玉米醇溶蛋白三维支架仿生矿化条件的优化和在微载体制备中的应用探索

发布时间：2018-03-22 15:01

本文选题：玉米蛋白　切入点：仿生矿化　出处：《上海交通大学》2015年硕士论文　论文类型：学位论文

【摘要】：组织工程在人类生物医学的发展中起着举足轻重的作用,而其中骨组织工程支架的制备和细胞三维培养的微载体技术扮演着重要的角色。本论文对玉米蛋白材料作为上述组织工程基质材料方面的应用进行了初步探索。首先将玉米蛋白制备成三维(3-D)多孔支架,尝试采用不同的仿生矿化条件将羟基磷灰石颗粒沉积在多孔支架表面,对其孔隙结构及表面化学组成等进行了表征。通过对3-D多孔支架的仿生矿化条件进行改进,获得了具有良好孔隙结构、表面均匀沉积了涂层且钙磷比接近羟基磷灰石的骨组织工程支架。使用环境友好技术将玉米蛋白制备成尺寸可控且圆整度高的微载体,评估了细胞贴壁率、细胞增殖情况,并与商业化微载体Cytodex1进行对比,发现细胞能够正常粘附并且增殖,细胞收获量达到了商业微载体水平,为进一步应用于组织工程提供了新型基质材料。方法:用磨具将玉米蛋白制备成5mm×5mm×15mm和Φ2mm×4mm的三维(3-D)多孔支架,并对其外观形貌和孔隙结构进行了表征。将该玉米蛋白多孔支架浸入5倍模拟体液(5×SBF)中,在不同的条件下进行仿生矿化。对仿生矿化后的材料也进行了孔隙率及表面形貌的表征,并使用能谱仪对羟基磷灰石涂层进行了元素分析(EDS)、计算其钙磷比,使用X射线衍射仪(XRD)确定涂层的物质是羟基磷灰石,通过穿孔实验评估了羟基磷灰石涂层的牢固程度,为动物成骨实验提供符合条件的样本。使用环境友好的甘油热塑法制备不同尺寸的微载体,并使用荧光显微镜观察所制备微载体的圆整度,测量其直径并进行统计。通过体外实验评价其抗酶解性能。种植Vero细胞评价细胞贴壁和增殖速度并与Cytodex1对比,扫描电子显微镜观察细胞在微载体上的生长状态。结果与讨论:最佳沉积条件为:将三维玉米蛋白多孔支架浸入5倍模拟体液中,置于全温摇床,在37℃,60rpm转速下仿生矿化3天,在一天半时,更换新鲜的5倍模拟体液,并将多孔支架上下翻转。所获得的玉米蛋白支架的各个表面均沉积到相对均匀的羟基磷灰石颗粒,颗粒直径在2-10μm,各个面的钙磷比在1.40-1.67之间。说明仿生矿化效果得到了改进。使用甘油作为加热介质,将玉米蛋白塑形。得到的微球直径可控,且分布均匀,圆整度良好。将Vero细胞种植在微载体表面,结果显示细胞能够正常贴壁,并能够增殖,两者均与商业广泛使用的微载体Cytodex1相当。说明玉米蛋白具有作为微载体的潜在新用途,不仅可用于疫苗生产目的的动物细胞的大规模培养,而且可拓展至组织工程领域。
[Abstract]:Tissue engineering plays an important role in the development of human beings in biomedical science, including the preparation of bone tissue engineering scaffolds and three-dimensional cell culture micro carrier technology plays an important role. This paper conducts a preliminary exploration of corn protein using the tissue engineering material as matrix materials. The preparation of corn protein the three-dimensional (3-D) porous scaffolds, using biomimetic mineralization conditions of different hydroxyapatite particles deposited on the surface of the porous scaffolds, pore structure and surface chemical composition were characterized. By improving the biomimetic mineralization conditions on 3-D porous scaffolds, obtained has good pore structure, uniform surface coating and deposition of calcium and phosphorus ratio of bone tissue engineering scaffolds. Close to hydroxyapatite using environmentally friendly technology of the corn protein prepared by size controllable and roundness of micro carrier, To evaluate the rate of cell adhesion, cell proliferation, and commercialization of microcarrier Cytodex1 were compared, found that normal cells can adhesion and proliferation, cell harvest reached commercial micro carrier level, provides a new matrix material for further application in tissue engineering. Methods: the three-dimensional corn protein preparation tools into 5mm * 5mm * 15mm * 4mm and 2mm diameter (3-D) porous scaffolds, and the morphology and pore structure were characterized. The corn protein scaffolds immersed in simulated body fluid 5 times (5 * SBF), biomimetic mineralization in different conditions. The biomimetic materials were also carried out after characterization of porosity and surface morphology, and the use of EDS to analyze elements of hydroxyapatite coating (EDS), calculate the ratio of calcium and phosphorus, using X ray diffraction (XRD) coating material is determined by experimental evaluation of hydroxyapatite, perforation of hydroxyl A strong degree of hydroxyapatite coating, to provide qualified samples for experimental animal bone. Microcarrier preparation of different sizes of glycerol heat using environmentally friendly plastic method, and fluorescence microscopy was used to prepare the micro carrier roundness, measure its diameter and statistics. By in vitro experiments to evaluate its anti enzyme properties Vero cell evaluation. Plant cell adherence and proliferation rate and compared with the Cytodex1, the growth state of scanning electron microscopy of cells on microcarrier. Results and conclusion: the best deposition conditions: three-dimensional porous scaffolds immersed in corn protein 5 times simulated body fluid, put the whole temperature shaker, at 37 degrees, 60rpm speed bionic the mineralization of 3 days, in a day and a half, 5 times simulated body fluid replacement of fresh, and the porous scaffold under flip. Each surface corn protein scaffold obtained were deposited onto hydroxyapatite particles relatively uniform, The particle diameter of 2-10 m, each surface of the calcium and phosphorus ratio in the range of 1.40-1.67. The biomimetic mineralization effect is improved. The use of glycerol as the heating medium, the corn protein shaping. Obtained microspheres with controllable diameter, and uniform distribution, good roundness. Vero cells grown on microcarriers. The results showed that the cell can be normal attached to the wall, and is capable of proliferation, microcarrier Cytodex1 both are widely used and quite. Commercial corn protein has potential use as a new micro carrier, not only can be used for vaccine production to large-scale culture of animal cells, and can be extended to the field of tissue engineering.

【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R318.08

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