甲壳素神经导管修复大鼠坐骨神经10mm缺损的实验研究

探讨壳聚糖导管乙酰化反应而成的甲壳素神经导管修复周围神经缺损的效果.先将脱乙酰度92.5%的壳聚糖经溶解、冷冻、成形、中和、干燥等步骤制成壳聚糖导管,再经乙酰化反应制备成甲壳素神经导管.以该神经导管桥接大鼠坐骨神经10mm缺损.术后16周,通过电生理、组织形态学等方法评价神经导管修复坐骨神经缺损的效果.结果显示,术后16周再生神经已通过甲壳素神经导管长入远端.坐骨神经干重新恢复连续性,再生神经具有电传导功能,并实现对靶肌肉的再支配.缺损组则未观察到神经再生.实验表明,壳聚糖导管乙酰化而成的甲壳素神经导管能有效修复周围神经缺损.     

人工组织神经移植物辅加神经再生素修复大鼠周围神经缺损的实验研究

目的　用人工组织神经移植物辅加神经再生素 (NRF)桥接修复大鼠周围神经缺损。　方法　用壳聚糖套管和聚乙醇酸纤维制成人工组织神经移植物 ,辅加促神经生长的中药有效组分NRF ,桥接大鼠坐骨神经缺损10mm。术后作足迹试验 ;2 4周时对再生神经进行电生理学测试、形态学观察和计量学统计。　结果　术后 2 4周内 ,动物未见炎症及排斥反应。实验组的再生神经在足迹试验、电生理学、形态学及计量学上优于硅胶管桥接组。结论　人工组织神经移植物辅加NRF与周围神经组织具有良好的生物相容性 ;它对缺损的神经修复有较好的桥梁和促进作用     

壳聚糖支架复合嗅鞘细胞修复坐骨神经损伤

背景:嗅鞘细胞可促进中枢神经损伤修复。将壳聚糖与胶原结合制备复合工程材料,已经广泛应用于组织工程化神经导管的构建。目的:探讨嗅鞘细胞复合壳聚糖用于治疗大鼠坐骨神经损伤的修复作用。方法:建立坐骨神经缺损大鼠模型,用联合培养的原代培养大鼠嗅鞘细胞和壳聚糖支架连接于缺损处,设为嗅鞘细胞结合壳聚糖组;单纯壳聚糖支架组用单纯壳聚糖支架连接缺损;空白单纯壳聚糖支架组不作任何处理。结果与结论:建模后1-4周分别检测大鼠坐骨神经功能指数,运动诱发电位潜伏期以及组织学检查发现,与嗅鞘细胞结合壳聚糖组大鼠坐骨神经功能指数显著升高(P0.05),运动诱发电位潜伏期显著低于单纯壳聚糖支架组和空白对照组(P0.01),且嗅鞘细胞结合壳聚糖组有新生的神经达到远侧端,周围少有炎症反应。说明嗅鞘细胞结合壳聚糖支架修复坐骨神经损伤效果较好。     

几丁糖与聚乳酸合成生物材料修复周围神经缺损

背景：单纯几丁糖材料制成的神经导管机械强度较差,易于塌陷,不利于再生神经的生长。 目的：观察几丁糖与聚乳酸复合物修复大鼠周围神经缺损的可行性。 方法：取30只SD大鼠,制作单侧坐骨神经缺损模型,随机均分为3组,分别采用自体神经、硅胶导管及几丁糖与聚乳酸复合导管修复神经缺损,修复后12周,观察桥接神经外观、表面粘连情况及有无神经瘤生成等,检测大鼠神经传导速度、动作电位波幅及潜伏期,苏木精-伊红染色观察坐骨神经桥接物中段神经再生轴突数量及再生神经横截面积,称量大鼠完整小腿三头肌湿质量。 结果与结论：修复后12周,3组再生神经均通过5 mm神经缺损间隙,硅胶管组形成神经瘤,其余两组均未出现神经瘤;自体神经组再生神经直径大于几丁糖-聚乳酸组、硅胶管组(P < 0.05),几丁糖-聚乳酸组再生神经直径大于硅胶管组(P < 0.05);几丁糖-聚乳酸组、自体神经组可见排列整齐的高密度再生轴突,再生轴突数量多于硅胶管组(P < 0.05),且神经传导速度、动作电位波幅、小腿三头肌湿质量显著大于硅胶管组(P < 0.05),潜伏期低于硅胶管组(P < 0.05)。表明几丁糖-聚乳酸复合导管可促进缺损周围神经的再生。  中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

壳聚糖材料在神经导引管桥接周围神经缺损中的应用

应用壳聚糖材料制备神经导引管作为神经再生室桥接大鼠坐骨神经缺损,观察对神经再生的作用。手术造成90只Wistar大鼠右后肢坐骨神经长约15mm的缺损,A组以含有NGF的壳聚糖神经导引管桥接神经缺损,B组单纯采用壳聚糖导管,C组则不用导管,以左侧正常坐骨神经作为正常对照,分别于术后4、12、24周进行大体及显微解剖观察、组织学检查、电镜观察和神经电生理测定。结果表明,A、B组在促进神经再生,加快血管化进程,再生神经纤维排列规律化,提高再生神经髓鞘化,加速再生神经功能重建等方面均优于C组。壳聚糖是制备神经导引管的理想材料。壳聚糖神经导引管可以为大鼠坐骨神经再生提供良好的再生微环境。     

NGF和GM_1联合应用对去细胞异种神经支架修复大鼠坐骨神经陈旧性缺损的影响

目的:研究神经生长因子(NGF)和单唾液酸四己糖神经节苷脂(GM1)联合应用对去细胞异种神经支架修复大鼠坐骨神经陈旧性缺损后神经再生和功能恢复的作用。方法:制作大鼠坐骨神经陈旧性缺损模型,用去细胞异种神经支架进行桥接修复。大鼠术侧小腿三头肌内分别注射NGF+GM1液或NS液,每日1次连续2周。于修复术后8或14周,检测计算大鼠坐骨神经运动传导速度(MNCV)恢复率和小腿腓肠肌复合动作电位(CMAP)波幅恢复率;辣根过氧化物酶(HRP)作为逆行神经轴突示踪剂注射于损伤神经,观察脊神经节内神经元HRP标记情况;检测小腿三头肌湿重及腓肠肌纤维平均截面积恢复率;免疫荧光染色等方法观察移植的神经支架内及其近、远端吻合口的神经纤维组织学特点。结果:在修复术后8周和12周的动物中都观察到:NGF+GM1组动物的MNCV恢复率、CMAP波幅恢复率、L3~L5脊神经节HRP标记细胞数、小腿三头肌湿重恢复率及腓肠肌纤维平均截面积恢复率均优于NS对照组(P﹤0.05);NGF+GM1组动物移植神经支架内再生神经纤维更加密集、排列规则整齐,神经支架内Schwann细胞(SC)大量增殖。结论:结果表明NGF及GM1联合去细胞异种神经支架可成功地修复周围神经陈旧性缺损,促进神经再生和功能恢复。     

大鼠坐骨神经缺损聚吡咯膜植入试验

了解大鼠神经组织对长期埋入其中的聚吡咯膜的生物学反应 ,同时观察聚吡咯涂层的硅胶管作为桥接物 ,修复周围神经缺损的可能性。在硅胶管内壁用电化学方法合成聚吡咯膜管涂层 ,并以桥接方法修复大鼠的坐骨神经缺损 10mm。术后 2 4周 ,对再生组织进行电生理学测试、组织形态学观察和计量学统计。在聚吡咯膜长期植入缺损神经期间 ,实验动物仅出现轻微炎症反应 ;聚吡咯膜管中可见到再生神经 ;聚吡咯膜管内再生的神经在电生理学、组织形态学及计量学方面的结果均略优于单纯的硅胶管桥接组。实验表明在体内周围神经组织对长期埋入的聚吡咯未产生不良反应。     

静电纺丝聚乳酸聚乙醇酸共聚物-丝素-胶原神经导管修复大鼠周围神经缺损

背景：周围神经缺损修复是临床上一大难题,由于自体神经移植有一定的局限性,人工神经修复材料是一种很有前途的选择。 目的：探讨静电纺丝聚乳酸聚乙醇酸共聚物(PLGA)-丝素-胶原纳米神经导管修复大鼠坐骨神经缺损的可能性。 方法：雌性SD大鼠36只,制备约10 mm的坐骨神经缺损,分别采用倒转自体神经、静电纺丝PLGA-丝素-胶原神经导管、单纯PLGA神经导管桥接,术后12周进行大体观察、神经电生理测定、光镜观察、透射电镜观察和图像分析对比,了解神经再生的情况。 结果与结论：静电纺丝法制备成的纳米神经导管管壁疏松多孔,能够模拟细胞外基质的结构。静电纺丝PLGA-丝 素-胶原神经导管组在促进坐骨神经再生、提高再生神经髓鞘化、加速再生神经功能重建等方面均优于单纯PLGA导管组,比自体神经移植组略差。     

移植壳聚糖导管修复大鼠坐骨神经缺损

目的:研究移植结合了碱性成纤维细胞生长因子(bFGF)的壳聚糖导管促进周围神经损伤再生的情况。方法:成年Wistar大鼠造成10mm坐骨神经缺损后,以壳聚糖导管(移植组,10只)作桥梁桥接神经两断端,以假手术组和单纯损伤组(造成10mm坐骨神经缺损后,不加以任何干预措施)各10只分别为阳性和阴性对照,术后通过肉眼观察和神经微丝(NF)、乙酰胆碱酯酶(AChE)免疫组织化学染色方法对损伤神经局部及远端靶肌肉运动终板的再生情况进行观察。结果:移植组大鼠术后3个月,新生的神经纤维已越过缺损部位并到达损伤远端。免疫组织化学染色显示:术后3个月,移植组大鼠坐骨神经缺损处再生组织内可观察到均匀、密集分布的NF免疫阳性纤维,且在腓肠肌终板区内可见AChE免疫阳性终末,至5个月时阳性染色明显增强。在术后3个月时可见新生的运动终板,但轮廓不规则、边界不清晰;而在5个月时,新生的运动终板的形态与密度均接近假手术组水平。结论:结合了bFGF的壳聚糖导管对缺损的坐骨神经修复具有良好的桥梁作用和促进神经生长及终板再生的作用。     

复合他克莫司聚乳酸-乙醇酸缓释导管修复大鼠胫神经缺损

背景：虽然单纯聚乳酸-乙醇酸导管修复大鼠神经缺损可部分恢复大鼠神经功能,但神经直径、再生纤维数量、髓鞘成熟度及功能恢复上均较自体神经移植差。 目的：观察复合他克莫司的聚乳酸-乙醇酸缓释导管修复大鼠胫神经缺损的可行性。 方法：制作SD大鼠右侧胫神经缺损模型,随机分为3组,分别植入自体胫神经、单纯聚乳酸-乙醇酸导管及复合他克莫司的聚乳酸-乙醇酸缓释导管修复。植入后3,6,12周行坐骨神经功能指数检查、电生理检查、组织学观测、腓肠肌湿质量测量。 结果与结论：植入后第6,12周复合他克莫司的聚乳酸-乙醇酸缓释导管组、自体胫神经组坐骨神经功能指数检查、电生理检查、组织学观测、腓肠肌湿质量测量结果优于单纯聚乳酸-乙醇酸导管组(P < 0.05),自体胫神经组、复合他克莫司的聚乳酸-乙醇酸缓释导管组比较差异无显著性意义。说明复合他克莫司的聚乳酸-乙醇酸缓释导管桥接修复大鼠胫神经缺损可明显促进断端神经的再生,在晚期功能恢复上取得接近自体神经移植的效果。 关键词：聚乳酸-乙醇酸;他克莫司;神经导管;大鼠;坐骨神经 doi:10.3969/j.issn.1673-8225.2012.12.003     

Functional recoveries of sciatic nerve regeneration by combining chitosan-coated conduit and neurosphere cells induced from adipose-derived stem cells

Suboptimal repair occurs in a peripheral nerve gap, which can be partially restored by bridging the gap with various biosynthetic conduits or cell-based therapy. In this study, we developed a combination of chitosan coating approach to induce neurosphere cells from human adipose-derived stem cells (ASCs) on chitosan-coated plate and then applied these cells to the interior of a chitosan-coated silicone tube to bridge a 10-mm gap in a rat sciatic nerve. Myelin sheath degeneration and glial scar formation were discovered in the nerve bridged by the silicone conduit. By using a single treatment of chitosan-coated conduit or neurosphere cell therapy, the nerve gap was partially recovered after 6 weeks of surgery. Substantial improvements in nerve regeneration were achieved by combining neurosphere cells and chitosan-coated conduit based on the increase of myelinated axons density and myelin thickness, gastrocnemius muscle weight and muscle fiber diameter, and step and stride lengths from gait analysis. High expressions of interleukin-1β and leukotriene B4 receptor 1 in the intra-neural scarring caused by using silicone conduits revealed that the inflammatory mechanism can be inhibited when the conduit is coated with chitosan. This study demonstrated that the chitosan-coated surface performs multiple functions that can be used to induce neurosphere cells from ASCs and to facilitate nerve regeneration in combination with a cells-assisted coated conduit.     

Use of a newly developed artificial nerve conduit to assist peripheral nerve regeneration across a long gap in dogs

There is now considerable evidence that peripheral nerves have the potential to regenerate if an appropriate microenvironment is provided. However, there are only a few reports of the successful use of artificial nerve conduits to repair major nerve defects more than 30 mm in length. In this study, we examined nerve regeneration across a long gap in the dog peroneal nerve using a novel artificial nerve conduit developed by our group. The conduit consists of a polyglycolic acid (PGA) collagen tube filled with laminin coated collagen fibers. In 12 dogs, the nerve conduit was implanted across an 80 mm gap in the left peroneal nerve. Three months after surgery, compound muscle action potentials (CMAPs) and somatosensory evoked potentials (SEPs) were detected. Evaluation of locomotor function revealed obvious limping for up to 3 months, but no marked difficulty in walking by 6 months. Microscopic observation of the regenerated nerve segment at 12 months showed numerous myelinated nerve fibers, which were smaller in diameter and enclosed in a thinner myelin sheath than normal axons. These results suggest that our artificial nerve conduit has potential usefulness in enhancing peripheral nerve regeneration, even across large gaps.     

Sciatic nerve repair by reinforced nerve conduits made of gelatin-tricalcium phosphate composites

This study proposes a biodegradable GGT composite nerve guide conduit containing genipin-cross-linked gelatin and tricalcium phosphate (TCP) ceramic particles in peripheral nerve regeneration. The proposed genipin-cross-linked gelatin annexed with TCP ceramic particles (GGT) conduit was dark bluish and round with a rough and compact surface. Water uptake and swelling tests indicated that the hydrated GGT conduit exhibited increased stability with not collapsing or stenosis. The GGT conduit had higher mechanical properties than the genipin-cross-linked gelatin without TCP ceramic particles (GG) conduit and served as a better nerve guide conduit. Cytotoxicity tests revealed that the GGT conduit was not toxic and that it promoted the viability and growth of neural stem cells. The experiments in this study confirmed the effectiveness of the GGT conduit as a guidance channel for repairing a 10-mm gap in rat sciatic nerve. Walking track analysis showed a significantly higher sciatic function index score and better toe spreading development in the GGT group than in the silicone group 8 weeks after implantation. Gross examination revealed that the diameter of the intratubular newly formed nerve fibers in GGT conduits exceeded those in silicone tubes after the implantation period. Histological observations revealed that the morphology and distribution patterns of nerve fibers in the GGT conduits at 8 weeks after implantation were similar to those of normal nerves. The quantitative results indicated the superiority of the conduits over the silicone tubes. Motor functional and histomorphometric assessments demonstrate that the proposed GGT conduit is a suitable candidate for peripheral nerve repair.     

生物材料构建神经导管修复周围神经损伤的临床应用

背景：显微外科技术及周围神经损伤修复技术的发展与神经导管材料密切相关。神经导管的构建特别是生物材料构建神经导管材料还有待进一步开发研究。 目的：探讨生物材料构建的神经导管在周围神经损伤修复中的应用及数据分析。 方法：SCI数据库中2001/2010检索有关神经导管在周围神经损伤修复中的应用的文献,检索词为“神经导管(nerve conduit);生物材料(biomaterials);周围神经损伤(peripheral nerve injury);神经再生(nerve regeneration);壳聚糖神经导管(chitosan/chitin nerve conduit);高分子神经导管(polymer/macromolecule nerve conduit);胶原神经导管(collagen nerve conduit)”,共检索文献183篇。 结果与结论：神经导管修复法是在神经断端之间留有一段间隙,利用神经导管在神经的远端和近端之间桥接,并创造相对密闭的环境,以充分发挥远端神经的趋化作用,同时阻隔外部的影响,减少瘢痕的产生。目前,已被用于制备神经导管材料分为非神经组织、非生物降解材料、可生物降解材料。随着分子生物学及其他相关技术的发展,探索寻找理想的材料构建神经导管来治疗周围神经损伤研究始终在进行中。     

A synthetic oxygen carrier in fibrin matrices promotes sciatic nerve regeneration in rats

Tissue-engineering nerve conduits have been studied for a long time in bridging large nerve defects. However, the low oxygen availability within the nerve conduits, which results in death of migratory Schwann cells (SC) or loss of the newly formed tissue’s function, is still an obstacle for axonal regeneration. Thus, it was hypothesized that an oxygen-enriched conduit would enhance axonal regeneration and functional recovery in vivo. To address this issue, perfluorotributylamine (PFTBA) enriched fibrin hydrogel was prepared and injected into collagen–chitosan conduits. The conduit containing PFTBA-enriched fibrin hydrogel was then used to bridge a 12-mm sciatic nerve defect in rats. The control rats were bridged with collagen–chitosan conduits filled with fibrin matrices without PFTBA. It was found that axonal regeneration and functional recovery in the combined PFTBA group were significantly higher than those in the control group without PFTBA. Further investigations showed that the mRNA and protein levels of S-100, brain-derived neurotrophic factor and nerve growth factor were enhanced by PFTBA at 1 and 3 weeks after surgery. However, the mRNA and protein levels of vascular endothelial growth factor were in a similar range between the combined PFTBA group and the control group without PFTBA. In addition, immunohistochemical results showed that the morphological appearances of regenerated nerve and survival of SC were enhanced by PFTBA at 4 and 12 weeks after surgery. In conclusion, PFTBA-enriched nerve conduit is capable of enhancing axonal regeneration, which provides a new avenue for achieving better functional recovery in the treatment of nerve defect.     

Bioactive poly(L-lactic acid) conduits seeded with Schwann cells for peripheral nerve regeneration.

This study attempted to enhance the efficacy of peripheral nerve regeneration using our previously tested poly(L-lactic acid) (PLLA) conduits by incorporating them with allogeneic Schwann cells (SCs). The SCs were harvested, cultured to obtain confluent monolayers and two concentrations (1 x 10(4) and 1 x 10(6) SC/ml) were combined with a collagen matrix (Vitrogen) and injected into the PLLA conduits. The conduits were then implanted into a 12 mm right sciatic nerve defect in rats. Three control groups were used: isografts, PLLA conduits filled with collagen alone and empty silicone tubes. The sciatic functional index (SFI) was calculated monthly through four months. At the end of second and fourth months, the gastrocnemius muscle was harvested and weighed for comparison and the graft conduit and distal nerve were harvested for histomorphologic analysis. The mean SFI demonstrated no group differences from isograft control. By four months, there was no significant difference in gastrocnemius muscle weight between the experimental groups compared to isograft controls. At four months, the distal nerve demonstrated a statistically lower number of axons mm2 for the high and low SC density groups and collagen control. The nerve fiber density was significantly lower in all of the groups compared to isograft controls by four months. The development of a "bioactive" nerve conduit using tissue engineering to replace autogenous nerve grafts offers a potential approach to improved patient care. Although equivalent nerve regeneration to autografts was not achieved, this study provides promising results for further investigation.     

合成材料神经导管与自体神经移植修复周围神经缺损的比较

背景：生物可降解材料制成的神经导管可在体内降解,避免出现的神经卡压等问题,因而受到越来越多的关注。 目的：比较自体神经移植与3种合成可生物降解材料神经导管在修复周围神经损伤的效果差异。 方法：通过电生理学检测,形态学观察等神经恢复效果评价方法,对比分析近年来常用的胶原神经导管、DL-乳酸-ε-己内酯神经导管、聚乙醇酸神经导管与自体神经移植修复周围神经缺损的效果。 结果与结论：虽然神经导管与自体神经移植相比在理论上有其优势的一面,但不同合成材料的神经导管之间在神经功能恢复中存在明显差异性,DL-乳酸-ε-己内酯神经导管修复效果与自体神经移植无明显差异,是较为理想的神经导管材料,聚乙醇酸神经导管因自身的因素影响其降解性能,在3种神经导管中的修复周围神经损伤效果最差,胶原神经导管需要交联剂改善其机械性能,其修复周围神经损伤效果居于前两者之间,因此,这3种神经导管在神经功能再生方面还有潜在的缺陷,不能完全替代自体神经移植,而且3者之间的性价比,还缺少足够的大样本长期随机对照实验结果来验证,还需要进一步的实验观察。     

Preparation of electrospun PLGA-silk fibroin nanofibers-based nerve conduits and evaluation in vivo

With advances in technical methodology, the grafting of biocompatible conduits may become a viable alternative for the reconstruction of nerve gaps. In this study, electrospinning was used to fabricate nerve conduits (NCs) from poly(L-lactide-coglycolide)-silk fibroin. Conduits or autograft nerves were employed to bridge 10 mm defects in the sciatic nerves of Sprague-Dawley rats. Six weeks after the operation, morphological and functional assessment showed that nerve conduits from PLGA-silk fibroin grafts promoted the regeneration of peripheral nerves. The effects were similar to those obtained using nerve autografts. This method offers a promising alternative to the use of nerve autografts.     

An in vivo study of tricalcium phosphate and glutaraldehyde crosslinking gelatin conduits in peripheral nerve repair

In order to modulate the mechanical properties of gelatin, we previously developed a biodegradable composite composed by tricalcium phosphate and glutaraldehyde crosslinking gelatin (GTG) feasible for surgical manipulation. In this study, we evaluated the in vivo applications of GTG conduit for peripheral nerve repair. The effect of sciatic nerve reconstruction was compared between resorbable permeable GTG conduits and durable impermeable silicone tubes. Traditional methods of assessing nerve recovery following peripheral nerve repair including histomorphometric and electrophysiologic features were conducted in our study. In addition, autotomy score and sciatic function index (SFI) in walking tract analysis were used as additional parameters for assessing the return of nerve function. Twenty-four weeks after sciatic nerve repair, the GTG conduits were harvested. Microscopically, regeneration of nerves was observed in the cross-section at the mid portion of all implanted GTG conduits. The cross-sectional area of regenerated nerve of the GTG group was significant larger than that of the silicone group. In the compound muscle action potentials (CMAP), the mean recovery index of CMAP amplitude was 0.24 +/- 0.02 for the silicone group, 0.41 +/- 0.07 for the GTG group. The mean SFI increased with time in the GTG group during the evaluation period until 24 weeks. Walking tract analysis showed a higher SFI score in the GTG group at both 12 and 24 weeks. The difference reached a significant level at 24 weeks. Thus, the histomorphometric, electrophysiologic, and functional assessments demonstrate that GTG can be a candidate for peripheral nerve repair.