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。
The regeneration and functional recovery of Peripheral nerve injury are worldwide clinicalchallenges currently. Clinically, direct end-to-end nerve sutures are proposed as treatmentsfor short nerve gap lesions（<30mm）. The standard technique for long-lesion gaps（>30mm）is to transplant autologous nerve grafts (autografts) from uninjured sites to the injured site toform a bridge between the two nerve stumps. Nevertheless, the use of autografts has anumber of disadvantages and limitations, including limited graft availability, secondarydeformities, and potential differences in the tissue structures and sizes, etc. Accordingly, it isimperative to develop alternatives to the conventional nerve autografts. Tissue engineeringtechnology has developed rapidly in recent years and there has been an increasing focus onthe tissue engineering grafts since they produced promising results in bridging long nervegaps. Tissue engineering grafts are generally developed by combining biomaterial scaffolds,seed cells and biologically active molecules. The inner microstructural properties of thescaffold are the predominant factors that determine the efficacy of a tissue engineering graftin bridging nerve gap lesions. In recent years, a variety of scaffolds with either oriented or random inner structures have been developed to bridge nerve gaps. Orientedmicro-structured scaffolds have been more advantageous than scaffolds with a random innerstructure for guiding the linear growth of axons across nerve gaps, which indicates thatphysical guiding cues are essential for guiding axon regeneration. To date, several scaffoldswith oriented structures, such as fibers and grooves, have been successfully fabricated andhave been shown to be capable of physical guiding the linear growth of regenerated axons tosome extent. However, these oriented structures still differ substantially from the guidingbasal lamina micro-channels in nerve autografts, which are considered the gold standardtechnique for treating peripheral nerve lesions. Since the basal lamina micro-channels innormal nerves are known to play a significant guiding role in the linear growth ofregenerated axons, extracellular matrix (ECM) based scaffolds with dimensions resemblingthe basal lamina micro-channels are expected to provide a promising alternative to autograftsfor briding nerve gaps. To date, however, little information has been obtained about scaffoldswith similar inner microstructures, and the efficacy of such scaffolds in bridging peripheralnerve gaps in vivo has never been examined.
In the current study, a collagen-chitosan scaffold (CCH) with longitudinally oriented porechannels and an interconnected porous structure was successfully fabricated by axiallyfreezing and subsequently freeze-drying collagen-chitosan suspensions. The optimumparameters for the fabrication process was determined regarding the raw material blend ratio,concentration of acetic acid and the velocity of freezing in the improved freeze-dryingtechnology. The mechanical property and degradationtion of the CCH scaffolds weremodified via post-fabrication cross-linking with genipin. The biomechanics of the scaffoldwas also fully examined before its usage in vivo. Subsequently, we evaluated its efficacy inbridging a30mm long sciatic nerve defect in Beagles by using a combination ofmorphological and functional techniques.
Part one: Fabrication of CCH scaffolds and determination of the optimum parametersfor the fabrication process
[Objectives] To fabricate the ECM based bionic scaffold with dimensions resembling the basal lamina micro-channels of normal nerve.
[Methods] The freeze-drying technique was improved to fabricate nerve guidance scaffoldsfrom collagen/chitosan. The inner structure was observed under the scanning electronicmicroscope. The pore sizes and interval porosity were examined to determine the optimumparameters for the fabrication process.
[Results] The novel CCH nerve guidance scaffold with different pore size andmicrostructure was fabricated using different velocity of freezing. The optimum velocity offreezing was examined to be2×10-5m/s (mean pore size:37.34±13.24μm). Considering themicrostructure and pore size of the CCH scaffold, the optimum concentration of acetic acidfor CCH fabrication was evaluated to be3mg/ml and the blend ratio was3:1subsequently.The pore size was ranging from24μm～102μm (mean pore size:49.85±19.85μm) andthe interval porosity was above90%.
Part two: Property modification and biomechanics evaluation of the CCH scaffolds
[Objectives] To modify the mechanical property and degradation of the CCH scaffold, andfurther evaluate the biomechanics of the modified CCH scaffolds.
[Methods] The tensile mechanical properties and degradation of the CCH scaffolds weremodified via post-fabrication cross-linking with genipin. The degradation kinetics of thecross-linked scaffolds was evaluated by incubating the scaffolds in PBS with or withoutlysozyme.
[Results] After cross-linking with genipin for48h, the CCH scaffolds showed a17.9±4.2%(without lysozyme) or20.1±4.6%(with lysozyme) reduction in dry weight relative to theoriginal weights after8weeks while the noncross-linked scaffolds showed29.6±4.8%(without lysozyme) or36.3±5.2%(with lysozyme) reduction. The cross-linked CCHscaffolds could withstand a higher average ultimate stress than the noncross-linked scaffoldsin either dry or wet condition, which meets the requirements of surgical procedures. Theseindicate that the cross-linked CCH scaffolds possessed the required mechanical stiffness andstability for successful surgical implantation and nerve regeneration. Part three: The efficacy of CCH scaffolds in bridging a30mm long sciatic nerve defectin Beagles
[Objectives] To investigation the efficacy of CCH scaffolds in bridging a long nerve gap inBeagles.
[Methods] The efficacy of the CCH scaffolds in bridging a30mm long sciatic nerve defectin Beagles was investigated by using a combination of morphological and functionaltechniques, including immunohistology, transmission electron microscope, electrophysiology,retrograde-labelling and behavioral tests.
[Results] Twelve weeks after implantation, the implanted CCH scaffolds were almostcompletely replaced by a large bundle of densely packed regenerated nerve fibers and thepenetration and remarkably linear growth of axons within the longitudinal micro-channels ofthe CCH scaffolds was also observed.24weeks after implantation, the functional tests alsoconfirmed that the CCH scaffolds achieved regeneration and functional recovery equivalentto that of an autograft, without exogenous delivery of regenerative agents or celltransplantation. These findings demonstrate that CCH scaffolds may be used as alternativesto nerve autografts for peripheral nerve regeneration.
引文
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