组织工程化人工神经实验研究

目的：研究组织工程化人工神经修复大鼠2.5cm长坐骨神经缺损的效果。方法：21只2月龄Lewis 1w雌性大鼠随机分成三个神经移植组,每组7只。A组：种植同源雪旺细胞并具有内部支架结构的胶原神经管,即组织工程化人工神经。B组：无雪旺细胞但具有内部支架结构的胶原神经管。C组：自体神经移植体。术后六个月,进行系列神经电生理监测,神经肌肉组织学观察,S－100和神经微丝蛋白（Neurofilament）免疫组化染色,轴突计数等检查。结果：在A组和C组移植神经上均能诱发出波幅明显的神经肌肉复合动作电位（CMAP）,再生轴突已通过移植神经全长,远端肌肉轻度萎缩。而B组中没有或仅记录到极小波幅的CMAP,移植神经远端结缔纤维组织增生,再生轴突罕见,所支配肌肉明显萎缩。结论：初步结果显示：组织工程化人工神经可用来修复大鼠长段神经缺损。     

组织工程化人工神经实验研究

目的 研究组织工程化人工神经修复大鼠2.5cm长坐骨神经缺损的效果。方法 21只2月龄Lewis lw雌性大鼠随机分成三个神经移植组,每组7只。A组:种植同源雪旺细胞并具有内部支架结构的胶原神经管,即组织工程化人工神经。B组:无雪旺细胞但具有内部支架结构的胶原神经管。C组:自体神经移植组。术后六个月,进行系列神经电生理监测,神经肌肉组织学观察,S-100和神经微丝蛋白(Neurofilament)免疫组化染色,轴突计数等检查。结果 在A组和C组移植神经上均能诱发出波幅明显的神经肌肉复合动作电位(CMAP),再生轴突已通过移植神经全长,远端肌肉轻度萎缩。而B组中没有或仅记录到极小波幅的CMAP,移植神经远端结缔纤维组织增生,再生轴突罕见,所支配肌肉明显萎缩。结论 初步结果显示:组织工程化人工神经可用来修复大鼠长段神经缺损。     

组织工程神经修复大鼠坐骨神经缺损的研究

目的观察组织工程神经修复SD大鼠1．5cm长坐骨神经缺损的效果。方法用甘油处理10只SD大鼠2．0cm长坐骨神经，制备成同种异体脱细胞基质，备用。取SD乳鼠10只，分离坐骨神经，去神经外膜后，剪成小碎块，在DMEM中培养3周，扩增后的细胞鉴定、备用。3个月龄的SD雌性大鼠40只，单纯随机分成4个神经移植组（A、B、C、D），每组10只。A组：用扩增的雪旺细胞加同种异体脱细胞基质桥接，即组织工程化人工神经组。B组：用元雪旺细胞但具有内部支架结构的同种异体脱细胞基质桥接。C组：自体神经移植组。D组；空白对照组。术后12周，进行一般情况、小腿三头肌湿重、再生神经的组织学观察。结果完成对40只大鼠（每组10只）的实验评估。所有大鼠伤口瑚愈合，元死亡。A、B、C组大鼠足部元溃疡形成，D组7只足部有溃疡形成，所有组实验侧小腿三头肌较健侧萎缩，但以D组最明显。小腿三头肌湿重、神经电生理监测A组、C组差异无统计学意义（P〉O．05），A、C组与B、D组差异有统计学意义（P〈O．05），B组与D组差异有统计学意义（P〈0．05）。A组和C组的胫前肌中均能诱发出波幅明显的神经肌肉复合动作电位（CMAP），B组、D组中则仅录到波幅很低的CMAP。A组和C组再生轴突已通过移植段神经全长，远端肌肉轻度萎缩。B组部分通过移植段神经；D组不能通过移植段神经，6例形成神经瘤。结论组织工程人工神经可用来修复大鼠长段神经缺损。     

组织工程化人工神经修复犬盆腔内脏运动神经缺损

目的探讨组织工程化人工神经修复盆腔内脏运动神经缺损的效果,为解决直肠癌手术盆腔植物神经损伤所致性功能障碍的治疗提供新思路和实验依据。方法以密度梯度离心法分离比格犬骨髓间充质干细胞（BMSCs）,体外培养和扩增后备用。制作比格犬盆腔内脏运动神经10mm缺损模型,分3组予以修复。A组：将BMSCs与胶原蛋白海绵混合移植于聚羟基乙酸和聚乳酸共聚物（PLGA）导管中构建组织工程化人工神经桥接盆腔内脏运动神经缺损段;B组：仅将胶原蛋白海绵移植于PLGA导管中桥接神经缺损段;C组：自体神经移植。术后12周移植段神经通过大体观察和免疫组织化学观察及电镜扫描、轴突计数等方法评价各组神经缺损修复的效果。结果术后12周,PLGA导管基本吸收,各组再生神经均能通过缺损区长至神经远端,A组再生神经纤维密度和神经结构与C组相近,均优于B组,差异有统计学意义（P〈0．05）。结论BMSCs与PLGA导管构建组织工程化人工神经用于修复盆腔内脏运动神经缺损效果好,与自体神经移植效果相当。     

雪旺细胞构建人工神经在大鼠长段神经缺损中的修复作用研究

[目的]研究雪旺细胞与PLGA构成的组织工程化人工神经修复大鼠周围神经缺损的效果。[方法]将雪旺细胞接种在内置polyglactin910纤维的PLGA中空管，构建成组织工程人工神经。将60只SD大鼠随机分3组，每组20只，建立大鼠坐骨神经缺损20mm的动物模型。A组用切下的自体神经段原位缝合，B组使用雪旺细胞组织工程人工神经进行修复，C组用未接种雪旺细胞的PLGA中空管进行修复。术后8、12周使用神经电生理和组织学观察分别进行效果评价。[结果]在大体观察、组织学观察中，B组的恢复表现与A组相近。在神经电生理检测、小腿三头肌肌肉重量测定、桥接物横切面神经纤维数量测定的结果分析中B组略低于A组但明显高于C组。B组大鼠桥接段远端辣根过氧化酶示踪后在脊髓前角可以看到被标记的神经元。透射电镜可见B组桥接物中段大量的再生神经纤维。[结论]用雪旺细胞和PLGA构建组织工程人工神经修复大鼠外周神经缺损，可以修复20mm的长段周围神经缺损，并可获得接近于大鼠自体神经修复的效果。     

小肠黏膜下层复合雪旺细胞构建组织工程化人工神经修复周围神经缺损的研究

目的小肠黏膜下层（small intestinal submucosa，SIS）复合雪旺细胞构建组织工程化人工神经并探讨其修复周围神经缺损的效果。方法SD大鼠60只随机分成三组，每组20只。构建右侧坐骨神经14mm缺损，A组：单纯SIS修复组；B组：复合雪旺细胞的SIS修复组；C组：自体神经移植对照组。术后不同时间段通过大体观察、电生理检测、组织学、透射电镜、图像分析、逆行示踪和小腿三头肌称重等方法分析评价修复效果。结果术后16周，B组移植段与近远段神经外观相似，未有神经瘤形成。组织学和透射电镜检查见B组再生神经组织可顺利通过缺损区，再生神经纤维排列整齐呈束状，含有大量的有髓神经纤维，与C组相似。电生理检测见第16周B组神经传导速度和复合动作电位的波幅均较第12周有所提高，与C组相比差异无统计学意义。真蓝逆行示踪证实B组和C组再生神经轴突轴浆运输功能恢复良好。图像分析证实B组再生神经组织面积百分比、有髓神经纤维密度和髓鞘厚度与C组相比差异无统计学意义，优于A组。B组和C组患侧小腿三头肌肌肉重量和恢复率均优于A组，C组优于B组，且差异有统计学意义。结论SIS复合雪旺细胞构建组织工程化人工神经能有效修复大鼠长距离坐骨神经缺损，有望成为自体神经的替代材料应用于周围神经缺损的修复。     

异种神经基膜管桥接周围神经缺损的初步研究

目的探索经化学萃取的去细胞神经基膜管支架用于异种移植桥接周围神经缺损的可行性.方法 SD大鼠 30只,随机分为5组,分别制作预溃变2周(溃变支架桥接组)和新鲜的兔胫神经进行化学萃取,形成去细胞神经基膜管支架(异体支架桥接组),兔新鲜胫神经束(异种神经移植组);切取自体神经15 mm原位移植(自体神经移植组),修复大鼠坐骨神经长15mm 的缺损.坐骨神经切断后不作移植修复,为对照组.术后6个月行坐骨神经功能指数(SFI)测定和疼痛试验,用美蓝染色、免疫组织化学、透射电镜等方法对吻合口、移植体中央和远侧神经段的再生神经纤维进行形态学观察,并对再生有髓纤维的数量、直径及髓鞘厚度等进行量化分析.结果术后3个月,仅有溃变支架桥接组、支架桥接组和自体神经移植组动物患肢行走逐渐恢复.6个月时,针刺患足可引起背部肌肉收缩和下肢屈曲反应;术后6个月行SFI检测,溃变支架桥接组为-36.2%±9.7%,支架桥接组为-30.7%±6.8 %,自体神经移植组为-33.9%±11.3%,各组间比较无统计学意义(P>0.05).组织学观察见溃变支架桥接组与支架桥接组移植体中央横切面再生神经呈微束状分布.透射电镜观察见再生神经纤维具有正常的形态和结构.图像分析结果显示溃变支架桥接组再生有髓纤维的数量和直径均达到自体神经移植组的水平(P>0.05);但溃变支架桥接组再生纤维的髓鞘厚度明显小于自体神经移植组(P<0.05).结论用化学方法萃取的兔神经基膜管支架能移植于大鼠,成功桥接周围神经缺损,为应用动物神经修复人体周围神经缺损提供了实验依据.     

构建生物人工神经修复周围神经缺损的实验研究

目的：探讨生物人工神经对周围神经再生的作用。方法：用自体雪旺氏细胞、Ⅳ型胶原及可降解的聚乳酸导管构建生物人工神经，桥接10mm缺损的鼠坐骨神经，对照组行自体神经移植，术后8周，2组行大体观察、组织学检查及神经电生理检查。结果：实验组再生神经纤维数、再生轴突恢复率、运动神经传导速度及复合肌肉动作电位振幅均同对照组相近。结论：实验构建的生物人工神经能有效的促进周围神经再生。     

几丁糖胶原复合膜及激活态雪旺细胞修复周围神经缺损的实验研究

目的研究几丁糖胶原复合膜、激活态雪旺细胞(activatedSchwanncell,ASC)促进周围神经再生的作用。方法将激活态雪旺细胞培养于几丁糖胶原复合膜后,将膜缝制成导管修复大鼠坐骨神经10mm的缺损(D组);并以自体神经移植(A组)、几丁糖胶原复合膜管(B组)及几丁糖胶原复合膜加脑源性神经营养因子[(brainderivedneurotrophicfactor,BDNF)C组]为对照。术后4、8、12周观察肢体运动,复合肌肉动作电位(CMAP)的波幅、潜伏期和运动神经传导速度(MNCV)。术后12周取材,样本染色观察神经轴突再生情况。结果几丁糖胶原复合膜加激活态雪旺细胞修复10mm神经缺损的效果优于几丁糖胶原复合膜加BDNF,与自体神经相似。结论几丁糖胶原复合膜加激活态雪旺细胞能有效地促进周围神经再生。     

人工神经的研究进展

目的 探讨组织工程化周围神经研制工作中神经导管材料和雪旺细胞移植的有关问题。方法 广泛查阅近年有关人工神经研究方面的文献,重点叙述在桥接神经缺损的移植体和雪旺细胞移植方面的研究进展。结果 硅胶、涤纶、聚四氟乙烯、聚交酯及几丁质等生物材料均可作为桥接神经缺损的神经导管,以可降解的生物材料较为。在神经导管内放置丝状内支架可增加再生轴突通过的距离。雪旺细胞经体外纯化和培养后仍具有生物活性,将雪旺细胞植入     

Muscle basal lamina: a new graft material for peripheral nerve repair

The suitability of muscle basal lamina as a graft material for the repair of peripheral nerves was investigated. Grafts were prepared by evacuating the myoplasm from muscles excised from rats and rabbits. This produced a material consisting mainly of basal lamina and connective tissue, with the basal lamina arranged as parallel tubes. Rat- and rabbit-derived graft material in 0.5-cm lengths was sutured into rat sciatic nerves, and 4-cm lengths of rabbit-derived graft material were interposed into rabbit sciatic nerves. For controls, 0.5-cm nerve autografts were grafted into rats and 4-cm autografts into rabbits. After 2 to 3 months, the success of the grafts was assessed functionally, electrophysiologically, and anatomically. By all these criteria the basal lamina grafts were as successful as nerve autografts; essentially the same number of axons of the same size grew through both graft types, animals recovered their limb function equally well, and the nerve conduction velocities and relative refractory periods were the same in both groups of animals. In rats, following both basal lamina and nerve autografts, the number of axons distal to the grafts was approximately the same as that proximal to them, but axon diameter and speed of conduction were significantly less than normal. The authors conclude that muscle basal lamina grafts are as effective as nerve autografts for repairing severed rat or rabbit peripheral nerves, and suggest that grafts prepared in this way may prove to be useful for nerve repair in humans.     

无细胞的异体神经修复鼠坐骨神经缺损

目的 通过化学萃取同种异体神经,去除髓鞘和雪旺细胞,形成无细胞基膜管后桥接鼠坐骨神经缺损,研究神经再生效果。方法 正常鼠坐骨神经用非变性生物剂处理后得到无细胞的基膜管,桥接鼠坐骨神经20mm缺损。实验分3组:无细胞基膜管移植组(A组),自体神经移植组(B组)和异体神经移植组(C组)。术后进行肌电图、光镜、电镜及图象分析仪检查。结果 A组再生神经有大量轴突通过移植体,术后2个月电生理检测再生神经的潜伏期及波幅低于B组(P<0.05),术后3个月2组差异无显著意义。髓鞘厚度在术后3个月时亦低于B组,差异有显著意义(P<0.05)。轴突直径及数目两组无差异。C组因无神经再生,结果无法测量。结论 这种无细胞基膜管移植体能支持轴突的生长和雪旺细胞的迁移,是一种良好的神经移植替代材料。     

骨髓干细胞诱导分化构建组织工程神经

[目的]探讨组织工程化神经修复周围神经缺损的作用。[方法]以DMEM为培养基体外诱导人骨髓基质干细胞分化为雪旺细胞，与细胞外基质及可降解聚乳酸导管构建组织工程化神经；建立坐骨神经缺损10mm的Wistar大鼠动物模型，A组：经诱导骨髓基质干细胞分化雪旺细胞与天然细胞外基质（extra cellular matrix，ECM）凝胶及可降解聚乳酸导管构建组织工程化神经桥接神经缺损；B组：单纯将ECM凝胶注入可降解聚乳酸导管桥接神经缺损；C组：自体神经移植组。术后12周进行神经电生理检测、新生神经组织学观察和轴突计数等检测坐骨神经功能恢复情况。[结果]1．诱导后骨髓基质干细胞呈梭形、胞核大、周围有光晕、突起细长呈纵形排列，GAFP及S-100免疫组织化学染色阳性。动物模型各组经移植术后12周，再生神经已通过缺损区长至神经远端。A组、C组组织学及电生理检测指标均优于B组（PAB=0．021，PBC=0．001），A组与C组无显著性差异（PAC=0．065）；A、B组聚乳酸导管降解吸收明显。[结论]骨髓基质干细胞在体外可诱导分化为雪旺氏细胞，利用其与细胞外基质及可降解聚乳酸导管构建组织工程化神经可以修复周围神经缺损。     

Acellular muscle with Schwann-cell implantation: an alternative biologic nerve conduit

Denatured or acellular muscle grafts are known to support axonal regeneration. With increasing gap length, failure of regeneration is evident, due to the lack of viable Schwann cells in the graft. The authors created a biologic nerve conduit, in a rat sciatic nerve model, by implanting cultured Schwann cells into an acellular gracilis muscle. Autologous nerve grafts and acellular muscle grafts without Schwann cells served as controls. After 6 weeks, regeneration was assessed clinically, histologically, and morphometrically. Polymerase chain reaction (PCR) analysis showed that the implanted Schwann cells remained viable within the graft. Good regeneration was noted in the muscle-Schwann cell group, while regeneration in the muscle grafts without Schwann cells was significantly impaired. The muscle-Schwann cell graft demonstrated systematic and organized regeneration, including the proper orientation of regenerated fibers. The number of axons regenerating through the muscle-Schwann cell grafts was significantly increased, compared with the acellular muscle without Schwann cells. Implantation of Schwann cells into acellular muscle thus provided a biologic conduit with large basal lamina tubes, as a pathway for regenerating axons. The positive effects of Schwann cells, producing neurotrophic and neurotropic factors, supported axonal regeneration.     

Peripheral nerve reconstruction with collagen tubes filled with denatured autologous muscle tissue in the rat model

Conventional nerve conduits lack cellular and extracellular guidance structures critical for bridging larger defects. In this study, an exogenous matrix for axonal regeneration was provided by pretreated muscle tissue. In 24 rats, 14-mm sciatic nerve segments were resected and surgically reconstructed using one of the following methods: autograft (AG); bovine type I collagen conduit; (MDM) collagen tube filled with modified denatured autologous muscle tissue. For 8 weeks, functional regeneration was evaluated by footprint and video gait analysis. Evaluation was complemented by electrophysiology, as well as qualitative and quantitative structural assessment of nerves and target muscles. Group AG was superior both structurally and functionally, showing higher axon counts, a more normal gait pattern, and less severe muscle atrophy. Fiber quality (fiber size and myelin thickness) was highest in group MDM, possibly related to the myelin-producing effect of muscular laminin. However, axon count was lowest in this group, and ultrastructural analysis of the denatured muscle tissue showed areas of incomplete denaturation that had acted as a mechanical barrier for regenerating axons. In light of these results, the often advocated use of muscular exogenous matrix for peripheral nerve reconstruction is reviewed in the literature, and its clinical application is critically discussed. In conclusion, combined muscle tubes may have a positive influence on nerve fiber maturation. However, muscle pretreatment is not without risks, and denaturation processes need to be further refined.     

去细胞异种神经复合神经生长因子修复神经缺损的实验研究

目的 应用含有神经生长因子(NGF)的去细胞异种神经基膜管作为神经移植替代物桥接大鼠坐骨神经缺损,观察其对神经再生的作用.方法 选用Wistar大鼠45只,随机分为3组,每组15只,于术制成右后肢坐骨神经长10 mm的神经缺损,取兔胫神经制成去细胞神经基膜管,电镜及HE染色观察神经基膜管超微结构,流式细胞仪检测去细胞前后神经主要组织相容性抗原Ⅱ(MHC Ⅱ)的变化情况.A组以含有NGF的去细胞异种神经基膜管桥接神经缺损,B组单纯采用去细胞异种神经基膜管桥接神经缺损,C组采用自体神经移植修复神经缺损.术后1个月行神经电生理检测即胫后肌群运动诱发电位,用HE染色、免疫组化染色、透射电镜等方法对移植体远端吻个口再生神经纤维进行形态学观察,并对再生有髓神经纤维的数量、密度、直径及雪旺细胞的密度进行量化分析.结果 移植前新鲜神经组MHC-Ⅱ检测值为72.14±19.88,去细胞组MHC-Ⅱ检测值为4.19±3.11,两组比较差异有统计学意义(t=3.817,P＜0.05);透射电镜观察显示为胶原性管道,无细胞成分.术后4周,处死前行运动诱发电位检测,神经传导速度A组为(21.16±2.31)m/s,B组为(13.37±1.89)m/s,C组为(21.43±2.18)m/s,A组与 C组比较差异无统计学意义(P＞0.05),A组与 B组比较差异有统计学意义(P＜0.05).组织学观察见3组移植体远端吻合口横切面再生神经纤维呈微束状,透射电镜观察再生神经纤维具有正常的形态和结构.A、C组再生神纤纤维数量及直径均优于B组,差异有统计学意义(P＜0.05).结论 经化学萃取的去细胞兔胫神经基膜管能够移植于大鼠,成功修复大鼠坐骨神经缺损,而且复合NGF的去细胞基膜管在神经修复质量上优于单纯的去细胞神经基膜管,更加接近自体神经移植的效果.     

Peripheral nerve regeneration by transplantation of bone marrow stromal cell-derived Schwann cells in adult rats

OBJECT: Bone marrow stromal cells (BMSCs) can be induced to form Schwann cells by sequentially treating the cells with beta-mercaptoethanol and retinoic acid, followed by forskolin and neurotrophic factors including heregulin. In this study the authors made artificial grafts filled with BMSC-derived Schwann cells (BMSC-DSCs) and transplanted them into the transected sciatic nerve in adult rats to evaluate the potential of BMSCs as a novel alternative method of peripheral nerve regeneration. METHODS: The BMSC-DSCs were suspended in Matrigel and transferred into hollow fibers (12 mm in length), which were transplanted into the transected sciatic nerve in adult Wistar rats. Six months after cell transplantation, electrophysiological evaluation and walking track analysis were performed. Results of these studies showed significant improvement in motor nerve conduction velocity and sciatic nerve functional index in the BMSC-DSC-transplanted group compared with the control group (Matrigel graft only). Immunohistochemical study data demonstrated that transplanted BMSCs labeled with retrovirus green fluorescent protein were positive for P0 and myelin-associated glycoprotein and had reconstructed nodes of Ranvier and remyelinated regenerated nerve axons. The number of regenerated axons in the axial section of the central portion of the graft was significantly greater in the transplanted group. Although BMSCs can differentiate into several types of cells, tumor formation did not occur 6 months after engraftment. CONCLUSIONS: Results in this study indicate that BMSC-DSCs have great potential to promote regeneration of peripheral nerves. The artificial graft made with BMSC-DSCs represents an alternative method for the difficult reconstruction of a long distance gap in a peripheral nerve.     

构建猕猴组织工程化周围神经的实验研究

目的评价组织工程化周围神经修复猕猴4cm尺神经缺损的实验效果,为临床研究提供资料。方法分别用6种移植物桥接4cm尺神经缺损。A组:自体BMSCs 去细胞同种异体神经支架;B组:自体SCs 去细胞同种异体神经支架;C组:自体BMSCs PLGA支架导管;D组:去细胞同种异体神经支架;E组:PLGA支架导管;F组:自体神经。通过功能学、神经电生理学及组织学研究评价各自的实验效果。结果A、B、C三种组织工程化神经实验组,术后6个月神经电生理和组织学检查,能引起小鱼际肌群产生复合动作电位的潜伏期、复合动作电位的最大振幅、神经传导速度和再生的神经纤维数目与自体神经移植组(F组)相比差异无显著性意义(P>0.05),但分别大于未加细胞的支架组(D、E组),差异有显著意义(P<0.05)。结论用自体源SCs或BMSCs作种子细胞与去细胞同种异体神经支架,或自体源BMSCs与PLGA支架导管构建不同的组织工程化周围神经,修复猕猴4cm尺神经缺损均取得较好的效果。