| A best substitute of blood vessel has been pursued for a long time and many materials have been tried such as autogenous artery or vein, artificial material, biomaterial, but no ideal substitute has been found up till now. Tissue engineering is a new and rapidly expanding field ,in which a series of techniques are being developed for culturing and reconstructing a variety of tissues both in vitro and in vivo. With these techniques, it is possible to reconstruct vascular model in vitro whose structure and function are similar to autogenous vessels.In our experiment human embryonic ECs and smooth muscle cells were used as the source of seeding cells, a number of degradable materials were screened, and different seeding methods were compared to find out the most suitable matrix material for perivascular model in vitro and the best route of inoculation. Meanwhile a bioreactor fit to be used in vascular culture in vitro was developed, on the basis of which, primary perivascular model in vitro was reconstructed with tissue-engineering techniques. Part I Objective: To provide the cells used in vascular tissue engineering experiments.Methods: Vascular ECs, smooth muscle cells and flbroblasts were cultured in vitro,underwent purification, identification and morpHological observation. Results: The3 type of cells treated accorded with vascular ECs, smooth muscle cells andflbroblasts respectively in morpHological properties. Conclusion: The cells culturedcould be used in further studies.Part IIObjective: different matrix materials for vessels were prepared and compared.Methods: The spongy collagen/chondroitin sulfate scaffold was crosslinked by EDC;the collagen/chitosan scaffold was crosslinked by glutaraldehyde and heat; Porcineacellular aorta were obtained through a cellular exrtaction procedure. Tubular porousvascular scaffolds were made under vacuum freeze condition in a special annularmodel. The tubular porous scaffolds have elasticity, stubbornness and hydropHilic,can maintain their shapes in culture medium for long time.Results: several matrix materials for vessels as tubular scaffold were obtain forfurther studies.Part IIIObjective: To screen the degradable material fit to be used in tissue-engineeringvessel construction and the best seeding method. Methods: VSMCS were seededinto several degradable matrix materials, and the growth of the VSMCs in them wasobserved respectively with MTT, HE and electroscope. Three seeding methods wereinvolved, Result: PGA, collagen/GAG, decalcifying bone were foundrelatively compatible with the cells and rotary seeding method has highestefficiency.Part IVThere are two difficulties in tissue engineering vessel construction: the implantedECS were prone to exfolication, due to lack of tolerance to blood flows, scouringand then induced secondary thrombasis, which decreased its long-term effect; the structural intensity of the tissue -engineering vessels obtained in vitro could not reach or approach that of normal vessels, due to lack of enough support of elastic media. In this experiment, we developed a new bioreactor for tissue-engineering construction in the light of the above-mentioned difficulties. It could imitate pulsation of human blood vessels and scouring effect of blood flow, which could make ECs gradually tolerate the shear stress from blood flow's scouring to decrease their exfoucation on the hand and accelerate growth, reproduction and differentiation of the smooth muscles cells on the other. The device could be put into cell incubator easily, which provide a comparatively endosed, sterile environment with enough oxygen and stable temperative for organ's construction in vitro like tissue-engineering blood vessel and cardiac valve. Part VIn this experiment, we used the vascular bioreactor developed by ourselves to study VECs' growth on PLGA mesh under shear stress. Primary experimental data were obtain for further researches on tissue-e... |
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