静电纺管状支架的制备及其在组织工程中的应用

发布时间：2018-05-21 21:07

本文选题：静电纺丝 + 组织工程　；参考：《东华大学》2013年博士论文

【摘要】：心血管疾病被认为是引起人类死亡的重要原因,其比例已经占全球死亡总数的30%,据统计在美国每年有14万例以上的外科血管植入手术,且多数患者的年龄大于50岁。由于世界上还没有一套理想的制备小口径血管组织的技术产品,医生只有取病人身体其他部位的不重要的静脉来替代硬化的冠状动脉,此类“二次手术”无疑是给病人引入了另一种痛苦。与血管疾病情况类似的是,外周神经损伤是临床上的常见疾患,主要由事故造成的创伤引起。仅美国每年就有约1万1千人因为神经受损而瘫痪,造成经济损失愈70亿美金,而外周神经手术则超过了5万例/年。这些修复神经断裂和缺损的手术也使用了病人其他部位相对不重要的神经来替代,实为权宜之计。有鉴于此,科研工作者们一直致力于研究可替代自体血管或神经的人造支架。20世纪80年代,组织工程学科的建立为人造小口径血管和神经导管的开发提供了新的思路。通过在可降解的人造组织工程支架上种植相应细胞,有可能获得功能化的组织或器官,并最终替代受损组织。而支架的最终降解,也避免了取出支架时的二次手术对病人造成的额外伤害。组织工程支架的众多成型方法中,静电纺丝法有着最广泛的使用。该方法获得的超细纤维能够有效模拟生命体中的细胞外基质,有利于细胞的黏附和增殖。在血管组织工程与神经组织工程中,使用静电纺丝法制备的管状支架仍有待改进。其中,血管支架难以兼具良好的力学与生物学性能,而神经导管支架的成型方法始终存有缺陷。为了解决这些问题,本文通过材料选择、纤维制备、支架成型、性能表征等方法,在制备出不同结构的管状支架并对这些支架进行系统研究的基础上,探讨其在血管与神经修复中的应用潜力。论文取得了一些具有重要学术价值和应用前景的研究成果。 1.血管支架的研究中,以获得天然蛋白-多糖为主要组成材料的血管支架为主要目的。蛋白-多糖静电纺纤维能够从材料组成和结构两方面模拟人体细胞外基质,以达到更好的组织修复效果。在制备管状纤维支架之前,先通过制备纤维膜进行性能表征,其中胶原蛋白与壳聚糖占比为75%(胶原蛋白／壳聚糖=4:1),并添加了25%的热塑性聚氨酯提高支架的力学性能。为使所获的共混纤维不溶于水溶液,使用戊二醛对其进行了交联,扫描电镜照片显示当交联时间为48小时的时候,纤维的形貌得到最佳保持。红外图谱证明,交联后的残留戊二醛可通过将样品长时间置于真空干燥箱中除去。生物相容性测试表明,内皮细胞和雪旺细胞在该类支架均能较快粘附和增殖。进一步对细胞生长形态进行观察,发现细胞的生长方向受纤维排列方向的影响较大。当纤维取向排列时,两种细胞均能够沿纤维排列的方向定向生长。基于此类共混纤维制备的管状支架具有较好的弹性,可满足体内血管对于力学性能的要求。 2.在获得了共混管状支架的基础上,对该成型方法加以改进,采用复合法制备出了蛋白-多糖-合成材料的管状支架。胶原蛋白和壳聚糖以4:1的比例共混作为支架的内、外层,而聚乳酸聚己内酯则被用作热塑性聚氦酯的替换材料,以期赋予支架更好的降解性能。该类复合支架的特点在于内外壁表面均为天然蛋白-多糖材料,避免了合成材料与细胞的直接接触,从而提高了支架的生物相容性。而中间的合成材料可用作加筋层,为支架提供了优良的力学性能。通过自制的体外动态细胞培养装置,培养内皮细胞7天后,将支架置于共聚焦显微镜下观察,可发现其表面被细胞铺满,形成了内皮细胞单层。考虑到血管结构的复杂性,除复合法外,又以管状支架中的纤维聚集形式为出发点,成功设计并制备出轴向取向、径向取向、层叠、梯度结构的管状纤维支架。这些支架各具特点,有望在不同方面满足血管支架的要求,其结构与成型方法可为下一代静电纺血管支架的发展提供重要借鉴。 3.神经导管的研究中,考虑到导管上的纤维若能沿导管的轴向方向排列,将有利于再生神经的生长,因此重点研究了轴向取向纤维导管支架的制备技术。从起初使用的卷绕-缝合法,到经过改进的滚轮法和转盘法,再到之后的旋转磁性导体棒-绝缘棒接收装置,成功制备出纤维沿支架轴向取向的管状支架。纤维受到电场、磁场以及拓扑结构的共同作用,定向沉积在绝缘棒上,令支架呈现出轴向取向的结构,而接收装置的旋转保证了管状支架壁厚的均匀性。此外,在静电纺丝开始前,对绝缘棒表面进行覆糖,待到纺丝结束后用水将糖溶去,可方便支架的取出。为验证该方法的普适性,使用多种聚合物进行静电纺丝,并通过扫描电镜和快速傅里叶转变法表征所获管状支架上纤维的取向度,发现不同材料的纤维均能够沿着支架的轴向方向取向排列,且在支架内壁表面的纤维具有最高的取向度。 4.获得轴向取向管状支架后,又对纤维自身的结构进行优化,目的在于使纤维本身就能够具有轴向取向的平行沟槽,将“轴向取向”这一特点增强。选择醋酸丁酸纤维素作为聚合物溶质,溶于二甲基乙酰胺和丙酮的混合溶剂后静电纺丝,成功制备出带有“轴向取向沟槽”结构的纤维。纤维制备完毕后,着重研究了该结构的成型机理。通过改变纺丝参数、使用不同分子量的聚合物材料、比较溶剂溶质性能、研究纺丝液挥发过程等方法,发现该轴向取向沟槽结构主要是由静电纺丝过程中,具有高挥发速度的溶剂迅速挥发后,所引起的相分离现象形成。聚合物流体暴露在空气中,在溶剂富集区形成了孔洞,而这些孔洞在电场力作用下被进一步拉伸、细化成沟槽结构。整个成型过程中,聚合物流体的粘度对于纤维形貌的影响起到了举足轻重的作用,而粘度又受到溶液浓度、聚合物分子量的直接影响。基于这些认识,使用相同方法对多种聚合物进行静电纺丝,并观察所获纤维的形貌,使这一成型机理得到了进一步的验证。 5.分别使用偏振红外、X射线衍射和力学拉伸等手段对纤维的理化性能进行了表征,并分别在光滑醋酸丁酸纤维素纤维和取向沟槽纤维上种植大鼠雪旺细胞,评价其体外生物相容性。研究发现,纤维表面带有的取向二级结构有利于雪旺细胞的黏附和增殖,尤其是早期黏附,细胞在培养8小时后呈现出典型的雪旺细胞“双极”形态。7天之后再观察,发现在取向沟槽纤维支架上,细胞的取向生长情况与增殖数量均优于光滑纤维。比较体外生物性能后,以这两种纤维构建神经导管支架,管外层使用了静电纺聚乳酸聚羟基乙酸纤维作为力学增强。将导管支架植入大鼠坐骨神经处,修复一段15毫米的神经缺损。术后12周剖开导管,观察神经再生情况,可发现再生神经的形成。电生理检查、髓鞘检查、腓肠肌检查和大鼠行走足印等结果显示,取向沟槽纤维构成的轴向取向管状支架可以进一步促进再生神经的生长。虽然其修复效果与自体神经相比仍有一定距离,但比光滑纤维支架有了明显提高。这些结果说明,具有“轴向取向沟槽结构”的纤维有利于神经愈合,在构建神经导管支架领域具有较好的应用潜力。
[Abstract]:Cardiovascular disease is considered to be an important cause of human death, which accounts for 30% of the total number of deaths in the world. According to statistics, there are more than 140 thousand cases of surgical vascular implantation in the United States each year, and most patients are older than 50. Only an unimportant vein of other parts of the patient's body is taken to replace the sclerotic coronary artery. This "two operation" is undoubtedly another pain to the patient. Similar to the vascular disease, peripheral nerve injury is a common clinical disease, mainly caused by the trauma caused by the accident. Only about 10 thousand and 1 in the United States. Thousands of people were paralyzed by nerve damage, resulting in more than $7 billion in economic loss and more than 50 thousand cases of peripheral nerve surgery. These repair of nerve breaks and defects have also used a relatively unimportant nerve to replace the other parts of the patient. It is an expedient measure.
In view of this, researchers have been working on the study of artificial scaffolds that can replace autologous blood vessels or nerves in the 80s.20 century. The establishment of the tissue engineering discipline provided a new idea for the development of artificial small caliber vessels and nerve conduits. It is possible to gain work by planting the corresponding cells on a biodegradable scaffold. An energetic tissue or organ, which eventually substitutes for the damaged tissue, and the final degradation of the scaffold avoids the additional damage to the patient from the two operation when the stent is removed.
The electrostatic spinning method is the most widely used method in many molding methods for the tissue engineering scaffold. The ultrafine fiber obtained by this method can effectively simulate the extracellular matrix in the life body, which is beneficial to the cell adhesion and proliferation. In the vascular tissue engineering and the neural tissue engineering, the tubular scaffold prepared by the electrostatic spinning method still needs to be improved. Among them, vascular stents are difficult to have good mechanical and biological properties, and the forming methods of the nerve conduit stents have always been defective. In order to solve these problems, this paper makes a systematic study of these scaffolds by means of material selection, fiber preparation, scaffolding molding and performance characterization. On the basis of this, we discussed its potential application in vascular and nerve repair. Some research results with important academic value and application prospect were obtained.
In the study of 1. vascular stents, the main purpose of the vascular scaffold is to obtain the natural protein polysaccharide as the main material. The protein polysaccharide electrostatic spun fiber can simulate the human extracellular matrix from two aspects of material composition and structure, so as to achieve better tissue repair effect. The fibrous membrane is prepared first by the preparation of the tubular fibrous scaffold. The ratio of collagen to chitosan was 75% (collagen / chitosan =4:1), and 25% of the thermoplastic polyurethane was added to improve the mechanical properties of the scaffold. The infrared map showed that the residual glutaraldehyde after cross linking could be removed in a vacuum drying box for a long time. Biocompatibility test showed that endothelial cells and Schwann cells were able to adhere and proliferate faster in the scaffold. The influence of the rectangle on the direction of fiber arrangement is great. When the fiber orientation is arranged, the two kinds of cells can grow along the direction of fiber arrangement. The tubular scaffold based on this kind of blend fiber has good elasticity, which can meet the requirements of the mechanical properties of the blood vessels in the body.
2. on the basis of obtaining the blend tubular scaffold, the molding method was improved. The tubular scaffold of protein polysaccharide synthetic material was prepared by the compound method. The collagen and chitosan were blended with 4:1 as the inner and outer layer of the scaffold. And polylactic acid polyhexyl ester was used as a replacement material for thermoplastic polyhelium. The scaffold has better degradation performance. The characteristic of this kind of composite scaffold is that the surface of the internal and external wall is natural protein - polysaccharide, which avoids the direct contact between the synthetic material and the cell, thus improving the biocompatibility of the scaffold. The intermediate synthetic material can be used as the stiffened layer and provides the excellent mechanical properties. Through the self-made body In the dynamic cell culture device, the endothelial cells were cultured for 7 days, and the scaffolds were observed under confocal microscopy. The surface of the cells was found to be covered with cells and formed a monolayer of endothelial cells. Considering the complexity of the vascular structure, the fiber aggregation in the tubular scaffold was the starting point except for the complex method. Tubular fiber scaffolds with radial orientation, cascading and gradient structure. These scaffolds have various characteristics and are expected to meet the requirements of vascular scaffolds in different aspects. The structure and molding methods can provide important reference for the development of the next generation of electrostatic spun stent.
In the study of 3. nerve conduits, considering that the fibers on the catheter were aligned along the axial direction of the catheter, it would be beneficial to the growth of the regenerative nerve. Therefore, the preparation techniques of the axially oriented fibrous stent were focused on. A body rod insulation rod receiving device has successfully prepared a tubular support with the axial orientation of the fiber support. The fiber is subjected to the joint action of electric field, magnetic field and topological structure. The fiber is directed to the insulation rod, making the bracket present an axial orientation structure, and the rotation of the receiving device ensures the uniformity of the wall thickness of the tubular support. In addition, the electrostatic spinning is used. In order to verify the universality of the method, a variety of polymers were used for electrostatic spinning to verify the universality of the method, and the orientation degree of the fibers on the tubular scaffold was characterized by scanning electron microscopy and rapid Fu Liye transformation, and the fiber of different materials was found. All dimensions are aligned along the axial direction of the support, and the fibers on the inner surface of the support have the highest orientation.
4. after the axial orientation tubular scaffold is obtained, the structure of the fiber is optimized. The purpose is to make the fiber itself have a parallel groove in the axial direction, and enhance the characteristic of "axial orientation". The fiber with "axial orientation groove" structure was successfully prepared. After the preparation of the fiber, the forming mechanism of the structure was emphatically studied. By changing the spinning parameters, using the polymer materials with different molecular weights, comparing the solute properties of the solvent and studying the volatilization process of the spinning solution, it was found that the axial orientation groove structure was mainly composed of static state. In the process of electrospinning, the phase separation phenomenon is caused by the rapid volatilization of the solvent with high volatilization. The polymer fluid is exposed to the air and formed holes in the solvent enrichment area. These holes are further stretched under the electric field force to refine the groove structure. In the whole process, the viscosity of the polymer fluid is to the fiber. The influence of the dimensional morphology plays an important role, and the viscosity is directly influenced by the concentration of the solution and the molecular weight of the polymer. Based on these knowledge, the same method is used to electrospun a variety of polymers, and the morphology of the obtained fibers is observed, so that the forming mechanism is further verified.
5. the physical and chemical properties of the fiber were characterized by polarizing infrared, X ray diffraction and mechanical stretching respectively. The rat Schwann cells were planted on the smooth cellulose acetate cellulose fiber and the orientation grooves fiber respectively, and the biocompatibility in vitro was evaluated. The study found that the orientation two structure with the surface of the fibrous surface was beneficial to Schwann fine. Cell adhesion and proliferation, especially early adhesion, showed a typical Schwann cell "bipolar" form.7 days after 8 hours of culture. It was found that the orientation growth and proliferation of the cells were superior to smooth fibers on the orientation grooved fiber scaffold. After comparing the biological properties of the cells in vitro, the two fibers were used to construct the nerve. The catheter stent was used as a mechanical enhancement by electrostatically spun poly (glycolic glycolic acid) fibers. The catheter stent was implanted into the rat sciatic nerve to repair a segment of the nerve defect of 15 millimeters. After 12 weeks, the catheter was opened to observe the regeneration of the nerve. Electrophysiological examination, myelin examination, gastrocnemius examination, and large amount of nerve were found. The results showed that the axially oriented tubular scaffold made of orientation grooves could further promote the growth of the regenerative nerve. Although the repair effect was still a certain distance compared with the autologous nerve, it was significantly better than the smooth fiber scaffold. These results suggest that the fiber with the "axial orientation groove structure" is beneficial. In nerve healing, it has a good potential in the field of constructing nerve conduit scaffolds.
【学位授予单位】：东华大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.11

【引证文献】