丝蛋白功能性水凝胶的制备和性能研究

发布时间：2018-08-05 12:41

【摘要】：心脏、血管、神经、肌肉、骨等作为人体重要的组织器官,对人体生命活动起着重要作用,意外创伤、疾病等原因导致的组织缺损和器官病变严重威胁人体健康,器官移植是目前最为有效的治疗方法,但存在供体严重缺乏的问题。因此,寻找更为有效的方法进行组织修复一直是再生医学领域的研究热点。综合性能优异的支架材料是提高组织修复效果的关键,丝蛋白(SF)由于具有良好的生物相容性、可降解性、可加工性及优良的机械性能,在组织工程领域获得了广泛的关注,特别是具有类细胞外基质纳米纤维结构的丝蛋白水凝胶正成为组织修复材料新的研究方向。溴化锂溶丝法是制备丝蛋白溶液的常用方法,然而所制备丝蛋白存在复杂的构象组成和纳米结构,难以精确控制。本研究通过溶剂体系的设计,调控溶液中最终丝蛋白的纳米结构,获得非晶态丝蛋白纳米短纤维溶液,为丝蛋白纳米纤维水凝胶的制备提供可靠基元。在此基础上,利用PEG浓缩的方法来提高丝蛋白纳米短纤维的浓度,以满足制备力学性能可控的丝蛋白凝胶的需要。用过氧化氢酶交联的方法最终获得力学性能可调的水凝胶支架材料。通过改变丝蛋白纳米短纤维、HRP、H2O2三者在溶液中的浓度,研究了水凝胶支架材料凝胶时间、二级结构、纳米结构、力学性能的变化情况。实验结果表明经PEG浓缩处理后溶液的二级结构并没有发生显著改变,HRP交联后丝蛋白的二级结构仍然为以无规线团为主的亚稳态结构。通过改变丝蛋白纳米短纤维、HRP和H2O2三者的比例,能够实现水凝胶的力学性能在600Pa-20kPa范围内的可控设计,满足不同组织修复的力学要求。因此通过丝蛋白结构的控制,利用HRP酶交联的方法,我们获得了力学性能可控范围更广,且含有纳米纤维结构的丝蛋白水凝胶材料。另一方面,考虑到课题组所制备高结晶丝蛋白纳米线负电荷大,可在电场下形成取向结构的特点,将高结晶丝蛋白纳米线和非晶态丝蛋白纳米短纤维以不同比例混合,在电场作用下进行HRP交联,制备内部具有纳米纤维取向结构的水凝胶,实现力学性能的进一步提高。结果表明,通过HRP交联和电场的共同作用,混合溶液中高结晶丝蛋白纳米线在向电源正极发生定向移动的同时非晶态丝蛋白纳米短纤维发生交联反应,形成一种具有不同力学性能的复合凝胶材料,丝蛋白纳米线与纳米短纤维溶液的混合比例和电场作用时间的改变能实现靠近电源正极复合电凝胶的力学性能在100 kPa-500kPa范围内变化,靠近电源负极复合电凝胶的力学性能在50kPa-200kPa范围内变化。综上所述,本研究通过HRP酶交联和电场的作用成功制备出具有不同纳米结构和不同力学性能的水凝胶,以此为基础通过改变丝蛋白、HRP和H2O2的浓度,混合溶液的比例,电场作用时间等实现了丝蛋白水凝胶力学性能的可调控性和结构的多级有序性。本研究为丝蛋白功能性水凝胶支架材料在组织修复中的应用奠定了一定的材料基础。
[Abstract]:The heart, the blood vessel, the nerve, the muscle, the bone, as the important tissue and organ of the human body, play an important role in the human life, the tissue defect and organ disease caused by the accidental trauma, the disease and other causes seriously threaten the human health. The organ transplantation is the most effective treatment method at present, but there is a serious shortage of donor. Tissue repair for effective methods has always been a hot topic in the field of regenerative medicine. The scaffolding material with excellent comprehensive performance is the key to improve the effect of tissue repair. Silk protein (SF) has received extensive attention in the field of tissue engineering because of its good biocompatibility, biodegradability, processability and excellent mechanical energy. The silk protein hydrogel with the structure of the extracellular matrix nanofibers is becoming a new research direction in the tissue repair materials. Lithium bromide dissolution is a common method to prepare the silk protein solution. However, the prepared silk protein has complex conformation and nanostructures. The nano structure of the final silk protein in the solution is controlled, and the amorphous silk protein nanofiber solution is obtained, which provides a reliable basis for the preparation of silk protein nanofiber hydrogel. On this basis, the concentration of silk protein nanofibers is improved by PEG concentration method to meet the needs of the preparation of silk protein gel with controllable mechanical properties. The hydrogel scaffold material was finally obtained by the method of catalase crosslinking. The gelation time, the two grade structure, the nano structure and the mechanical properties of the hydrogel scaffold were studied by changing the concentration of the silk protein nanoscale, HRP, and H2O2 three in the solution. The experimental results showed that the solution was dissolved after PEG concentration treatment. The two stage structure of the liquid did not change significantly. The two grade structure of the HRP crosslinked silk protein was still a metastable structure based on the random line. By changing the ratio of silk fibroin nanoscale, HRP and H2O2 three, the controllable design of the mechanical properties of the hydrogel in the 600Pa-20kPa enclosure could be realized to meet the force of different tissue repair. Therefore, through the control of the silk protein structure and using the method of HRP enzyme crosslinking, we obtained the silk protein hydrogel materials with more controllable mechanical properties and nanofiber structure. On the other hand, considering the large negative charge of the nanowires with high crystal protein, the orientation structure can be formed under the electric field. The high crystalline silk protein nanowires and amorphous silk protein nanofibers were mixed in different proportions, and the HRP was crosslinked under the action of the electric field to prepare the hydrogel with nanofiber orientation structure. The results showed that the high crystalline egg in the mixed solution was highly crystallized in the mixed solution through the interaction of HRP and the electric field. The nanowires have a crosslinking reaction to the non crystalline silk protein nanoscale at the same time to the positive pole of the power supply, forming a composite gel with different mechanical properties. The mixing ratio of the silk protein nanowires and the nanofilm solution and the modification of the electric field action time can achieve the compound electrogel near the power cathode. The mechanical properties change within the range of 100 kPa-500kPa, and the mechanical properties of the composite electrogels are changed in the range of 50kPa-200kPa. To sum up, this study successfully prepared the water condensate with different nanostructures and different mechanical properties through the HRP enzyme crosslinking and the effect of the electric field. On this basis, the silk protein, HRP and H were changed. The concentration of 2O2, the proportion of mixed solution and the action time of electric field have realized the regulation of the mechanical properties of the silk protein hydrogel and the multi-level order of the structure. This study laid a certain material foundation for the application of silk protein functional hydrogel scaffold materials in the tissue repair.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O648.17

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