高仿真组织工程神经修复材料修复比格犬长节段坐骨神经缺损的应用研究

发布时间：2018-05-17 09:12

本文选题：比格犬 + 周围神经损伤　；参考：《第四军医大学》2013年博士论文

【摘要】：周围神经损伤、再生及其功能修复是世界性临床医学难题。尤其是在临床工作中对于短节段（30mm）神经损伤，手术能够直接将损伤的神经对端吻合，使近端再生的神经纤维能够朝向远端发育生长。但是特别对于长节段（30mm）的神经缺损，临床上不能进行无张力直接缝合，目前的国际上的治疗金标准是将未损伤的非重要区域的自体神经移植到受损伤区、来修复桥接损伤的神经。但是，临床上手术应用自体神经移植受到各种条件和因素限制：主要包括供体区必需进行二次手术，但是供区神经又存在来源不足，医源性造成继发的供区神经功能丧失，以及供区神经在组织的结构和尺寸的大小上与需修补神经匹配等问题。综上所述，必须有效研发能够有效代替自体神经的移植替代物，同时这是目前迫切需要解决的难题。近些年来，组织工程发展速度十分迅猛，制备组织工程周围神经早已经成为新的研究热点。运用组织工程学方法制备组织工程移植物修复桥接长节段神经缺损收获得越来越多的关注。组织工程移植物一般由三部分构成：支架材料、种子细胞和相应活性因子。其中支架材料作为种子细胞和相关神经营养因子的载体，是组成神经损伤修复微环境的主要结构，其结构构建和性能改进是制约周围神经缺损修复的关键问题，其组成结合及内部微结构是影响组织工程移植物修复长节段神经损伤的关键因素。最新的研究表明，内部结构具有定向微结构的支架材料，在修复桥接神经缺损时比无定向结构的支架更加适于引导再生的神经轴突定向生长，从而促进再生神经纤维通过损伤区到达远端，进一步验证了支架内部结构的物理引导作用对于神经再生过程中的重要性，分析可能原因是因其内部定向结构模拟了正常神经基底膜的轴向微管结构。但是文献报道的支架材料在原料组成及内部结构上，与神经神经基底膜的组成和结构还有较大差距，需要进一步研究和改进。鉴于大量实验证实神经基底膜微管结构引导再生神经轴突定向生长的关键作用，如果能够依据神经组织的成分组成和结构来选择和制备组织工程支架，，使该支架具备一定的类似神经组织的特性，有可能会获得理想的移植替代修复结果。然而，截止目前为止，国内外仅有少数具有类似结构的支架研究被报道，而应用其在实验动物体内长节段周围神经缺损至今未见研究报道。在本实验中，选用神经基底膜基质的主要材料——胶原-壳聚糖做为神经支架的主要原料，应用本实验组改良并获得国家专利的梯度冷冻干燥技术，制备具有轴向微管结构的三维多孔神经支架材料，该支架在组成及内部结构方面高度模拟神经基底膜。并从原料配比、冰醋酸浓度以及冷淋速度等三个方面对改良的梯度冷冻干燥技术工艺进行横向对比研究，筛选出最佳的制备工艺指标。同时为了使支架能够满足体内移植的需求，应用新型低细胞毒性交联剂——京尼平进行化学交联，改善其机械强度和降解速度，并确定其最佳交联参数。同时进行相关生物力学测试，证实其具有良好的生物力学，适于体内移植修复。最后，应用免疫组织学、透射电镜、神经电生理、逆行示踪技术等方法，从形态学和功能学两方面综合评价支架修复桥接格犬坐骨神经30mm缺损效果，结果证实其修补缺损神经效果接近于自体神经移植。相关具体内容如下：第一部分神经支架的制备工艺参数确定目的：制备并构建组成、结构高度仿真的组织工程神经支架。方法：以胶原-壳聚糖为原料，应用改良的梯度冷冻干燥技术制备仿真支架，扫描电镜观察其结构，评测支架孔径、孔隙率等基本性能，优化其制备参数。结果：本实验中同时采用不同冷淋速度方法来制备各种孔径大小和结构的支架材料。选择速度为2×10~(-5)m/s的冷凝速度时，支架材料内部的微管直径逐渐增大，效果达到最佳，平均孔径为37.34±13.24μm，其内部微管呈轴向平行规律排列。在综合考虑仿生神经支架内部结构和孔径的前提条件下，证实冰醋酸浓度为3mg/ml，冷淋速度为2×10~(-5)m/s为制备最佳参数。同时进一步确定以胶原：壳聚糖（C：CH）=3：1制备的神经支架材料具有最佳的三维仿生结构和良好的性能，孔径在24μm～102μm之间，平均孔径为49.85±19.85μm；孔隙率90%以上，上述结构在神经再生过程中，可以起到瘢痕屏障的保护作用，能够在不影响神经营养物质互相交通的情况下，有效阻止瘢痕纤维的长入，保护再生纤维的顺利向远端通过。同时还能够满足理想支架对孔径的要求：小到能够物引导再生神经轴突的定向生长；大到能够支持足够有效的血管化及相关再生支持细胞的不断渗入。第二部分支架材料改性及生物力学评测目的：通过改进支架的机械性能及生物降解性，达到适应体内移植需求，并进行生物力性评价为体内移植提供实验依据和基础。方法：选用低生物毒性交联剂——京尼平进行化学交联改性，通过测定交联率、机械拉力、降解率等指标检验交联参数对CCH支架性能的影响。结果：应用低生物毒性交联剂——京尼平（1wt%）交联48h，结果显示：未交联的支架材料在纯PBS液中共孵育8w后重量减少29.6±4.8%。而经过Genipin交联的支架材料在相同条件下重量仅减少原来的17.9±4.2%，重量丢失明显少于未交联组。在溶菌酶溶液组中，未交联的支架重量减少36.3±5.2%，明显多于交联组支架的20.1±4.6%。未交联和经Genipin交联后的CCH支架分别在干燥、湿润的状态下进行拉力实验，结果显示：干燥组的拉伸应力均高于湿润组，而相应的拉伸应变则为干燥状态下小于湿的状态，证明交联处理能够改善支架材料的生物力学性能，同时使支架材料在神经轴突修复再生过程中能够保持稳定的内部结构，有效的配合神经轴突的再生。第三部分支架材料修复比格犬坐骨神经缺损有效性评价目的：评价支架材料修复长阶段神经缺损的有效性。方法：应用免疫组织学、透射电镜、神经电生理、逆行示踪技术等方法，从形态学和功能学两方面综合评价支架材料桥接比格犬坐骨神经30mm的修复效果。结果：术后12w在支架材料组,尽管新生髓鞘比较纤细，但外形结构完整，在髓鞘周围可见基底膜完整的雪旺细胞，同时还可以观察到完整的再生血管结构和良好排列的髓鞘板层结构，修复效果接近自体神经移植组。同时神经电生理、逆行示踪等方法，从功能学的不同侧面综合支持材料桥接神经损伤的修复效果。在术后24w支架材料组的运动神经传导速度、潜伏期和波幅与自体神经移植结果接近，两组之间差异无显著性意义；逆行示踪标记后，在脊髓前角和背根神经节可检测到与自体神经移植组数量相当的荧光金标记阳性神经元，胶原-壳聚糖支架桥接的坐骨神经缺损功能性修复效果接近自体神经移植。
[Abstract]:Peripheral nerve injury, regeneration and functional repair are the world's clinical medical problems. Especially in clinical work, the operation can directly anastomosed the injured nerve to the injured nerve to the distal segment (30mm) nerve injury, so that the nerve fibers regenerated near the end can grow toward the distal end, but especially for the long segment (30mm) nerve defect. There is no tension free direct suture on the bed. The current international standard of treatment is to transplant autologous nerves from undamaged areas to the damaged area to repair the injured nerve. However, the application of autologous nerve graft in clinical surgery is limited by various conditions and factors: two times, mainly including the donor area, are required. The operation, but the source of the donor nerve has a shortage of sources, iatrogenic loss of the donor nerve function secondary to the donor area, and the matching of nerve in the structure and size of the donor nerve in the tissue and size. In summary, it is necessary to develop an effective substitute for the autologous transplanting substitutes. At the same time, it is urgently needed. In recent years, the development of tissue engineering has been developing rapidly, and the preparation of tissue engineering peripheral nerve has become a new research hotspot. Tissue engineering method is used to prepare tissue engineering graft for repairing long segmental nerve defects. Scaffold materials, seed cells and corresponding active factors, in which scaffold material is the carrier of seed cells and related neurotrophic factors, is the main structure to form neural damage repair microenvironment. Its structure construction and performance improvement are the key problems that restrict the repair of peripheral nerve defect. Its composition and internal microstructure are the influence of tissue workers. The latest research shows that the internal structure has a directional microstructural scaffold material, which is more suitable for guiding the regeneration of the axon to direct the growth of the axon in repairing the bridged nerve defect than the non directional scaffold, thus promoting the regeneration of the regenerated nerve fibers to the distal end through the damaged area. The importance of the physical guidance of the internal structure of the stent to the process of nerve regeneration is verified. The possible reason is that the axial microtubule structure of the normal nerve basement membrane is simulated because of its internal orienting structure. However, the composition and structure of the scaffold material in the material composition and internal structure of the material and the nerve nerve basement membrane are reported in the literature. There is still a big gap, and further research and improvement are needed. In view of the fact that a large number of experiments have proved the key role of the nerve basement membrane microtubule structure to guide the directional growth of the axon, it is possible to select and prepare a tissue engineering scaffold based on the composition and structure of the nerve tissue, so that the scaffold has some characteristics similar to the nerve tissue. However, only a small number of scaffolding studies with similar structures have been reported at home and abroad, and there have been no reports on the application of it to the long segment of peripheral nerve defects in experimental animals.
In this experiment, the main material of the nerve basement membrane matrix, collagen chitosan, was used as the main raw material of the nerve scaffold. The experimental group was used to improve and obtain the national patent gradient freeze drying technology to prepare the three-dimensional porous nerve scaffold with the axial microtubule structure. The scaffold was high mould in the composition and internal structure. The modified gradient freeze-drying technology was compared in three aspects, such as the ratio of raw materials, glacial acetic acid concentration and the speed of cold drenching. In order to meet the needs of the body transplantation, a new type of low cytotoxic crosslinking agent, geniping, should be used to make the stent meet the needs of the body transplantation. Chemical crosslinking was performed to improve the mechanical strength and degradation rate, and to determine the best cross-linking parameters. At the same time, the biomechanical tests were carried out to prove that it had good biomechanics and suitable for the transplantation in vivo. Finally, the methods of immunohistochemistry, transmission electron microscopy, neuroelectrophysiology, retrograde tracing technique were used in the two party of morphology and function. The results showed that the repair of the bridged canine sciatic nerve 30mm defect was close to that of the autologous nerve graft.
Part 1 Determination of preparation parameters of neural scaffolds
Objective: to prepare and construct tissue-engineered neural scaffolds with high degree of organization and structure.
Methods: using the collagen chitosan as the raw material, the modified gradient freeze drying technology was used to prepare the simulation scaffold. The structure was observed by scanning electron microscope, and the basic properties of the scaffold diameter and porosity were evaluated, and the parameters of the preparation were optimized.
Results: in this experiment, different cooling rate methods were used to prepare the scaffold materials of various sizes and structures. The microtubule diameter of the stent material increased gradually when the velocity of 2 * 10~ (-5) m/s was selected. The average diameter was 37.34 + 13.24 mu m, and the internal microtubule was arranged in the axial parallel law. Under the condition of the internal structure and aperture of the bionic nerve scaffold, it was confirmed that the concentration of glacial acetic acid was 3mg/ml and the cooling rate was 2 * 10~ (-5) m/s as the best parameter. At the same time, it was further determined that the scaffold materials prepared with collagen: C:CH =3:1 had the best three-dimensional bionic structure and good performance, and the pore size was 24 mu. Between M and 102 mu, the average pore size is 49.85 + 19.85 mu m and the porosity is more than 90%. In the process of nerve regeneration, the structure can play a protective role in the scar barrier. It can effectively prevent the long entry of scar fibers and protect the regenerated fibers from the distal end without affecting the mutual traffic of the nerve nutrients. The requirements for the aperture of the ideal scaffold are satisfied: small enough to guide the directional growth of the regeneration of the axon; large enough to support sufficient and effective vascularization and the continuous infiltration of the associated regenerative support cells.
The second part is the modification and biomechanical evaluation of scaffold materials.
Objective: to improve the mechanical performance and biodegradability of the scaffold to meet the needs of transplantation in vivo, and to provide the basis and basis for the evaluation of biological force for the body transplantation.
Methods: a low biological toxic crosslinking agent, genipine, was modified by chemical crosslinking. The effects of crosslinking parameters on the performance of CCH scaffold were tested by measuring the cross-linking rate, mechanical pull and degradation rate.
Results: using a low biotoxic crosslinking agent, genipine (1wt%) crosslinked 48h, the results showed that the weight of the stents without cross linking was reduced by 29.6 + 4.8%. in the pure PBS solution for 8W and the weight of the scaffold crosslinked by Genipin only decreased by 17.9 + 4.2% in the same condition, and the weight loss was obviously less than that of the non crosslinked group. In the enzyme solution group, the weight of the non crosslinked scaffold decreased by 36.3 + 5.2%, obviously more than 20.1 + 4.6%. of the crosslinked scaffold and the CCH scaffold after Genipin crosslinking, and the tensile tests were carried out in the dry and wet state respectively. The results showed that the tensile stress of the drying group was higher than that of the wet group, while the corresponding tensile strain was in the dry state. It is proved that the cross-linking treatment can improve the biomechanical properties of the scaffold material and can maintain the stable internal structure during the repair and regeneration of the axon, and effectively cooperate with the regeneration of the axon.
Third parts of scaffold materials for repairing beagle dogs' sciatic nerve defects
Objective: To evaluate the effectiveness of scaffold materials in repairing long-term nerve defects.
Methods: with the methods of immunohistochemistry, transmission electron microscopy, neuroelectrophysiology, and retrograde tracing technique, the effect of the scaffold material bridging the 30mm of the sciatic nerve of beagle dogs was evaluated from two aspects of morphology and function.
Results: after the operation, 12W in the scaffold material group, although the newborn myelin sheath was thin, but the shape and structure was complete, the intact Schwann cells in the basement membrane were visible around the myelin sheath, and the complete regenerative vascular structure and the well arranged myelin lamellar structure were also observed. The repair effect was close to the autologous nerve transplantation group. In the 24W stent material group, the motor nerve conduction velocity, the latency and amplitude were close to the results of autologous nerve transplantation, and there was no significant difference between the two groups. The retrograde tracer markers were in the anterior horn of the spinal cord and the dorsal root ganglion. The effect of collagen chitosan scaffold bridged on the functional repair of the sciatic nerve defect was close to that of the autologous nerve graft.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

【参考文献】