周围神经再生中人工再生管道的研究进展

周围神经损伤造成的长段神经缺损的临床修复一直是临床工作中的一个难题。随着对神经再生理论认识的深入 ,应用人工再生管道做桥接物 ,引导再生轴突长向靶器官已取得了一定的进展。本文对各种人工再生管道的特点以及在神经再生中的作用进行了综述     

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。     

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。     

Engineering bi-layer nanofibrous conduits for peripheral nerve regeneration

Trauma injuries often cause peripheral nerve damage and disability. A goal in neural tissue engineering is to develop synthetic nerve conduits for peripheral nerve regeneration having therapeutic efficacy comparable to that of autografts. Nanofibrous conduits with aligned nanofibers have been shown to promote nerve regeneration, but current fabrication methods rely on rolling a fibrous sheet into the shape of a conduit, which results in a graft with inconsistent size and a discontinuous joint or seam. In addition, the long-term effects of nanofibrous nerve conduits, in comparison with autografts, are still unknown. Here we developed a novel one-step electrospinning process and, for the first time, fabricated a seamless bi-layer nanofibrous nerve conduit: the luminal layer having longitudinally aligned nanofibers to promote nerve regeneration, and the outer layer having randomly organized nanofibers for mechanical support. Long-term in vivo studies demonstrated that bi-layer aligned nanofibrous nerve conduits were superior to random nanofibrous conduits and had comparable therapeutic effects to autografts for nerve regeneration. In summary, we showed that the engineered nanostructure had a significant impact on neural tissue regeneration in situ. The results from this study will also lead to the scalable fabrication of engineered nanofibrous nerve conduits with designed nanostructure. This technology platform can be combined with drug delivery and cell therapies for tissue engineering.     

Biomaterials for the development of peripheral nerve guidance conduits

Currently, surgical treatments for peripheral nerve injury are less than satisfactory. The gold standard of treatment for peripheral nerve gaps >5 mm is the autologous nerve graft; however, this treatment is associated with a variety of clinical complications, such as donor site morbidity, limited availability, nerve site mismatch, and the formation of neuromas. Despite many recent advances in the field, clinical studies implementing the use of artificial nerve guides have yielded results that are yet to surpass those of autografts. Thus, the development of a nerve guidance conduit, which could match the effectiveness of the autologous nerve graft, would be beneficial to the field of peripheral nerve surgery. Design strategies to improve surgical outcomes have included the development of biopolymers and synthetic polymers as primary scaffolds with tailored mechanical and physical properties, luminal "fillers" such as laminin and fibronectin as secondary internal scaffolds, surface micropatterning, stem cell inclusion, and controlled release of neurotrophic factors. The current article highlights approaches to peripheral nerve repair through a channel or conduit, implementing chemical and physical growth and guidance cues to direct that repair process.     

Fabrication and evaluation of microgrooved polymers as peripheral nerve conduits

Cell alignment plays an important role in the repair of damaged peripheral nerves. The aligned Schwann cells could direct the axonal outgrowth during nerve reconstruction. One way of aligning Schwann cells is to use surface grooves in micrometric dimensions. In this study, microgrooves on chitosan or poly(d,l-lactide) (PLA) were fabricated and the behaviors of Schwann cells and glial cell line C6 on these surfaces were examined. It was found that Schwann cells and C6 cells could be successfully aligned by the microgrooves, and express the genes related to the production of neurotrophic factors. The polymer conduits with microgrooves on the inner surface were implanted in rats to repair the damaged sciatic nerve. The microgrooved conduits were demonstrated to enhance peripheral nerve regeneration as compared to the smooth conduits.     

合成可生物降解神经导管修复损伤周围神经：生物相容性良好

背景：临床对周围神经损伤进行修复治疗的时候,可以利用自体神经进行治疗或者利用不同材质的神经导管进行治疗。 目的：探索合成可生物降解材料神经导管在周围神经损伤修复中的应用效果。 方法：48只新西兰大白兔,随机等分为3组,自体神经移植组、硅胶导管组和可降解神经导管组。各组动物切除10 mm坐骨神经,构建坐骨神经缺损动物模型,并分别利用自体神经、硅胶导管以及可降解神经导管进行坐骨神经修复。 结果与结论：造模后3周,硅胶导管组兔运动神经传导效果、小腿三头肌肌肉湿质量恢复率比自体神经移植组差,但可降解神经导管组兔运动神经传导效果、小腿三头肌肌肉湿质量恢复率与自体神经移植组接近。造模后12周时,自体神经移植组中存在大量呈均匀排列的有髓神经纤维,可降解神经导管组中可见大量分布不均匀的再生有髓神经纤维,而硅胶导管组中存在少量呈不均匀排列的髓神经纤维。表明合成可生物降解材料神经导管在周围神经损伤修复中可以获得与自体神经较为接近的良好效果。  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     

New artificial nerve conduits made with photocrosslinked hyaluronic acid for peripheral nerve regeneration

For peripheral nerve regeneration, three-dimensional distribution and growth of cells within the porous scaffold are of clinical significance. We demonstrate that cultured rat Schwann cells and neurospheres grow in vitro on new artificial nerve conduits made with photocrosslinked hyaluronic acid (HA). HA tubular conduits have an inner diameter of 1.2 mm with porous nano-structure of 50 microm. After 3 weeks of cultivation, HA conduits remained circular with a round lumen, and construct of cell-conduits maintained the size and shape of the original architecture of the tube. HA itself has the function to facilitate a pathway for cellular and axonal ingrowth during peripheral nerve regeneration. These findings provide the feasibility of using the HA conduits for better cell adhesion and differentiation, leading to axonal regeneration in peripheral nerve reconstruction.     

Addition of fibronectin to alginate matrix improves peripheral nerve regeneration in tissue-engineered conduits

Schwann cell (SC) transplantation has been proposed to encourage peripheral nerve regeneration, but an optimal SC-carrying matrix would be needed. The aim of this study was to characterize how the addition of fibronectin to alginate would affect the outcome of nerve regeneration promoted by Schwann cells embedded in this matrix. Genetically labeled rat SCs were obtained by lacZ gene transduction. SCs were suspended in alginate hydrogel matrix with/without addition of liquid fibronectin, and their viability and growth in the different types of matrices were assessed in vitro by AlamarBlue assay. In vivo assessment of SC transplantation in the matrix was carried out with poly-3-hydroxybutyrate (PHB) conduits to bridge a sciatic nerve gap. The grafted conduits were harvested at 2, 3, and 6 weeks and assessed for the presence of labeled SCs in relation to regrowing axons. The amount and rate of axonal regeneration were assessed by quantitative immunohistochemistry. Addition of fibronectin to alginate hydrogel improved SC viability and growth profile in vitro. X-Gal staining confirmed that SCs transplanted in PHB conduits were viable throughout the time course, and that the labeled SCs were clearly associated with regenerating axons. The regeneration rate was enhanced when liquid fibronectin was added to the alginate matrix. Furthermore, the presence of SCs also enhanced regeneration and there was an additive effect when both SCs and fibronectin were combined with alginate. In conclusion, the addition of fibronectin to alginate hydrogel matrix contributed to improve nerve regeneration, supporting SC viability and augmenting their effect on axonal growth when transplanted in a bioengineered nerve conduit.     

The effect of pulse-released nerve growth factor from genipin-crosslinked gelatin in schwann cell-seeded polycaprolactone conduits on large-gap peripheral nerve regeneration

Different lag-time of pulse-released nerve growth factor (NGF) from genipin-crosslinked gelatin within polycaprolactone (PCL) conduits was evaluated in large-gap peripheral nerve repair. In this study, 10% (w/v) gelatin was mixed with NGF, crosslinked with 0%, 0.1%, 0.5%, and 1% (w/v) genipin, and then sucked into the wall of PCL conduits. These controlled-release nerve conduits were named NCL (non-crosslink), LCL (low crosslink), MCL (medium crosslink), and HCL (high crosslink), respectively. The NGF releasing character showed four distinctive curves, including initial burst within 5 days, pulse releasing at 5-20 days, pulse releasing at 10-25 days, and steadily releasing. The bioactivity of the released NGF was shown by neurite outgrowth of PC12 cells after culturing in all groups. Finally, the controlled-release conduits were seeded with 9 x 10(3) Schwann cells. Conduits were used to bridge a 15-mm rat sciatic nerve defect, and the results were compared with the isografts (control group). Eight weeks after implantation, morphological analysis revealed that LCL, MCL, and HCL groups were similar to autograft treatment in the numbers and area of myelinated axons. The LCL group, although insignificant, showed a trend to have the highest myelinated axon counts of the conduit-treated groups. Thus, comparing the different NGF release characteristics among NCL, MCL, and LCL groups, we concluded that a high concentration of NGF at 5-10 days in LCL groups is needed in bridging a 15-mm peripheral nerve injury.     

Regenerative potential of silk conduits in repair of peripheral nerve injury in adult rats

Various attempts have been made to develop artificial conduits for nerve repair, but with limited success. We describe here conduits made from Bombyx mori regenerated silk protein, and containing luminal fibres of Spidrex^®, a silk-based biomaterial with properties similar to those of spider silk. Assessment in vitro demonstrated that Spidrex^® fibres support neurite outgrowth. For evaluation in vivo, silk conduits 10 mm in length and containing 0, 100, 200 or 300 luminal Spidrex^® fibres, were implanted to bridge an 8 mm gap in the rat sciatic nerve. At 4 weeks, conduits containing 200 luminal Spidrex^® fibres (PN200) supported 62% and 59% as much axon growth as autologous nerve graft controls at mid-conduit and distal nerve respectively. Furthermore, Spidrex^® conduits displayed similar Schwann cell support and macrophage response to controls. At 12 weeks, animals implanted with PN200 conduits showed similar numbers of myelinated axons (81%) to controls, similar gastrocnemius muscle innervation, and similar hindpaw stance assessed by Catwalk footprint analysis. Plantar skin innervation was 73% of that of controls. PN200 Spidrex^® conduits were also effective at bridging longer (11 and 13 mm) gaps. Our results show that Spidrex^® conduits promote excellent axonal regeneration and function recovery, and may have potential for clinical application.     

Comparison and characterization of multiple biomaterial conduits for peripheral nerve repair

Four biomaterial tubes, poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone fumarate) (PCLF), a neutral oligo[(polyethylene glycol) fumarate] (OPF) hydrogel or a positively charged oligo[(polyethylene glycol) fumarate] (OPF⁺) hydrogel with a PCLF sleeve, have previously been shown to have benefits for nerve repair. However, no direct comparison to identify the optimal material have been made. Herein, these nerve tubes were implanted in a rat sciatic nerve model and nerve regeneration was quantified and compared by using accepted nerve assessment techniques. Using standard statistical methods, no significant differences of individual parameters were apparent between groups despite PCLF showing a tendency to perform better than the others. Using a mean–variance based ranking system of multiple independent parameters, statistical differences became apparent. It was clear that the PLCF tube supported significantly improved nerve regeneration and recovery compared to the other three biomaterial conduits. The ability to simultaneously compare a number of regenerative parameters and elucidate the best material from the combination of these individual parameters is of importance to the nerve regeneration area and has implications for the tissue engineering field. By using this method of comparison, a number of biomaterial constructs may be compared under similar conditions and the optimal construct elucidated using the minimal number of animals and materials.     

骨髓间充质干细胞与新型神经导管复合材料修复周围神经缺损

背景：随着医学模式的不断发展进步,人们对于外周神经缺损的治疗以及后期康复水平提出更高的要求,这就使得以干细胞培养为基础的神经组织工程技术为神经缺损治疗提供了新策略。 目的：探讨骨髓间充质干细胞与新型神经导管复合材料修复周围神经缺损的可行性。 方法：选取清洁级纯种新西兰大白兔50只,采用随机数字表法分为实验组与对照组,每组25只,于兔前肢桡骨中段约15 mm处造成外周神经损伤,造模后1周,实验组外周神经损伤处移植骨髓间充质干细胞联合新型神经导管复合材料,对照组外周神经损伤处移植骨髓间充质干细胞。移植后4周,取神经损伤处的神经纤维长约5 mm,进行苏木精-伊红染色和扫描电镜观察,对比两组再生神经组织内神经纤维的密度和直径。 结果与结论：实验组神经纤维密度明显高于对照组,神经纤维直径明显低于对照组,组间相比差异有显著性意义(P < 0.05);实验组再生神经组织表面细胞数较多,细胞生长状态良好,体积较大,胞体发出多个突起,并且细胞之间相互连接交织成网状,其轴突较长和较粗,呈典型的神经元样细胞表现,且生长密度和状态优于对照组,可见骨髓间充质干细胞与新型神经导管复合材料可以用于修复周围神经缺损,且效果确切。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

胶原/丝素蛋白神经导管修复周围神经缺损的研究与应用进展

BACKGROUND: Peripheral nerve defect due to limb dysfunction has always been the difficulty faced by the medical profession. Ideal materials and processing technology for constructing a tissue engineering scaffold targeting peripheral nerve repair are still in research stage. OBJECTIVE: To review the research progress in peripheral nerve repair using collagen/silk fibroin nerve conduits. METHODS:In this paper, the first author retrieved the PubMed and CNKI from 2003 to 2016 to search articles regarding methods of constructing artificial nerve scaffolds and selection of raw materials. Data from these articles were collected, summarized and analyzed. RESULTS AND CONCLUSION: Forty-six articles were included for final analysis. Collagen and its degradation products trigger no inflammatory response in the host because of high biocompatibility and biodegradability. However, its use is largely limited by its rapid degradation and poor physical performance. Silk fibroin has a high flexibility and biocompatibility, and exhibits a slow degradation in vivo. As a rapid prototyping technique, three-dimensional printing can print various forms of scaffolds within a short time, characterized as high-quality pore structure and large-scale production. Given these, the collagen/silk fibroin nerve conduit prepared using the three-dimensional printing technology can maintain the biocompatibility and even improve the mechanical properties of the raw materials. Until now, more investigations on nerve repair using collagen or silk fibroin have been done, and we have never stopped improving the production process of these scaffolds. Therefore, the collagen/silk fibroin scaffold prepared using the three-dimensional printing technology is expected to become the main candidate for the repair of peripheral nerve defects.      

Promoting regeneration of peripheral nerves in-vivo using new PCL-NGF/Tirofiban nerve conduits

Poly(ε-caprolactone) (PCL) scaffolds were modified by grafting nerve growth factor (NGF) and Tirofiban (TF), a clinical anti-thrombosis drug, as a new biomaterial for producing nerve conduits to promote the regeneration of sciatic nerves. The successful grafting of NGF and TF onto PCL scaffolds was confirmed by FTIR and ESCA spectra. In-vitro growths of the PC12 cells in PCL-NGF and PCL-NGF/TF scaffolds, determined by MTS, were significantly higher (P < 0.05, n = 4) than those in the PCL scaffolds following three days of cultivation. Interestingly, this study evaluation of the PCL, PCL-NGF, and PCL-NGF/TF nerve conduits in a 12 mm long gap of the rat sciatic nerve defect model that the gastrocnemius muscle mass of the tested rats in the PCL-NGF/TF groups significantly exceeded those in the PCL-NGF and PCL group. In the rats that had been implanted with PCL-NGF/TF conduits, the generated nerves passed through those conduits, expressing beta-III tubulin (TB), growth association protein-43 (GAP-43) and myelin basic protein (MBP) along their longitudinal axis, and the proximal and distal nerve ends of the rats were successfully connected. Those that had been implanted with PCL and PCL-NGF conduits did not exhibit these effects, as revealed by an immunochemical study of the expressions of the proteins in the conduits. Moreover, counting within the dorsal horn of the spinal cord (C(5)) demonstrated that the numbers of CTB-HRP-labeled neurons in the rats that had been implanted with PCL-NGF/TF conduits were significantly higher than those in the other groups. In this study, in-vivo examinations of the use of newly designed PCL-NGF/TF conduits to promote the generation of nerves in a defective rat model significantly increased the gastrocnemius muscle mass, and led to the successful regeneration of nerves that bridged a 12 mm long defected gap of nerves in rats. However, more rats must be tested to confirm the efficacy the newly designed nerve conduits.     

PDLLA/chondroitin sulfate/chitosan/NGF conduits for peripheral nerve regeneration

Biodegradable PDLLA/Chondroitin sulfate/Chitosan(PDLLA/CS/CHS) nerve conduits with potentially good biocompatibility and good mechanical property feasible for surgical manipulation have been developed in our previous work. The purpose of this study was to investigate their possible application in repairing damaged nerves and the effect of nerve growth factor (NGF). The PDLLA/CS/CHS/NGF nerve conduits were prepared by immobilizing NGF onto the PDLLA/CS/CHS nerve conduits with carbodiimide. Adult Sprague-Dawley (SD) rats weighing 200-250 g were used as the animal model. The conduits were employed to bridge the 10 mm defects in the sciatic nerve of the SD rats. Nerve conduction velocities (NCVs) were clearly detected in both nerve conduits after 3 months of implantation, indicating a rapid functional recovery for the disrupted nerves. The results of histological sections showed that the internal sides of the conduits were compact enough to prevent the connective tissues from ingrowth. Combined with the strong mechanical properties, good nerve regeneration ability and non-toxicity of its degradation products, PDLLA/CS/CHS nerve conduits would be expected to be useful materials to repair nerve damage and NGF can effectively promote the regeneration of peripheral nerve defect.     

静电纺丝聚乳酸聚乙醇酸共聚物-丝素-胶原神经导管修复大鼠周围神经缺损

背景：周围神经缺损修复是临床上一大难题,由于自体神经移植有一定的局限性,人工神经修复材料是一种很有前途的选择。 目的：探讨静电纺丝聚乳酸聚乙醇酸共聚物(PLGA)-丝素-胶原纳米神经导管修复大鼠坐骨神经缺损的可能性。 方法：雌性SD大鼠36只,制备约10 mm的坐骨神经缺损,分别采用倒转自体神经、静电纺丝PLGA-丝素-胶原神经导管、单纯PLGA神经导管桥接,术后12周进行大体观察、神经电生理测定、光镜观察、透射电镜观察和图像分析对比,了解神经再生的情况。 结果与结论：静电纺丝法制备成的纳米神经导管管壁疏松多孔,能够模拟细胞外基质的结构。静电纺丝PLGA-丝 素-胶原神经导管组在促进坐骨神经再生、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。     

Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration

The human amnion consists of the epithelial cell layer and underlying connective tissue. After removing the epithelial cells, the resulting acellular connective tissue matrix was manufactured into thin dry sheets called amnion matrix sheets. The sheets were further processed into tubes, amnion matrix tubes (AMTs), of varying diameters, with the walls of varying numbers of amnion matrix sheets with or without a gelatin coating. The AMTs were implanted into rat sciatic nerves. Regenerating nerves extended in bundles through tubes of 1-2 mm in diameter and further elongated into host distal nerves 1-3 weeks after implantation. Morphometrical analysis of the regenerated nerve cable at the middle of each amnion matrix tube 3 weeks after implantation was performed. The average numbers of myelinated axons were almost the same (ca. 80-112/10(4) microm(2)) in AMTs of 1-2 mm in diameter, as in the normal sciatic nerve (ca. 95/10(4) microm(2)). No myelinated fibers were found in AMTs composed of multiple thin tubes of 0.2 mm in diameter. The myelinated axons were thinner in implanted tubes than those in the normal sciatic nerve. The rate of occurrences of myelinated axons less than 4 microm in diameter was significantly higher in the AMTs, whereas axons in the normal sciatic nerve were diverse in distribution, with the highest population at 8-12 microm in diameter. Reinnervation to the gastrocnemius muscle was demonstrated electrophysiologically 9 months after implantation. It was concluded that the extracellular matrix sheet from the human amnion is an effective conduit material for peripheral nerve regeneration.     

Muscle recovery after repair of short and long peripheral nerve gaps using fibrin conduits

Peripheral nerve injuries with loss of nervous tissue are a significant clinical problem and are currently treated using autologous nerve transplants. To avoid the need for donor nerve, which results in additional morbidity such as loss of sensation and scarring, alternative bridging methods have been sought. Recently we showed that an artificial nerve conduit moulded from fibrin glue is biocompatible to nerve regeneration. In this present study, we have used the fibrin conduit or a nerve graft to bridge either a 10 mm or 20 mm sciatic nerve gap and analyzed the muscle recovery in adult rats after 16 weeks. The gastrocnemius muscle weights of the operated side were similar for both gap sizes when treated with nerve graft. In contrast, muscle weight was 48.32 ± 4.96% of the contra-lateral side for the 10 mm gap repaired with fibrin conduit but only 25.20 ± 2.50% for the 20 mm gap repaired with fibrin conduit. The morphology of the muscles in the nerve graft groups showed an intact, ordered structure, with the muscle fibers grouped in fascicles whereas the 20 mm nerve gap fibrin group had a more chaotic appearance. The mean area and diameter of fast type fibers in the 20 mm gap repaired with fibrin conduits were significantly (P < 0.01) worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. Furthermore, the 10 mm gaps repaired with either nerve graft or fibrin conduit showed similar muscle fiber size. These results indicate that the fibrin conduit can effectively treat short nerve gaps but further modification such as the inclusion of regenerative cells may be required to attain the outcomes of nerve graft for long gaps.