去细胞异体神经的形态结构及移植后的组织相容性

目的:为面神经缺损寻找一种理想的异体神经移植物。方法:取Wistar大鼠胫神经,经Triton X-100和脱氧胆酸钠溶液进行化学去细胞处理。将处理后的神经行组织学染色和免疫组织化学染色;并行异体移植修复面神经缺损,观察其组织相容性。结果:去细胞神经为一中空的神经基质管,其中的细胞和髓鞘成分被有效清除,神经基底膜被保留;异体移植后无明显炎症反应,无排斥和吸收反应,能引导宿主轴突和Schwann细胞增殖。结论:去细胞异体神经移植物具有良好的仿生性和组织相容性,可能用于修复面神经缺损。     

脱细胞同种异体神经移植物修复大鼠坐骨神经缺损的实验研究

目的 观察脱细胞处理的同种异体神经移植物修复大鼠坐骨神经缺损的作用。方法 用组织工程学方法制备的大鼠同种异体神经移植物桥接大鼠坐骨神经缺损，并对再生神经进行电生理学功能测试，光镜、电镜观察移植物内的再生神经，并进行统计分析。结果 术后13周内，动物未见炎症及排斥反应。用脱细胞处理的同种异体神经移植物修复神经缺损，再生神经的传导功能、纤维数量、轴突直径、有髓纤维占有的面积与自体神经移植对照及计量学上统计分析均无显著性差异。结论 脱细胞处理的同种异体神经移植物具有良好的组织相容性，它对缺损的坐骨神经再生有促进作用。     

脱细胞处理的同种异体神经移植研究进展

周围神经损伤的修复是创伤外科难题之一,筛选促进神经再生理想的移植物,是解决这一难题的关键。近年来,在修复周围神经缺损研究中采用脱细胞处理的同种异体神经作为移植物,已取得促进神经再生的效果,展示了较好的应用前景。本文对脱细胞处理移植体的脱细胞处理方法,脱细胞处理后的细胞外基质所含的生物活性物质,以及这些物质的对促进神经再生的生物效应进行综述,以期为此种移植体的深入研究提供参考资料。     

人工组织神经移植物对大鼠陈旧性坐骨神经缺损(60天)的修复作用

目的 观察人工组织神经移植物对陈旧性大鼠坐骨神经缺损的修复作用.方法 切除成年SD大鼠部分左侧坐骨神经,饲养60d形成陈旧性坐骨神经缺损后,以人工组织神经移植物修复缺损,同时设自体神经修复和不修复两对照组.修复术后3个月做神经-肌复合电位、腓肠肌湿重及再生神经形态学检测.结果 人工组织神经移植物修复组实验侧的运动神经传导速度、腓肠肌湿重、移植物远侧再生神经有髓神经纤维髓鞘厚度等结果与自体神经修复组相似.不修复对照组则未记录到神经-肌复合电位,未观察到再生神经纤维结构,腓肠肌湿重明显小于人工组织神经移植组和自体神经移植组.结论 人工组织神经移植物在一定程度上能修复缺损60d的大鼠坐骨神经.     

神经导管生物材料在神经修复中的应用

背景：神经导管是由天然或人工合成材料制成的、用于桥接神经断端的组织工程支架材料,具有引导和促进神经再生作用。 目的：总结近年来常用的神经导管生物材料在神经修复中的应用。 方法：由作者应用计算机检索维普数据库中与神经导管生物材料在神经修复中应用有关的文章,检索时限2002-01/ 2010-12。检索关键词：神经导管;生物材料;神经损伤;神经修复;神经再生。纳入标准：与神经导管生物材料在神经修复中应用有关的文章。排除标准：重复研究或较陈旧文献。根据纳入排除标准共保留相关文献30篇。 结果与结论：非生物降解材料由于其不可吸收性和对再生神经的远期不良影响使临床应用受到限制。生物降解材料在神经再生完成后可在体内降解吸收,无需二次手术取出,但目前未能利用生物降解材料完全仿制出具有天然神经结构的支架。生物衍生材料生物相容性好、排异反应小,可提供细胞外基质、胶原,起支架作用,但缺血后存在管形塌陷、再生不良、吸收瘢痕组织、增生及粘连等问题。神经导管生物材料在神经修复中的应用前景广阔,但单用一类材料难以制作出理想的神经导管生物材料,通过结合各类材料的优点,与神经营养因子、细胞外基质成分和许旺细胞等联合应用,制备新型具有生物活性的导管材料,将有利于神经修复进一步发展。     

无细胞神经支架复合骨髓间充质干细胞构建组织工程人工神经修复坐骨神经缺损

背景：作者已经成功制备了无细胞神经移植物,并且复合骨髓间充质干细胞构建组织工程人工神经桥接大鼠坐骨神经缺损。 目的：无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复大鼠坐骨神经缺损后运动功能的恢复。 方法：成年雄性SD大鼠构建大鼠坐骨神经15 mm缺损模型,分别应用组织工程人工神经、组织工程神经支架或自行神经桥接坐骨神经缺损。桥接后20周再生神经电生理学测定,手术侧胫骨前肌湿质量、腓肠肌组织学及透视电镜分析。 结果与结论：桥接20周后,组织工程人工神经与自体神经移植组胫骨前肌湿质量比较,差异无显著性意义(P > 0.05),神经干传导速度为(30.56±2.15)m/s。结果提示,无细胞神经移植物复合骨髓间充质干细胞构建的组织工程人工神经桥接大鼠坐骨神经缺损后,可以促进再生神经运动功能的恢复。     

纳米导管的制备及其在神经修复中的应用

纳米导管可以效仿细胞外基质的纤维结构,为细胞的存活、分化等提供合适的微环境。电纺技术是较为成熟的制备纳米导管的方法。多项细胞生长与增殖的实验表明纳米导管的生物相容性良好,对免疫系统也未见明显影响。在神经修复领域,实验证实纳米导管对于神经细胞与神经突的再生,神经胶质细胞的吸附与生长都具有促进作用;此外纳米导管还可以用来作为神经营养因子的载体,为神经修复过程创造适宜的生物微环境。     

异体神经移植研究进展

周围神经断裂缺损过大时，不宜直接拉拢缝合，否则，由于增加神经干张力，将导致神经干内的血液循环障碍而不利于神经再生。神经缺损超过10mm时，尽管远段神经的趋向性导引，但神经再生失败；这时需用移植物来桥接、促进和引导神经再生。桥接物大体分为抻经组织和非神经组织，后包括问皮管、硅胶管、自体静脉、变性骨骼肌…等。自体抻经移植修复神经缺损，神经再生效果和机能恢复相对而言较为理想，但自体神经来源有限，且引起供区感觉、运动的障碍，限制了临床应用自体抻经移植物来桥接神经缺损。因免疫排斥反应之故，异体神经移植必须借助于免疫抑制剂方能应用于临床。根据近年来有关献报道神经移植后神经再生机制，本就异体神经移植研究进展综述如下。     

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.     

干细胞及神经导管支架修复神经缺损

背景：组织工程的发展为神经缺损的修复提供了可能,种子细胞与导管支架制成的复合体是构建组织工程神经的核心。 目的：从干细胞的组织工程应用及构建具有良好生物相容性的导管支架材料角度,探索如何更好的修复神经损伤。 方法：以“干细胞,神经损伤,修复,神经导管,神经支架材料”为中文关键词,以“stem cells,nerve damage,repair,nerve guide conduit material,scaffold materials,nerve tissue engineering”为英文关键词,采用计算机检索CNKI和Medline数据库1996-01/2011-01有关不同来源干细胞和导管支架材料修复神经缺损的相关文章,排除重复研究或Meta分析类文章,筛选纳入30篇文献进行评价。 结果与结论：移植神经干细胞可以在神经系统存活、增殖、迁移,在不同部位分化为相应的细胞,因此给神经修复领域带来新的希望。另外,随着生物材料的发展,神经导管材料修复神经缺损也取得了优良的效果,具有良好的应用前景。将神经干细胞复合导管可降解生物材料有望能更好的满足神经支架的要求,达到修复和重建的目的。     

Bridging extra large defects of peripheral nerves: possibilities and limitations of alternative biological grafts from acellular muscle and Schwann cells

Defects of peripheral nerves are bridged with autologous nerve grafts. Tissue-engineered nerve grafts offer a laboratory-based alternative to overcome limited donor nerve availability. Our objective was to evaluate whether a graft made from acellular muscle enriched with cultivated Schwann cells can bridge extra large gaps where conventional conduits usually fail. Our well-established rat sciatic nerve model was used with an increased gap length of 50 mm. The conduits consisted of freeze-thawed or chemically extracted homologous acellular rat rectus muscles and implanted Schwann cells. Autologous nerve grafts were used for control purposes. Biocompatibility of the grafts was demonstrated by Schwann cell settlement, revascularization, and macrophage recruitment. After 12 weeks regeneration was assessed clinically, histologically, and morphometrically. The control group showed superior results regarding axon counts, histologic appearance, and functional recovery compared with the muscle grafts. The chemically extracted conduits completely failed to support nerve regeneration. They were not stable enough to bridge longer nerve gaps with an expanded regeneration time. On the basis of morphological parameters freeze-thawed muscle grafts were, however, able to support peripheral nerve regeneration even over the extralong distance of 50 mm, and therefore are of potential benefit for new therapeutic strategies.     

A bioengineered peripheral nerve construct using aligned peptide amphiphile nanofibers

Peripheral nerve injuries can result in lifelong disability. Primary coaptation is the treatment of choice when the gap between transected nerve ends is short. Long nerve gaps seen in more complex injuries often require autologous nerve grafts or nerve conduits implemented into the repair. Nerve grafts, however, cause morbidity and functional loss at donor sites, which are limited in number. Nerve conduits, in turn, lack an internal scaffold to support and guide axonal regeneration, resulting in decreased efficacy over longer nerve gap lengths. By comparison, peptide amphiphiles (PAs) are molecules that can self-assemble into nanofibers, which can be aligned to mimic the native architecture of peripheral nerve. As such, they represent a potential substrate for use in a bioengineered nerve graft substitute. To examine this, we cultured Schwann cells with bioactive PAs (RGDS-PA, IKVAV-PA) to determine their ability to attach to and proliferate within the biomaterial. Next, we devised a PA construct for use in a peripheral nerve critical sized defect model. Rat sciatic nerve defects were created and reconstructed with autologous nerve, PLGA conduits filled with various forms of aligned PAs, or left unrepaired. Motor and sensory recovery were determined and compared among groups. Our results demonstrate that Schwann cells are able to adhere to and proliferate in aligned PA gels, with greater efficacy in bioactive PAs compared to the backbone-PA alone. In vivo testing revealed recovery of motor and sensory function in animals treated with conduit/PA constructs comparable to animals treated with autologous nerve grafts. Functional recovery in conduit/PA and autologous graft groups was significantly faster than in animals treated with empty PLGA conduits. Histological examinations also demonstrated increased axonal and Schwann cell regeneration within the reconstructed nerve gap in animals treated with conduit/PA constructs. These results indicate that PA nanofibers may represent a promising biomaterial for use in bioengineered peripheral nerve repair.     

Use of skeletal muscle tissue in peripheral nerve repair: review of the literature

The management of peripheral nerve injury continues to be a major clinical challenge. The most widely used technique for bridging defects in peripheral nerves is the use of autologous nerve grafts. This technique, however, necessitates a donor nerve and corresponding deficit. Many alternative techniques have thus been developed. The use of skeletal muscle tissue as graft material for nerve repair is one example. The rationale regarding the use of the skeletal muscle tissue technique is the availability of a longitudinally oriented basal lamina and extracellular matrix components that direct and enhance regenerating nerve fibers. These factors provide superiority over other bridging methods as vein grafts or (non)degradable nerve conduits. The main disadvantages of this technique are the risk that nerve fibers can grow out of the muscle tissue during nerve regeneration, and that a donor site is necessary to harvest the muscle tissue. Despite publications on nerve conduits as an alternative for peripheral nerve repair, autologous nerve grafting is still the standard care for treatment of a nerve gap in the clinical situation; however, the use of the skeletal muscle tissue technique can be added to the surgeon's arsenal of peripheral nerve repair tools, especially for bridging short nerve defects or when traditional nerve autografts cannot be employed. This technique has been investigated both experimentally and clinically and, in this article, an overview of the literature on skeletal muscle grafts for bridging peripheral nerve defects is presented.     

干细胞与周围神经损伤修复

目前周围神经缺损的修复仍是临床的一大难题。周围神经系统具有再生的潜能,但在自体神经移植中受到取材长度的限制,近年来许多学者将体外培养扩增的大量雪旺细胞种植到神经导管中,以引导周围神经再生,然而在适当条件下难以维持大量的雪旺细胞,雪旺细胞传代后形态和功能逐渐改变。体外培养的干细胞,尤其是中枢来源的干细胞,具有低免疫原性,可以分化为雪旺细胞,移植至受损部位后可促进神经再生。本文综述了干细胞在修复周围神经损伤中的应用及其在再生过程中所发挥的作用。     

Collagen nerve conduits--assessment of biocompatibility and axonal regeneration

Bridging of nerve gaps is still a major problem in peripheral nerve surgery. Alternatively to autologous nerve grafts tissue engineering of peripheral nerves focuses on biocompatible conduits to reconstruct nerves. Such non-neural conduits fail to support regeneration over larger gaps due to lacking viable Schwann cells that promote regeneration by producing growth factors and cell guiding molecules. This problem may be overcome by implantation of cultivated Schwann cells into suitable scaffolds. In the present experiments we tested a collagen type I/III tube as a potential nerve guiding matrix. Revascularization, tolerance and Schwann cell settlement were evaluated by light, fluorescence and scanning electron microscopy after different implantation times. The conduits were completely revascularized between day 5 and 7 post-operatively and well integrated into the host tissue. Implanted Schwann cells adhered, survived and proliferated on the inner surface of the conduits. Nevertheless, bridging a 2 cm gap of the sciatic nerve of adult Wistar rats with these collagen/Schwann cell conduits led to a disappointing regeneration compared to controls with autologous grafts. From these results, we conclude that a sufficient biocompatibility of bioartificial nerve conduits is a necessary prerequisite, however, it remains only one of several parameters important for peripheral nerve regeneration.     

Low-intensity-ultrasound-accelerated nerve regeneration using cell-seeded poly(D,L-lactic acid-co-glycolic acid) conduits: an in vivo and in vitro study

This study investigated the effects of low intensity ultrasound on seeded Schwann cells within poly(DL-lactic acid-co-glycolic acid) (PLGA) conduits by in vitro and in vivo trials for peripheral nerve regeneration. The possible differences in the ultrasonic effects when using biodegradable and non-biodegradable materials as the conduits were also studied, using silicone rubber tubes as comparisons. In the in vitro study, seeded Schwann cells were cultured in serum deprivation culture medium that simulated the environment of mechanical trauma on injury nerve site. After 12, 24, and 48 h, only the PLGA conduit groups exposed to 0.05 W/cm(2), 3 min/treatment of ultrasound exhibited decreased LDH release and increased MTT values compared to the sham groups. Based on the results of the in vitro experiment in LDH and MTT testing, the silicone conduits with seeded Schwann cells group was ignored in the in vivo study. The PLGA nerve conduits seeded with Schwann cells (9 x 10(3) cells) were implanted to 15-mm right sciatic nerve defects in rats. Each conduit received 12 ultrasonic treatment sessions over 2 weeks after 1 day of rest. Ultrasound was applied as follows: frequency, 1MHz; intensity, 0.3 W/cm(2) (SATP); treatment, 5 min/day. Implanted graft specimens were harvested for histological analysis at 8 weeks following surgery. PLGA groups (with and without Schwann cells) treated with pulsed ultrasonic stimulation were found to have significantly greater number and area of regenerated axons at the mid-conduit of implanted grafts, as compared to the sham groups. Ultrasonic stimulation on silicone groups was found to induce a mass of fibrous tissues that covered the nerve conduits and retarded axon regeneration.     

Regeneration of acutely and chronically injured descending respiratory pathways within post-traumatic nerve grafts

Central respiratory neurons, which are acutely axotomized by peripheral nerve grafts implanted at the level of the descending respiratory pathways within the C2 spinal cord, can regenerate their axons within the grafts and still transmit normal physiological messages [Decherchi et al., 1996. Exp. Neurol. 137, 1-14]. The present work investigated the extent to which mature central neurons, acutely or chronically axotomized by a spinal lesion, still maintain the potential to regenerate an axon following post-traumatic nerve grafting within supra-lesional spinal structures and remained functional. This study is an extension of earlier work employing the more chronic lesions, that investigated whether respiratory neurons chronically axotomized by a spinal cord injury can retain the ability to regenerate their axonal process within a post-traumatic peripheral nerve graft. Here implantation was performed into the supra-lesional ventrolateral part of the ipsilateral C2 spinal cord (at the level of the descending respiratory pathways) previously hemisected at the C3 level. In the present study, these post-traumatic peripheral nerve grafts were performed either acutely (group I, n=15, 2.5 h post-injury: acute conditions) or chronically (group II, n=17, 3 weeks; group III, n=6, 3 months: chronic conditions) after the injury.Electrophysiological recording of teased filaments (n=2362) within the post-traumatic peripheral nerve grafts revealed the presence of regenerated nerve fibers with spontaneous unitary impulse traffic (graft units, n=954) in all animals. These graft units were respiratory (n=247) and non-respiratory (n=707). Respiratory discharges originated from central respiratory neurons which remained functional with preserved afferent connections. Except for the group III, post-traumatic C2 peripheral nerve grafts of the groups I and II contained a significantly higher occurrence rate (13.2+/-2% and 11.6+/-1.9%) of respiratory units than C2 spinal peripheral nerve grafts (5.9+/-1.6%) realized without previous CNS injury.The main conclusion of our study is that for a prolonged period of 3 weeks following a spinal cord injury, central respiratory neurons have the potential to remain functional and to regenerate their axonal process within post-traumatic peripheral nerve grafts inserted rostrally to the spinal damage. This indicates that supra-lesional post-traumatic nerve grafts may constitute an efficient delayed strategy for inducing axonal regrowth of chronically axotomized adult central neurons. This suggests that surgical intervention which is not always possible immediately after a spinal cord injury may be satisfactorily carried out after an appropriate delay.     

以组织工程建构技术修复周围神经损伤的难点

背景：组织工程构建技术是近年来周围神经损伤修复的重要方法之一,在周围神经治疗领域有着良好的前景。 目的：总结近年来利用组织工程学构建技术对周围神经损伤修复的研究进展。 方法：应用计算机检索万方数据库、CNKI和PubMed数据库中1995年1月至2011年12月关于组织工程构建技术的文章,在标题和摘要中以“组织工程,神经导管支架,生物活性,周围神经损伤”或“Tissue engineering,Nerve scaffold,Bioactivity,Peripheral nerve defect”为检索词进行检索,初检得到156篇文献,最终纳入56篇文献进行综述。 结果与结论：周围神经损伤组织工程修复的两个要素是神经支架材料的选择和生物功能化。构建神经的支架材料包括可降解和非可降解两大类,通常需要具有三维多孔结构和相应的孔隙率及比表面积,其力学性能、表面活性、生物相容性和导电性等直接影响神经损伤修复的效果;生物功能化的主要生物活性因子包括支持细胞,种子蛋白和神经营养因子,将这些生物活性因子接种在神经导管支架材料上,促进受损神经的修复与功能替代。组织工程技术应用于周围神经损伤修复与再生的研究重点在于导管、细胞与生长因子的综合应用。组织工程技术与生物技术的联合应用将成为周围神经损伤修复的研究热点。     

The effect of high outflow permeability in asymmetric poly(dl-lactic acid-co-glycolic acid) conduits for peripheral nerve regeneration

This study attempted to accelerate the peripheral nerve regeneration, using the high outflow rate of asymmetric poly(dl-lactic acid-co-glycolic acid) (PLGA) nerve conduits. Asymmetric PLGA nerve conduits of monomer ratio 85/15 were prepared by immersion-precipitation method to serve as possible materials. In this study, mandrels were immersed into a 20% (wt/wt) of PLGA/1,4-dioxane solution and precipitated in a non-solvent bath followed by freeze-drying. Different concentrations of isopropyl alcohol (95%, 40% and 20%) were used as precipitation baths where non-asymmetric (95%) and asymmetric (40% and 20%) conduits could easily form. The asymmetric nerve conduits that consisted of macrovoids on the outer layer, and interconnected micropores in the inner sublayer, possessed characters of larger outflow rate than inflow rate. The asymmetric conduits were implanted to 10mm right sciatic nerve defects in rats. Autografts, silicone and non-asymmetric PLGA conduits were performed as the control and the contrast groups. Implanted graft specimens of all groups were harvested for histological analysis at 4 and 6 weeks following surgery. The asymmetric PLGA conduits maintained a stable supporting structure and inhibited exogenous cells invasion during entire regeneration process. Asymmetric PLGA conduits were found to have statistically greater number of regenerated axons at the midconduit and distal nerve site of implanted grafts, as compared to the silicone and non-asymmetric groups at 4 and 6 weeks. Of interest was that the results of 4 weeks in asymmetric groups were better than the non-asymmetric groups at 6 weeks in number of axons. According to the results of permeability, the asymmetric structure in the conduit wall seemed to enhance the removal of the blockage of the waste drain from the inner inflamed wound in the early stage, which may have improved the efficacy of the peripheral nerve regeneration. The asymmetric structure could be adequately employed in the future as optimal nerve conduits in peripheral nerve regeneration.     

The regeneration of transected sciatic nerves of adult rats using chitosan nerve conduits seeded with bone marrow stromal cell-derived Schwann cells

Autologous nerve grafts have been the 'gold standard' for treatment of peripheral nerve defects that exceed the critical gap length. To address issues of limited availability of donor nerves and donor site morbidity, we have fabricated chitosan conduits and seeded them with bone marrow stromal cell (BMSC)-derived Schwann cells as an alternative. The derived Schwann cells used were checked for fate commitment. The conduits were tested for efficacy in bridging the critical gap length of 12 mm in sciatic nerves of adult rats. By three months post-operation, mid-shank circumference, nerve conduction velocity, average regenerated myelin area, and myelinated axon count, in nerves bridged with BMSC-derived Schwann cells were similar to those treated with sciatic nerve-derived Schwann cells (p > 0.05) but significantly higher than those bridged with PBS-filled conduits (p < 0.05). Evidence is thus provided in support of the use of chitosan conduits seeded with BMSC-derived Schwann cells to treat critical defects in peripheral nerves. This provides the basis to pursue BMSC as an autologous source of Schwann cells for transplantation therapy in larger animal species.