The advances in nerve tissue engineering: From fabrication of nerve conduit to in vivo nerve regeneration assays

Peripheral nerve damage is a common clinical complication of traumatic injury occurring after accident, tumorous outgrowth, or surgical side effects. Although the new methods and biomaterials have been improved recently, regeneration of peripheral nerve gaps is still a challenge. These injuries affect the quality of life of the patients negatively. In the recent years, many efforts have been made to develop innovative nerve tissue engineering approaches aiming to improve peripheral nerve treatment following nerve injuries. Herein, we will not only outline what we know about the peripheral nerve regeneration but also offer our insight regarding the types of nerve conduits, their fabrication process, and factors associated with conduits as well as types of animal and nerve models for evaluating conduit function. Finally, nerve regeneration in a rat sciatic nerve injury model by nerve conduits has been considered, and the main aspects that may affect the preclinical outcome have been discussed.     

神经生长因子和神经碎片套管对修复损伤神经的实验研究

目的探讨富含神经生长因子（NGF）和神经碎片的生物套管在周围神经损伤再生修复过程中的作用。 方法建立动物模型,将SD大鼠随机分成4组,分别为A对照组、B自体神经外膜小间隙吻合单纯添加自体神经碎片组、C自体神经外膜小间隙吻合同时添加NGF和自体神经碎片组、D生物套管吻合同时添加NGF和自体神经碎片组;观察各组一般形态、显微镜下吻合口处神经恢复形态以及组织学表现。采用方差分析比较各组有髓神经纤维数、施旺细胞数以及运动神经传导速度（MNCV）值的组间差异,组间的两两比较采用LSD-t检验。 结果（1）A组大鼠在第8周时足底溃疡出现明显自愈,右下肢开始出现自主活动,部分足底溃疡的大鼠在第12周时仍未出现明显的自愈,右下肢无活动;其余实验组出现右下肢足底溃疡的大鼠在第8周时出现明显的自愈,并且右下肢表现出自主活动。第12周时A组大鼠右下肢足趾仍然呈挛缩状态,不能自由活动,而其余3组右下肢足趾大部分恢复自主活动。（2）纵切面见B组、C组、D组所构建的小间隙管腔内均可见到再生神经纤维。第12周时,A组有髓神经纤维数目为（22.30±4.66）根／400视野,B组有髓神经纤维数目为（51.60±4.45）根/400视野,C组有髓神经纤维数目为（56.20±4.66）根/400视野,D组有髓神经纤维数目为（59.20±5.81）根/400视野,组间差异具有统计学意义（F=298.48,P<0.001）;抗S-100染色单位视野下,A组施旺细胞数目为（16.00±2.24）根/400视野,B组施旺细胞数目为（21.00±3.40）根/400视野,C组施旺细胞数目为（30.20±3.03）根/400视野,D组施旺细胞数目为（34.80±3.35）根/400视野,组间差异具有统计学意义（F=197.63,P<0.001）。术后第12周时,A组MNCV值为（7.57±1.79）m/s,B组MNCV值为（11.49±1.12）m/s,C组MNCV值为（13.86±2.03）m/s,D组MNCV值为（20.16±2.48）m/s,组间比较差异具有统计学意义（F=188.80,P<0.001）。D组对周围神经损伤的修复后的有髓神经纤维数目、施旺细胞数目以及MNCV均具有明显优势,与A组、B组、C组比较,差异均具有统计学意义（t=26.55、5.45、2.15,P<0.001、<0.001、=0.034;t=21.87、16.05、5.35,P均<0.001;t=23.19、15.97、11.60,P均<0.001）。 结论本实验证实自体神经碎片与NGF的联合应用,明显提高周围神经再生微环境中神经损伤修复效果。     

合成材料神经导管与自体神经移植修复周围神经缺损的比较

背景：生物可降解材料制成的神经导管可在体内降解,避免出现的神经卡压等问题,因而受到越来越多的关注. 目的：比较自体神经移植与3种合成可生物降解材料神经导管在修复周围神经损伤的效果差异. 方法：通过电生理学检测,形态学观察等神经恢复效果评价方法,对比分析近年来常用的胶原神经导管、DL-乳酸-ε-己内酯神经导管、聚乙醇酸神经导管与自体神经移植修复周围神经缺损的效果. 结果与结论：虽然神经导管与自体神经移植相比在理论上有其优势的一面,但不同合成材料的神经导管之间在神经功能恢复中存在明显差异性,DL-乳酸-ε-己内酯神经导管修复效果与自体神经移植无明显差异,是较为理想的神经导管材料,聚乙醇酸神经导管因自身的因素影响其降解性能,在3种神经导管中的修复周围神经损伤效果最差,胶原神经导管需要交联剂改善其机械性能,其修复周围神经损伤效果居于前两者之间,因此,这3种神经导管在神经功能再生方面还有潜在的缺陷,不能完全替代自体神经移植,而且3者之间的性价比,还缺少足够的大样本长期随机对照实验结果来验证,还需要进一步的实验观察.     

Functional recovery guided by an electrospun silk fibroin conduit after sciatic nerve injury in rats

The aim of this study was to evaluate the regenerative capacity of a newly developed nerve guidance conduit using electrospun silk fibroin (SFNC) implanted in a 10‐mm defect of the sciatic nerve in rats. After evaluating the physical properties and cytocompatibility of SFNC in vitro, rats were randomly allocated into three groups: defect only, autograft and SFNC. To compare motor function and abnormal sensation among groups, ankle stance angle (ASA) and severity of autotomy were observed for 10 weeks after injury. Immunostaining with axonal neurofilament (NF) and myelin basic protein (MBP) antibodies were performed to investigate regenerated nerve fibres inside SFNC. ASA increased significantly in the SFNC group at 1, 7 and 10 weeks after injury compared to the defect only group (p < 0.05). At one week, mean ASA of the SFNC group was significantly higher than that of the autograft group (p < 0.05). Onset and severity of autotomy decreased significantly in the SFNC group compared to other groups (p < 0.05). Autotomy in the SFNC group started at 4 weeks and maximally reached toe level. However, the defect only and autograft groups first showed autotomy at 2 and 1 weeks following injury, respectively, and then reached the sole level. Well myelinated nerve fibres stained with NF and MBP were found inside SFNC. In conclusion, SFNC could be helpful in restoring motor function and preventing abnormal sensations after nerve injury. Copyright © 2012 John Wiley & Sons, Ltd.     

Surgical repair of a 30 mm long human median nerve defect in the distal forearm by implantation of a chitosan-PGA nerve guidance conduit

This paper describes a clinical case study in which a chitosan/polyglycolic acid nerve guidance conduit (chitosan-PGA NGC) was utilized to repair a 30 mm long median nerve defect in the right distal forearm of a 55 year-old male patient. Thirty-six months after the nerve repair, the palm abduction of the thumb and the thumb-index digital opposition recovered, facilitating the patient to accomplish fine activities, such as handling chopsticks. Static two-point discrimination measured 14, 9 and 9 mm in the thumb, index and middle fingers of the right hand. Reproducible compound muscle action potentials were recorded on the right abductor pollicis. The ninhydrin test, a classical method for assessing sympathetic nerve function, showed partial recovery of the perspiration function of the injured thumb, index and middle fingers. This repair case suggested a possible strategy for the clinical reconstruction of extended defects in human peripheral nerve trunks by the implantation of chitosan-PGA NGCs.     

Regenerative effect of adipose tissue‐derived stem cells transplantation using nerve conduit therapy on sciatic nerve injury in rats

This study proposed a biodegradable GGT nerve conduit containing genipin crosslinked gelatin annexed with tricalcium phosphate (TCP) ceramic particles for the regeneration of peripheral nerves. Cytotoxicity tests revealed that GGT‐extracts were non‐toxic and promoted proliferation and neuronal differentiation in the induction of stem cells (i‐ASCs) derived from adipose tissue. Furthermore, the study confirmed the effectiveness of a GGT/i‐ASCs nerve conduit as a guidance channel in the repair of a 10‐mm gap in the sciatic nerve of rats. At eight weeks post‐implantation, walking track analysis showed a significantly higher sciatic function index (SFI) (P < 0.05) in the GGT/i‐ASC group than in the autograft group. Furthermore, the mean recovery index of compound muscle action potential (CMAP) differed significantly between GGT/i‐ASCs and autograft groups (P < 0.05), both of which were significantly superior to the GGT group (P < 0.05). No severe inflammatory reaction in the peripheral nerve tissue at the site of implantation was observed in either group. Histological observation and immunohistochemistry revealed that the morphology and distribution patterns of nerve fibers in the GGT/i‐ASCs nerve conduits were similar to those of the autografts. These promising results achieved through a combination of regenerative cells and GGT nerve conduits suggest the potential value in the future development of clinical applications for the treatment of peripheral nerve injury. Copyright © 2012 John Wiley & Sons, Ltd.     

穴位电针刺激对面神经再生过程中表情肌组织神经生长因子mRNA表达的影响

目的 观察穴位电针刺激对面神经再生过程中表情肌组织神经生长因子（ＮＧＦ）表达的影响。方法 日本大耳白兔面神经压榨伤后,电针刺激翳风、颧冢、地仓、颊车、四白、阳白、合谷穴,每天３０ｍｉｎ,２周为１疗程。经１、２、３疗程治疗后,在电生理学及病理形态学证实穴位电针刺激能促进面神经再生基础上,应用原位杂交及ＲＴ－ＰＣＲ技术检测地刺组及对照组作表情肌组织中ＮＧＦ ｍＲＮＡ表达水平的变化。结果 两组动物的保情     

A polylactic acid non‐woven nerve conduit for facial nerve regeneration in rats

This study developed a biodegradable nerve conduit with PLA non‐woven fabric and evaluated its nerve regeneration‐promoting effect. The buccal branch of the facial nerve of 8 week‐old Lewis rats was exposed, and a 7 mm nerve defect was created. A nerve conduit made of either PLA non‐woven fabric (mean fibre diameter 460 nm), or silicone tube filled with type I collagen gel, or an autologous nerve, was implanted into the nerve defect, and their nerve regenerative abilities were evaluated 13 weeks after the surgery. The number of myelinated neural fibres in the middle portion of the regenerated nerve was the highest for PLA tubes (mean ± SD, 5051 ± 2335), followed by autologous nerves (4233 ± 590) and silicone tubes (1604 ± 148). Axon diameter was significantly greater in the PLA tube group (5.17 ± 1.69 µm) than in the silicone tube group (4.25 ± 1.60 µm) and no significant difference was found between the PLA tube and autograft (5.53 ± 1.93 µm) groups. Myelin thickness was greatest for the autograft group (0.65 ± 0.24 µm), followed by the PLA tube (0.54 ± 0.18 µm) and silicone tube (0.38 ± 0.12 µm) groups, showing significant differences among the three groups. The PLA non‐woven fabric tube, composed of randomly‐connected PLA fibres, is porous and has a number of advantages, such as sufficient strength to maintain luminal structure. The tube has demonstrated a comparable ability to induce peripheral nerve regeneration following autologous nerve transplantation. Copyright © 2012 John Wiley & Sons, Ltd.     

神经预变性促进周围神经桥接后神经再生的初步研究

目的探讨经过体内预变性的神经用于周围神经缺损桥接修复的效果。方法 SD大鼠20只,制作右侧坐骨神经压榨伤动物模型,3 d后,取坐骨神经用于对侧行神经桥接,在桥接后0 d、3 d、7 d、14 d取材,应用ED1和NF200染色比较双侧神经再生速度及神经纤维内巨噬细胞浸入情况。结果压榨伤后3 d,远端神经纤维内见大量ED1染色阳性巨噬细胞侵入,NF200阳性染色成棒状或碎片状;神经桥接后3 d、7 d、14 d,预变性组与对照组桥接远端均可见大量ED1染色阳性巨噬细胞侵入,经过预变性的神经内神经再生速度明显提高。结论 经过体内预变性的神经用于周围神经缺损桥接修复可明显促进神经再生,其机制可能是巨噬细胞的早期侵入,有利于神经生长抑制物的清除。     

分米波对大鼠再生神经NGF mRNA表达的影响

目的研究分米波对周围神经损伤后再生神经中神经生长因子(NGF)表达的影响。方法选用60只SD大鼠，随机分为实验组和对照组各30只，均于右侧坐骨神经中段切除5mm神经组织，硅胶管桥接神经缺损，形成神经再生室，实验组术后进行局部分米波辐射。于术后1，2，4和8周取材，行大体和光镜观察，并进行免疫组化检测及RT-PCR定量分析。结果实验组NGF阳性染色颗粒及积分光密度值(IOD)明显高于对照组。实验组各时间点再生神经纤维中NGFmRNA的含量均明显高于对照组(P＜0、01)，同一神经纤维中．NGF mRNA表达量近侧明显高于远侧(P＜0．05)。实验组再生神经纤维中NGF mRNA于术后1周即有表达，术后2～4周达到高峰，术后8周后开始下降。结论分米波能增加神经纤维中NGF mRNA的表达，促进周围神经再生。     

Recent advances in de novo design strategy for practical lead identification

De novo design programs such as LEGEND, LUDI, and LeapFrog can identify novel structures that are predicted to fit the active site of a target protein. However, in the conventional de novo design strategy, the output structures obtained from the programs can be problematic with regard to synthetic accessibility and binding affinity prediction. Thus it has been practically difficult to obtain novel lead compounds that are appropriate for medicinal chemists through the de novo design strategy. Since the late 1990s, several new strategies for lead identification have been reported and the successful examples have been disclosed. One of the strategies is validation of small fragments, which can be substructures of de novo ligands, by using NMR, X-ray, or MS spectra. Another method is prioritization of output structures obtained from de novo design programs by chemical accessibility. This review describes these new strategies with practical applications and future perspectives of de novo design.     

Measurement of motor nerve conduction velocity of the sciatic nerve in patients with piriformis syndrome: a magnetic stimulation study

OBJECTIVE: To assess the motor nerve conduction of the sciatic nerve by a magnetic stimulation method in patients with piriformis syndrome. DESIGN: Prospective study. SETTING: An electrodiagnostic laboratory in a university hospital. PARTICIPANTS: Twenty-three patients with piriformis syndrome and 15 healthy persons for control. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Motor nerve conduction velocity (MNCV) of the sciatic nerve was measured at the gluteal segment by magnetic stimulation proximally at L5 and S1 roots and distally at sciatic nerve at gluteal fold and recording at the corresponding muscles. Diagnostic sensitivities were measured in the magnetic stimulation method and the conventional nerve conduction, long latency reflex, and needle electromyography studies. RESULTS: The mean MNCV of the sciatic nerve +/- standard deviation at the gluteal segment in L5 component was 55.4+/-7.8 m/s in patients with piriformis syndrome, which was slower than the mean value of 68.1+/-10.3 m/s obtained in healthy controls (P=.014). The MNCV of the sciatic nerve in S1 component showed no significant difference between the patients and controls (P=.062). A negative relation was found between the disease duration and the MNCV values of sciatic nerves in patients with piriformis syndrome (r=-.68, P<.01). The diagnostic sensitivity by magnetic stimulation is .467. CONCLUSIONS: Magnetic nerve stimulation provides a painless, noninvasive, and objective method for evaluation of sciatic nerve function in patients with piriformis syndrome.     

穴位电针刺激对面神经再生过程中神经营养因子-3mRNA表达的影响

目的　观察穴位电针刺激对面神经再生过程中表情肌及面神经组织神经营养因子 - 3(NT - 3)mRNA表达的影响。方法　日本大耳白兔面神经压榨伤后 ,电针刺激翳风、颧　、地仓、颊车、四白、阳白、合谷穴 ,每天 30min ,2周为 1疗程。经 1,2 ,3疗程治疗后 ,应用原位杂交及RT -PCR技术检测针刺组及对照组表情肌及面神经组织中 (NT - 3)mRNA表达水平的变化。结果　两组动物的表情肌及面神经组织中 ,NT - 3mRNA表达高峰在神经损伤后第 6周 ;原位杂交和RT -PCR技术均显示伤后第 3,6 ,9周针刺组表情肌组织 ,第 6 ,9周神经组织NT - 3mRNA的表达均明显高于对照组 (P <0 .0 5 ,0 .0 1)。结论　穴位电针刺激促进面神经再生的机理可能与表情肌及面神经组织中的NT - 3表达增强有关。     

海藻纤维膜片促进大鼠损伤坐骨神经的再生

背景：课题组和青岛大学高分子材料研究所合作研制的海藻纤维生物膜,具有优良的生物相容性,常被用作制备各种复合材料。目的：观察海藻纤维膜片包绕覆盖神经断端吻合口对大鼠坐骨神经损伤后再生的影响。方法：切断36只雄性Wistar大鼠右侧坐骨神经,随机分组：对照组行神经外膜端端吻合;实验组行神经外膜端端缝合,将海藻纤维膜片包绕并覆盖神经吻合口远近端各约0.5 cm,形成封闭再生室。术后观察海藻纤维膜片降解吸收规律及缝合处粘连情况,组织学切片行苏木精-伊红染色、锇酸染色、白细胞介素2及白细胞介素4免疫组织化学染色。结果与结论：术后4-6周,实验组海藻纤维膜片逐渐被降解吸收,与周围组织粘连较少,炎性细胞浸润程度较轻,纤维组织增生较少。两组术后1,7,14 d的白细胞介素2及白细胞介素4含量比较差异无显著性意义。实验组术后6周再生神经纤维分布规则且大小较为均一,其神经纤维数量、轴突大小及髓鞘厚度等指标均显著优于对照组（P 〈0.05）。表明海藻纤维膜片具有良好的生物降解性和组织相容性,其包绕覆盖坐骨神经形成的神经再生密闭室可促进大鼠损伤坐骨神经再生。     

兔颞骨内面神经缺损人工室修复的功能性研究

目的：通过测试再生神经及肌肉的功能，探讨神经再支配情况。方法：选用48只成年大耳白兔，横断左侧颞骨内面神经，神经近端分别用几丁质管和硅胶管桥接，术后1个月、3个月和5个月进行面部行为学观察以及电生理检查。结果：两种人工室5个月组比3个月组睑裂缩小，上唇自发运动有力，叩鼻后长须更明显偏向外侧，电极刺激神经后面肌收缩剧烈。神经电生理显示随着修复时间的延长，潜伏期逐渐缩短，动作电位波幅逐渐增高，神经-肌肉传导速度逐渐增快，两种人工室之间差异无显著性。结论：人工室修复颞骨内面神经缺损神经肌肉功能恢复良好，肌肉重获神经再支配。     

Critical analysis of the value of the rabbit median nerve model for biomedical research on peripheral nerve grafts

The rabbit has been proposed to represent an animal model that allows studying peripheral nerve regeneration across extended gap lengths. We describe here our experiences with the rabbit median nerve model and the obstacles it comes along with. This short communication is meant to inform the community and to prevent other researcher from investing time and animal lives in a model with low translational power.     

去细胞异体神经材料修复不同长度周围神经缺损

目的:为开发新的神经组织工程材料,研究去细胞异体神经修复周围神经缺损的神经再生效果。方法:采用神经电生理功能学指标和免疫组织化学染色等形态学方法,对1,2和3cm长度神经缺损修复后的神经再生进行检测。结果:再生神经纤维流畅地通过了不同长度的移植体,各组动物电生理功能均有不同程度的恢复。随移植材料长度的延长神经再生效果减低,3cm长度缺损在修复后20周再生有髓纤维数为(77±20)根/104μm2。结论:去细胞异体神经材料能够成功修复3cm内的周围神经缺损。     

脑源性神经营养因子对面神经损伤的修复作用

目的探讨局部应用脑源性神经营养因子(BDNF)对面神经损伤的修复作用。方法将成年新西兰兔面神经上颊支切除5mm,在断端间置入硅胶再生室,随机实验组室内注入BDNF,对照组用生理盐水。术后4周和8周进行电生理学、组织病理学观察和形态学定量分析。结果术后4周,两组电刺激面神经很少能引发面肌兴奋,有髓轴索计数,轴索直径和面积t检验,两组差异有显著性意义(P<0.05)。术后8周面神经复合肌动作电位(CMAP)BDNF组运动神经传导速度(MCV)明显短于对照组,有髓轴索计数,轴索直径和面积BDNF组大于对照组(P<0.05)。结论局部应用BDNF可促进面神经损伤的修复。     

Ultrasound-guided blockade of the lateral femoral cutaneous nerve: technical description and review of 10 cases

Blockade of the lateral femoral cutaneous nerve (LFCN) is performed for therapeutic management of meralgia paresthetica and as a regional anesthetic technique. The conventional technique is associated with high failure rates secondary to variable LFCN anatomy. We describe a technique for blockade of the LFCN using ultrasound guidance. A cross-sectional view of the LFCN was obtained by identifying the anterior superior iliac spine, then moving a 14-to-7MHz linear array ultrasound probe in a medial caudal direction until the nerve was encountered. The needle was advanced to the LFCN under ultrasound guidance via a lateral to medial approach. Injection using dynamic ultrasound demonstrated excellent perineural spread. Ten subjects underwent successful blockade of the LFCN with this technique. Five subjects were obese. Use of ultrasound for precise needle placement allowed low injection volumes to be utilized. Theref were no complications. Ultrasound guidance can facilitate blockade of the LFCN for diagnostic and therapeutic purposes and may be particularly beneficial with patients with challenging surface anatomic landmarks, or when low volume injections are desired.     

Recent advances in targeted delivery of non-coding RNA-based therapeutics for atherosclerosis

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