Hydrogel composition and laser micropatterning to regulate sciatic nerve regeneration

Treatment of peripheral nerve injuries has evolved over the past several decades to include the use of sophisticated new materials endowed with trophic and topographical cues that are essential for in vivo nerve fibre regeneration. In this research, we explored the use of an advanced design strategy for peripheral nerve repair, using biological and semi‐synthetic hydrogels that enable controlled environmental stimuli to regenerate neurons and glial cells in a rat sciatic nerve resection model. The provisional nerve growth conduits were composed of either natural fibrin or adducts of synthetic polyethylene glycol and fibrinogen or gelatin. A photo‐patterning technique was further applied to these 3D hydrogel biomaterials, in the form of laser‐ablated microchannels, to provide contact guidance for unidirectional growth following sciatic nerve injury. We tested the regeneration capacity of subcritical nerve gap injuries in rats treated with photo‐patterned materials and compared these with injuries treated with unpatterned hydrogels, either stiff or compliant. Among the factors tested were shear modulus, biological composition, and micropatterning of the materials. The microchannel guidance patterns, combined with appropriately matched degradation and stiffness properties of the material, proved most essential for the uniform tissue propagation during the nerve regeneration process.     

Nanofibrous nerve conduit‐enhanced peripheral nerve regeneration

Fibre structures represent a potential class of materials for the formation of synthetic nerve conduits due to their biomimicking architecture. Although the advantages of fibres in enhancing nerve regeneration have been demonstrated, in vivo evaluation of fibre size effect on nerve regeneration remains limited. In this study, we analyzed the effects of fibre diameter of electrospun conduits on peripheral nerve regeneration across a 15‐mm critical defect gap in a rat sciatic nerve injury model. By using an electrospinning technique, fibrous conduits comprised of aligned electrospun poly (ε‐caprolactone) (PCL) microfibers (981 ± 83 nm, Microfiber) or nanofibers (251 ± 32 nm, Nanofiber) were obtained. At three months post implantation, axons regenerated across the defect gap in all animals that received fibrous conduits. In contrast, complete nerve regeneration was not observed in the control group that received empty, non‐porous PCL film conduits (Film). Nanofiber conduits resulted in significantly higher total number of myelinated axons and thicker myelin sheaths compared to Microfiber and Film conduits. Retrograde labeling revealed a significant increase in number of regenerated dorsal root ganglion sensory neurons in the presence of Nanofiber conduits (1.93 ± 0.71 × 10³ vs. 0.98 ± 0.30 × 10³ in Microfiber, p < 0.01). In addition, the compound muscle action potential (CMAP) amplitudes were higher and distal motor latency values were lower in the Nanofiber conduit group compared to the Microfiber group. This study demonstrated the impact of fibre size on peripheral nerve regeneration. These results could provide useful insights for future nerve guide designs. Copyright © 2012 John Wiley & Sons, Ltd.     

Functional collagen conduits combined with human mesenchymal stem cells promote regeneration after sciatic nerve transection in dogs

Numerous studies have focused on the development of novel and innovative approaches for the treatment of peripheral nerve injury using artificial nerve guide conduits. In this study, we attempted to bridge 3.5‐cm defects of the sciatic nerve with a longitudinally oriented collagen conduit (LOCC) loaded with human umbilical cord mesenchymal stem cells (hUC‐MSCs). The LOCC contains a bundle of longitudinally aligned collagenous fibres enclosed in a hollow collagen tube. Our previous studies showed that an LOCC combined with neurotrophic factors enhances peripheral nerve regeneration. However, it remained unknown whether an LOCC seeded with hUC‐MSCs could also promote regeneration. In this study, using various histological and electrophysiological analyses, we found that an LOCC provides mechanical support to newly growing nerves and functions as a structural scaffold for cells, thereby stimulating sciatic nerve regeneration. The LOCC and hUC‐MSCs synergistically promoted regeneration and improved the functional recovery in a dog model of sciatic nerve injury. Therefore, the combined use of an LOCC and hUC‐MSCs might have therapeutic potential for the treatment of peripheral nerve injury.     

Recent advances in artificial nerve conduit design: Strategies for the delivery of luminal fillers

Artificial nerve conduits offer an attractive alternative to nerve autografts for the repair of peripheral nerve injuries and several commercially-available conduits are currently on the market. However, at present, utilization of these conduits is limited to the repair of nerve gaps less than 3 cm in length. Thus, current research is focused on how best to design artificial conduits with improved nerve regeneration potential over longer distances. Successful nerve regeneration necessitates that the cells, extracellular matrix components, and growth factors involved interact in a highly specific manner that is tightly coordinated. Combinatorial approaches that take into account these interactions and conduits that utilize supportive factors, such as neurotrophins and stem cells, may be key components of the next generation of artificial conduits. Additionally, design strategies that combine physical cues for contact guidance and biochemical signals to enhance cellular function have shown promise. This review highlights recent advances in artificial nerve conduit design, focusing on the use of luminal fillers, with special focus on the various techniques for accessory cell and/or growth factor delivery into artificial nerve conduits.     

NGF／PLGA复合神经导管修复大鼠周围神经缺损的实验研究

目的：应用神经生长因子（NGV）、聚乳酸和聚羟基乙酸的共聚物（PLGA）和牛血清白蛋白（BSA）制成NGF／PLGA复合神经导管。检测其综合性能和了解其修复大鼠周围神经缺损的可能性。方法：体外模拟体内环境，检测它的降解时间及用ELISA的方法来检测NGF的释放情况；手术造成大鼠坐骨神经约10mm的缺损，分别采用自体神经移植（A组）、NGF／PLGA复合神经导管桥接（B组）和单纯PLGA导管（C组）桥接，术后4、8、12周进行大体观察、神经电生理测定、HE染色、变色酸2R一亮绿髓鞘染色、电镜观察和图像分析对比。结果：在体外NGF／PLGA复合神经导管能在体外释放NGF约18天，约在14周左右导管降解完毕。NGF／PLGA神经导管组在促进坐骨神经再生、再生神经纤维排列规律化、提高再生神经髓鞘化、加速再生神经功能重建等方面均优于单纯PLGA导管组。比自体神经移植组略差。结论：NGF，PLGA复合神经导管具有良好的组织相容性，对大鼠坐骨神经缺损具有良好的桥梁作用和促神经生长的作用，效果接近自体神经移植。     

生物材料构建神经导管修复周围神经损伤的临床应用

背景:显微外科技术及周围神经损伤修复技术的发展与神经导管材料密切相关。神经导管的构建特别是生物材料构建神经导管材料还有待进一步开发研究。目的:探讨生物材料构建的神经导管在周围神经损伤修复中的应用及数据分析。方法:SCI数据库中2001/2010检索有关神经导管在周围神经损伤修复中的应用的文献,检索词为"神经导管(nerve conduit);生物材料(biomaterials);周围神经损伤(peripheral nerve injury);神经再生(nerve regeneration);壳聚糖神经导管(chitosan/chitin nerve conduit);高分子神经导管(polymer/macromolecule nerve conduit);胶原神经导管(collagen nerve conduit)",共检索文献183篇。结果与结论:神经导管修复法是在神经断端之间留有一段间隙,利用神经导管在神经的远端和近端之间桥接,并创造相对密闭的环境,以充分发挥远端神经的趋化作用,同时阻隔外部的影响,减少瘢痕的产生。目前,已被用于制备神经导管材料分为非神经组织、非生物降解材料、可生物降解材料。随着分子生物学及其他相关技术的发展,探索寻找理想的材料构建神经导管来治疗周围神经损伤研究始终在进行中。     

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.     

超声在周围神经损伤诊断和治疗中的应用进展

既往诊断外周神经损伤主要依赖运动感觉功能检查、电生理及MR检查。高频超声诊断外周神经损伤具有实时、动态、直观、无创、可重复性强等优点,有利于发现损伤部位及判断损伤类型,为术前评估和术后随访等提供重要信息。低强度超声（LIUS）能促进施万细胞增殖及大鼠损伤神经再生。虽然LIUS治疗外周神经损伤仍然处于实验阶段并存在许多问题有待解决,但其未来广阔的临床应用空间仍值得期待。     

以组织工程建构技术修复周围神经损伤的难点

背景：组织工程构建技术是近年来周围神经损伤修复的重要方法之一,在周围神经治疗领域有着良好的前景。目的：总结近年来利用组织工程学构建技术对周围神经损伤修复的研究进展。方法：应用计算机检索万方数据库、CNKI和PubMed数据库中1995年1月至2011年12月关于组织工程构建技术的文章,在标题和摘要中以“组织工程,神经导管支架,生物活性,周围神经损伤”或“TiSSMeengineering,Nervescaffold。Bioactivity,Peripheralnervedefect”为检索词进行检索,初检得到156篇文献,最终纳入56篇文献进行综述。结果与结论：周围神经损伤组织工程修复的两个要素是神经支架材料的选择和生物功能化。构建神经的支架材料包括可降解和非可降解两大类,通常需要具有三维多孔结构和相应的孔隙率及比表面积,其力学性能、表面活性、生物相容性和导电性等直接影响神经损伤修复的效果;生物功能化的主要生物活性因子包括支持细胞,种子蛋白和神经营养因子,将这些生物活性因子接种在神经导管支架材料上,促进受损神经的修复与功能替代。组织工程技术应用于周围神经损伤修复与再生的研究重点在于导管、细胞与生长因子的综合应用。组织工程技术与生物技术的联合应用将成为周围神经损伤修复的研究热点。     

Enhanced in vivo survival of Schwann cells by a synthetic oxygen carrier promotes sciatic nerve regeneration and functional recovery

Local hypoxia in the early stages of peripheral nerve injury is a challenge for axonal regeneration. To address this issue, perfluorotributylamine (PFTBA)‐based oxygen carrying fibrin hydrogel was prepared and injected into Schwann cell (SC)‐seeded collagen‐chitosan conduits to increase oxygen supply to SCs within the conduits. The conduit containing PFTBA‐SC gel was then applied to bridge a 15‐mm sciatic nerve defect in rats. It was observed that most of the GFP‐labeled SCs initially seeded in the PFTBA hydrogel remained alive for approximately 28 days after their in vivo implantation. The number of SCs was significantly higher in the PFTBA‐SC scaffold than that in the SC scaffold without PFTBA. In addition, nerve regeneration and functional recovery were examined after nerve injury repair. We found that the PFTBA‐SC scaffold was capable of promoting axonal regeneration and remyelination of the regenerated axons. Further studies showed the PFTBA‐SC scaffold was able to accelerate the recovery of motor and sensory function of the regenerating nerves. Electrophysiological analysis showed area under the curve of compound muscle action potential and nerve conduction velocity were also improved, and gastrocnemius muscle atrophy was partially reversed by PFTBA‐SC scaffold. Furthermore, microvessel density analysis showed PFTBA‐SC composites were beneficial for microvascular growth, which provided sustained oxygen for regenerating nerve in the later stages of nerve regeneration. In conclusion, enhanced survival of SCs by PFTBA is capable of promoting sciatic nerve regeneration and functional recovery, which provides a new avenue for achieving better functional recovery in the treatment of peripheral nerve injuries. Copyright © 2016 John Wiley & Sons, Ltd.     

周围神经损伤后修复再生的研究进展

周围神经损伤是手显微外科的常见病,其治疗及功能恢复一直都是手显微外科的难题。周围神经缺损后如何促进再生修复,提高神经缺损的治疗效果,使患者功能恢复较好,一直是临床研究的重点、热点、难点。近年来随着基础研究的不断深入,人们对周围神经解剖及其再生微环境的认识,周围神经损伤治疗方法已经由药物治疗、手术治疗发展到基因工程等,为周围神经损伤患者的治疗提供了更好的治疗思路。本文就周围神经缺损后周围神经修复的方法作一综述。     

周围神经术前肌电图结果与术中所见的比较研究及误诊原因分析

目的 探讨肌电图(EMG)检查对周围神经损伤的诊断意义，分析误诊的原因。方法 收集2000年1月至2003年4月行手术治疗的周围神经损伤患者63例(69条神经)，按神经损伤特点分为开放性周围神经损伤组、闭合性周围神经损害组、臂丛损伤组和神经修复后再生组，各组患者均于术前进行肌电图检测，并将检测结果与术中所见进行比较、分析。结果 开放性周围神经损伤组术前EMG对神经完全损伤的诊断符合率为73.08％，与术中所见结果比较，差异有统计学意义。闭合性周围神经损害组对受损神经的定性、定位诊断，均在术中得到证实。臂丛损伤组的大体定位正确率达96．30％，完全符合率达70．37％；对臂丛完全根性损伤的检出率为68．52％，与磁共振的检出率(55．56％)相比，差异无统计学意义。神经修复后再生组5条神经，EMG结果与术中所见3条符合，2条不符合。69条神经中，EMG检查完全符合率为71.01％，基本符合率为85．51％，完全不符率为13．04％；假阳性率为4．49％，假阴性率为22．73％。结论 EMG检查对损伤神经的定位、定性诊断及神经修复后再生状况的评价在临床诊治中具有重要的指导意义，但可出现假阳性及假阴性结果，且以运动诱发电位的假阴性为多。术前EMG与磁共振检查相结合，可提高对臂丛神经完全根性损伤的检出率。     

Regenerative effects of human embryonic stem cell‐derived neural crest cells for treatment of peripheral nerve injury

Surgical intervention is the current gold standard treatment following peripheral nerve injury. However, this approach has limitations, and full recovery of both motor and sensory modalities often remains incomplete. The development of artificial nerve grafts that either complement or replace current surgical procedures is therefore of paramount importance. An essential component of artificial grafts is biodegradable conduits and transplanted cells that provide trophic support during the regenerative process. Neural crest cells are promising support cell candidates because they are the parent population to many peripheral nervous system lineages. In this study, neural crest cells were differentiated from human embryonic stem cells. The differentiated cells exhibited typical stellate morphology and protein expression signatures that were comparable with native neural crest. Conditioned media harvested from the differentiated cells contained a range of biologically active trophic factors and was able to stimulate in vitro neurite outgrowth. Differentiated neural crest cells were seeded into a biodegradable nerve conduit, and their regeneration potential was assessed in a rat sciatic nerve injury model. A robust regeneration front was observed across the entire width of the conduit seeded with the differentiated neural crest cells. Moreover, the up‐regulation of several regeneration‐related genes was observed within the dorsal root ganglion and spinal cord segments harvested from transplanted animals. Our results demonstrate that the differentiated neural crest cells are biologically active and provide trophic support to stimulate peripheral nerve regeneration. Differentiated neural crest cells are therefore promising supporting cell candidates to aid in peripheral nerve repair.     

组织工程化神经导管修复外周神经损伤

背景：神经导管技术理论上采用生物或非生物材料预制成合适的管状支架,桥接神经断端两侧,在提供神经再生微环境的同时通过神经诱导、营养作用促进神经再生.目的：观察组织工程化神经导管修复外周神经损伤的临床效果.方法：选择24例陈旧性上肢神经损伤患者,以患者自愿原则分2组治疗：试验组采用组织工程化神经导管修复,对照组采用自体周围体表感觉神经移植修复.治疗后随访6个月观察患者肢体神经损伤功能修复效果.结果与结论：随访6个月后,两组肢体远端感觉运动功能与目测类比疼痛评分均较治疗前改善（P 〈0.05）,且试验组效果更好（P 〈0.05）;两组损伤侧感觉与运动神经传导速度均较治疗前改善（P 〈0.05）,且两组间差异无显著性意义.说明组织工程化神经导管材料符合神经修复导管支架的要求,临床应用疗效肯定.     

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.     

Macrophage monocarboxylate transporter 1 promotes peripheral nerve regeneration after injury in mice

Peripheral nerves have the capacity for regeneration, but the rate of regeneration is so slow that many nerve injuries lead to incomplete recovery and permanent disability for patients. Macrophages play a critical role in the peripheral nerve response to injury, contributing to both Wallerian degeneration and nerve regeneration, and their function has recently been shown to be dependent on intracellular metabolism. To date, the impact of their intracellular metabolism on peripheral nerve regeneration has not been studied. We examined conditional transgenic mice with selective ablation in macrophages of solute carrier family 16, member 1 (Slc16a1), which encodes monocarboxylate transporter 1 (MCT1), and found that MCT1 contributed to macrophage metabolism, phenotype, and function, specifically in regard to phagocytosis and peripheral nerve regeneration. Adoptive cell transfer of wild-type macrophages ameliorated the impaired nerve regeneration in macrophage-selective MCT1-null mice. We also developed a mouse model that overexpressed MCT1 in macrophages and found that peripheral nerves in these mice regenerated more rapidly than in control mice. Our study provides further evidence that MCT1 has an important biological role in macrophages and that manipulations of macrophage metabolism can enhance recovery from peripheral nerve injuries, for which there are currently no approved medical therapies.     

Delivery of chondroitinase ABC and glial cell line‐derived neurotrophic factor from silk fibroin conduits enhances peripheral nerve regeneration

Nerve conduits are a proven strategy for guiding axon regrowth following injury. This study compares degradable silk–trehalose films containing chondroitinase ABC (ChABC) and/or glial cell line‐derived neurotrophic factor (GDNF) loaded within a silk fibroin‐based nerve conduit in a rat sciatic nerve defect model. Four groups of silk conduits were prepared, with the following silk–trehalose films inserted into the conduit: (a) empty; (b) 1 µg GDNF; (3) 2 U ChABC; and (4) 1 µg GDNF/2 U ChABC. Drug release studies demonstrated 20% recovery of GDNF and ChABC at 6 weeks and 24 h, respectively. Six conduits of each type were implanted into 15 mm sciatic nerve defects in Lewis rats; conduits were explanted for histological analysis at 6 weeks. Tissues stained with Schwann cell S‐100 antibody demonstrated an increased density of cells in both GDNF‐ and ChABC‐treated groups compared to empty control conduits (p < 0.05). Conduits loaded with GDNF and ChABC also demonstrated higher levels of neuron‐specific PGP 9.5 protein when compared to controls (p < 0.05). In this study we demonstrated a method to enhance Schwann cell migration and proliferation and also foster axonal regeneration when repairing peripheral nerve gap defects. Silk fibroin‐based nerve conduits possess favourable mechanical and degradative properties and are further enhanced when loaded with ChABC and GDNF. Copyright © 2014 John Wiley & Sons, Ltd.     

周围神经损伤后再生的药物调控研究

目的　探讨神康灵对损伤的坐骨神经再生的作用。方法　采用 2 8只成年体重为 2 0 0g的Wistar大鼠 ,随机分成 2组 (实验组和对照组 ) ,分别于术后 4周和 6周 ,通过肌电图检测坐骨神经运动诱发电位的传导速度和波幅 ;组织学检测有髓神经轴突数目、横截面积 ,从而探讨神康灵对坐骨神经损伤后再生的作用。结果　实验组神经传导速度、再生的有髓神经纤维横截面积、数目均优于对照组。结论　神康灵对坐骨神经损伤后的再生有明显的促进作用。     

Neurotrophin releasing single and multiple lumen nerve conduits.

Tissue engineering strategies for nerve repair employ polymer conduits termed guidance channels and bridges to promote regeneration for peripheral nerve injury and spinal cord injury, respectively. An approach for fabrication of nerve conduits with single and multiple lumens capable of controlled release of neurotrophic factors was developed. These conduits were fabricated from a mixture of poly(lactide-co-glycolide) (PLG) microspheres and porogen (NaCl) that was loaded into a mold and processed by gas foaming. The porosity and mechanical properties of the constructs were regulated by the ratio of porogen to polymer microsphere. The neurotrophin, nerve growth factor (NGF), was incorporated into the conduit by either mixing the protein with microspheres or encapsulating the protein within microspheres prior to gas foaming. A sustained release was observed for at least 42 days, with the release rate controlled by method of incorporation and polymer molecular weight. Released NGF retained its bioactivity, as demonstrated by its ability to stimulate neurite outgrowth from primary dorsal root ganglion (DRG). In vivo results indicate that conduits retain their original architecture, and allow for cellular infiltration into the channels. Polymer conduits with controllable lumen diameters and protein release may enhance nerve regeneration by guiding and stimulating neurite outgrowth.     

Engineered neural tissue with Schwann cell differentiated human dental pulp stem cells: potential for peripheral nerve repair?

Despite the spontaneous regenerative capacity of the peripheral nervous system, large gap peripheral nerve injuries (PNIs) require bridging strategies. The limitations and suboptimal results obtained with autografts or hollow nerve conduits in the clinic urge the need for alternative treatments. Recently, we have described promising neuroregenerative capacities of Schwann cells derived from differentiated human dental pulp stem cells (d‐hDPSCs) in vitro . Here, we extended the in vitro assays to show the pro‐angiogenic effects of d‐hDPSCs, such as enhanced endothelial cell proliferation, migration and differentiation. In addition, for the first time we evaluated the performance of d‐hDPSCs in an in vivo rat model of PNI. Eight weeks after transplantation of NeuraWrap? conduits filled with engineered neural tissue (EngNT) containing aligned d‐hDPSCs in 15‐mm rat sciatic nerve defects, immunohistochemistry and ultrastructural analysis revealed ingrowing neurites, myelinated nerve fibres and blood vessels along the construct. Although further research is required to optimize the delivery of this EngNT, our findings suggest that d‐hDPSCs are able to exert a positive effect in the regeneration of nerve tissue in vivo . Copyright © 2017 John Wiley & Sons, Ltd.