静电纺丝构建蚕丝蛋白基支架及其应用于神经修复的研究
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摘要
纳米级的纤维材料是生物体系统的基本组成结构。如DNA双螺旋的直径大约是2 nm,螺距为3.5 nm左右,细胞骨架丝的直径是30 nm左右。纳米纤维材料与细胞外基质的微观结构存在一致性,因此其在生物医药领域的应用具有先天优势,目前加工纳米纤维材料的方法很多,如模板法、拉伸法、自组装法等,但由于各种条件限制,静电纺丝被认为是最有可能实现连续及大量制备纳米纤维材料的工艺技术。通过静电纺丝工艺可以将聚合物加工成直径为3 nm的纤维,小到3 nm的纤维其截面仅有6-7个大分子。
近年来,利用天然材料制备纳米产品引起了广泛的注意,如将蚕丝纤维蛋白加工制备成微纳米纤维组织工程支架。蚕丝是一种天然高分子蛋白质,近年来获得各领域学者浓厚的研究兴趣,这主要归因于其优异的特性(强力和生物相容性)和潜在的应用价值,如用于智能纺织品、防护服、过滤材料、缓释材料和组织工程,特别是在组织工程领域的研究成为了热点。
本课题选取桑蚕丝素蛋白(BSF)和柞蚕丝素蛋白(TSF)为主要实验对象,旨在研究丝素蛋白(SF)的静电纺丝性能,获得SF基微纳米纤维材料,在对材料形态、结构和性能研究的基础上,考察材料与神经类细胞的生物相容性并探索材料用于神经损伤修复的可行性。研究发现:
纺丝溶剂的不同不仅会影响到纤维形态,而且对纤维材料聚集态结构及热性能产生影响。如以甲酸为溶剂所纺BSF微纳米纤维的直径远远小于以HFIP为溶剂所纺纤维,而且含有更多的β-折叠结构。此外,通过不同醇溶液及不同浓度乙醇溶液后处理实验发现,BSF微纳米纤维网的结构转变不受醇分子大小及水分含量的影响,分析认为溶剂极性、材料形态、初始结构及结晶结构将决定有机溶剂后处理是否能够促进蚕丝蛋白发生构象转变;通过在纺丝液中加入EDC,或进行EDC/NHS乙醇溶液后处理,均能明显提高BSF微纳米纤维材料的物理机械性能和尺寸稳定性。
以六氟异丙醇(HFIP)为溶剂,通过静电纺丝方法纺制多种比例BSF/TSF共混微纳米纤维非织造网,研究发现随TSF含量的增加BSF/TSF共混纤维的直径显著降低;BSF/TSF共混纤维中两种成分在宏观融为一体,但结构分析表明两者在微观上是相分离的,即不能形成共晶结构;星形胶质细胞培养实验表明,BSF/TSF共混材料与常用神经细胞培养用聚赖氨酸包被板(PLL)在支持细胞的黏附、生长与增殖方面没有显著差异,显示出与星形胶质细胞良好的生物相容性。
细胞培养实验表明:丝素蛋白微纳米纤维材料支持神经元、神经干、嗅鞘和雪旺细胞的黏附、铺展、生长发育和增殖,其支持效果与PLL板相近;与BSF纤维材料相比,TSF纤维材料更支持神经元细胞的存活及提高了神经元的复杂度;与BSF纤维材料相比,神经干细胞在TSF纤维材料上黏附、增殖和迁移更快,并且细胞的分布更均匀,而培养的雪旺细胞对材料成分并不敏感,因此TSF微纳米纤维材料更适合于神经元和神经干细胞的生长;与微米级BSF纤维材料相比,纳米级BSF纤维材料对嗅鞘细胞和雪旺细胞的生长与增殖更加有利,这主要表现在:嗅鞘细胞在纳米级BSF纤维材料上能够岩纤维排列并伸出胞突,细胞增殖速度更快;雪旺细胞在纳米级BSF纤维材料上能够相互连接,形成类Büngner带,细胞的纯度更高,细胞胞突较长。
通过旋转收集装置的旋转与平移可以制得平行于轴向或周向的BSF微纳米纤维导管;导管SD大鼠体内实验表明:导管支架材料植入老鼠体内后无明显不良反应,两个月和四个月后均可发现再生神经通过整个导管,老鼠右腿运动及感觉功能得到部分恢复,再生修复效果TSF导管优于BSF导管。
Nanoscale polymeric fibrous materials are the fundamental building unit of live systems. For example, the diameter of double helix DNA molecules is 2 nm, and the cytoskeleton filament is 30 nm. Nano-scale fibrous materials are consistency in the aspect of microstructure with the extracellular matrix (ECM). To date, many distinct techniques have proved successful in preparing nanoscale fibrous materials, such as self-assembly, phase separation, template method etc; however, electrospinning is considered to be more likely to achieve continuous and mass production of nano-fiber material. Polymer can be electrospun to produce nanofibers of with diameters as small as 3 nm, and the 3 nm diameter fibers have only 6 or 7 molecules across the fiber.
In recent years, there has been significant increasing interest in the utilization of natural materials for novel nano-products, such as silk fibroin nanofibrous tissue engineered scaffolds. Silkworm silk, a protein-base natural biopolymer, has received keen interest in various areas due to its unique properties (strength, biocompatibility) and vast potential applications such as smart textiles, protective clothing, filter materials, sustained-release material and tissue engineering, especially in the field of tissue engineering has become the hot spot.
This research deals with fabrication of tissue scaffolds from Bombyx mori silk fibroin (BSF) and Tussah silk fibroin (TSF) for its abundant supply and excellent mechanical and biological property. The purpose of this study is to prepare silk fibroin (SF)-based nanofibrous scaffold, determine the property of electrospun silk fibroin nanofibers; to study the biocompatibility with nerve cells (neuron, neural stem cell (NSCs), Olfactory ensheathing cells (OECs), Schwann cells (SCs)), and to bridge 10-mm sciatic nerve defects of rats with silk fibroin-based artificial nerve tube.
The BSF/TSF blends nanofibers were prepared by electrospinning with the solvent of HFIP, and the average diameters of BSF/TSF blend fibers increased from 404 to 1977nm, with the increase of BSF content in blend solutions, and the relationship between the average diameters of BSF/TSF and BSF content was proved to be linear correlation. Results from FTIR, TG-DTA and X-ray diffraction showed BSF and TSF were still immiscible even dissolved in hexafluoroisopropanol (HFIP) after electrospinning and ethanol treatment.
Methanol, ethanol, isopropanol and different aqueous ethanol solution all were proved effective in inducing conformation transition of BSF nanofibers, it suggested that the material morphology, initial structure, crystalline structure determine if the conformation transition of silk fibroin when immersion in organic solution, and hydrophobic interaction is the driving force of structural change. The two methods of addition of EDC in electrospin solution, or EDC/NHS ethanol system were effective in improving mechanical integrity and stability.
Analysis of the morphology and number of nerve cells cultured on silk fibroin nanofibers indicated that cell adhesive, complexity, and proliferation was more obvious on TSF and BSF nanofibers, importantly, cell gather to form büngner band which is important for nerve regeneration.
SF nanofiber-based artificial nerve conduits were prepared by electrospinning and applied to bridge 10mm long sciatic nerve defect of rats. The present study shows that the electrospun silk fibroin tubes are successful in bridging a 10mm gap in the sciatic nerve of the rat, especially TSF tubes demonstrated a superior repair results.
近年来,利用天然材料制备纳米产品引起了广泛的注意,如将蚕丝纤维蛋白加工制备成微纳米纤维组织工程支架。蚕丝是一种天然高分子蛋白质,近年来获得各领域学者浓厚的研究兴趣,这主要归因于其优异的特性(强力和生物相容性)和潜在的应用价值,如用于智能纺织品、防护服、过滤材料、缓释材料和组织工程,特别是在组织工程领域的研究成为了热点。
本课题选取桑蚕丝素蛋白(BSF)和柞蚕丝素蛋白(TSF)为主要实验对象,旨在研究丝素蛋白(SF)的静电纺丝性能,获得SF基微纳米纤维材料,在对材料形态、结构和性能研究的基础上,考察材料与神经类细胞的生物相容性并探索材料用于神经损伤修复的可行性。研究发现:
纺丝溶剂的不同不仅会影响到纤维形态,而且对纤维材料聚集态结构及热性能产生影响。如以甲酸为溶剂所纺BSF微纳米纤维的直径远远小于以HFIP为溶剂所纺纤维,而且含有更多的β-折叠结构。此外,通过不同醇溶液及不同浓度乙醇溶液后处理实验发现,BSF微纳米纤维网的结构转变不受醇分子大小及水分含量的影响,分析认为溶剂极性、材料形态、初始结构及结晶结构将决定有机溶剂后处理是否能够促进蚕丝蛋白发生构象转变;通过在纺丝液中加入EDC,或进行EDC/NHS乙醇溶液后处理,均能明显提高BSF微纳米纤维材料的物理机械性能和尺寸稳定性。
以六氟异丙醇(HFIP)为溶剂,通过静电纺丝方法纺制多种比例BSF/TSF共混微纳米纤维非织造网,研究发现随TSF含量的增加BSF/TSF共混纤维的直径显著降低;BSF/TSF共混纤维中两种成分在宏观融为一体,但结构分析表明两者在微观上是相分离的,即不能形成共晶结构;星形胶质细胞培养实验表明,BSF/TSF共混材料与常用神经细胞培养用聚赖氨酸包被板(PLL)在支持细胞的黏附、生长与增殖方面没有显著差异,显示出与星形胶质细胞良好的生物相容性。
细胞培养实验表明:丝素蛋白微纳米纤维材料支持神经元、神经干、嗅鞘和雪旺细胞的黏附、铺展、生长发育和增殖,其支持效果与PLL板相近;与BSF纤维材料相比,TSF纤维材料更支持神经元细胞的存活及提高了神经元的复杂度;与BSF纤维材料相比,神经干细胞在TSF纤维材料上黏附、增殖和迁移更快,并且细胞的分布更均匀,而培养的雪旺细胞对材料成分并不敏感,因此TSF微纳米纤维材料更适合于神经元和神经干细胞的生长;与微米级BSF纤维材料相比,纳米级BSF纤维材料对嗅鞘细胞和雪旺细胞的生长与增殖更加有利,这主要表现在:嗅鞘细胞在纳米级BSF纤维材料上能够岩纤维排列并伸出胞突,细胞增殖速度更快;雪旺细胞在纳米级BSF纤维材料上能够相互连接,形成类Büngner带,细胞的纯度更高,细胞胞突较长。
通过旋转收集装置的旋转与平移可以制得平行于轴向或周向的BSF微纳米纤维导管;导管SD大鼠体内实验表明:导管支架材料植入老鼠体内后无明显不良反应,两个月和四个月后均可发现再生神经通过整个导管,老鼠右腿运动及感觉功能得到部分恢复,再生修复效果TSF导管优于BSF导管。
Nanoscale polymeric fibrous materials are the fundamental building unit of live systems. For example, the diameter of double helix DNA molecules is 2 nm, and the cytoskeleton filament is 30 nm. Nano-scale fibrous materials are consistency in the aspect of microstructure with the extracellular matrix (ECM). To date, many distinct techniques have proved successful in preparing nanoscale fibrous materials, such as self-assembly, phase separation, template method etc; however, electrospinning is considered to be more likely to achieve continuous and mass production of nano-fiber material. Polymer can be electrospun to produce nanofibers of with diameters as small as 3 nm, and the 3 nm diameter fibers have only 6 or 7 molecules across the fiber.
In recent years, there has been significant increasing interest in the utilization of natural materials for novel nano-products, such as silk fibroin nanofibrous tissue engineered scaffolds. Silkworm silk, a protein-base natural biopolymer, has received keen interest in various areas due to its unique properties (strength, biocompatibility) and vast potential applications such as smart textiles, protective clothing, filter materials, sustained-release material and tissue engineering, especially in the field of tissue engineering has become the hot spot.
This research deals with fabrication of tissue scaffolds from Bombyx mori silk fibroin (BSF) and Tussah silk fibroin (TSF) for its abundant supply and excellent mechanical and biological property. The purpose of this study is to prepare silk fibroin (SF)-based nanofibrous scaffold, determine the property of electrospun silk fibroin nanofibers; to study the biocompatibility with nerve cells (neuron, neural stem cell (NSCs), Olfactory ensheathing cells (OECs), Schwann cells (SCs)), and to bridge 10-mm sciatic nerve defects of rats with silk fibroin-based artificial nerve tube.
The BSF/TSF blends nanofibers were prepared by electrospinning with the solvent of HFIP, and the average diameters of BSF/TSF blend fibers increased from 404 to 1977nm, with the increase of BSF content in blend solutions, and the relationship between the average diameters of BSF/TSF and BSF content was proved to be linear correlation. Results from FTIR, TG-DTA and X-ray diffraction showed BSF and TSF were still immiscible even dissolved in hexafluoroisopropanol (HFIP) after electrospinning and ethanol treatment.
Methanol, ethanol, isopropanol and different aqueous ethanol solution all were proved effective in inducing conformation transition of BSF nanofibers, it suggested that the material morphology, initial structure, crystalline structure determine if the conformation transition of silk fibroin when immersion in organic solution, and hydrophobic interaction is the driving force of structural change. The two methods of addition of EDC in electrospin solution, or EDC/NHS ethanol system were effective in improving mechanical integrity and stability.
Analysis of the morphology and number of nerve cells cultured on silk fibroin nanofibers indicated that cell adhesive, complexity, and proliferation was more obvious on TSF and BSF nanofibers, importantly, cell gather to form büngner band which is important for nerve regeneration.
SF nanofiber-based artificial nerve conduits were prepared by electrospinning and applied to bridge 10mm long sciatic nerve defect of rats. The present study shows that the electrospun silk fibroin tubes are successful in bridging a 10mm gap in the sciatic nerve of the rat, especially TSF tubes demonstrated a superior repair results.
引文
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