| Artificial blood vessels with different size are widely used to replace damaged or diseased arteries or veins in clinic.Artificial blood vessels are usually made from synthetic materials including Dacon and ePTEE.The use of small artificial vessels(diameter<5 mm)is limited because of frequent thrombosis and occlusion of small diameter grafts.Several surface modifications have been developed to enhance patency.However,due to the lack of cell structures,secretion,and biological activities,keeping high patency of small artificial vessels in long-term is still a challenge.The current improvements on vessel engineering offered a new avenue to overcome the limitation of artificial materials.There are two major steps in vessel tissue engineering,choosing of seed cells and seeding these cells on artificial blood vessels.Cord blood-derived stem cells are the cells can self-renew to produce more stem cells,and have potential to differentiate into diverse specialized cell types.Therefore,cord blood-derived stem cells are one of the best candidate seed cells for artificial vessels.Extracellular matrix could support seed cell growth in a later stage.Ammonium phosphate zwitterions belongs to phophatidylcholines,which have a hydrophilic ion and a hydrophobic long carbon chain and usually form a lipid bilayer-like structure.It has been shown with anti-coagulation and anti-platelet aggregation effect.In this study,I first generated endothelial progenitor cells(EPCs)from cord blood-derived stem cells by in vitro induced differentiation and prepared by co-precipitation.Then EPCs were seeded on and co-cultured with the vascular bioscaffold to form tissue-engineered vessels.Finally,the functions of these artificial vessels were determined by in vivo and in vitro assays.By doing these,I have developed a novel tissue engineering method to generate artificial vessels with improved performance.This thesis includes following four sections:Part ?.Culture,identification,and labeling of endothelial progenitor cells differentiated from cord blood-derived stem cellsObjective:Generate methods for in vitro inducing cord blood-derived stem cells differentiation into EPCs.Set up procedure to label and identify induced EPCs as seed cells for tissue-engineered vessels.Methods:Commercially available human cord blood-derived stem cells were cultured with EGM-2 medium in the flasks pre-coated with fibronectin,and induced to differentiate into EPCs.The proliferation,cell morphology,gene expression,angiogenesis,and growth factors and NO secretion of induced EPCs were determined.Results:EPCs could be generated from monocytes.After 5 days cultured in EGM-2 medium,cells formed colonies and showed a high proliferation rate.The morphology of induced cells was similar to endothelial cells and uptake of Dil-ad-LDL and UEA-1 was observed.Induced cells expressed high level of endothelial cell surface markers CD 133 and vWF,and low level of CD34.Induced EPCs formed capillary vessel-like network when cultured in soft agar.Furthermore,NO production was observed in induced EPCs.Conclusion:Adherent culture could be used for isolation and purification EPCs differentiated from cord blood-derived stem cells.Induced EPCs exhibited similar phenotypes and characters with endothelial progenitor cells,and proliferated at a high rate.Cord blood-derived stem cells induced EPCs could be used for generating tissue-engineered vessels.Part ?.Preparation of decellularized human great saphenous veinsObjective:Generate extracellular matrix for tissue-engineered vessels by treating human great saphenous veins with buffers containing Triton X-100 or SDS,DNase,and RNase.Methods:Treat human great saphenous veins with buffers containing Triton X-100 or SDS,DNase,and RNase to generate decellularized vessel matrix.Investigate the structure and morphology of the decellularized vessel matrix by using inverted microscope and scanning electron microscope.Results:All cells of human great saphenous veins were removed by Trixon X-100 or SDS,DNase,and RNase treatment,while extracellular matrix.fractions,including elastic fibers,collagenous fiber,and glycosaminoglycan were resided.Mechanical strength and plasticity were mainly maintained.Immunogenicity was decreased after decellularized treatment,proved by MHCI immunostaining.Conclusion:The treatment of Trixon X-100 or SDS,DNase,and RNase decellularized all cellular fractions from human great saphenous veins.The low immunogenicity and high mechanical strength and plasticity of decellularized human great saphenous veins made it appropriate as a material for tissue-engineering vessel in the further investigation.Part ?.Modification of the decellularized great saphenous vein and the artificial blood vessel with ammonium phosphate zwitterionsObjective:To explore the feasibility of the decellularized great saphenous vein and artificial blood vessel modified as tissue engineering stent with ammonium phosphate zwitterions.Methods:Application of co-precipitation method to combine the ammonium phosphate zwitterions mixed with polyurethane and decellularized great saphenous vein build with the method in Section Two and commercialized artificial blood vessels.Detected by hemolysis,complex calcium,platelet aggregation,in vitro cytotoxicity and each physical performance testing.Results:Compared to control group,hemolysis rate of artificial blood vessels and decellularized great saphenous vein modified applying co-precipitation method is low.The time from fibrinogen into fibrin is longer significantly.The aggregation of blood platelet would reduce clearly.There is without cytotoxicity in vitro.Implant materials are used in composite medical.And it shows a higher superiority in performance of decellularized artery in the detection of various physics compared to control group.Conclusion:Combining AP zwitterionic mixed with polyurethane with artificial blood vessels commercialized and decellularized great saphenous vein in the second way applying co-precipitation method can improve hemolysis and recalcificatic and platelet aggregation without cytotoxicity.There is an improvement in a certain degree to decellularized great saphenous vein physical properties.Part ?.Endothelial progenitor cells seeding decellularized scaffolds modified with polyurethane ionomer nanoparticlesObjective:To investigate the adhesion,growth,and distribution of EPCs after the short period seeding on decellularized scaffolds modified with polyurethane ionomer nanoparticles.Methods:The cord blood-derived stem cells differentiation into EPCs were marked with CM-DiI and made into suspensions.Place decellularized scaffolds modified with polyurethane ionomer nanoparticles in a home-made bioreactor.Take the scaffolds composited with EPCs after one day,two days,one week and two weeks.By using light and electron microscopy,the adhesion,growth and distribution of EPCs on decellularized scaffolds modified with polyurethane ionomer nanoparticles in different raising time were compared.Using the fluorescence microscope to check the adhesion and growth of CM-Dil staining cells.Using the MTT method and NO kits to test the influences on cell viability and cell function.Also,biomechanical analyzer was used to test the mechanical properties of tissue engineered blood vessel patches after modification.Results:With EPCs seeding on the stents,HE staining showed that the EPCs cover the inner wall of the modified stents smoothly and widely.The scanning electron microscopy also confirmed that cells covering in the tissue engineered blood vessel had same structure as the common EPCs,linked closely together and grew well.The staining analysis indicated that CM-DiI and FITC-vWF fluorescence double-staining cells exist in patch wall.This demonstrates that the cultivation of EPCs can differentiate into endothelial cells and involve in the process.Induced cells expressed high level of endothelial cell surface markers vWF,also proved the cells grew well on the decellularized scaffolds modified with polyurethane ionomer nanoparticles.Conclusion:All the tests showed that induced cells grew well on the stents.This has no adversely effects on cell activity and function.Tissue engineered blood vessel patches of EPCs compounding decellularized scaffolds modified with polyurethane ionomer nanoparticles is successfully fabricated,and is expected to be further used for vascular tissue engineered research. |
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