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.     

Development and characterization of novel agar and gelatin injectable hydrogel as filler for peripheral nerve guidance channels

Injectable hydrogels are becoming of increasing interest in the field of tissue engineering thanks to their versatile properties and to the possibility of being injected into tissues or devices during surgery. In peripheral nerve tissue engineering, injectable hydrogels having shear‐thinning properties are advantageous as filler of nerve guidance channels (NGCs) to improve the regeneration process. In the present work, gelatin‐based hydrogels were developed and specifically designed for the insertion into the lumen of hollow NGCs through a syringe during surgery. Injectable hydrogels were obtained using an agar–gelatin 20:80 weight ratio, (wt/wt) blend crosslinked by the addition of genipin (A/GL_GP). The physicochemical properties of the A/GL_GP hydrogels were analysed, including their injectability, rheological, swelling and dissolution behaviour, and their mechanical properties under compression. The hydrogel developed showed shear‐thinning properties and was applied as filler of NGCs. The A/GL_GP hydrogel was tested in vitro using different cell lines, among them Schwann cells which have been used because they have an important role in peripheral nerve regeneration. Viability assays demonstrated the lack of cytotoxicity. In vitro experiments showed that the hydrogel is able to promote cell adhesion and proliferation. Two‐ and three‐dimensional migration assays confirmed the capability of the cells to migrate both on the surface and within the internal framework of the hydrogel. These data show that A/GL_GP hydrogel has characteristics that make it a promising scaffold material for tissue engineering and nerve regeneration. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

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.     

脉冲电磁场对周围神经再生的影响

目的:探讨脉冲电磁场对周围神经再生的影响及其作用机理。方法:以60只Wistar大鼠左侧坐骨神经重度钳夹伤为模型,术后随机将大鼠均分为治疗组和对照组,治疗组给予脉冲电磁场治疗。术后不同时期观测大鼠伤肢功能神经恢复情况、电生理指标和组织学检查。结果:脉冲电磁场治疗促进伤肢功能的恢复,加速了损伤神经远段Wallerian变性进程,促进雪旺氏细胞增殖,促进轴索及髓鞘再生,加速运动神经传导速度的恢复。结论:脉冲电磁场可能是通过对周围神经再生过程中多环节的调控和促进,促进周围神经再生和功能恢复的。     

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.     

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.     

Nerve lengthening and subsequent end‐to‐end repair yield more favourable outcomes compared with autograft repair of rat sciatic nerve defects

Outcomes of end‐to‐end nerve repairs are more successful compared with outcomes of repairs bridged by nerve grafts. However, end‐to‐end repairs are not always possible for large nerve gaps, as excessive tension may cause catastrophic failure. In this study, we built on previous nerve‐lengthening studies to test the hypotheses that gradual lengthening of the proximal stump across a large nerve gap enables an end‐to‐end repair and such a repair results in more favourable regenerative outcomes than autografts, which represent the gold standard in bridging nerve gaps. To test these, we compared structural and functional outcomes in Lewis rats after repair of sciatic nerve gaps using either autografts or a novel compact internal fixator device, which was used to lengthen proximal nerve stumps towards the distal stump over 2 weeks, prior to end‐to‐end repair. Twelve weeks after the initial injury, outcomes following nerve lengthening/end‐to‐end repair were either comparable or superior in every measure compared with repair by autografting. The sciatic functional index was not significantly different between groups at 12 weeks. However, we observed a reduced rate of contracture and corresponding significant increase in paw length in the lengthening group. This functional improvement was consistent with structural regeneration; axonal growth distal to the injury was denser and more evenly distributed compared with the autograft group, suggesting substantial regeneration into both tibial and peroneal branches of the sciatic nerve. Our findings show that end‐to‐end repairs following nerve lengthening are possible for large gaps and that this strategy may be superior to graft‐based repairs.     

Poly‐3‐hydroxybutyrate strips seeded with regenerative cells are effective promoters of peripheral nerve repair

Peripheral nerve injuries are often associated with loss of nerve tissue and require a graft to bridge the gap. Autologous nerve grafts are still the 'gold standard' in reconstructive surgery but have several disadvantages, such as sacrifice of a functional nerve, neuroma formation and loss of sensation at the donor site. Bioengineered grafts represent a promising approach to address this problem. In this study, poly‐3‐hydroxybutyrate (PHB) strips were used to bridge a 10 mm rat sciatic nerve gap and their effects on long‐term (12 weeks) nerve regeneration were compared. PHB strips were seeded with different cell types, either primary Schwann cells (SCs) or SC‐like differentiated adipose‐derived stem cells (dASCs) suspended in a fibrin glue matrix. The control group was PHB and fibrin matrix without cells. Functional and morphological properties of the regenerated nerve were assessed using walking track analysis, EMGs, muscle weight ratios and muscle and nerve histology. The animals treated with PHB strips seeded with SCs or dASCs showed significantly better functional ability than the control group. This correlated with less muscle atrophy and greater axon myelination in the cell groups. These findings suggest that the PHB strip seeded with cells provides a beneficial environment for nerve regeneration. Furthermore, dASCs, which are abundant and easily accessible, constitute an attractive cell source for future applications of cell therapy for the clinical repair of traumatic nerve injuries. Copyright © 2015 John Wiley & Sons, Ltd.     

经颅磁电刺激促进周围神经再生的电生理学研究

目的;研究经颅磁电刺激后损伤坐骨神经的电生理学变化,探索一种促进周围神经功能恢复的有效治疗方法。方法：用电生理学方法观察磁电刺激对周围神经恢复的影响,并与对照组进行比较。结果：磁电刺激组受损坐骨神经的潜伏期较对照组缩短,差异有显著意义。实验组的波幅较对照组增高,神经传导速度也较对照组增快,但无显著差异。结论：经颅磁电刺激可能具有促进受损周围神经再生和传导功能恢复的作用。     

A laminin‐2‐derived peptide promotes early‐stage peripheral nerve regeneration in a dual‐component artificial nerve graft

The DLTIDDSYWYRI motif (Ln2‐P3) of human laminin‐2 has been reported to promote PC12 cell attachment through syndecan‐1; however, the in vivo effects of Ln2‐P3 have not been studied. In Schwann cells differentiated from skin‐derived precursors, the peptide was effective in promoting cell attachment and spreading in vitro. To examine the effects of Ln2‐P3 in peripheral nerve regeneration in vivo, we developed a dual‐component poly(p‐dioxanone) (PPD)/poly(lactic‐co‐glycolic acid) (PLGA) artificial nerve graft. The novel graft was coated with scrambled peptide or Ln2‐P3 and used to bridge a 10 mm defect in rat sciatic nerves. The dual‐component nerve grafts provided tensile strength comparable to that of a real rat nerve trunk. The Ln2‐P3‐treated grafts promoted early‐stage peripheral nerve regeneration by enhancing the nerve regeneration rate and significantly increased the myelinated fibre density compared with scrambled peptide‐treated controls. These findings indicate that Ln2‐P3, combined with tissue‐engineering scaffolds, has potential biomedical applications in peripheral nerve injury repair. Copyright © 2012 John Wiley & Sons, Ltd.     

Cell‐free artificial implants of electrospun fibres in a three‐dimensional gelatin matrix support sciatic nerve regeneration in vivo

Surgical repair of larger peripheral nerve lesions requires the use of autologous nerve grafts. At present, clinical alternatives to avoid nerve transplantation consist of empty tubes, which are only suitable for the repair over short distances and have limited success. We developed a cell‐free, three‐dimensional scaffold for axonal guidance in long‐distance nerve repair. Sub‐micron scale fibres of biodegradable poly‐ε‐caprolactone (PCL) and collagen/PCL (c/PCL) blends were incorporated in a gelatin matrix and inserted in collagen tubes. The conduits were tested by replacing 15‐mm‐long segments of rat sciatic nerves in vivo. Biocompatibility of the implants and nerve regeneration were assessed histologically, with electromyography and with behavioural tests for motor functions. Functional repair was achieved in all animals with autologous transplants, in 12 of 13 rats that received artificial implants with an internal structure and in half of the animals with empty nerve conduits. In rats with implants containing c/PCL fibres, the extent of recovery (compound muscle action potentials, motor functions of the hind limbs) was superior to animals that had received empty implants, but not as good as with autologous nerve transplantation. Schwann cell migration and axonal regeneration were observed in all artificial implants, and muscular atrophy was reduced in comparison with animals that had received no implants. The present design represents a significant step towards cell‐free, artificial nerve bridges that can replace autologous nerve transplants in the clinic. Copyright © 2017 John Wiley & Sons, Ltd.     

生物粘接端端缝合法修复外周神经的实验研究

目的　研究生物粘接端端缝合修复外周神经的早期效果。方法　选用 Wister大鼠 32只 ,在双目放大镜下分别用生物粘接端端缝合法和单纯端端缝合法修复坐骨神经损伤 ,2周后进行组织形态学、电生理学、坐骨神经功能指数检测评定。结果　生物粘接端缝合组各项指标均优于对照组 ,其神经纤维的生长速度、数量明显优于单纯缝合组 ,SFI的恢复优于单纯缝合组。结论　生物粘接端端缝合法可以促进神经再生 ,加快神经再生速度与神经功能恢复。     

Identification of critical growth factors for peripheral nerve regeneration

Growth factors are essential for the repair and regeneration of tissues and organs, including injured peripheral nerves. However, the expression changes of growth factors during peripheral nerve regeneration have not been fully elucidated. To obtain a global view of alternations of growth factors during the regeneration process, we explored previously achieved sequencing data of rat sciatic nerve stumps at 0 h, 1 d, 4 d, 7 d, and 14 d after nerve crush injury and screened differentially expressed upstream growth factors using Ingenuity Pathway Analysis (IPA) bioinformatic software. Differentially expressed growth factors were then subjected to Gene Ontology (GO) annotation and Kyoto Enrichment of Genes and Genomes (KEGG) pathway analysis. Regulatory networks of the differentially expressed growth factors in axon growth-related biological processes were constructed. Pivotal growth factors involved in axon growth were identified and validated by qRT-PCR. Our current study identified differentially expressed growth factors in the injured nerve stumps after peripheral nerve injury, discovered key growth factors for axon growth and nerve regeneration, and might facilitate the discovery of potential therapeutic targets of peripheral nerve injury.

Growth factors are essential for the repair and regeneration of tissues and organs, including injured peripheral nerves.     

Efficacy of multi-wave locked system laser therapy on nerve regeneration after crushing in Wister rats

[Purpose] To investigate the efficacy of the multi-wave locked system laser therapy on the regeneration of peripheral nerve injuries by evaluating the functional, electrophysiological, and morphological changes of the crushed sciatic nerve in Wistar rats. [Materials and Methods] Sixty male Wistar rats (200–250 g) were randomly assigned to control negative, control positive, or laser groups and subjected to no laser therapy or crushing, to crushing without laser therapy, or crushing followed by multi-wave locked system laser therapy five times/week for four weeks (power=1 W, energy density=10 J/cm², total energy=100 J), respectively. Functional, electrophysiological, and morphometric analyses were performed before and 7, 15, 21, and 28 days after crushing. The sciatic functional index, compound motor action potential amplitude, motor nerve conduction velocity, and nerve and myelin sheath diameters were measured. [Results] The sciatic functional index value decreased significantly, while the compound motor action potential amplitude, motor nerve conduction velocity, nerve diameter, and myelin sheath diameter increased significantly in the laser group post-treatment compared to the values in the control groups. [Conclusion] Multi-wave locked system laser therapy was effective in accelerating the regeneration of crushed sciatic nerves in Wistar rats.     

应用人工神经修复大鼠坐骨神经损伤的实验研究

目的观察应用复合碱性成纤维细胞生长因子(bFGF)-壳聚糖导管修复大鼠周围神经损伤的效果。方法成年Wistar大鼠25 只分为假手术组(n=10)、单纯损伤组(n=5)和人工神经修复组(n=10)。假手术组仅暴露坐骨神经5 mm,单纯损伤组暴露坐骨神经并切断,人工神经修复组以复合bFGF-壳聚糖导管桥接缺损。术后对实验动物的坐骨神经进行大体观察,对运动进行行为观察,以及组织化学和免疫组织化学方法评价神经再生情况和靶肌肉恢复情况。结果术后5 周,与单纯损伤组相比,人工神经修复组运动功能有一定程度恢复。人工神经修复组再生的坐骨神经已经通过bFGF-壳聚糖导管越过缺损并与远端相连接。免疫组织化学方法显示,坐骨神经再生段可观察到神经微丝(NF)阳性和S-100 阳性纤维。应用Masson 染色方法,可观察到人工神经修复组腓肠肌去纤维化程度相对于单纯损伤组有明显改善。结论应用bFGF-壳聚糖导管能有效修复周围神经损伤并使大鼠运动功能得到改善。     

Phosphate glass fibres promote neurite outgrowth and early regeneration in a peripheral nerve injury model

Three‐dimensional (3D) scaffolds, which are bioactive and aid in neuronal guidance, are essential in the repair and regeneration of injured peripheral nerves. In this study, we used novel inorganic microfibres guided by phosphate glass (PG). PG fibres (PGfs) were aligned on compressed collagen that was rolled into a nerve conduit. In vitro tests confirmed that adult dorsal root ganglion (DRG) neurons showed active neurite outgrowth along the fibres, with a maximum number and length of neurites being significantly higher than those cultured on tissue culture plastic. In vivo experiments with nerve conduits that either contained PGfs (PGf/Col) or lacked them (Col) were conducted on transected sciatic nerves of rats for up to 12 weeks. One week after implantation, the PGf/Col group showed many axons extending along the scaffold, whereas the Col group showed none. Eight weeks after implantation, the PGf/Col group exhibited greater recovery of plantar muscle atrophy than the Col group. Electrophysiological studies revealed that some animals in the PGf/Col group at 6 and 7 weeks post‐implantation (5.3% and 15.8%, respectively) showed compound muscle action potential. The Col group over the same period showed no response. Motor function also showed faster recovery in the PGf/Col group compared to the Col group up to 7 weeks. However, there was no significant difference in the number of axons, muscle atrophy or motor and sensory functions between the two groups at 12 weeks post‐implantation. In summary, phosphate glass fibres can promote directional growth of axons in cases of peripheral nerve injury by acting as physical guides. Copyright © 2012 John Wiley & Sons, Ltd.     

坐骨神经损伤后脊髓肝细胞生长因子mRNA及蛋白的表达

目的 研究坐骨神经损伤后再生过程中脊髓肝细胞生长因子（HGF）mRNA和蛋白的表达及经时变化规律，探讨周围神经损伤的机制。方法 采用原位杂交及免疫组化技术检测坐骨神经损伤后再生过程中脊髓HGF的mRNA及蛋白的表达。结果 大鼠坐骨神经损伤模型损伤侧的神经细胞胞质颗粒阳性染色强于未损伤侧；神经损伤后第3，7和14天，感觉。运动和副交感神经元内杂交信号明显增强，以第7天的变化最为显著。HGF蛋白的表达均于坐骨神经损伤后第1周开始增强，第2周时达峰值，然后下降。结论 周围神经损伤后，内源性HGF mRNA和蛋白表达增强，对神经元具有保护作用。     

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.