低晶不溶性丝蛋白支架的制备及体外细胞实验研究

发布时间：2018-01-07 04:03

本文关键词：低晶不溶性丝蛋白支架的制备及体外细胞实验研究　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：蚕丝因具有良好的细胞相容性、优良的机械性能和可生物降解性而成为一种普适性的生物材料,在组织工程领域引起了广泛的关注。然而不同组织的修复对支架的微观形貌、力学性能及降解性能等都有着不同的要求,调控支架的结构和性能使其与人体组织相匹配成为关键问题。新鲜丝蛋白溶液可采用冷冻干燥法制备多孔材料,但其成孔性一般较差,不利于细胞的生长、增殖及迁移。本文先对新鲜丝蛋白溶液进行预处理,即缓慢浓缩再稀释获得纳米纤维丝蛋白溶液,然后用该溶液制备丝蛋白支架,观察丝蛋白材料处理前后微观形貌的变化。实验结果表明,缓慢浓缩处理可以诱导丝蛋白溶液中纳米球向纳米纤维转变,明显提高支架的成孔性从而可获得具有良好孔结构的丝蛋白材料。在此基础上,向缓慢浓缩处理的丝蛋白溶液中添加不同含量的甘油制备结构可调的支架,并对支架的二级结构、稳定性、降解性能等进行研究。实验结果表明甘油可以改变丝蛋白的二级结构,使丝蛋白由无规结构向silk I和silk II结构转变,提高支架的稳定性,甘油含量对力学性能有着一定的调控作用,从而通过添加甘油及改变其含量实现了对支架结构和性能的调控。另一方面,基于丝蛋白分子自组装原理,在不添加任何有机试剂的条件下,通过改变分子运动性进一步实现对丝蛋白支架结构和性能的调控。将丝蛋白纳米纤维溶液与新鲜丝蛋白溶液按丝蛋白干重比1:15进行混合,通过改变混合溶液的冷冻-解冻次数或对其进行50oC温度处理来调节丝蛋白在溶液中的组装形态,随后采用冷冻干燥法制备不同的丝蛋白多孔支架。结果表明,随着冷冻-解冻次数的增加,支架中silk II含量增加,压缩模量增大,而温度处理则有利于支架中silk I结构增加,压缩模量相应降低。因此,反复冷冻-解冻和温度处理也可以实现对二级结构和力学性能的调控,获得性能不同的支架。最后,将SD大鼠骨髓间充质干细胞(r BMSCs)在支架上进行体外细胞培养,研究不同性能的丝蛋白多孔支架对r BMSCs增殖和分化的影响。结果表明细胞在所有支架上增殖情况良好,且实验组增殖情况明显优于对照组。DNA含量结果进一步证明了支架良好的细胞相容性,且支架模量越低,细胞增殖情况越好。随后选取不同力学性能的支架复合r BMSCs进行体外培养,研究力学性能变化对干细胞分化的影响,结果表明细胞在模量在8~17k Pa的丝蛋白支架上优先向成肌细胞分化,而在压缩模量在1~7k Pa的支架上向内皮细胞分化。综上所述,本论文通过温和的方法调控丝蛋白的纳米结构、亲水作用及分子运动性,从而制备微观结构、二级结构、力学性能可控的丝蛋白多孔支架,以实现不同的功能,提高了丝蛋白在不同组织修复领域的适用性。
[Abstract]:Because silk has a good biocompatibility, excellent mechanical properties and biodegradability and become a kind of universal biological material, has attracted wide attention in the field of tissue engineering. However, the morphology of different tissues of stent repair, mechanical properties and degradation properties have different requirements, structure the performance of the control and support of human tissue matching becomes a key issue. The fresh silk fibroin solution can be prepared by freeze drying method of porous materials, but the hole is generally poor, is not conducive to cell growth, proliferation and migration. The article focuses on the fresh silk fibroin solution pretreatment, slowly concentrated and then diluted to obtain nano fiber protein solution, preparation of silk fibroin protein and then the solution was observed, the morphology of silk protein materials before and after processing. The experimental results show that the slow concentration could induce silk protein Change of nanoparticles solution to nano fiber, improve stent porosity can be obtained with good pore structure of silk protein materials. On this basis, adding different content to the silk fibroin solution slowly in glycerol concentration treatment preparation support structure can be adjusted, and the two level structure, the stability of support study on degradation performance, etc. The experimental results show that glycerol can change the two level structure of silk protein, the silk protein changed from random structure to silk I and silk II structure, improve the stability of the stent, the glycerol content plays a role in control of certain mechanical properties, and by adding glycerol and change its content to achieve the regulation on the structure and properties of the scaffold. On the other hand, based on the principle of molecular self-assembly of silk protein, without adding any organic reagent conditions, by changing the molecular motion of the further realization of the silk scaffold structure Regulation and performance. The silk fibroin nanofiber solution and fresh silk protein fibroin solution according to dry weight ratio of 1:15 were mixed by changing the mixed solution of freezing thawing cycles or the 50oC temperature to adjust the assembly structure of silk protein in solution, followed by freeze drying method of silk fibroin porous scaffold preparation different. The results show that with the increase of freezing thawing cycles, increase the silk content of II stent, compression modulus increases, while the temperature is conducive to the increase of silk in I frame structure, compression modulus decreased. Therefore, repeated freezing and thawing temperature treatment can achieve the control of the two level structure and mechanical properties the performance of different support. Finally, the SD of rat bone marrow mesenchymal stem cells (R BMSCs) were cultured on the scaffold, study the different properties of silk fibroin porous scaffold on the proliferation of BMSCs and R The effect of differentiation. The results showed that the cell proliferation in all scaffolds, and cell growth in experimental group was significantly better than the control group.DNA content results prove that support good cell compatibility, and support the lower modulus, the proliferation of R BMSCs composite scaffold is better. Then choosing different mechanical properties in vitro, influence the study on mechanics behavior of the differentiation of stem cells. The results show that the silk scaffold modulus in 8~17k Pa cells in preference to differentiation into muscle cells, and the compression modulus in support of Pa 1~7k points to the endothelial cells. In summary, the gentle method in this paper through the nano structure regulation of silk protein, and hydrophilic interaction molecular motion, thus preparing the micro structure, two level structure of silk fibroin porous scaffold, controllable mechanical properties, in order to achieve different functions, improve the silk protein in different tissue repair The applicability of the complex domain.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R318.08

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