Improvement of peripheral nerve regeneration by a tissue-engineered nerve filled with ectomesenchymal stem cells

Ectomesenchymal stem cells (EMSCs) originate from the cranial neural crest. They are a potential source of neuronal and Schwann cells (SCs) of the peripheral nervous system (PNS) during embryonic development. The third passage of EMSCs enzymatically isolated from the mandibular processes of Sprague-Dawley rats were cultured in forskolin and bovine pituitary extract for 6 days to generate functional Schwann cell phenotypes. Next, 10-mm defects in the sciatic nerves were bridged with an autograft, tissue-engineered nerve filled with differentiated cells in collagen, or a PLGA conduit alone in 18 rats, and the nerve defects of another four rats were left untreated. The regenerated nerves were evaluated by the sciatic functional index (SFI) monthly and by histological analysis 4 months after grafting. The recovery index of the sciatic nerve improved significantly in the autograft and tissue-engineered nerve groups, both of which were superior to the PLGA group. In animals transplanted with the EMSCs, there was greater regeneration than with conduit alone during the same period of implantation. These results show that when EMSCs are transplanted to a peripheral nerve defect they differentiate into supportive cells that contribute to the promotion of axonal regeneration.     

The use of undifferentiated bone marrow stromal cells for sciatic nerve regeneration in rats

In recent years, cell transplantation has become a focus of attention and reliable outcomes have been achieved in regeneration of the sciatic nerve. The effect of undifferentiated bone marrow stromal cells (BMSCs) on peripheral nerve regeneration was studied using a rat sciatic nerve regeneration model. A 10-mm sciatic nerve defect was bridged using an inside-out vein graft (IOVG) filled with undifferentiated BMSCs (2 × 10(7)cells/ml). In the control group, the vein was filled with phosphate buffer saline alone. The regenerated fibres were studied 4, 8 and 12 weeks after surgery. Assessment of nerve regeneration was based on functional (walking track analysis), histomorphometric and immunohistochemical (Schwann cell detection by S100 expression) criteria. The functional study confirmed significant recovery of regenerated axons in the IOVG/BMSC group (P<0.05). Quantitative morphometric analyses of regenerated fibres showed the number and diameter of myelinated fibres in the IOVG/BMSC group were significantly higher than in the control group (P<0.05). This demonstrates the potential for using undifferentiated BMSCs in peripheral nerve regeneration without the limitations of donor-site morbidity associated with isolation of Schwann cells. It also reduces costs because the interval between tissue collection and cell injection is reduced and the laboratory procedures are simpler compared to undifferentiated BMSCs.     

Sciatic nerve regeneration induced by transplantation of in vitro bone marrow stromal cells into an inside-out artery graft in rat

Traumatic injury to peripheral nerves results in considerable motor and sensory disability. Several research groups have tried to improve the regeneration of traumatized nerves by invention of favorable microsurgery. Effect of undifferentiated bone marrow stromal cells (BMSCs) combined with artery graft on peripheral nerve regeneration was studied using a rat sciatic nerve regeneration model. A 10-mm sciatic nerve defect was bridged using an artery graft (IOAG) filled with undifferentiated BMSCs (2 × 10⁷ cells/mL). In control group, the graft was filled with phosphated buffer saline alone. The regenerated fibers were studied 4, 8 and 12 weeks after surgery. Assessment of nerve regeneration was based on behavioral, functional (Walking Track Analysis), electrophysiological, histomorphometric and immuohistochemical (Schwann cell detection by S-100 expression) criteria. The behavioral, functional and electrophysiological studies confirmed significant recovery of regenerated axons in IOAG/BMSC group (P < 0.05). Quantitative morphometric analyses of regenerated fibers showed the number and diameter of myelinated fibers in IOAG/BMSC group were significantly higher than in the control group (P < 0.05). This demonstrates the potential of using undifferentiated BMSCs combined with artery graft in peripheral nerve regeneration without limitations of donor-site morbidity associated with isolation of Schwann cells. It is also cost saving due to reduction in interval from tissue collection until cell injection, simplicity of laboratory procedures compared to differentiated BMSCs and may have clinical implications for the surgical management of patients after facial nerve transection.     

Functional histopathological and morphometric study of the use of gangliosides in nerve regeneration in rats after axonotmesis

The purpose of the present functional, histopathological and morphometric study was to evaluate, qualitatively and quantitatively, the function of gangliosides (GM1, GD1a, GD1b e GT1b) in peripheral nerve regeneration. An experimental model was used with 96 male albino Wistar rats (Rattus norvegicus, Muridae). After the sciatic nerves of young adults had been crushed for 2 min using haemostatic tweezers, the rats were divided into experimental and control groups. The 48 animals in the experimental group received subcutaneous dorsal injections of gangliosides for 15 days after the surgical action, while the 48 control animals were injected with a saline solution. A functional, histopathological and morphometric evaluation of the sciatic function index (SFI) was made on 12 rats from both groups at 8, 15, 30 and 60 days. As a result of the methodology employed and the functional, histopathology and morphometric analyses, it was concluded that the administration of exogenous gangliosides seems to enhance nerve regeneration, because it stimulated the proliferation of Schwann cells and perhaps reduced the presence of inflammatory ones, seeming to promote nerve regeneration after axonotmesis.     

Prefabricated nerve conduits advance histomorphological and functional outcomes in nerve regeneration of the sciatic nerve of the rat

Bridging a nerve defect is sometimes necessary to achieve nerve regeneration after injury. Different methods and conduit designs have been considered, but only isograft transplants or prefabricated conduits are available. This study presents a comparison of prefabricated conduits and isograft transplants in rats, with the aim of making suggestions for clinical settings. In rats of inbred strains LEW and DA, a 1.5 cm defect of the sciatic nerve was reconstructed by isograft (n = 10) or conduit (n = 10). Untreated rats (n = 10), sham-operated rats (n = 10) and nerves of the non-operated contralateral limb served as controls. Regeneration was evaluated by histomorphological examination and with walking track analysis of the ankle stance angle (ASA) and the sciatic functional index (SFI). After 16 weeks, myelinization and ASA in the conduit group were significantly superior to that in the isograft group. There was no significant difference in SFI between the groups. Reconstruction in the isograft group showed a negative impact on the non-operated side. Conduits and isografts did not reach the morphological or functional levels of untreated or sham-operated animals. The results suggest preferential conduits should be used for nerve reconstruction.     

神经生长因子对下齿槽神经再生影响的实验研究

目的 探讨外源性神经生长因子（ｎｅｒｖｅ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ＮＧＦ）对成年大白兔下齿槽神经再生的影响。方法 选用２４只成年日本大耳白兔,在双侧下齿槽神经各造成８ｍｍ缺损,神经近远端用硅胶管桥接,右侧硅小室 内注入外源性ＮＧＦ 作实验侧,左侧注入生理盐水为对照侧。对实验侧和对照侧的再生有髓神经纤维数目、传导速度、纤维横断面积和髓鞘厚度作相应比较。结果 ①ＮＧＦ能使有髓神经纤维数目明显增加;     

The effects of low level laser treatment on recovery of nerve conduction and motor function after compression injury in the rat sciatic nerve

An animal study is presented examining the effect of low level laser (LLL) treatment on nerve regeneration following axonotmesis. Twenty animals received a standardised injury to the right sciatic nerve using a time, load and length sequence (10 min, 150 N. 5 mm) known to cause extensive axonal degeneration of the rat sciatic nerve. The LLL treatment was administered using a hand-held laser probe in light contact with the skin on the dorsal aspect of the hind leg overlying the site of the axonotmesis injury to the sciatic nerve. A group of 10 animals were treated with 6J of LLL (GaAlAs 830 nm) daily for a period of 28 d. Ten more animals were treated daily with a sham exposure setting and served as controls. Nerve function was assessed by a recognised method of walking tract print analysis; the "Sciatic Functional Index" (SFI), and nerve regeneration was assessed by recording the evoked compound action potentials (cAP) in the common peroneal nerve. At 21 d post-injury, the laser-treated group had a significantly lower median SFI than the sham laser-treated group, indicating that the real laser treatment had improved functional recovery in the nerve. However, no differences were found between the evoked cAP parameters that were measured in the laser-treated and sham laser-treated groups. Histological examination reiterated the lack of difference between the two groups. Consequently, the effects of LLL on recovery must have occurred more peripherally to the point measured.     

纤维蛋白胶联合几丁聚糖-胶原导管修复兔面神经损伤的效果

目的:探讨纤维蛋白胶联合几丁聚糖-胶原导管修复兔面神经损伤的效果。方法:30只健康中国大耳白兔随机分成3组,分别为显微外科吻合组(Ⅰ组)、纤维蛋白胶粘合组(Ⅱ组)、纤维蛋白胶联合几丁聚糖-胶原导管粘合组(Ⅲ组)。手术制作兔右侧面神经下颊支损伤模型,分别采用显微外科吻合、纤维蛋白胶粘合以及在几丁聚糖-胶原导管内用纤维蛋白胶粘合技术进行修复。术后16周进行大体观察、神经电生理检测、组织学观察、图像分析,采用SPSS 17.0统计软件对各组数据进行方差分析(one way ANOVA),以评价神经再生恢复情况。结果:16周时肉眼观察3组吻合口处均见神经再生。几丁聚糖-胶原导管吸收明显,能抑制吻合口周围纤维结缔组织形成。Ⅰ组再生神经周围软组织粘连较Ⅱ组和Ⅲ组严重;3个组神经肌肉功能恢复良好,口轮匝肌动作电位潜伏期和复合神经肌肉动作电位振幅(M波)检测结果经统计学分析无统计学意义(P>0.05);组织学观察3组均可见不同程度纤维结缔组织和新生毛细血管增生及炎性细胞浸润,无论是HE低倍镜还是Gomori银染高倍镜观察,纤维蛋白胶和几丁聚糖-胶原导管作用明显;图像分析结果提示3组轴突再生率不同(P<0.05),Ⅱ组、Ⅲ组均高于Ⅰ组,其中Ⅱ组明显高于Ⅰ组且差异有统计学意义(P<0.01);3个组再生轴突恢复比相似(P>0.05)。结论:几丁聚糖-胶原导管生物相容性良好,与纤维蛋白胶联合应用修复兔面神经损伤操作简便、效果肯定。     

Uncultured undifferentiated adipose-derived nucleated cell fractions combined with inside-out artery graft accelerate sciatic nerve regeneration and functional recovery

Effects of transplantation of adipose-derived nucleated cell fractions (ADNCs) on sciatic nerve regeneration were studied. A 10-mm sciatic nerve defect was bridged using artery graft filled with ADNCs. In control group, artery graft was filled with saline alone. Regenerated nerve fibres were studied for 12 weeks. In sham-operated group, sciatic nerve was only exposed and manipulated. Behavioural and functional studies confirmed faster recovery of regenerated axons in ADNCs transplanted animals than in control group (P < 0.05). At the end of study period, animals in ADNCs transplanted group achieved a sciatic functional index (SFI) value of −31.6 ± −3.14, whereas in control group a value of −42.5 ± −3.7 was found. Gastrocnemius muscle mass in ADNCs transplanted animals was found to be significantly higher than that in control group (P = 0.001). Morphometric indices of regenerated fibres showed the number and diameter of myelinated fibres to be significantly higher in ADNCs transplanted animals than in control group (P = 0.001). On immunohistochemistry, there was more positive staining of S100 in the ADNCs transplanted animals than in control group. ADNCs transplantation into an artery graft could be considered a readily accessible technique that improves functional recovery of sciatic nerve.     

医用OB胶联合几丁聚糖-胶原导管修复兔面神经损伤的实验研究

目的:探讨医用OB胶联合几丁聚糖--胶原导管修复兔面神经损伤的效果.方法:30只健康中国大耳白兔随机分为3组,分别为显微外科吻合组(1组)、医用OB胶黏合组(Ⅱ组)和医用OB胶几丁聚糖--胶原导管黏合组(Ⅲ组).手术制作兔右侧面神经下颊支损伤模型,分别采用显微外科吻合、医用OB胶黏合以及在几丁聚糖--胶原导管内OB胶黏合技术进行修复.术后16周进行大体观察、神经电生理检测、组织学观察、图像分析,采用SPSS 17.0软件包对各组数据进行方差分析,评价神经再生恢复情况.结果:16周时,肉眼观察3组吻合口处均见神经再生,可抵抗一定拉力,几丁聚糖--胶原导管吸收明显,能抑制吻合口周围纤维结缔组织形成,Ⅰ组和Ⅱ组再生神经周围软组织黏连较Ⅲ组严重;3组神经肌肉功能恢复良好,口轮匝肌动作电位潜伏期无显著差异(P>0.05),但Ⅲ组的复合神经肌肉动作电位振幅(M波)显著高于Ⅱ组(P<0.05),其余两两比较均无显著差异(P>0.05);组织学观察,3组均可见不同程度纤维结缔组织、新生毛细血管增生及炎性细胞浸润,无论是光学低倍镜还是Gomori银染色高倍镜观察,Ⅲ组的几丁聚糖--胶原导管作用明显;图像分析结果提示,3组轴突再生率不同(P<0.05),Ⅱ组、Ⅲ组均高于Ⅰ组,其中Ⅱ组显著高于Ⅰ组且差异显著(P<0.01);再生轴突恢复比不同(P<0.05),Ⅰ组显著高于Ⅱ组(P<0.05),其余两两比较无显著差异(P>0.05).结论:几丁聚糖--胶原导管生物相容性良好,与医用OB胶联合应用修复兔面神经损伤操作简便、效果肯定.     

Peripheral nerve regeneration following transection injury to rat sciatic nerve by local application of adrenocorticotropic hormone

The objective of this study was to assess local effect of adrenocorticotropic hormone (ACTH) on the functional recovery of the sciatic nerve in a transection model. Sixty male healthy white Wistar rats were randomized into four experimental groups of 15 animals each: In the sham-operated group (SHAM), the sciatic nerve was exposed and manipulated. In the transected group (TC), the left sciatic nerve was transected and the cut nerve ends were fixed in the adjacent muscle. In the silicone graft group (SIL) a 10-mm defect was made and bridged using a silicone tube. The graft was filled with phosphated-buffer saline alone. In the treatment group a silicone tube (SIL/ACTH) was filled with 10 μL ACTH (0.1 mg/mL). Each group was subdivided into three subgroups of five animals each and regenerated nerve fibres were studied at 4, 8 and 12 weeks post operation. Behavioral testing, functional, gastrocnemius muscle mass and morphometric indices showed earlier regeneration of axons in SIL/ACTH than in SIL group (p < 0.05). Immunohistochemistry clearly showed more positive location of reactions to S-100 in SIL/ACTH than in SIL group. ACTH improved functional recovery and morphometric indices of sciatic nerve. This finding supports role of ACTH after peripheral nerve repair and may have clinical implications for the surgical management of patients after nerve transection.     

An experimental study on regeneration of the inferior alveolar nerve after lyophilized nerve homografting in the rabbit

This study was designed to evaluate the differences between the regenerative process in cases of autogenous nerve grafting and lyophilized homologous nerve grafting. Rabbit inferior alveolar nerves (10 mm lengths) were resected and replaced with lyophilized homologous segments from the sciatic nerve. On the opposite side, the resected nerves were autogenously grafted. The experimental subjects were divided into autogenous nerve-graft and the lyophilized nerve-graft groups. Results. 1. Regenerating axons appeared in the autogenous-graft group 2 weeks after the operation and 4 weeks after the operation in the homografted lyophilized group. The difference in regeneration between the 2 groups was significant. 2. Regenerating axons in the autogenously grafted nerves made contact with remaining Schwann cells and endneural tubes. Axons in the homografted lyophilized nerves invaded along newly infiltrated Schwann cells and empty tube skeletal structures. The number of regenerating axons from outside the skeletal structure was greater than the number of regenerating axons from inside the skeletal structure. 3. In the case of autogenous grafting, nerve fibers of diameters greater than 3 microns increased 66.7% after 24 weeks; the corresponding figure for homografted lyophilized nerves was 48.4%. 4. In instances of autogenous grafting, 16 weeks after surgery, the ratio of distal proximal myelinated nerve fibers had grown. In cases of homografted lyophilized nerves, this tendency to increase continued until the twenty-fourth postsurgical week. 5. In both groups, it remained possible to record nerve action potentials 12 weeks after surgery. The sensory nerve conduction velocity of autogenously grafted nerves increased gradually to approach control values 24 weeks after surgery. That of homografted lyophilized nerves recovered more slowly. 6. Increases in number of nerve fibers with a diameter of more than 3 microns were proportional to the rate at which sensory nerve conduction velocity recovered.     

Ultrastructural characteristics of axons in traumatic neuromas of the human lingual nerve

AIMS: To determine the ultrastructural characteristics of axons in traumatic neuromas of the human lingual nerve during the surgical removal of lower third molar teeth and to establish whether any characteristics were different between patients with dysesthesia and patients without dysesthesia. METHODS: Transmission electron microscopy was used to determine the ultrastructural morphological characteristics of human lingual nerve neuromas (n = 34) removed at the time of microsurgical nerve repair. From a sample population of myelinated and nonmyelinated fibers within the neuromas, fiber diameter, myelin thickness, g-ratio, and the number of mitochondria per axon were quantified. Comparisons were made with normal control lingual nerve specimens (n = 8) removed at the time of organ donor retrieval. RESULTS: Significant differences in ultrastructural morphology were found between the neuromas and control nerves. The neuromas contained a higher proportion of small (2- to 8-microm diameter) myelinated nerve fibers than controls, and the mean myelinated fiber diameter was significantly lower in neuromas than in controls. Mean myelin sheath thickness was significantly thinner in neuromas (0.6 +/- 0.1 microm) than in controls. However, the g-ratio, which is a measure of the myelination status of the nerve fibers in relation to their diameter, was found to be similar in each group, suggesting a normal process of myelination in the damaged axons. Nonmyelinated axon diameter was also significantly smaller in the neuromas than in the controls, and Schwann cells were found to sheathe more nonmyelinated axons in neuromas than in controls. The ratio of nonmyelinated to myelinated axons was significantly higher in neuromas than in controls. However, no significant differences were found between patients with dysesthesia and those without dysesthesia. CONCLUSION: Damage to the lingual nerve results in marked changes to axon diameter, myelin sheath thickness, and Schwann cell-axon relationships. These ultrastructural changes could contribute to the altered electrophysiological properties of axons trapped within neuromas. However, no significant differences in the ultrastructural characteristics studied were found between specimens from patients with or without symptoms of dysesthesia.     

兔骨髓间充质干细胞修复面神经缺损的初步研究

目的:初步观察骨髓间充质干细胞(MSCs)在面神经缺损修复中的作用。方法:抽取兔骨髓液经Percoll液离心得到单个核细胞,经体外培养、纯化后获得兔骨髓MSCs。15只新西兰白兔的两侧面神经上颊支分别造成12mm的缺损,一侧硅胶管套管连接后植入MSCs,另一侧单纯硅胶管套管。于术后第4、8、16周行大体观察,测定神经传导速度,处死动物后作组织学检查。结果:第4周时双侧管内未见明显再生神经纤维;第8周和16周时,双侧管内可见再生神经纤维。神经电生理测定和有髓神经纤维计数结果显示,MSCs侧优于单纯套管侧(P<0.05)。结论:MSCs可促进面神经缺损的修复,提示MSCs可应用于修复外周神经缺损。     

人牙髓干细胞来源外泌体促进周围神经损伤修复的实验研究

目的:研究人牙髓干细胞(human dental pulp stem cells,hDPSCs)来源外泌体对周围神经损伤修复的作用.方法:将36只成年SD大鼠随机分为2组,实验组为坐骨神经损伤后hDPSCs来源外泌体注射组,对照组为坐骨神经损伤后PBS注射组.分别于损伤后第3、7、14天观察大鼠的行为学改变,取材后进行...     

翻转静脉双桥接兔面神经缺损的实验研究

目的:探讨利用双桥接技术和翻转静脉再生室修复兔面神经缺损的可行性。方法:将实验用30只健康中国大耳白兔随机分成3组,分别为显微外科吻合组(Ⅰ组)、导管双桥接组(Ⅱ组)、翻转静脉双桥接组(Ⅲ组)。手术制作兔右侧面神经下颊支损伤模型,分别采用双桥接技术和原位神经吻合进行修复。术后16周进行大体观察、神经电生理检测、组织学观察、图象分析,以评价神经再生恢复情况。结果:16周时几丁聚糖—胶原再生室吸收明显且无异物反应,IOVG基本完全吸收形成类神经外膜样结构。3个组的神经传导速度(NCV)和复合神经肌肉动作电位振幅(M波)检测结果经统计学分析无显著性差异。图象分析结果提示3个组轴突再生率相同,再生轴突成熟度Ⅰ组与Ⅲ组均高于Ⅱ组而且差异有统计学意义(P<0.05)。结论:利用翻转静脉和双桥接技术联合修复周围神经缺损方法简单、效果肯定。     

Evaluation of nerve growth factor (NGF) treated mesenchymal stem cells for recovery in neurotmesis model of peripheral nerve injury

Background

Peripheral nerve damages are a relatively common type of the nervous system injuries. Although peripheral nerves show some capacity of regeneration after injury, the extent of regeneration is not remarkable. The present study aimed to evaluate the effect of NGF treated mesenchymal stem cells on regeneration of transected sciatic nerve.

应用纤维蛋白胶和医用OB胶修复兔面神经损伤的实验研究

目的:探讨用纤维蛋白胶或医用OB胶修复兔面神经的损伤效果。方法:手术制作实验用健康30只新西兰大耳白兔右侧面神经下颊支损伤模型,随机分成3组,分别为显微外科吻合组(Ⅰ组)、纤维蛋白胶粘合组(Ⅱ组)、医用OB胶粘合组(Ⅲ组),分别进行修复。术后16周进行大体观察、神经电生理检测、组织学观察、图像分析,评价神经再生恢复情况。结果:16周时肉眼观察3个组吻合口处均见神经再生,可抵抗一定拉力,Ⅰ组和Ⅲ组修复神经的周围软组织粘连较Ⅱ组严重;与Ⅰ组相比,Ⅱ组轴突再生率和再生轴突恢复比均有提高。结论:纤维蛋白胶和医用OB胶均有较好的生物相容性,可修复神经损伤,纤维蛋白胶对神经损伤的修复效果更佳。     

Advances in bioengineered conduits for peripheral nerve regeneration

Although resorbable NGCs have been developed for peripheral nerve grafting, there has been little published on their use as a material for trigeminal nerve repair. Advances in engineered guidance channels and modifications to the single-lumen conduit with growth-permissive substrates, ECM proteins, neurotrophic factors, and supportive Schwann or stem cells, and anisotropic placement of these within the NGC may translate from animal models to clinical human use in the future. A great deal of research is still needed to optimize the presently available NGCs, and their use in peripheral trigeminal nerve repair and regeneration remains yet to be explored. Bioengineered NGCs and additives remain promising alternatives to autogenous nerve grafting in the future. They can incorporate all of the developing strategies for peripheral nerve regeneration that develop in concert with the ever-increasing understanding of regenerative mechanisms. The use of nanomaterials also may resolve the numerous problems associated with traditional conduit limitations by better mimicking the properties of natural tissues. Since cells directly interact with nanostructured ECM proteins, the biomimetic features of anisotropic-designed nanomaterials coupled with luminal additive ECMs, neurotrophic factors, and Schwann cells may provide for great progress in peripheral nerve regeneration.     

胶质细胞源性神经营养因子在自体静脉桥修复面神经缺损中促进神经再生的效应

目的 旨在研究胶质细胞源性神经营养因子(GDNF)在自体静脉桥修复面神经缺损中促进神经再生的效应.方法选用36只成年雄性新西兰大白兔作为实验动物,切除双侧10mm长面神经颊支,制作面神经缺损动物模型.以同侧面后静脉作为神经再生管道,修复面神经缺损.静脉管腔内分别注入生理盐水(A组,n=16)或GDNF溶液(B组,n=1...