组织工程周围神经的构建及其应用基础研究

发布时间：2018-03-05 21:38

本文选题：壳聚糖　切入点：组织工程　出处：《山东大学》2012年硕士论文　论文类型：学位论文

【摘要】：本课题旨在研制组织工程周围神经,以解决长段神经损伤修复中供体神经来源缺乏问题。包括导管材料制备与表征、导管制作、神经生长因子的固定化、种子细胞的分离培养,并对所构建的组织工程周围神经进行了系统的功能评价,以明确各影响因素在坐骨神经损伤后修复过程中的作用。壳聚糖(chitosan, CS)是一种天然的聚阳离子多糖,具有无毒、生物可降解、不会引起排异反应等特点。但由于其脆性较高,不易加工,因而应用受到一些限制。聚乳酸(polylactide, PLA)具有优良的机械力学性能,对人体无毒、无刺激、可以被人体吸收,因而已被美国食品和药品管理局(Food and Drug Administration,FDA)批准用作药物控释载体及修复材料。但PLA存在亲水性及组织相容性差,降解产物会引起局部酸性炎症等不足。本课题采用接枝共聚法制备壳聚糖／聚乳酸的复合材料,以达到整合两者优点,克服两者不足的目的。静电自组装(electrostatic self-assembly, ESA)技术已成为一种日益引起广大生物材料工作者兴趣的新型制备方法,它是基于相反电荷电解质相互吸附的超薄膜形成技术。该技术操作简单、成膜稳定性好、不受基材形状和大小的限制。现己在生物材料改性、药物缓释、生物传感器制备等领域广泛应用。本课题采用静电自组装技术,将壳聚糖及肝素固定在5/0手术缝合线表面,制备神经引导丝。并通过1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDC)交联将神经生长因子(nerve growth factor, NGF)固定于神经引导丝及导管支架材料表面,以达到缓释NGF的作用。组织工程神经导管的种子细胞中雪旺细胞是最理想的,其作用包括分泌多种神经营养因子和细胞外基质,表达粘附分子,引导轴突再生等。但雪旺细胞本身是一种终末期细胞,体外培养的增殖能力差,很难达到移植所需的理想数量,且自体取材易造成新的损伤,故使其在临床使用中受到一定的限制。骨髓来源的干细胞是较为理想的雪旺细胞替代细胞。本课题采用骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)作为种子细胞。随着交通方式的改变及机械化大生产的发展,当前每年都有大量人员遭受周围神经损伤。显微外科手术仍是现阶段临床上广泛采用的治疗手段,对于5mm以上的神经损伤只能采用自体移植方法,但神经来源有限,且会造成新的损伤。因此研究者开始将着眼于采用组织工程技术修复长距离的神经损伤。该领域重点问题包括：①神经导管材料选择和表面形态修饰。②神经导管与神经营养因子的联合应用。③选择并添加合适的种子细胞。本课题的工作即在此领域展开,主要包括： 1.壳聚糖／聚乳酸复合材料的构建及性能研究采用接枝共聚法制备壳聚糖／聚乳酸复合材料,并利用固态核磁、单反射红外光谱、X射线衍射及扫描电子显微镜对其理化性质进行表征。测定壳聚糖氨基取代度、乳酸聚合度、孔隙率、溶胀性能、体内外降解速率、体外降解pH变化及细胞亲和性、组织相容性。结果表明,随乳酸与壳聚糖质量比的增大,复合材料孔隙率和溶胀性能逐渐减小,体外降解pH呈波动性变化,且随乳酸量的增大波动性逐渐增大,体内外降解质量减少随乳酸量的增大逐渐增大,在所设的三种比例下,复合材料的细胞亲和性和组织相容性都较好。 2.组织工程神经的构建 2.1壳聚糖／聚乳酸复合材料神经导管的制各采用自制模具,利用冷冻干燥及高温真空干燥方法制备复合材料导管支架。并对其力学性能及超微结构进行测定。结果表明,复合材料导管与壳聚糖导管相比可有效提高力学性能,且导管支架呈瓦片状,能够有效防止周围组织的长入,同时有利于损伤神经进行物质交换,能够为神经的再生提供良好的微环境。 2.2神经引导丝的静电自组装修饰采用可降解的聚乙醇酸5/0手术缝合线为载体,在其表面制备聚乳酸涂层,利用静电自组装技术在其表面制备壳聚糖／肝素多层膜结构,并通过红外光谱、X射线光电子能谱及扫描电子显微镜进行表征。结果表明,神经引导丝表面已形成壳聚糖／肝素多层膜。并且在复合神经导管后,可有效地增大导管的内表面积。 2.3NGF的交联固定采用EDC交联方式在神经引导丝及导管支架上固定NGF,以达到在神经修复期间缓释NGF的作用。结果表明,经释放前期其呈现突释,但随时间的延长其逐渐呈平稳释放；且由于表面积大的原因,神经导管固定NGF的量明显多于神经引导丝。 2.4BMSCs作为种子细胞的分离及荧光标记采用市售试剂盒,按照密度梯度离心法分离大鼠BMSCs与并与神经导管复合构建组织工程神经,应用于大鼠坐骨神经损伤修复,采用Hoechst33258对BMSCs进行标记,结合免疫组化方法检测其分化状态。结果表明在手术6w后BMSCs依然存活,并且有nestin的表达,表明其已经分化为类神经元细胞。 3.组织工程神经对大鼠坐骨神经损伤修复的效果观察及初步机制研究采用大鼠坐骨神经损伤模型,以神经电生理指数,坐骨神经功能指数(SFI),神经的纤维密度、直径及nestin免疫组化为考察指标,对比研究了空白组、造模组、自体移植组、组织工程神经组、导管复合NGF组、导管复合干细胞组、固定有NGF的引导丝组、静电自组装修饰后的引导丝组、吡咯喹啉醌组、复合材料各组、壳聚糖组、聚乳酸组修复坐骨神经损伤的效果,按效果从优到劣的排序是：自体移植组、组织工程神经组、NGF组、干细胞组、生长因子引导丝组、自组装引导丝组、吡咯喹啉醌组、复合材料-Ⅱ组、复合材料-Ⅰ组、复合材料-Ⅲ组、壳聚糖组、聚乳酸组、造模组。荧光标记和免疫组织化学检测表明,nestin在组织工程神经组及干细胞组均有表达,表明BMSCs向神经干细胞的方向进行了分化,推测这是所构建的组织工程神经具有良好修复作用的机制。本研究取得的主要研究成果有： 1.制备了壳聚糖／聚乳酸复合材料并对其理化性能,体内外降解性能,细胞亲和性及组织相容性进行评价,为制备适宜的组织工程材料提供依据。 2.利用静电自组装技术修饰神经引导丝,在其表面制备肝素／壳聚糖膜,并对其理化性质进行表征；利用EDC交联固定NGF,体外检测显示其可缓释NGF；利用模具制作壳聚糖／聚乳酸复合材料支架。研究表明,复合材料-Ⅱ组的力学性能和微观结构更有利于损伤神经的修复。 3.评价了各影响因素在大鼠周围神经损伤后修复中的效果。结果显示各组修复情况排序为：自体移植组、组织工程神经组、NGF组、干细胞组、生长因子引导丝组、自组装引导丝组、吡咯喹啉醌组、复合材料-Ⅱ组、复合材料-Ⅰ组、复合材料-Ⅲ组、壳聚糖组、聚乳酸组、造模组。结果表明,各影响因素的作用排序为：NGF、种子细胞、神经引导丝、吡咯喹啉醌。结果表明,通过接枝共聚法制备的壳聚糖/聚乳酸导管材料支架,在结合骨髓间充值干细胞及生长因子后,所制备组织工程周围神经对坐骨神经损伤具有明显的修复作用。
[Abstract]:This paper aims at the development of peripheral nerve tissue engineering, to solve the repair of long nerve injury in the donor nerve. The lack of sources including preparation and characterization, fabrication of catheter catheter materials, immobilized NGF, isolation and culture of seed cells, and to construct tissue-engineered peripheral nerves function evaluation system, in order to clear the influence factors in the process of repairing effect on sciatic nerve injury.
Chitosan (chitosan, CS) is a natural cationic polysaccharide, is non-toxic, biodegradable, will not cause the characteristics of rejection. But because of its high brittleness, easy processing, and the application of some restrictions. Polylactic acid (polylactide, PLA) has better mechanical properties, non-toxic to humans. No stimulation, can be absorbed by the body, which has been the United States Food and Drug Administration (Food and Drug Administration, FDA) for use as drug delivery carriers and repair materials. But PLA is hydrophilic and biocompatibility, degradation products can cause local inflammation. The acid was prepared by graft copolymerization the composite chitosan / poly lactic acid, in order to achieve the integration of the two advantages, to overcome the deficiencies of the two.
Electrostatic self-assembly (electrostatic self-assembly ESA) technology has become a new business has attracted considerable interest in biological material workers preparation method, it is the formation of thin film technology based on the mutual adsorption of oppositely charged electrolyte. This technology has the advantages of simple operation, good film-forming stability, is not affected by the shape and size of the substrate is in the limit. Instead, biological materials, drug delivery, wide application of biosensor and other fields. This paper adopts electrostatic self-assembly technology, chitosan and heparin immobilized on 5/0 suture surface, preparation of nerve guidance wire. And through 1- ethyl - (3- methyl propyl two amino carbonyl imine hydrochloride (two) (1- 3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, EDC) the crosslinking of nerve growth factor (nerve growth, factor, NGF) is fixed on the guide wire and catheter surface nerve scaffold, in order to achieve the sustained release effect of NGF.
Schwann cell seed cells in tissue engineering nerve conduit is the most ideal, including its role in secretion of various neurotrophic factors and extracellular matrix, the expression of adhesion molecules, and axon regeneration. But Schwann cell itself is a kind of end-stage cells, the proliferation ability is poor in vitro, it is difficult to achieve the desired number required for transplantation, and autografts are apt to cause new damage, so it is limited in clinical use. Bone marrow stem cells is an ideal alternative to Schwann cells cells. The bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) as seed cells.
With the development and change of mechanized production mode of transportation, the current year a large number of peripheral nerve injury. Microsurgery is widely used at this stage is still in clinical treatment, only for more than 5mm of nerve injury by autologous transplantation method, but the nerve is limited and will cause new damage. Therefore researchers began to focus on using tissue engineering techniques for repairing nerve injury in long distance. The main issues in the field include: nerve conduit materials selection and modification of surface morphology. In combination of nerve conduit with neurotrophic factor. The select and add suitable seed cells. This research work in this field, including:
Study on the construction and properties of 1. chitosan / polylactic acid composites
Preparation of chitosan / polylactic acid composite material prepared by graft copolymerization, and the use of solid-state NMR, single reflection infrared spectroscopy, X ray diffraction and scanning electron microscope on its physicochemical properties were characterized. The determination of chitosan amino substitution degree, degree of polymerization of lactic acid, porosity, swelling properties, in vitro degradation rate, degradation in vitro and pH changes cell affinity, tissue compatibility. The results show that with the increase of the quality of lactic acid and chitosan ratio, composite porosity and swelling properties decreased gradually, in vitro degradation of pH fluctuated, with the increase in volatility increases the amount of lactic acid, reduced with the increase of the quality of in vivo and in vitro degradation of lactic acid increased gradually, in three the proportion of the composite material, cell affinity and tissue compatibility are good.
2. construction of tissue engineering nerve
The production of 2.1 chitosan / polylactic acid composite nerve conduits
Using homemade mould for drying and high temperature vacuum drying method using frozen composite material conduit. And the mechanical properties and ultrastructure were measured. The results show that the composite chitosan catheter and catheter compared can effectively improve the mechanical properties, and the conduit a shingle, can effectively prevent the surrounding tissue ingrowth, at the same time to damage nerve material exchange, can provide a good microenvironment for nerve regeneration.
Self assembling decoration of 2.2 nerve guide wire
The use of biodegradable polyglycolic acid 5/0 suture as the carrier, on the surface of the preparation of polylactic acid coating, electrostatic self-assembly technique to prepare chitosan / heparin multilayer film structure on the surface, and through the infrared spectrum, X ray photoelectron spectroscopy and scanning electron microscopy were characterized. The results showed that the nerve guide wire the surface has formed chitosan / heparin multilayer film. And the composite nerve conduit, can effectively increase the tube surface area.
Crosslinking fixation of 2.3NGF
The EDC guide wire and catheter cross bracket is fixed in NGF to achieve the function of nerve, release NGF in nerve repair period. The results showed that the burst release was released early, but with time prolonging the gradually stable release; and because the table causes a large area, nerve conduit was significantly more than the fixed NGF nerve guide wire.
Isolation and fluorescence labeling of 2.4BMSCs as seed cells
Using commercially available kits, in accordance with the density gradient centrifugation of rat with BMSCs and composite nerve conduit in nerve tissue engineering, applied to the repair of sciatic nerve injury in rats, using Hoechst33258 to mark BMSCs, combined with the immunohistochemical method to detect the differentiation status. Results show that the BMSCs is still alive in 6W after surgery, and the expression of nestin, indicating that it has differentiated into neuron like cells.
The effect of 3. tissue engineering nerve on the repair of sciatic nerve injury in rats and its preliminary mechanism
The rat model of sciatic nerve injury, the nerve electrophysiological index, sciatic nerve function index (SFI), nerve fiber density, diameter and nestin immunohistochemistry as investigation index, comparative study of the blank group, model group, autograft group and nerve tissue engineering composite duct group, NGF group, catheter composite stem the cell group, fixed with NGF guide wire group, guide wire group electrostatic self-assembly of pyrroloquinoline quinone group, composite group, chitosan group, polylactic acid group and repair of sciatic nerve injury effect, according to the effect in descending order: autograft group, tissue engineering nerve group, NGF group, stem cell growth factor group, guide wire group, self assembling guide wire group, pyrroloquinoline quinone group, composite II group, composite material and composite material - I group, III group, chitosan group, poly lactic acid group, model group showed fluorescence labeling and immunohistochemical detection of nestin. It is expressed in tissue-engineered nerve and stem cell groups, indicating that BMSCs has differentiated into the direction of neural stem cells. It is presumed that this tissue engineering nerve has good repair effect.
The main achievements of this study are as follows:
1., chitosan / PLA composites were prepared, and their physicochemical properties, in vitro and in vivo degradability, cell affinity and histocompatibility were evaluated, providing a basis for preparing suitable tissue engineering materials.
2. using the electrostatic self-assembly technique modified microfilament, preparation of heparin / chitosan film on its surface, and physicochemical properties were characterized by EDC; cross-linked NGF, in vitro testing showed that the sustained-release NGF; making chitosan / polylactic acid composite scaffold using a mold. The research shows that the mechanical properties and the microstructure is more composite materials of group II to the repair of nerve injury.
3. evaluation of various factors in the repair of peripheral nerve injury in rats. The results showed the repair order: autograft group, tissue engineering nerve group, NGF group, stem cell growth factor group, guide wire group, self assembling guide wire group, pyrroloquinoline quinone group, composite II group composite materials, composite materials - - I group, III group, chitosan group, poly lactic acid group and model group. The results show that the influence of the order: NGF, seed cells, nerve guide wire, pyrroloquinoline quinone.
The results showed that the chitosan / polylactic acid catheter scaffolds prepared by graft copolymerization combined with bone marrow restores stem cells and growth factors, and the tissue-engineered peripheral nerve tissue had significant repair effect on sciatic nerve injury.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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