再生丝素纤维的制备及其在人工韧带中的应用

发布时间：2019-02-19 21:10

【摘要】：前交叉韧带(Anterior cruciate ligament,ACL)是膝关节内的核心结构,对膝关节的稳定性起着重要作用。目前我国因运动和交通事故等造成ACL损伤的现象明显增多,其损伤断裂会导致膝关节稳定性差等问题的出现,严重损伤还会影响膝关节功能,因此我国对ACL修复材料市场需求量急剧增加。丝素蛋白(SF)因其良好的可塑性、生物相容性,可以克服早期移植物的免疫排斥反应而成为制备生物医用材料的理想素材。发挥再生SF纤维可降解性以及天然SF纤维优良力学性质的优势,本文拟制备以SF为原料的可降解型再生SF纤维复合韧带。采用氯化钙(Ca Cl2)-甲酸(FA)溶解体系在常温下破坏原纤内的结晶结构分纤溶解蚕丝,利用湿法纺丝、静电纺丝技术制备再生SF纤维,探讨不同溶解参数对溶液溶解性的影响以及溶液性质对再生加工过程、纤维形貌及力学性能的影响。在此研究基础上,通过体外降解和细胞培养实验验证再生SF纤维的降解性及生物相容性。该溶解体系下溶液中的原纤结构为制备高性能再生纤维提供保障,故选用再生SF长丝、静电纺再生SF纳米纤维以及天然SF纤维为原料构建再生SF纤维复合韧带,为开拓以SF为基材的韧带修复材料提供新思路。传统溶解方法(Ca Cl2-C2H5OH-H2O)极大地破坏了蚕丝的原纤结构,甚至是分子结构,严重影响了再生SF材料的性能,同时对中性盐浓度、溶解温度以及溶解时间的要求更高。本文采用Ca Cl2-FA溶解体系通过强极性离子产生的水化作用使纤维溶胀、体积增大,削弱了分子间氢键作用,可以常温条件下分纤溶解蚕丝并破坏原纤内的β-折叠结晶结构达到溶解蚕丝的目的。在SF溶液及干燥膜中都能清晰观察到原纤结构,溶液中的原纤尺寸随Ca Cl2浓度、溶解温度的增加呈现减小的趋势。该溶解方法能获得粘度更高的溶液,SF在溶液中的溶解度会随着Ca Cl2浓度、溶解温度的增加而增加。此外,该溶解法制备的SF膜材料为水不溶性物质,去离子水浸泡可以诱导其由无规卷曲向β-折叠结构转变。溶液中的原纤结构在湿法纺丝过程中经过凝固浴并受到剪切作用重组成纤维,以水为凝固浴、质量分数为15.0 wt.%的SF溶液湿法纺丝,4倍牵伸后可获得直径为10.8±1.4μm、断裂应力达358.4±43.2 MPa、断裂伸长率达24.8±4.2%的SF长丝,力学性能有明显提高。此外,水浴凝固具有对环境无污染、低成本、易操作等优点。将Ca Cl2-FA溶解体系获得的SF溶液干燥成膜后溶解于甲酸得到仍保留原纤结构的SF溶液进行静电纺丝,采用自制滚筒收集装置获得取向纳米纤维,并与细胞外基质(ECM)具有相似的尺寸结构。讨论不同Ca Cl2浓度制备得到的纺丝液、不同SF浓度对成纤性的影响,分析滚筒转速和拉伸对纤维取向性的影响。结果表明SF质量分数为6.0 wt.%、Ca Cl2质量分数为5.0 wt.%、滚筒转速为1000 rpm获得直径为221±20nm的纤维,1倍牵伸后纤维的断裂应力和断裂伸长率分别达到了18.6±3.8 MPa和15.1±2.5%,纤维局部取向度明显改善,力学性能较Ca Cl2-C2H5OH-H2O溶解方法的纤维有较大幅度提高。再生SF纤维在PBS溶液中降解相对缓慢,降解60天,SF纳米纤维重量损失15%,纤维间部分出现粘连现象;SF长丝重量损失8.9%,降解只发生在纤维表面、出现部分裂痕。而蛋白酶XIV溶液对材料的降解能力明显增强,降解48h时,SF纳米纤维几乎完全降解,SF长丝重量损失61.5%,纤维主体分解。取向排列的SF纳米纤维较乱序纳米纤维可以在短时间内明显促进细胞的大量生长,SF长丝和天然SF纤维也利于细胞生长粘附,具有良好的生物相容性。基于高性能再生SF纤维的制备,采用立式锭子编织机,以SF长丝为轴纱,天然SF纤维编织物、静电纺SF纳米纤维包裹的形式构建再生SF纤维复合韧带,结构稳定,断裂强力达2581.7±23.7 N,满足人体ACL的要求。不同SF纤维在酶溶液中呈现出降解差异,再生SF纤维的快速降解为细胞再生提供有利的生长空间,降解速度慢、性质稳定的天然SF纤维支架保证了韧带材料的力学要求,实现了不同材料降解速度差异化的设计要求。本文采用Ca Cl2-FA溶解体系获得的再生SF纤维表现出良好的性能,开发设计出以SF纤维为原料、既保证力学要求又充分发挥其良好生物相容性和可降解性的人工韧带,为制备韧带修复材料提供一定的实验依据。
[Abstract]:Anterior cruciate ligament (ACL) is the core structure in the knee joint and plays an important role in the stability of the knee joint. At present, the phenomenon of ACL injury caused by sports and traffic accidents in our country is obviously increased, and the damage and fracture can cause the problems of poor stability of the knee joint and the like, and the serious injury can also affect the function of the knee joint, so the demand of the ACL repair material market is rapidly increased. the silk fibroin (SF) has good plasticity and biocompatibility, and can overcome the immune rejection reaction of the early graft and become the ideal material for preparing the biological medical material. It is proposed to prepare the degradable regenerated SF-fiber composite ligament with SF as the raw material to play the advantage of the degradability of the regenerated SF fibers and the excellent mechanical properties of the natural SF fibers. a calcium chloride (Ca 2)-formic acid (FA) dissolving system is adopted to destroy the crystalline structure of the original fiber at normal temperature to dissolve the silk, and the regenerated SF fiber is prepared by wet spinning and electrostatic spinning technology, The effects of different dissolution parameters on the solubility of the solution and the effect of the solution properties on the regeneration process, the morphology and the mechanical properties of the solution were discussed. On the basis of this study, the degradation and biocompatibility of the regenerated SF fibers were verified by in vitro degradation and cell culture experiments. the original fiber structure in the solution under the dissolution system is used for preparing the high-performance regenerated fiber, so that the regenerated SF filament, the electrostatic spinning regenerated SF nano-fiber and the natural SF fiber are used as the raw materials to construct the regenerated SF-fiber composite ligament, in order to provide a new thought for the development of the ligament repair material with the SF as the base material. The traditional dissolution method (Ca 2-C2H5OH-H2O) greatly destroys the original fiber structure of the silk, and even the molecular structure, and the performance of the regenerated SF material is seriously affected, and meanwhile, the requirement of the neutral salt concentration, the dissolution temperature and the dissolution time is higher. In this paper, the dissolution system of Ca 2-FA is used to make the fiber swell, the volume is increased, the intermolecular hydrogen bond is weakened by the hydration of the strong polar ion, and the silk can be dissolved in the fiber at normal temperature and the silk-folded crystal structure in the original fiber can be destroyed to achieve the purpose of dissolving the silk. The original fiber structure can be clearly observed in the SF solution and the dry film, and the size of the original fiber in the solution changes with the concentration of Ca 2 and the increase of the dissolution temperature. The dissolution method can obtain a solution with higher viscosity, and the solubility of the SF in the solution increases with the increase of the concentration of Ca 2 and the dissolution temperature. In addition, the SF film material prepared by the dissolution method is a water-insoluble substance, and the deionized water is soaked to induce the SF film material to be changed from the random curl to the crotch-folding structure. the original fiber structure in the solution is subjected to a coagulation bath in a wet spinning process and is subjected to shearing action to form a fiber, and the water is a coagulating bath, and the mass fraction is 15. 0wt. The mechanical properties of SF-filament with a diameter of 10. 8-1.4. m, a fracture stress of 358. 4-43. 2 MPa and an elongation of break of 24. 8-4. 2% can be obtained after 4-fold drawing, and the mechanical properties of the SF-filament are obviously improved. in addition, the water bath solidification has the advantages of no pollution to the environment, low cost, easy operation and the like. The SF solution obtained by the Ca 2-FA dissolving system is dried to form a film, then dissolved in formic acid to obtain the SF solution which still retains the original fiber structure to carry out electrostatic spinning, and the self-made roller collecting device is adopted to obtain the oriented nano-fiber and has a similar size structure as the extracellular matrix (ECM). The influence of different SF concentration on the fiber orientation was discussed, and the effect of roller speed and tensile on the fiber orientation was analyzed. The results show that the SF mass fraction is 6.0 wt.%, the mass fraction of Ca 2 is 5.0 wt. The fiber with the diameter of 221-20nm was obtained at the speed of 1000 rpm. The fracture stress and the elongation at break of the fiber were 18. 6-3. 8 MPa and 15. 1-2.5%, respectively. The local orientation of the fiber was obviously improved, and the mechanical properties of the fiber with higher mechanical properties were greatly improved than that of the Ca-2-C2H5OH-H2O dissolving method. The degradation of the regenerated SF fibers in the PBS solution was relatively slow, the degradation was 60 days, the weight loss of the SF nano-fiber was 15%, the fiber-to-fiber part had a blocking phenomenon, the weight loss of the SF filament was 8. 9%, and the degradation only occurred on the surface of the fiber, and a partial crack appeared. and the degradation ability of the protease XIV solution to the material is obviously enhanced, and when the degradation 48h, the SF nano-fiber is almost completely degraded, the weight loss of the SF filament is 61.5%, and the fiber body is decomposed. The SF nano-fiber with the orientation arrangement can obviously promote the large-volume growth of the cells in a short time, and the SF filaments and the natural SF fibers are also beneficial to cell growth and adhesion, and have good biocompatibility. based on the preparation of high-performance regenerated SF fiber, a vertical spindle knitting machine is adopted, the SF filament is a shaft yarn, a natural SF fiber braid and an electrostatic spinning SF nano-fiber are wrapped, the regenerated SF fiber composite ligament is constructed, the structure is stable, the breaking strength is up to 2581.7 to 237.N, and the requirements of the human body ACL are met. the different SF fibers exhibit a degradation difference in the enzyme solution, the rapid degradation of the regenerated SF fiber provides an advantageous growth space for cell regeneration, the degradation speed is slow, and the stable natural SF fiber support ensures the mechanical requirements of the ligament material, and the design requirements of differential speed differentiation of different materials are realized. In this paper, the regenerated SF fibers obtained by the Ca 2-FA dissolving system show good performance, and the development of artificial ligament with SF fiber as the raw material, which not only guarantees the mechanical requirements but also gives full play to its good biocompatibility and degradability, provides some experimental basis for the preparation of the ligament repair material.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08

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