丝素蛋白质电凝胶的制备及其性能研究
发布时间：2019-03-01 18:46
【摘要】：蚕丝丝素蛋白质水凝胶具有良好的生物相容性、生物降解性、保水性和孔隙连通性等,已被研究用于软组织工程修复、药物缓释、基因工程等领域,为当前生物医用材料和再生医学领域的研究热点。作为软组织修复材料,丝素蛋白质水凝胶常通过交联、接枝或机械作用等方法制备,但存在耗时较长、凝胶机械性能差、引入有机溶剂、形貌难以调控等缺点,难以满足临床应用的要求。因此,迫切需要建立一种新型的丝素水凝胶制备方法,构筑性能良好且又绿色温和的丝素水凝胶。针对这一问题,本研究着眼于丝素蛋白质大分子的理化性能特性,采用绿色、温和环保的静电场组装技术,通过调节丝素水溶液的pH值和外加低压电场,快速有效地制备性能优异的丝素水凝胶,为软组织修复工程提供一种新型的丝素支架。在深入理解丝素大分子的理化特性和目标材料性能的基础上,本研究设想通过pH值(pI附近)和低压电场电泳的协同作用,调控丝素蛋白质大分子的组装行为,降低或抵消丝素大分子之间和之内的相互排斥作用,强化丝素大分子之间的亲疏水相互作用和相互纠缠、抱合的程度,获得稳定的二级结构(β-sheets);同时,改变部分丝素大分子的排列方式,获得机械性能优异、稳定性好的丝素电凝胶。通过改变稳定直流电压、丝素蛋白质溶液浓度和pH获得丝素蛋白质电凝胶制备优化条件:稳定直流电压24 V,丝素溶液浓度为5wt%,pH值为4。通过吸水率、压缩强度等机械性能的测试来评价丝素蛋白质电凝胶;采用扫描电镜(SEM)观察丝素电凝胶的形貌;傅立叶红外光谱(FTIR)、X射线衍射(XRD)、拉曼光谱(Raman)等表征了凝胶的聚集态结构和二级结构;通过体外模型药物的装载和释放实验探究了凝胶的药物缓释性能;最后通过体外蛋白酶XIV的降解实验和对大鼠肾小管上皮细胞相容性的实验,评价了丝素蛋白质电凝胶的生物降解行为和细胞相容性。研究结果表明,采用调节pH值和低压电场协同作用制备的丝蛋白质电凝胶(SF pH-e-gel)较自然形成的丝蛋白凝胶(SF gel)和调节pH值制备的丝蛋白凝胶(SF pH-gel)具有更显著的机械性能。压缩强度约为7.90MPa,压缩模量约为70.0MPa,远高于SF gel的0.25MPa和1.0MPa,并且其含水率可达约390%。其次,SEM结果表明,SF pH-e-gel材料孔呈规整的圆形、孔径较小且孔壁较厚,与传统丝素凝胶的层状或片状孔结构不同。FTIR、XRD、Raman等表征结果表明,SF pH-e-gel内β-sheet结构的含量较低,而α-helix的含量较其他两种凝胶高,silk I型结晶的含量明显高于另外两凝胶。再次,以罗丹明B作为模型药物的缓释实验结果表明,当丝素溶液的药物初始浓度为10μg/mL时,SF pH-e-gel的药物包覆率可达87.5%;在120h内,药物的释放量约为60%。而在蛋白酶XIV作用下,SF pH-e-gel体外降解28d,降解量约为80%,表明SF pH-e-gel支架具备良好的可生物降解性。最后,选择大鼠肾小管上皮细胞作为模型细胞,接种于SF pH-e-gel支架上,通过不同时间点细胞形态和活力测试,结果表明凝胶支架能支持上皮细胞的黏附、分化和增殖,具有良好的细胞相容性。通过本文的研究,建立了一种绿色环保、快速有效制备丝素蛋白质凝胶的新方法,所制备凝胶具备良好的机械性能、优异的生物相容性、药物缓释性和生物降解性;阐明了SF pH-e-gel的形成机理。其主要形成机制:通过调控丝素蛋白质的pH值,降低或消除丝素大分子之间或分子内的排斥力,使分子链相互靠近;在静电场的作用下,丝素大分子被牵拉、伸直,在热力学作用下,进一步相互纠缠,形成较多的α-helix结构;同时,在丝素大分子相互靠近、分子链热运动和亲疏水性相互作用的共同影响下,形成稳定的β-sheet结构,并与上述的α-helix结构一起组装成结晶含量较高、不溶于水的固体凝胶。主要以α-helix结构形成的silk I型结晶,使凝胶弹性模量降低;以β-sheet结构形成的silk II型结晶,起到提高凝胶强度和模量的作用。本研究制备的丝素蛋白质电凝胶改善了传统丝素蛋白质凝胶机械性能差的问题,表现出良好的生物相容性和生物降解性,为组织工程中软组织的修复提供了一种新型的生物材料。
[Abstract]:Silk fibroin hydrogel has good biocompatibility, biodegradability, water retention and pore connectivity. It has been used in the fields of soft tissue engineering repair, drug slow release, gene engineering and so on. As a soft tissue repair material, the silk fibroin hydrogel is prepared by a method such as cross-linking, grafting or mechanical action, but has the disadvantages of long time consumption, poor gel mechanical property, introduction of organic solvent, difficult regulation of appearance and the like, and is difficult to meet the requirements of clinical application. Therefore, it is urgent to establish a new method for preparing silk fibroin hydrogel, which is a good and green silk fibroin hydrogel. aiming at the problem, the research focuses on the physical and chemical properties of the silk fibroin macromolecules, adopts a green, mild and environment-friendly electrostatic field assembly technology, and rapidly and effectively prepares the silk fibroin hydrogel with excellent performance by adjusting the pH value of the silk fibroin aqueous solution and the applied low-voltage electric field, And provides a novel silk fibroin bracket for soft tissue repair engineering. On the basis of in-depth understanding of the physical and chemical properties of the silk fibroin and the properties of the target material, this study envisages the co-action of the pH value (near pI) and the low-voltage electric field electrophoresis to control the assembly behavior of the silk fibroin macromolecules. the mutual exclusion between the silk fibroin macromolecules is reduced or eliminated, the interaction of the hydrophilic and hydrophobic water between the silk fibroin macromolecules and the entanglement of the silk fibroin macromolecules are enhanced, the degree of the binding is improved, and the stable secondary structure is obtained; and meanwhile, the arrangement mode of the partial silk fibroin macromolecules is changed, And the silk fibroin electrogel with excellent mechanical performance and good stability is obtained. The optimized conditions were obtained by changing the stable DC voltage, the concentration of the fibroin solution and the pH. The optimized conditions were as follows: the stable DC voltage is 24 V, the concentration of the silk fibroin solution is 5% by weight, and the pH value is 4. The electrogel of silk fibroin was evaluated by the tests of water absorption, compressive strength and other mechanical properties. The morphology of the silk fibroin gel was observed by scanning electron microscope (SEM), Fourier transform infrared (FTIR), X-ray diffraction (XRD), The aggregation state structure and the secondary structure of the gel are characterized by Raman and the like; the drug sustained-release performance of the gel is explored through the loading and release experiments of the in-vitro model drug; and finally, the experiment of the degradation experiment of the in vitro protease XIV and the compatibility of the renal tubular epithelial cells of the rat is carried out, The biodegradability and cell compatibility of silk fibroin electrogel were evaluated. The results show that the SF gel and SF-gel prepared by the synergistic effect of pH and low-voltage electric field have more significant mechanical properties than that of the naturally-formed silk fibroin gel (SF gel) and the adjusted pH value. The compressive strength is about 7.90 MPa, the compression modulus is about 70.0 MPa, much higher than that of the SF gel of 0.25 MPa and 1.0 MPa, and the water content can reach about 390%. The results showed that the pore size of SF-e-gel material was regular, the pore diameter was small and the pore wall was thick, and it was different from the lamellar or sheet-like pore structure of the traditional silk fibroin gel. The results of FTIR, XRD and Raman show that the content of the S-sheet in the SF-e-gel is low, and the content of the HCO3-help is higher than that of the other two gels, and the content of the sikI-type crystals is higher than that of the other two gels. The results of the sustained-release experiment with rhodamine B as a model drug show that the drug-coating rate of SF-e-gel can reach 87.5% when the initial concentration of the drug in the solution is 10. m u.g/ mL, and the release amount of the drug is about 60% in 120 h. Under the action of protease XIV, the degradation of SF-e-gel in vitro was about 80%, indicating that the SF-e-gel scaffold had good biodegradability. In the end, the rat renal tubular epithelial cells were selected as model cells, seeded on the SF pH-e-gel scaffold, and the cell morphology and viability were tested by different time points. The results showed that the gel scaffold can support the adhesion, differentiation and proliferation of the epithelial cells, and has good cell compatibility. In this paper, a new method of green environmental protection and rapid and effective preparation of silk fibroin gel is established. The prepared gel has good mechanical properties, excellent biocompatibility, drug release and biodegradability, and the formation mechanism of SF pH-e-gel is clarified. The main forming mechanism is that by regulating the pH value of the silk fibroin protein, the repulsive force between the silk fibroin macromolecules or the molecules is reduced or eliminated, so that the molecular chains are close to each other; under the action of the electrostatic field, the silk fibroin macromolecules are pulled and straightened, and the silk fibroin macromolecules are further entangled with each other under the action of thermodynamics; And meanwhile, under the mutual influence of the mutual proximity of the silk fibroin macromolecules, the thermal movement of the molecular chain and the hydrophobic interaction, a stable l-sheet structure is formed, and is assembled into a solid gel with higher crystal content and is insoluble in water together with the carbon-help structure described above. Silk type I crystals, which are mainly formed by the structure of the Si-helix structure, are used to reduce the elastic modulus of the gel, and the silk type II crystal formed by the structure of the Si-sheet structure plays a role in improving the strength and the modulus of the gel. The silk fibroin electrogel prepared by the method improves the problem of poor mechanical property of the traditional silk fibroin gel, has good biocompatibility and biodegradability, and provides a novel biological material for the repair of soft tissue in the tissue engineering.
【学位授予单位】：武汉纺织大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ427.26
本文编号：2432700
[Abstract]:Silk fibroin hydrogel has good biocompatibility, biodegradability, water retention and pore connectivity. It has been used in the fields of soft tissue engineering repair, drug slow release, gene engineering and so on. As a soft tissue repair material, the silk fibroin hydrogel is prepared by a method such as cross-linking, grafting or mechanical action, but has the disadvantages of long time consumption, poor gel mechanical property, introduction of organic solvent, difficult regulation of appearance and the like, and is difficult to meet the requirements of clinical application. Therefore, it is urgent to establish a new method for preparing silk fibroin hydrogel, which is a good and green silk fibroin hydrogel. aiming at the problem, the research focuses on the physical and chemical properties of the silk fibroin macromolecules, adopts a green, mild and environment-friendly electrostatic field assembly technology, and rapidly and effectively prepares the silk fibroin hydrogel with excellent performance by adjusting the pH value of the silk fibroin aqueous solution and the applied low-voltage electric field, And provides a novel silk fibroin bracket for soft tissue repair engineering. On the basis of in-depth understanding of the physical and chemical properties of the silk fibroin and the properties of the target material, this study envisages the co-action of the pH value (near pI) and the low-voltage electric field electrophoresis to control the assembly behavior of the silk fibroin macromolecules. the mutual exclusion between the silk fibroin macromolecules is reduced or eliminated, the interaction of the hydrophilic and hydrophobic water between the silk fibroin macromolecules and the entanglement of the silk fibroin macromolecules are enhanced, the degree of the binding is improved, and the stable secondary structure is obtained; and meanwhile, the arrangement mode of the partial silk fibroin macromolecules is changed, And the silk fibroin electrogel with excellent mechanical performance and good stability is obtained. The optimized conditions were obtained by changing the stable DC voltage, the concentration of the fibroin solution and the pH. The optimized conditions were as follows: the stable DC voltage is 24 V, the concentration of the silk fibroin solution is 5% by weight, and the pH value is 4. The electrogel of silk fibroin was evaluated by the tests of water absorption, compressive strength and other mechanical properties. The morphology of the silk fibroin gel was observed by scanning electron microscope (SEM), Fourier transform infrared (FTIR), X-ray diffraction (XRD), The aggregation state structure and the secondary structure of the gel are characterized by Raman and the like; the drug sustained-release performance of the gel is explored through the loading and release experiments of the in-vitro model drug; and finally, the experiment of the degradation experiment of the in vitro protease XIV and the compatibility of the renal tubular epithelial cells of the rat is carried out, The biodegradability and cell compatibility of silk fibroin electrogel were evaluated. The results show that the SF gel and SF-gel prepared by the synergistic effect of pH and low-voltage electric field have more significant mechanical properties than that of the naturally-formed silk fibroin gel (SF gel) and the adjusted pH value. The compressive strength is about 7.90 MPa, the compression modulus is about 70.0 MPa, much higher than that of the SF gel of 0.25 MPa and 1.0 MPa, and the water content can reach about 390%. The results showed that the pore size of SF-e-gel material was regular, the pore diameter was small and the pore wall was thick, and it was different from the lamellar or sheet-like pore structure of the traditional silk fibroin gel. The results of FTIR, XRD and Raman show that the content of the S-sheet in the SF-e-gel is low, and the content of the HCO3-help is higher than that of the other two gels, and the content of the sikI-type crystals is higher than that of the other two gels. The results of the sustained-release experiment with rhodamine B as a model drug show that the drug-coating rate of SF-e-gel can reach 87.5% when the initial concentration of the drug in the solution is 10. m u.g/ mL, and the release amount of the drug is about 60% in 120 h. Under the action of protease XIV, the degradation of SF-e-gel in vitro was about 80%, indicating that the SF-e-gel scaffold had good biodegradability. In the end, the rat renal tubular epithelial cells were selected as model cells, seeded on the SF pH-e-gel scaffold, and the cell morphology and viability were tested by different time points. The results showed that the gel scaffold can support the adhesion, differentiation and proliferation of the epithelial cells, and has good cell compatibility. In this paper, a new method of green environmental protection and rapid and effective preparation of silk fibroin gel is established. The prepared gel has good mechanical properties, excellent biocompatibility, drug release and biodegradability, and the formation mechanism of SF pH-e-gel is clarified. The main forming mechanism is that by regulating the pH value of the silk fibroin protein, the repulsive force between the silk fibroin macromolecules or the molecules is reduced or eliminated, so that the molecular chains are close to each other; under the action of the electrostatic field, the silk fibroin macromolecules are pulled and straightened, and the silk fibroin macromolecules are further entangled with each other under the action of thermodynamics; And meanwhile, under the mutual influence of the mutual proximity of the silk fibroin macromolecules, the thermal movement of the molecular chain and the hydrophobic interaction, a stable l-sheet structure is formed, and is assembled into a solid gel with higher crystal content and is insoluble in water together with the carbon-help structure described above. Silk type I crystals, which are mainly formed by the structure of the Si-helix structure, are used to reduce the elastic modulus of the gel, and the silk type II crystal formed by the structure of the Si-sheet structure plays a role in improving the strength and the modulus of the gel. The silk fibroin electrogel prepared by the method improves the problem of poor mechanical property of the traditional silk fibroin gel, has good biocompatibility and biodegradability, and provides a novel biological material for the repair of soft tissue in the tissue engineering.
【学位授予单位】：武汉纺织大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ427.26
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