The Study Of Zein-based Tissue Engineering Of Vascular Implants And 3D Cell Culture Material

Tissue engineering is an important development direction of biomedicine. Vascular tissue engineering and 3D cell culture are the important parts of it. In developed countries, cardiovascular disease is the leading killer. In recent years, with the improvement of people’s living standard, the domestic incidence of cardiovascular disease also showed an increasing trend. The researchers hope to develop an artificial blood vessel with good biocompatibility and biodegradable, in order to solve the current clinical problems. 3D cell culture technology is a bridge between monolayer cell culture and animal experiments, through simulation environment in vivo to show advantages of cell culture with intuition and controllability. And it can be used for drug toxicity screening test. More importantly, the three-dimensional cell culture and tissue engineering can provide adequate amounts of cells and tissue blocks for organ regeneration.Previous studies indicate that zein has good cell compatibility with NIH3T3 cells, human umbilical vein endothelial cells, and it can be used for bone defect filling material. In the field of vascular tissue engineering, we not only need to study cell adhesion, diffusion capacity, also need to test the cell ability of resistance to fluid shear stress. Higher cell retention can accelerate the rate of formation of the endothelial layer, which is conducive to the formation of resisting thrombus, and improve the success rate of operation. We coated the material surface with microspheres of zein, and the microspheres were controlled in certain diameters. We hope to improve the cell ability of resistance to fluid shear stress. After the experiment, we found that immersed in water for 26 hours and under fluid shear stress for 6 hours, microstructure of zein 3 film(the film is composed of microsphere with the diameter bigger than 500 nm) is still clearly visible, no significant difference with the initial state. However, microspheres structure on zein 2 film(the film is composed of microsphere with the diameter smaller than 500nm) was disappeared. That is to say, the two-dimensional coating with high roughness and large size zein microspheres is the highest stability of surface morphology. Three kinds of zein films with different roughness showed different resistance to fluid shear stress. The retention of EA.hy926 cells and NIH3T3 cells on zein 3 film with highest roughness is higher than other two kinds of films. Compared with EA.hy926 cell, NIH3T3 cell is more sensitive to surface roughness. The NIH3T3 cell retention of zein 2 film with lower roughness increased significantly. While the ability of fluid flow stress resistance of EA.hy926 cells on zein 3 film with highest roughness can be enhanced significantly. We can improve the ability of cell resistance of fluid shear stress by controlling the microstructure of material surface.Since the biodegradation of zein, it can be applied to vascular tissue engineering. We prepare zein tube by repeated coating, and find some way to improve its characteristic. After be treated under 60, 90 and 121℃, the enzymatic degradation rate of these samples are significant difference in vitro. And the sequence is in accordance with the processing temperature. In vivo, we also found the same phenomenon, along with the increase of treatment temperature, the degradation rate decreased. On the other hand, after moist heat treatment, the mechanical strength of samples increases with the temperature. However, after the temperature above 90 ℃, enhancement of mechanical strength is not significant. We test the prepared zein tube under fluid shear stress for 4 weeks. The tubes can maintain structural integrity without leakage. By controlling the heat treatment temperature, the degradation rate and mechanical strength of the zein tubes can be controlled. These results are in favor of zein tube which is applied to the field of artificial vascular in the future.Three dimensional cell culture including film stack, microspheres stack and porous scaffolds. At the same time, the materials which are used in microfluidic system and cell culture dishes are important cell culture materials. We found that pure zein film has good transparency in the dry state. So we intend to use as cell culture material, in order to replace the polystyrene. However, the swelling make it to be opaque and cells can not be observed. So we invent the moist heat treatment(121 ℃, 100% humidity and 103.4 KPa, 20 minutes). This method can reduce the swelling and degradation rate, and after swelling the film can still remain transparency. After analyzing FT-IR and XRD spectra, we found that the heat moisture treatment make the proportion of α-helical structure decreased and β-folding structure increased. NIH3T3 cells were used to validate the film cell compatibility. Compared with 48 well culture plates, the treated and untreated zein film have no significant difference. The characters improvement can extend the range of zein applications, especially for cell culture substrate and microfluidic device.Zein as a natural biodegradable material can be used in the field of microcarrier, too. If it can be prepared into the required microcarrier, large-scale cell proliferation and tissue engineering can be combined. We invent three methods for preparing the microcarrier, the first method is to cut the film into rectangular particle directly, and after moist heat treatment to make it right angle bend. The second method is used for the preparation of microcarrier with diameter between 50 and 500 μm. We firstly use the process of glycerol heating polymerization to prepare the microspheres. The third kind of method is used for the preparation of microcarrier with diameter between 500 and 3000 μm. It is also the first time to prepare zein microspheres use of acid-base neutralization drop. Cell proliferation experiments were conducted on three kinds of microcarriers. We used Vero cells to test the biocompatible of the microcarrier which was prepared by the first and second method. And NIH3T3 cell is used to test the microcarriers of zein which is prepared by the third method. These two kinds of cells can adherent and proliferation on microcarrier, and cover the surface of the microcarriers. Three methods are the non-toxic, environmental protection and easy amplification. Compared with the phase separation process for preparing microspheres, these methods are more convenient, lower cost and non-toxic reagents. Also, the solvent which used in the preparation can be recycled.