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。     

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。     

胶质细胞源性神经营养因子基因修饰的雪旺氏细胞修复大鼠周围神经缺损的实验研究

目的　从转基因角度探讨治疗周围神经损伤的有效方法。方法　成年Wister大鼠 4 8只 ,平均分为 3组。切断大鼠坐骨神经并形成 10mm长缺损 ,用硅胶管桥接两侧断端 ,管腔内植入胶质细胞源性神经营养因子 (glialcell linederivedneurotrophicfactor,GDNF)修饰的雪旺氏细胞 (schwanncells,SCs) ,正常SCs修复组和单纯硅胶管修复组作为对照 ,分别于术后 4、8、12和 16周对各组动物进行大体观察 ,肌电图测量 ,组织学切片观察 ,再生神经的神经电生理检测 ,GDNF免疫组化检测 ,组织学切片 ,观察和图像分析。结果　GDNF SCs组动物的神经传导速度、有髓神经纤维密度、神经组织面积、髓鞘厚度均显著优于SCs组和硅胶管组。结论　将GDNF基因修饰的雪旺氏细胞移植修复周围神经缺损 ,使局部释放的GDNF维持神经元存活 ,加快轴突再生速度以促进周围神经再生 ,此方法为将来治疗周围神经损伤提供了线索。     

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。     

PLGA神经导管联合ADSCs与自体神经组织修复大鼠坐骨神经缺损的实验研究

目的:探讨PLGA神经导管联合ADSCs与自体神经组织碎屑修复大鼠坐骨神经缺损的修复效果。方法:32只SD大鼠平均分成4组,无菌条件下切断右侧的坐骨神经,制成1 0衄长的大鼠坐骨神经缺损模型,A组采用PLGA神经导管连接缺损神经进行修复,B组由内置自体神经组织碎屑的PLGA神经导管连接,C组由内置ADSCs与自体神经组织碎屑的PLGA神经导管连接,D组采用自体神经移植方式。12周后通过对再生神经桥接体的一般观察、HE染色、免疫荧光染色、甲苯胺蓝染色来评价神经的再生情况;通过肌电图、腓肠肌的HE和Masson染色来评价神经对靶器官的再支配情况。结果:术后12周,各组的神经导管均已降解,切断神经通过神经导管向两端生长。一般观察、肌电图、组织学观察结果均提示PLGA神经导管联合ADSCs与自体神经组织碎屑组C组的修复效果显著优于内置神经组织碎屑的PLGA神经导管组B组和空导管组A组的修复效果,但仍稍差于自体神经移植组D组。结论:PLGA神经导管联合ADSCs与自体神经组织可以比较有效地修复周围神经缺损,为周围神经缺损的修复提供了一种新的方法。     

碱性成纤维细胞生长因子对组织工程化外周神经的影响

目的　研究碱性成纤维细胞生长因子 (bFGF)和肝素与乳兔许旺细胞、去细胞基膜管、构成的复合型组织工程化外周神经桥接体修复兔正中神经缺损的效果。　方法　新西兰兔 48只 ,建立左侧上臂正中神经 3 0mm缺损模型 ,随机分为 4组 ,分别用去细胞基膜管种植许旺细胞并复合bFGF及肝素 (Hep)的桥接体 (A组 )、去细胞基膜管种植许旺细胞的桥接体 (B组 )、去细胞基膜管复合bFGF及Hep桥接体 (C组 )、自体神经 (D组 )修复神经缺损 ,于术后 1、3个月分别进行大体观察 ,Masson三色染色光镜观察神经再生、神经内胶原纤维形成及血管形成 ,3个月检测各组桥接体运动神经传导速度 ,并行透射电镜检查 ,称量指浅屈肌肌肉湿重 ,观察神经功能恢复。　结果　去细胞基膜管种植许旺细胞并复合bFGF及Hep的桥接体组 (A组 )神经再生及功能指标 (再生有髓神经纤维密度、平均髓鞘厚度、有髓纤维直径、运动神经传导速度、肌肉湿重恢复率 )与自体神经移植 (D组 )比较 ,差异无显著性 (P >0 .0 5 )。　结论　bFGF及肝素与许旺细胞、去细胞神经基膜管构成的复合型组织工程神经桥接体修复神经缺损能提高神经再生质量。     

静脉体基底膜许旺细胞和NGF复合移植桥接神经缺损的实验研究

目的观察静脉体、基底膜、许旺细胞和神经生长因子复合移植桥接神经缺损的作用。方法采用健康白兔40只，分为4组，每组10条坐骨神经。A组为静脉体、基底膜、许旺细胞和神经生长因子复合移植组，B组为自体静脉组， C组为人羊膜基底膜组，D组为自体神经移植组。均修复坐骨神经1.5 cm缺损，术后3 个月观察肢体运动，肌电图动作电位波幅、潜伏期和传导速度，并常规HE染色、免疫组化SP 法染色显示髓磷脂碱性蛋白（MBP）及Cajal吡啶银染色，镜下观察移植体段神经束面积、有髓神经纤维密度、直径、轴突直径等，做统计学处理。结果静脉体、基底膜、许旺细胞和神经生长因子复合移植组的上述指标与自体神经移植组比较，差异无显著性(P＞0.05)，与静脉和人羊膜基底膜组比较，差异有显著性(P＜0.05)。结论（1 ）静脉体、基底膜、许旺细胞和神经生长因子复合移植起到了自体神经移植桥接神经缺损的作用；（2）移植体内有较粗大成熟的有髓神经纤维轴突再生为有效神经再生，再生有髓神经纤维直径和轴突直径与功能恢复密切相关。     

静脉体基底膜许旺细胞和NGF合移植桥接神经缺损的实验研究

目的 观察静脉体、基底膜、许旺细胞和神经生长因子复合移植桥接神经缺损的作用。方法 采用健康白兔40只，分为4组，每组10条坐骨神经。A组为静脉体、基底膜、许旺细胞和神经生长因子复合移植组，B组为自体静脉组，C组为人羊膜基底膜组，D组为自体神经移植组。均修复坐骨神经1.5cm缺损，术后3个月观察肢体运动，肌电图动作电位波幅、潜伏期和传导速度，工常规HE染色、免疫组化SP法梁色显示髓磷脂碱性蛋白（MBP）及Cajal吡啶银染色，镜下观察移植体段神经束面积、有髓神经纤维密度、直径、轴突直径等，做统计学处理。结果 静脉体、基底膜、许旺细胞和这在子复合移植组的上述指标与自体神经移植组比较，差异无显著性（P＞0.05），与静脉和人羊膜基底膜组比较，差异有显著性（P＜0.05）。结论 （1）静脉体、基底膜、许旺细胞和神经生长因子复合移植起到了自体神经移植桥接神经缺损的作用；（2）移植体内有较粗大成熟的有髓神经纤维轴突再生为有效神经再生，再生有髓神经纤维直径和轴突直径与功能恢复密切相关。     

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。     

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。     

雪旺细胞复合细胞外基质凝胶移植对神经元逆行性死亡保护作用的研究

目的　雪旺细胞 (SC)和细胞外基质 (ECM)凝胶复合 ,修复周围神经缺损 ,研究 SC的成活和对背根神经节内神经元逆行性死亡的保护作用。　方法　 1将 SC制成 1× 10 8/ ml的 ECM凝胶后 ,置入 PL A中空纤维管中 ,修复大鼠 10 mm坐骨神经缺损 ,以单纯 ECM凝胶组和生理盐水组作为对照。术后 8、 12周 ,组织学检测 ,评价其效果。 2将Brd U标记的 SC复合 ECM凝胶后 ,置入 PL A中空纤维管中 ,修复大鼠坐骨神经缺损 ,术后 3、6周取材 ,Brd U免疫组织化学染色 ,观察 SC存活情况。　结果　 1SC和 ECM凝胶复合移植 ,多数细胞可存活至 3周 ,部分细胞可存活至 6周。2 SC和 ECM凝胶复合组、ECM凝胶组 ,L5背根神经节内成活的神经元数量分别是健侧的 83.5 %和 81.3% ,显著高于单纯生理盐水组的 72 .9% (P<0 .0 5 )。　结论　 SC和 ECM凝胶复合移植 ,多数细胞能成活并能使背根神经节内 83.5 %神经元受到保护而免于死亡     

A new artificial nerve graft containing rolled Schwann cell monolayers

This study hypothesized that introducing high numbers of Schwann cells in monolayers via a novel rolled graft architecture would promote robust nerve regeneration. The objective was to place adherent Schwann cells in artificial nerve grafts and to assess regeneration through the Schwann cell-laden grafts compared with that through acellular grafts and autografts. Schwann cells were isolated from neonatal Fisher rats. Small intestinal submucosa (SIS) was harvested from adult Fisher rats, cut into 7 mm x 8 cm pieces, and pinned out. Schwann cells were plated onto the strips, allowed to reach confluence, and subsequently rolled into a laminar structure and implanted across a 7-mm gap in the rat sciatic nerve (n = 12). Control animals received SIS conduits without Schwann cells (n = 11) or autograft repair (n = 12). At 10.5 weeks, functional regeneration through the Schwann cell-laden grafts, measured by both sciatic function index and extensor postural thrust testing, exceeded that through the cell-free grafts and approached that achieved through autografts. These results highlight the role of Schwann cells in nerve regeneration. Regenerative results approaching autograft levels in the Schwann cell-laden group suggest that this methodology may ultimately be useful in clinical nerve repair.     

Spatially controlled delivery of neurotrophic factors in silk fibroin-based nerve conduits for peripheral nerve repair

Restoration with sufficient functional recovery after long-gap peripheral nerve damage remains a clinical challenge. Silk has shown clinical promise for numerous tissue engineering applications due to its biocompatibility, impressive mechanical properties, and Food and Drug Administration approval. The aim of this study was to evaluate the efficacy of silk fibroin--based nerve guides containing glial cell line-derived neurotrophic factor (GDNF) in a long-gap sized (15 mm) rat sciatic nerve defect model. Four groups of nerve conduits were prepared: (1) silk conduits with empty silk microspheres, (2) silk conduits with GDNF-loaded silk microspheres uniformly distributed in the conduit wall, (3) silk conduits with GDNF-loaded silk microspheres in a controlled manner with the highest GDNF concentration at the distal end, and (4) isograft. After 6 weeks, the nerve grafts were explanted, harvested, and fixed for histologic analysis. Nerve tissue stained with the S-100, and neuroendocrine marker PGP 9.5 antibodies demonstrated a significantly increased density of nerve tissue in the GDNF-treated groups compared with the empty microsphere (control) group (P < 0.05). GDNF-treated animals with a higher concentration of GDNF in the distal portion possessed a significantly higher density of PGP 9.5 protein middle conduit part than comparison to GDNF uniform-treated animals (P < 0.05). Silk-based nerve conduits possess optimal mechanical and degradative properties, rendering them potentially useful in peripheral nerve repair. This study demonstrates that novel, porous silk fibroin--based nerve conduits, infused with GDNF in a controlled manner, represent a potentially viable conduit for Schwann cell migration and proliferation in the regeneration of peripheral nerves.     

Sciatic nerve repair with tissue engineered nerve: Olfactory ensheathing cells seeded poly(lactic-co-glygolic acid) conduit in an animal model

Background and Aim:

Synthetic nerve conduits have been sought for repair of nerve defects as the autologous nerve grafts causes donor site morbidity and possess other drawbacks. Many strategies have been investigated to improve nerve regeneration through synthetic nerve guided conduits. Olfactory ensheathing cells (OECs) that share both Schwann cell and astrocytic characteristics have been shown to promote axonal regeneration after transplantation. The present study was driven by the hypothesis that tissue-engineered poly(lactic-co-glycolic acid) (PLGA) seeded with OECs would improve peripheral nerve regeneration in a long sciatic nerve defect.

Materials and Methods:

Sciatic nerve gap of 15 mm was created in six adult female Sprague-Dawley rats and implanted with PLGA seeded with OECs. The nerve regeneration was assessed electrophysiologically at 2, 4 and 6 weeks following implantation. Histopathological examination, scanning electron microscopic (SEM) examination and immunohistochemical analysis were performed at the end of the study.

Results:

Nerve conduction studies revealed a significant improvement of nerve conduction velocities whereby the mean nerve conduction velocity increases from 4.2 ΁ 0.4 m/s at week 2 to 27.3 ΁ 5.7 m/s at week 6 post-implantation (P < 0.0001). Histological analysis revealed presence of spindle-shaped cells. Immunohistochemical analysis further demonstrated the expression of S100 protein in both cell nucleus and the cytoplasm in these cells, hence confirming their Schwann-cell-like property. Under SEM, these cells were found to be actively secreting extracellular matrix.

Conclusion:

Tissue-engineered PLGA conduit seeded with OECs provided a permissive environment to facilitate nerve regeneration in a small animal model.     

Sciatic nerve regeneration by microporous nerve conduits seeded with glial cell line-derived neurotrophic factor or brain-derived neurotrophic factor gene transfected neural stem cells

Neurotrophic factors such as the glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) promote nerve cell survival and regeneration, but their efficacy in repairing a longer gap defect of rat sciatic nerve (15 mm) has not been established. In this study, two recombinant mammalian vectors containing either rat GDNF gene or BDNF gene were constructed and each was transfected into neural stem cells (NSCs). It was found that the transfection of GDNF or BDNF gene into NSCs led to significantly enhanced expression of GDNF or BDNF mRNA. The amount of GDNF or BDNF protein secreted from the transfected NSCs showed a 3.3-fold or 2.5-fold increase than that from nontransfected NSCs, respectively. The regeneration capacity of rat sciatic nerve in a poly(D,L-lactide) conduit seeded with GDNF or BDNF-transfected NSCs was evaluated by the histology, functional gait, and electrophysiology after 8 weeks of implantation. It was observed that the degree of myelination and the size of regenerated tissue in the conduits seeded with GDNF- and BDNF-transfected NSCs were higher than those seeded with the nontransfected NSCs. Conduits seeded with GDNF-transfected NSCs had the greatest number of blood vessels. The functional recovery assessed by the functional gait and electrophysiology was significantly improved for conduits seeded with GDNF or BDNF-transfected NSCs. It was concluded that the genetically modified NSCs may have potential applications in promoting nerve regeneration and functional recovery.     

组织工程化人工神经修复长段神经缺损实验的初步报告

目的 研究组织化人工神经修复大鼠2．5cm长坐骨神经缺损的效果。方法 90只2个月月龄的Lewis1W雌性大鼠,按手术先后顺序随机分成3个神经移植组,每组30只。A组：用种植同源雪旺细胞并具有内部支架结构的胶原神经管桥接,即组织工程化人工神经组。B组：用无雪旺细胞但具有内部支架结构的胶原神经管桥接,即对照组。C组：自体神经移植组。术后6月,进行神经电生理监测,神经肌肉组织学观察;用S－100和神经微丝蛋白免疫组化染色后,行轴突计数等检测。结果 完成对21只大鼠（每组7只）的实验评估。从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）,再生轴突已通过移植段神经全长,远端肌肉轻度萎缩。B组中则没有或仅记录到波幅很低的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 组织工程化人工神经可用来修复大鼠长段神经缺损。     

嗅鞘细胞-胶质细胞源性神经营养因子基因工程细胞移植对坐骨神经再生的作用

目的　研究胶质细胞源性神经营养因子 (GDNF)基因修饰的嗅鞘细胞 (OECs)移植对周围神经再生的作用。方法　将 40只SD大鼠随机分为 4组 ,每组 10只 ,切除 3mm右侧坐骨神经并用硅胶管套接修复 ,管内分别给予生理盐水、体外培养纯化的OECs、GDNF和OECs GDNF基因工程细胞 ,术后 6周 ,分别行电生理、组织学检查和辣根过氧化物酶 (HRP)逆行示踪 ,观察各种处理对神经元存活和纤维再生的影响。结果　术后 6周 ,各组修复神经均有不同程度再生。电生理及组织化学结果显示治疗组优于对照组 ,差异有显著性。结论　OECs GDNF移植能促进周围神经再生 ,且效果优于单纯OECs或GDNF移植。     

A Novel Approach to Align Adult Neural Stem Cells on Micropatterned Conduits for Peripheral Nerve Regeneration: A Feasibility Study

There is a strong need for nerve-tissue engineering using the guide conduit and Schwann cells or neural stem cells (NSCs) with regeneration potential for injured peripheral nerves. In this study, micropatterned poly( d , l -lactide) (PLA) conduits were fabricated by microlithography and solvent-casting. The PLA conduits were seeded with the novel green fluorescent protein (GFP)-positive adult mouse NSCs obtained using the patented method of one of the authors. About 85% of the seeded NSCs were successfully aligned on the micropatterned conduits within 72 h and expressed the genes related to the production of neurotrophic factors. Gene expressions for the neurotrophic factors, such as nerve growth factor and brain-derived neurotrophic factor were upregulated by the micropatterned conduits at 72 h. The micropatterned PLA conduits seeded with the aligned NSCs were used to bridge the 10-mm sciatic nerve gaps in rats and were found to facilitate nerve repair and functional recovery during a period of 6 weeks compared with the nonseeded group. This model can be used to study the role of adult NSCs in peripheral-nerve regeneration in the future.