation,collagen fibers in the wall of tissue engineered blood vessels were more densely packed and circumferentially aligned.And elastic plate exhibited the wave shape,thus increasingly mimicking the middle layer of a native blood vessel.The inner surface of explanted HBTEVs and HDPCA showed positive staining for CD31.The SMC density and the thickness of tunica media in the walls of HBTEV resembled those in the wall of native arteries at 4-wk.However,the distribution of the?-SMA~+cells in the wall of HDPCAs was dispersed.SEM images of inner surface showed that a cell monolayer was observed on the inner surface of HDPCA and HBTEV at 4-wk,but the junctions between ECs of HDPCA were not as tight as those of HBTEV.Mechanical properties of tissue-engineered blood vessels were similar to that of native. |
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