周围神经支架材料的制备及其修复神经缺损的实验研究

发布时间：2018-05-03 22:01

本文选题：复合支架 + 神经　；参考：《重庆医科大学》2015年博士论文

【摘要】：周围神经缺损的修复与功能重建,一直是外科领域致力解决的难题之一。周围神经缺损,会导致肢体严重的感觉、运动障碍,致残率极高,给患者、家庭及社会带来沉重的经济负和精神上的痛苦,因此,需找到最佳的神经修复材料迫在眉睫。自体神经移植到目前仍是公认的神经缺损修复的金标准,但存在供体神经支配区永新的久性失神经功能障碍,且神经供体来源有限。多年来,无数学者致力于此,分别尝试了多种生物材料(如静脉、肌肉,去细胞神经等)、人工合成材料(如硅胶管、聚羟乙酸等)材料,虽取得不少成就,但仍难以突破现实临床应用的瓶颈,近年来,同种神经异种在神经缺损修复过程中取了得了较满意的效果,但同种供体来源同样有限,且成本费用极高,临床应用受限,这促使我们努力寻找到广阔的修复材料来源,为周围神经缺损提供更为有效的修复手段。该论文设计了人工合成神经支架和异种去细胞组织工程神经支架修复神经缺损,希望能为周围神经缺损的修复提供新的思路。实验分为以下两个部分：1、将二氧化硅纳米颗粒掺入到胶原蛋白溶液中,构建不同浓度的多孔状胶原状蛋白结构,寻找最适合周围神经再生所需的最理想支架。我们对制备支架的各项生物学特性进行了详细研究,如形态,化学成分,润湿性,孔隙率,支架膨胀率和降解性。支架内进行雪旺细胞的培养,用于评价胶原/二氧化硅复合材料对神经再生的生物活性的影响,并对支架内雪旺细胞DNA含量进行测定。我们成功将二氧化硅纳米颗粒掺入胶原构成支架,二氧化硅纳米颗粒的掺入可以提高支架疏水性,降低孔隙率、膨胀率和降解速率。此外,雪旺细胞在多孔的支架内比单纯胶原更容易贴附、增殖。复合支架内的细胞数量和DNA含量随着纳米颗粒浓度的增加而先增加后降低。与其它配比组相比,25微克/毫升的二氧化硅浓度适合细胞的贴附和增殖,其支架内DNA含量最高。这些结果表明,掺入二氧化硅纳米颗粒的多孔胶原支架有可能用作植入支架材料,促进周围神经的修复与再生。2、用化学去细胞方法萃取异种神经支架AXN,体外培养大鼠BMSCs,与AXN构建组织工程化神经；雌性Vistar大鼠60只,建立右侧坐骨神经10mm缺损修复模型,随机分成3组(20只/组),A组：骨髓基质干细胞与异种去细胞神经支架复合构建组织工程神经桥接坐骨神经缺损；B组：单纯异种去细胞神经支架桥接坐骨神经缺损；C组：自体神经移植修复坐骨神经缺损组。分别于术后4周、12周进行干细胞的转归、神经移植免疫学检测、神经电生理检测、再生神经组织学观察、扫描电镜观察和患肢小腿三头肌肌纤维横径测量等方法评判坐骨神经功能恢复情况。该课题成功获取了大鼠BMSCs,并用BrdU标记,免疫组织化学染色和流式细胞仪检测培养的BMSCs表达CD44(+)、CD90(+)和CD34(-),BrdU具有较好的初始标记率,其可达88.36%。去细胞神经与新鲜神经相比较细胞和髓鞘被彻底清除,保持了原有的三维仿生结构,免疫原成份被清除,流式细胞仪对去细胞神经支架组织MHC Ⅱ的检测结果表明异种神经经化学去细胞处理后免疫原性明显减弱,可供神经移植使用。3.移植术后4周,流式细胞仪检测外周血CD3+、CD4+和CD8+T细胞数量A、B、C组间均无统计学差异,取移植段切片荧光显微镜下可见再生纤维束状排列,其间散在有BrdU标记的细胞核,S-100免疫组化染色检测BrdU标记细胞S-100蛋白表达阳性；术后12周通过神经电生理肌电图检测显示,电刺激可通过移植的神经支架到达远端的效应感受器,记录该神经支配的肌肉所产生的运动诱发电位,通过移植神经段的传导速度B组动物较A组动物和C组动物略慢,A组和C组无统计学差异。神经再生组织学检查显示有再生的神经纤维通过移植段神经,移植段内有纵行排列分布的SCs。A组、C组组织形态学、电生理检测及小腿三头肌肌纤维横径指标均优于B组,A组与C组无显著性差异。
[Abstract]:The repair and function reconstruction of peripheral nerve defect has always been one of the difficult problems to be solved in the field of surgery. The peripheral nerve defect can cause severe feeling, dyskinesia, high disability rate, heavy economic negative and mental pain for the patients, family and society. Therefore, it is urgent to find the best material for nerve repair. Autologous nerve transplantation is still a recognized gold standard for repair of nerve defects, but there is a permanent denervation dysfunction in the donor nerve area of Yongxin, and the source of the nerve donor is limited. Many scholars have tried this for years, and have tried a variety of biomaterials (such as static veins, muscles, cell nerves, etc.), and artificial synthetic materials (such as silicon, such as silicon) Although a lot of achievements have been made in rubber tube and polyoacetic acid, it is still difficult to break through the bottleneck of practical clinical application. In recent years, the same kind of nerve xenograft has taken a satisfactory effect in the process of nerve defect repair, but the source of the same donor is also limited, and the cost and cost are very high and the clinical application is limited. The restorative materials provide more effective repair methods for peripheral nerve defects. This paper designs artificial synthetic nerve scaffolds and xenoacellular tissue engineering nerve scaffolds to repair nerve defects, and hopes to provide new ideas for the repair of peripheral nerve defects. The experiment is divided into two parts: 1, silica nanoparticles The particles were added into the collagen solution to construct a porous protein structure with different concentrations to find the most ideal scaffold for the regeneration of peripheral nerves. We studied the biological properties of the scaffolds in detail, such as morphology, chemical composition, wettability, porosity, stent expansion rate and degradation. The culture of Schwann cells was used to evaluate the effect of collagen / silica composite on the biological activity of nerve regeneration and to determine the DNA content of Schwann cells in the scaffold. We successfully mixed silica nanoparticles into the scaffolds. The incorporation of silica nanoparticles can improve the hydrophobicity of the scaffolds, reduce the porosity, and expand. In addition, Schwann cells are more easily attached and proliferate in porous scaffolds than pure collagen. The number of cells and DNA content in the composite scaffold increase first and then decrease with the increase of the concentration of nanoparticles. Compared with the other matching groups, the concentration of 25 microgram / ml of two silicon oxide is suitable for cell attachment and proliferation, and its scaffold The content of internal DNA is the highest. These results suggest that the porous collagen scaffold doped with silica nanoparticles may be used as a scaffold material to promote the repair and regeneration of peripheral nerve,.2, AXN, BMSCs in vitro, and tissue engineering nerve in vitro, and 60 female Vistar rats. The right sciatic nerve 10mm defect repair model was established and divided randomly into 3 groups (20 rats / groups). Group A: bone marrow stromal cells and xenogeneic nerve scaffolds to construct tissue engineering nerve bridging sciatic nerve defect; group B: simple xenoacellular nerve scaffold bridged sciatic deity defect; group C: autologous nerve graft for the repair of sciatic deity After 4 weeks and 12 weeks, the changes of stem cells, neural transplantation immunology, neurophysiological test, regenerative nerve histology, scanning electron microscopy and transverse diameter measurement of triceps muscle fiber were used to evaluate the recovery of sciatic nerve function. The BMSCs of rats was successfully obtained, and the BrdU standard was used. BMSCs expressed CD44 (+), CD90 (+) and CD34 (-), and BrdU had better initial labeling rate, which could reach 88.36%. to cell nerve and fresh nerve. The cells and myelin sheath were thoroughly removed, and the original three-dimensional biomimetic structure was maintained. The immunogen components were cleared and flow cytometry was used. The results of the detection of MHC II in the cellular nerve scaffold showed that the immunogenicity of the xenogeneic nerve was obviously weakened after the chemical removal of the cell. 4 weeks after the transplantation of.3., the flow cytometry was used to detect the number of CD3+, CD4+ and CD8+T cells in the peripheral blood, A, B, C, and the fluorescence microscope of the transplantation section was seen under the fluorescence microscope. The regenerated fibers were arranged in a fascicular arrangement, scattered in the nucleus with BrdU markers, and S-100 immunohistochemical staining was used to detect the positive expression of S-100 protein in the BrdU labeled cells. Electroelectromyography detected by the electrophysiological electromyography at 12 weeks after the operation showed that the electrical stimulation could reach the distal effector through the transplantation of the nerve scaffold to record the muscle of the innervated muscle. The motor evoked potential of the B group was slightly slower than that of the A group and the C group through the conduction velocity of the transplanted nerve segment. There was no statistical difference between the group A and the C group. The regeneration of the nerve regeneration histology showed that the regenerated nerve fibers passed the segmental nerve, the SCs.A group was arranged in a longitudinal arrangement, the morphology of the C group, the electrophysiological test and the calf. The transverse diameter index of triceps muscle fiber was better than that of group B, and there was no significant difference between group A and group C.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08;R651

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