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。     

冻融联合优化化学法制备粗大去细胞同种异体神经

背景：异体神经移植修复神经缺损需要面对和解决的是宿主免疫排斥反应问题。因此,如何避免、减轻免疫排斥反应是同种异体神经移植获得成功的关键因素。 目的：探索新的异体神经预处理方法,清除犬周围神经中的许旺细胞和髓鞘,保留完整的基底膜,建立粗大异体神经移植物的预处理方法,获得去细胞异体神经移植物。 方法：取健康成年杂种犬游离双侧坐骨神经,以冻融联合优化化学法对神经进行预处理,光、电镜观察其结构特征,组织学染色及Western blot分析其成分。 结果与结论：预处理后的去细胞神经的延展性和神经外膜的弹韧性良好,许旺细胞和髓鞘被彻底清除,基底膜保留完整,去细胞神经为一没有细胞、髓鞘及其碎片的空的神经基膜管。结果表明该方法有效的清除了周围神经中主要抗原成分许旺细胞及髓鞘,并且保留了促神经再生的重要成分基底膜,可作为制备组织工程化神经较理想的方法。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;组织工程全文链接：     

种植施万细胞的脱细胞同种异体神经移植物对大鼠坐骨神经缺损的修复作用

目的观察种植施万细胞的脱细胞同种异体神经移植物,桥接大鼠坐骨神经缺损后的神经再生。方法应用酶反复消化法与差速贴壁法体外分离培养乳鼠施万细胞;显微注射法将施万细胞种植到脱细胞同种异体神经移植物内;再应用种植施万细胞的脱细胞同种异体神经移植物桥接大鼠坐骨神经10 mm缺损。光镜、透射电镜和扫描电镜观察再生神经的形态结构、有髓神经纤维数量、平均髓鞘厚度并进行统计学分析。结果光、电镜观察到实验组(SCs+ARSN)的施万细胞在再生神经纤维中互相连结纵行排列成类似Büngner带样细胞链,对照组(ARSN)未见到施万细胞的链状排列。实验组再生有髓神经纤维的髓鞘厚度较对照组均匀且较厚,有髓神经纤维数量和平均髓鞘厚度明显多于对照组(P<0.05)。结论种植施万细胞的脱细胞同种异体神经移植物对缺损的坐骨神经再生有更加有效的促进作用。     

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。     

去细胞神经与自体神经移植桥接修复大鼠坐骨神经缺损的对比研究

目的观察同种异体去细胞神经与自体神经移植桥接修复大鼠坐骨神经缺损的神经再生情况。方法制备大鼠同种异体去细胞神经及大鼠坐骨神经缺损模型,修复12周后应用HE染色,Bielschowsky改良染色,Weil氏铁明矾苏木素染色,光镜下观察神经外膜上的微血管数和微血管面积百分比,计数单位面积的轴突数目,远端轴突密度/近端轴突密度为再生神经通过率,计数单位面积的有髓神经纤维数目和有髓神经纤维的直径。结果在坐骨神经纤维的再生神经纤维通过率、有髓神经纤维密度和直径、桥接体微血管的数目和微血管面积百分比等再生指标上,自体神经移植组略优于化学去细胞神经组。结论同种异体去细胞神经移植可促进神经再生,但仍然不如自体移植效果好。     

脱细胞同种异体神经移植物促进大鼠运动功能恢复的实验研究

目的　检测脱细胞同种异体神经移植物桥接大鼠坐骨神经 10mm缺损后运动功能的恢复。方法　用脱细胞同种异体神经移植物桥接大鼠坐骨神经 10mm缺损 ,术后 12周、16周运用电生理及AchE结合镀银染色检测再生神经传导速度和腓肠肌的运动终板。自体神经移植作为对照组。结果　实验组术后 12周、16周再生神经传导速度与对照组相比 ,无显著性差异 ;术后 12周组织化学染色可见腓肠肌内有呈AchE阳性的运动终板 ;16周运动终板AchE阳性反应加深 ,并整齐地排列于腓肠肌的中上部形成终板带 ,经结合镀银染色后可见再生的神经束及发出的分支与运动终板相连。结论　脱细胞同种异体神经移植物桥接大鼠坐骨神经缺损具有促进其运动功能恢复的作用     

脱细胞异体神经移植物桥接大鼠坐骨神经缺损促进神经-肌结构重建和功能恢复的实验研究

目的 探讨脱细胞同种异体神经移植物桥接大鼠坐骨神经缺损对神经-肌结构重建和功能恢复的作用。方法用脱细胞同种异体神经移植物桥接大鼠坐骨神经10mm缺损;称量术后12周、24周实验组手术侧胫骨前肌湿重,并与对照组术侧该肌湿重进行比较;用电生理学方法检测再生神经传导速度及其对胫骨前肌的再支配作用;用AChE和AChE结合镀银染色观察胫骨前肌内神经和运动终板的再生。结果 术后12周、24周实验组胫骨前肌湿重与对照组相比无显差异;再生神经传导速度与对照组相比,也无显差异;术后12周肌内见有AChE阳性反应的运动终板;24周运动终板AChE阳性反应加深,并整齐地排列于胫骨前肌的中上部形成终板带,经结合镀银染色后可见再生的神经束及其发出的分支与运动终板相连;肌电显示再生神经已支配胫骨前肌的运动。结论 脱细胞同种异体神经移植物桥接大鼠坐骨神经缺损具有促进神经一肌结构重建和运动功能恢复的作用。     

干细胞与脱细胞异体神经支架在周围神经长段缺损中的应用

背景:将种子细胞植入合适的载体支架可以构建具有生物活性及相应功能的组织工程神经桥接物,干细胞与脱细胞异体神经构成的组织工程移植物正成为周围神经长段缺损研究领域的重要移植材料,并已初步显示出良好的应用前景。目的:综述近年来干细胞与脱细胞异体神经支架在周围神经长段缺损中的应用。方法:第一、二作者应用计算机检索1998年1月至2014年2月PubMed数据库、中国期刊全文数据库有关干细胞与脱细胞异体神经支架在周围神经长段缺损中应用的文章,英文检索词"stem cells,peripheral nerve defect,acellular allogeneic nerves";中文检索词"干细胞,周围神经缺损,脱细胞异体神经"。共检索到1 013篇相关文献,其中97篇文献符合纳入标准。结果与结论:干细胞因组织损伤后释放的各种趋化因子吸引以及其自身趋化作用聚集到损伤部位,分泌大量的营养物质,促进机体损伤神经功能的修复。干细胞可以在周围环境的诱导和内在分化偏向共同作用下分化并代替人体内损伤或死亡的神经细胞。此外,干细胞联合组织工程材料移植,减少胶质瘢痕的形成也是促进周围神经损伤修复的因素。干细胞可以增强神经突触之间的联系,建立新的神经环路。神经干细胞具有分化为其他神经细胞的潜力,但其分化与调控的确切机制尚不明了。对于如何改善微环境,使更多的干细胞分化为神经元与少突胶质细胞并维持细胞活性尚缺乏有效的方法。故有效的抑制移植早期的免疫排斥反应,应成为研究的重点所在。神经移植后如何提高神经再生速度和质量,维持靶器官的组织结构与功能更需要长期的摸索。     

异种神经脱细胞移植物修复周围神经缺损的实验研究

目的 探讨异种神经脱细胞移植物桥接大鼠坐骨神经缺损后的神经再生及其再生过程中免疫排斥反应. 方法用脱细胞兔周围神经作为移植物桥接大鼠坐骨神经1 cm缺损;术后3、5、8、11、15天检测血液中淋巴细胞占白细胞百分比;3个月后取移植物及腓肠肌,用甲苯胺蓝、乙酰胆碱酯酶（AchE）、琥珀酸脱氢酶（SDH）组化染色,光、电镜观察神经再生及腓肠肌运动终板的恢复情况. 结果术后大鼠血液中淋巴细胞占白细胞的百分比与正常大鼠相比较无显著性差异,3个月后大鼠术侧下肢足趾能分开,行走时后蹬动作有力,针刺足底有逃避反应,桥接物内见有大量再生的坐骨神经纤维,腓肠肌肌纤维上见有呈AchE阳性的运动终板和神经纤维.结论 异种神经脱细胞移植物桥接大鼠坐骨神经缺损具有促进其再生的作用.     

神经束膜细胞的发育及其在周围神经损伤修复中的作用

正周围神经的膜性结构包括神经外膜、神经束膜和神经内膜。其中,神经束膜包裹着神经内大小不等的神经纤维束。神经束膜又分内、外两层,外层为结缔组织,内层由多层扁平上皮样细胞构成,称神经束膜细胞。束膜细胞之间有紧密连接,每层上皮样细胞都有基膜[1]。传统观点认为神经束膜上皮仅仅对进出神     

组织工程化脱细胞神经基质移植体的研究进展

组织工程化的细胞外基质成分、细胞或通道的基底物质已经显示出支持轴突再生和功能恢复的巨大潜能。脱细胞处理的神经基质移植体具有良好的仿生性和生物相容性,包含较丰富的促进神经生长的蛋白等成分并适合于种子细胞在其内迁移生长,移植后免疫排斥反应较低,为轴突再生提供适宜的微环境,能够有效地促进轴突再生,是修复周围神经缺损的适宜移植物,将在周围神经损伤修复中占明显优势。     

Bone marrow stromal cells differentiated into functional Schwann cells in injured rats sciatic nerve

Bone marrow stromal cells (MSCs) have been shown to differentiate into various lineage cells including neural cells in vitro and in vivo. We therefore examined whether MSCs can differentiate into Schwann cells in injured peripheral nerves, After cultured in vitro, PKH-67-labeled MSCs were injected into the mechanically injured rat sciatic nerves. Three weeks after injection, immunofluorescent examinations were carried out. MSCs had been incorporated around the injured nerves and differentiated into Schwann cells. MSCs had accumulated mainly in the epineurium around the injured nerve. The incorporated cells partially expressed GFAP, S-100, and P75. These results confirmed the possibility that MSCs have the ability to differentiate into Schwann cells, and that injection of MSCs into the injured peripheral nerve would help repair damaged nerve.     

An anatomical study of porcine peripheral nerve and its potential use in nerve tissue engineering

Current nerve tissue engineering applications are adopting xenogeneic nerve tissue as potential nerve grafts to help aid nerve regeneration. However, there is little literature that describes the exact location, anatomy and physiology of these nerves to highlight their potential as a donor graft. The aim of this study was to identify and characterise the structural and extracellular matrix (ECM) components of porcine peripheral nerves in the hind leg. Methods included the dissection of porcine nerves, localisation, characterisation and quantification of the ECM components and identification of nerve cells. Results showed a noticeable variance between porcine and rat nerve (a commonly studied species) in terms of fascicle number. The study also revealed that when porcine peripheral nerves branch, a decrease in fascicle number and size was evident. Porcine ECM and nerve fascicles were found to be predominately comprised of collagen together with glycosaminoglycans, laminin and fibronectin. Immunolabelling for nerve growth factor receptor p75 also revealed the localisation of Schwann cells around and inside the fascicles. In conclusion, it is shown that porcine peripheral nerves possess a microstructure similar to that found in rat, and is not dissimilar to human. This finding could extend to the suggestion that due to the similarities in anatomy to human nerve, porcine nerves may have utility as a nerve graft providing guidance and support to regenerating axons.     

Peripheral nerve regeneration using acellular nerve grafts

Long gap peripheral nerve injuries usually require a graft to facilitate axonal regeneration into the distal nerve stump. The use of autografts is often limited because of graft availability and donor-site morbidity. We investigated whether acellular nerve allografts would provide an appropriate channel for the promotion and induction of sciatic nerve regeneration in rats. Axons sprouted from the proximal portion and reached the distal portion in the 1 cm-long grafts by 1 month. The number of axons in the regenerated nerves was similar to that of normal nerves at 1 month. Loading the grafts with betaNGF and VEGF increased the number and mean diameter of axons and neovascularization in the regenerated nerves at 1 month. The motor conduction velocity increased over time and reached 63 +/- 10% of that of normal nerves at 6 months. The nerve injuries treated with the acellular grafts had a significant improvement in motor, nociception, and proprioception function compared to untreated nerves. The results from this study suggest that acellular nerve allografts may be a useful biomaterial for functional peripheral nerve regeneration.     

同种异体神经复合体修复兔周围神经缺损

背景：应用种植许旺细胞的去细胞同种异体神经复合体修复周围神经缺损,探索其对神经再生及功能恢复有更好的促进作用,并且免疫原性非常小。 目的：用种植胎兔许旺细胞的去细胞同种异体神经复合体修复兔缺损的坐骨神经,观察移植神经周围免疫细胞的变化及功能恢复。 　 方法：48只新西兰白兔随机分成实验组和对照组。两组动物均切除一段坐骨神经,造成2.0 cm长的缺损,实验组用种植胎兔许旺细胞的同种异体神经复合体修复坐骨神经;对照组仅用去细胞同种异体神经修复。移植后1,4,8周光镜观察移植段坐骨神经周围肌肉组织中免疫细胞的浸润情况,计数每个高倍视野免疫细胞的数量。移植后4,8,16周大体观察兔的足部溃疡形成及愈合情况,大体观察神经愈合情况;肌电图检查桥接段坐骨神经的传导速度。 结果与结论：手术区局部均未出现明显的排斥反应,实验组足部溃疡愈合情况优于对照组。移植后1周移植段坐骨神经周围肌肉组织中有大量淋巴细胞及巨噬细胞浸润,实验组明显多于对照组(P < 0.05);移植后4周,浸润的免疫细胞两组均较1周后明显减少,实验组减少更明显。移植后8周,浸润的免疫细胞更加减少,但两组间比较差异无显著性意义(P > 0.05)。移植后4周时,两组均未见明显的神经传导,8,16周神经传导速度实验组均优于对照组(P < 0.05)。提示,种植许旺细胞的去细胞同种异体神经复合体免疫原性非常小,对神经再生及功能恢复有更好的促进作用。     

In vivo proliferation,migration and phenotypic changes of Schwann cells in the presence of myelinated fibers

Following injury to a peripheral nerve, changes in the behavior of Schwann cells help to define the subsequent microenvironment for regeneration. Such changes, however, have almost exclusively been considered in the context of Wallerian degeneration distal to an injury, where loss of axonal contact or input is thought to be critical to the changes that occur. This supposition, however, may be incorrect in the proximal stumps where axons are still in contact with their cell bodies. In this work, we studied aspects of in vivo Schwann cell behavior after injury within the microenvironment of proximal stumps of transected rat sciatic nerves, where axons are preserved. In particular we studied this microenvironment proximal to the outgrowth zone, in an area containing intact myelinated fibers and a perineurial layer, by using double immunolabelling of Schwann cell markers and 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells.In normal sciatic nerve, Schwann cells were differentiated, in an orderly fashion, into those associated with unmyelinated fibers that labeled with glial fibrillary acidic protein (GFAP) and those associated with myelinated fibers that could be identified by individual axons and myelin sheaths. After sciatic nerve transection, there was rapid and early expansion in the population of GFAP-labeled cells in proximal stumps that was generated in part, by de novo expression of GFAP in Schwann cells of myelinated fibers. Schwann cells from this population also underwent proliferation, indicated by progressive rises in BrdU and GFAP double labeling. Finally, this Schwann cell pool also developed the property of migration, traveling to the distal outgrowth zone, but also with lateral penetration into the perineurium and epineurium, while in intimate contact with new axons.The findings suggest that other signals, in the injured proximal nerve stumps, beyond actual loss of axons, induce 'mature' Schwann cells of myelinated axons to dedifferentiate into those that up-regulated their GFAP expression, proliferate and migrate with axons.     

Tissue-engineered peripheral nerve grafting by differentiated bone marrow stromal cells

Bone marrow stromal cells are multipotential stem cells that contribute to the differentiation of tissues such as bone, cartilage, fat and muscle. In the experiment, we found that bone marrow stromal cells can be induced to differentiate into cells expressing characteristic markers of Schwann cells, such as S-100 and glial fibrillary acidic protein, promoting peripheral nerve regeneration. Tissue-engineered bioartificial nerve grafting of rats by differentiated bone marrow stromal cells was applied for bridging a 10 mm-long sciatic nerve defect. Twenty-eight inbred strains of female F344 rats weighing 160 approximately 200 g were randomly divided into four nerve grafting groups, with seven rats in each group. Differentiated bone marrow stromal cell-laden group: poly(lactic-co-glycolic) acid tubes with an intrinsic framework were seeded with syngeneic bone marrow stromal cells which were induced for 5 days; Schwann cell-laden group: poly(lactic-co-glycolic) acid tubes with an intrinsic framework were seeded with syngeneic Schwann cells; acellular group: poly(lactic-co-glycolic) acid tubes were only filled with an intrinsic framework; autografts group. Three months later, a series of examinations was performed, including electrophysiological methods, walking track analysis, immunohistological staining of nerves, immunostaining of S-100 and neurofilament, and axon counts. The outcome indicated that bone marrow stromal cells are able to differentiate into Schwann-like cells and Schwann-like cells could promote nerve regeneration. Bone marrow stromal cells may be potentially optional seed cells for peripheral nerve tissue engineering because of abilities of promoting axonal regeneration.