神经导管修复周围神经损伤的研究进展

随着神经修复技术特别是显微外科的发展，神经损伤修复的质量有了进一步的提高；利用神经导管桥接神经断端以实现修复周围神经损伤是目前的一个研究热点。本综述了神经导管修复周围神经损伤的发展历史，分析比较了非神经组织、非生物降解材料、可生物降解材料神经导管在神经损伤修复中的效果，讨论了导管的形态及导管内微环境对神经再生的影响。     

A new nerve guide conduit material composed of a biodegradable poly(phosphoester)

There is a resurgence of interest in the development of degradable and biocompatible polymers for fabrication of nerve guide conduits (NGCs) in recent years. Poly(phosphoester) (PPE) polymers are among the attractive candidates in this context, in view of their high biocompatibility, adjustable biodegradability, flexibility in coupling fragile biomolecules under physiological conditions and a wide variety of physicochemical properties. The feasibility of using a biodegradable PPE, P(BHET-EOP/TC), as a novel NGC material was investigated. Two types of conduits were fabricated by using two batches of P(BHET-EOP/TC) with different weight-average molecular weights (Mw) and polydispersity indexes (PI). The polymers as well as conduits were non-toxic to all six types of cells tested, including primary neurones and neuronally differentiated PC12 cells. After in situ implantation in the sciatic nerve of the rat, two types of conduits triggered a similar tissue response, inducing the formation of a thin tissue capsule composed of approximately eight layers of fibroblasts surrounding the conduits at 3 months. Biological performances of the conduits were examined in the rat sciatic nerve model with a 10 mm gap. Although tube fragmentation, even tube breakage, was observed within less than 5 days post-implantation, successful regeneration through the gap occurred in both types of conduits, with four out of 10 in the Type I conduits (Mw 14,900 and PI 2.57) and 11 out of 12 in the Type II conduits (Mw 18,900 and PI 1.72). The degradation of conduits was further evidenced by increased roughness on the tube surface in vivo under scanning electron microscope and a mass decrease in a time-dependent manner in vitro. The Mw of the polymers dropped 33 and 24% in the Type I and II conduits, respectively, in vitro within 3 months. Among their advantages over other biodegradable NGCs, the PPE conduits showed negligible swelling and no crystallisation after implantation. Thus, these PPE conduits can be effective aids for nerve regeneration with potential to be further developed into more sophisticated NGCs that have better control of the conduit micro-environment for improved nerve regeneration.     

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.     

Peripheral nerve regeneration by microbraided poly(L-lactide-co-glycolide) biodegradable polymer fibers

Tiny tubes with fiber architecture were developed by a novel method of fabrication upon introducing some modification to the microbraiding technique, to function as nerve guide conduit and the feasibility of in vivo nerve regeneration was investigated through several of these conduits. Poly(L-lactide-co-glycolide) (10:90) polymer fibers being biocompatible and biodegradable were used for the fabrication of the conduits. The microbraided nerve guide conduits (MNGCs) were characterized using scanning electron microscopy to study the surface morphology and fiber arrangement. Degradation tests were performed and the micrographs of the conduit showed that the degradation of the conduit is by fiber breakage indicating bulk hydrolysis of the polymer. Biological performances of the conduits were examined in the rat sciatic nerve model with a 12-mm gap. After implantation of the MNGC to the right sciatic nerve of the rat, there was no inflammatory response. One week after implantation, a thin tissue capsule was formed on the outer surface of the conduit, indicating good biological response of the conduit. Fibrin matrix cable formation was seen inside the MNGC after 1 week implantation. One month after implantation, 9 of 10 rats showed successful nerve regeneration. None of the implanted tubes showed tube breakage. The MNGCs were flexible, permeable, and showed no swelling apart from its other advantages. Thus, these new poly(L-lactide-co-glycolide) microbraided conduits can be effective aids for nerve regeneration and repair and may lead to clinical applications.     

A composite poly-hydroxybutyrate-glial growth factor conduit for long nerve gap repairs

There is considerable evidence that peripheral nerves have the potential to regenerate in an appropriate microenvironment. We have developed a novel artificial nerve guide composed of poly 3-hydroxybutyrate (PHB) filled with glial growth factor (GGF) suspended in alginate hydrogel. Gaps of 2-4 cm in rabbit common peroneal nerve were bridged using a PHB conduit containing either GGF in alginate hydrogel (GGF) or alginate alone (Alginate), or with an empty PHB conduit (Empty). Tissues were harvested 21, 42 and 63 days post-operatively. Schwann cell and axonal regeneration were assessed using quantitative immunohistochemistry. At 21 days, addition of GGF increased significantly the distance of axonal and Schwann cells regeneration in comparison with that observed in Alginate and Empty conduits for both gap lengths. The axons bridged the 2-cm GGF conduits gap by 63 days, with a comparable rate of regeneration seen in 4-cm conduits. Schwann cells and axonal regeneration quantity was similar for both gap lengths in each group. However, at all time points the quantity of axonal and Schwann cells regeneration in GGF grafts was significantly greater than in both Alginate and Empty conduits, the latter showing better regeneration than Alginate conduits. The results indicate an inhibitory effect of alginate on regeneration, which is partially reversed by the addition of GGF to the conduits. In conclusion, GGF stimulates a progressive and sustainable regeneration increase in long nerve gap conduits.     

Promoting peripheral nerve regeneration with biodegradable poly (DL-lactic acid) films

Regeneration and repair of peripheral nerve injury has always been a major problem in the clinic. The conventional technique based on suturing the nerve ends to each other coupled with the implantation of nerve conduits outside is associated with postoperative adhesions and scar problems. Recently, a novel biodegradable poly (DL-lactic acid) (PDLLA) film has been introduced. This novel anti-adhesion film has a porous structure with better mechanical properties, better flexibility, and more controllable degradation as compared to traditional non-porous nerve conduits. However, little is known about the effects of such PDLLA films on regeneration and repair of peripheral nerve injury in vivo. In this study, we evaluated the effects of PDLLA films implantation after sciatic nerve transection and anastomosis on subsequent sciatic nerve regeneration in vivo, using a rat sciatic nerve injury model. Sciatic nerve transection surgery coupled with direct suturing only, suturing and wrapping with traditional nerve conduits, or suturing and wrapping with PDLLA films was performed on adult Wistar rats. The additional wrapping with PDLLA films inhibited the nerve adhesion after 12 weeks recovery from surgery. It also increased the compound muscle action potentials and tibialis and gastrocnemius muscle wet weight ratio following 8 weeks recovery from surgery. Regenerated nerve fibers were relatively straight and the aligned structure was complete in rats with implantations of PDLLA films. The results suggested that PDLLA films can improve the nutritional status in the muscles innervated by the damaged nerves and promote nerve regeneration in vivo.     

神经导管修复周围神经损伤的研究进展

对神经导管在周围神经损伤的修复和再生的应用及研究进展作一综述。主要包括两方面内容：神经导管材料设计及导管内微环境构建。     

Heparin-Binding-Affinity-Based Delivery Systems Releasing Nerve Growth Factor Enhance Sciatic Nerve Regeneration

The controlled delivery of nerve growth factor (NGF) to the peripheral nervous system has been shown to enhance nerve regeneration following injury, although the effect of release rate has not been previously studied with an affinity-based delivery system (DS). The goal of this research was to determine if the binding site affinity of the DS affected nerve regeneration in vivo using nerve guidance conduits (NGCs) in a 13-mm rat sciatic nerve defect. These DSs consisted of bi-domain peptides that varied in heparin-binding affinity, heparin and NGF, which binds to heparin with moderate affinity. Eight experimental groups were evaluated consisting of NGF with DS, control groups excluding one or more components of the DS within silicone conduits and nerve isografts. Nerves were harvested 6 weeks after treatment for analysis by histomorphometry. These DSs with NGF resulted in a higher frequency of nerve regeneration compared to control groups and were similar to the nerve isograft group in measures of nerve fiber density and percent neural tissue, but not in total nerve fiber count. In addition, these DSs with NGF contained a significantly greater percentage of larger diameter nerve fibers, suggesting more mature regenerating nerve content. While there were no differences in nerve regeneration due to varying peptide affinity with these DSs, their use with NGF enhanced peripheral nerve regeneration through a NGC across a critical nerve gap.     

The behavior of neuronal cells on tendon-derived collagen sheets as potential substrates for nerve regeneration

Peripheral nervous system injuries result in a decreased quality of life, and generally require surgical intervention for repair. Currently, the gold standard of nerve autografting, based on the use of host tissue such as sensory nerves is suboptimal as it results in donor-site loss of function and requires a secondary surgery. Nerve guidance conduits fabricated from natural polymers such as collagen are a common alternative to bridge nerve defects. In the present work, tendon sections derived through a process named bioskiving were studied for their potential for use as a substrate to fabricate nerve guidance conduits. We show that cells such as rat Schwann cells adhere, proliferate, and align along the fibrous tendon substrate which has been shown to result in a more mature phenotype. Additionally we demonstrate that chick dorsal root ganglia explants cultured on the tendon grow to similar lengths compared to dorsal root ganglia cultured on collagen gels, but also grow in a more oriented manner on the tendon sections. These results show that tendon sections produced through bioskiving can support directional nerve growth and may be of use as a substrate for the fabrication of nerve guidance conduits.     

可降解神经套接管制备及临床试验流程专家共识

周围神经损伤是一种常见临床疾病,损伤后的有效修复是减少损伤并发症、改善预后的重要措施.临床上治疗单纯横断伤或微小缺损的神经损伤多采用显微外科对位缝合方式吻合神经断端,但临床修复效果难达到预期.基于周围神经选择性再生学说,研究者发现小间隙套接缝合的修复效果明显好于传统的神经外膜缝合方法.因此,研发一种用来桥接周围神经断端...     

微球体的制备及其在修复神经系统损伤研究中的应用

利用组织工程技术制备神经支架应用于神经系统损伤的修复已经成为神经系统再生研究领域的重点,其中利用生物材料以微球体形式,持续缓慢释放活性物质,有利于神经系统再生的微环境改善,也是研究的热点。本文就微球体制备的相关材料、制备方法、检测及在神经系统损伤修复中的作用等进行综述,并进行了总结和展望。     

Nerve guide material made from fibronectin: assessment of in vitro properties

We have previously used orientated mats of fibronectin as conduits to repair short gaps in peripheral nerves. Here we describe the in vitro properties of a new material in the form of large cables produced from a fibronectin-enriched solution with potential as a conduit for longer nerve defects. Large cables of fibronectin were made up to 14 cm long x 1.5 cm in diameter. When freeze dried, scanning electron microscopy revealed a predominant fiber orientation. Dried cables hydrated rapidly to 1.6 and 4.8 times their original length and diameter, respectively. Once hydrated these cables had pores that ranged from 10 to 100 microm through which Schwann cells and fibroblasts were able to grow in vitro and align with the axis of the fibrils by contact guidance. Furthermore, the porosity of the cable was enhanced by the natural dissolution of protein over a 3-week duration in culture with cells, such that 50- to 200-microm pores were observed. This study suggests that large fibronectin cables are a suitable alternative to the original fibronectin mats to guide components of the peripheral nerves and so to act as conduits with potential use in guiding regeneration across long nerve defects.     

A bioengineered peripheral nerve construct using aligned peptide amphiphile nanofibers

Peripheral nerve injuries can result in lifelong disability. Primary coaptation is the treatment of choice when the gap between transected nerve ends is short. Long nerve gaps seen in more complex injuries often require autologous nerve grafts or nerve conduits implemented into the repair. Nerve grafts, however, cause morbidity and functional loss at donor sites, which are limited in number. Nerve conduits, in turn, lack an internal scaffold to support and guide axonal regeneration, resulting in decreased efficacy over longer nerve gap lengths. By comparison, peptide amphiphiles (PAs) are molecules that can self-assemble into nanofibers, which can be aligned to mimic the native architecture of peripheral nerve. As such, they represent a potential substrate for use in a bioengineered nerve graft substitute. To examine this, we cultured Schwann cells with bioactive PAs (RGDS-PA, IKVAV-PA) to determine their ability to attach to and proliferate within the biomaterial. Next, we devised a PA construct for use in a peripheral nerve critical sized defect model. Rat sciatic nerve defects were created and reconstructed with autologous nerve, PLGA conduits filled with various forms of aligned PAs, or left unrepaired. Motor and sensory recovery were determined and compared among groups. Our results demonstrate that Schwann cells are able to adhere to and proliferate in aligned PA gels, with greater efficacy in bioactive PAs compared to the backbone-PA alone. In vivo testing revealed recovery of motor and sensory function in animals treated with conduit/PA constructs comparable to animals treated with autologous nerve grafts. Functional recovery in conduit/PA and autologous graft groups was significantly faster than in animals treated with empty PLGA conduits. Histological examinations also demonstrated increased axonal and Schwann cell regeneration within the reconstructed nerve gap in animals treated with conduit/PA constructs. These results indicate that PA nanofibers may represent a promising biomaterial for use in bioengineered peripheral nerve repair.     

干细胞与周围神经损伤修复

目前周围神经缺损的修复仍是临床的一大难题。周围神经系统具有再生的潜能,但在自体神经移植中受到取材长度的限制,近年来许多学者将体外培养扩增的大量雪旺细胞种植到神经导管中,以引导周围神经再生,然而在适当条件下难以维持大量的雪旺细胞,雪旺细胞传代后形态和功能逐渐改变。体外培养的干细胞,尤其是中枢来源的干细胞,具有低免疫原性,可以分化为雪旺细胞,移植至受损部位后可促进神经再生。本文综述了干细胞在修复周围神经损伤中的应用及其在再生过程中所发挥的作用。     

Peripheral nerve regeneration with sustained release of poly(phosphoester) microencapsulated nerve growth factor within nerve guide conduits

Prolonged delivery of neurotrophic proteins to the target tissue is valuable in the treatment of various disorders of the nervous system. We have tested in this study whether sustained release of nerve growth factor (NGF) within nerve guide conduits (NGCs), a device used to repair injured nerves, would augment peripheral nerve regeneration. NGF-containing polymeric microspheres fabricated from a biodegradable poly(phosphoester) (PPE) polymer were loaded into silicone or PPE conduits to provide for prolonged, site-specific delivery of NGF. The conduits were used to bridge a 10 mm gap in a rat sciatic nerve model. Three months after implantation, morphological analysis revealed higher values of fiber diameter, fiber population and fiber density and lower G-ratio at the distal end of regenerated nerve cables collected from NGF microsphere-loaded silicone conduits, as compared with those from control conduits loaded with either saline alone, BSA microspheres, or NGF protein without microencapsulation. Beneficial effects on fiber diameter, G-ratio and fiber density were also observed in the permeable PPE NGCs. Thus, the results confirm a long-term promoting effect of exogenous NGF on morphological regeneration of peripheral nerves. The tissue-engineering approach reported in this study of incorporation of a microsphere protein release system into NGCs holds potential for improved functional recovery in patients whose injured nerves are reconstructed by entubulation.     

Nerve growth factor expression by PLG-mediated lipofection

Biomaterials capable of efficient gene delivery provide a fundamental tool for basic and applied research models, such as promoting neural regeneration. We developed a system for the encapsulation and sustained release of plasmid DNA complexed with a cationic lipid and investigated their efficacy using in vitro models of neurite outgrowth. Sustained lipoplex release was obtained for up to 50 days, with rates controlled by the fabrication conditions. Released lipoplexes retained their activity, transfecting 48.2+/-8.3% of NIH3T3 cells with luciferase activity of 3.97x10(7)RLU/mg. Expression of nerve growth factor (NGF) was employed in two models of neurite outgrowth: PC12 and primary dorsal root ganglia (DRG) co-culture. Polymer-mediated lipofection of PC12 produced bioactive NGF, eliciting robust neurite outgrowth. An EGFP/NGF dual-expression vector identified transfected cells (GFP-positive) while neurite outgrowth verified NGF secretion. A co-culture model examined the ability of NGF secretion by an accessory cell population to stimulate DRG neurite outgrowth. Polymer-mediated transfection of HEK293T with an NGF-encoding plasmid induced outgrowth by DRG neurons. This system could be fabricated as implants or nerve guidance conduits to support cellular and tissue regeneration. Combining this physical support with the ability to locally express neurotrophic factors will potentiate regeneration in nerve injury and disease models.     

Performance modification of chitosan membranes induced by gamma irradiation

Trauma of the nervous system often results in permanent functional loss because the spontaneous regeneration of nerves is very difficult. Thus, various methods have been developed to facilitate the restoration of damaged nerve. The biodegradable nerve conduit is one of the most promising methods for nerve regeneration. Chitosan, a natural polysaccharide that has excellent biocompatibility and biodegradability, can be used as conduit material. But, nerves regenerated by nerve conduits made from chitosan have some problems, for example, with their mechanical properties. This article shows that the mechanical properties of chitosan film were markedly improved by selected doses of gamma radiation and cell culturing experiments on the surface of the irradiated chitosan film indicated that the film still has excellent biocompatibility.     

A synthetic oxygen carrier in fibrin matrices promotes sciatic nerve regeneration in rats

Tissue-engineering nerve conduits have been studied for a long time in bridging large nerve defects. However, the low oxygen availability within the nerve conduits, which results in death of migratory Schwann cells (SC) or loss of the newly formed tissue’s function, is still an obstacle for axonal regeneration. Thus, it was hypothesized that an oxygen-enriched conduit would enhance axonal regeneration and functional recovery in vivo. To address this issue, perfluorotributylamine (PFTBA) enriched fibrin hydrogel was prepared and injected into collagen–chitosan conduits. The conduit containing PFTBA-enriched fibrin hydrogel was then used to bridge a 12-mm sciatic nerve defect in rats. The control rats were bridged with collagen–chitosan conduits filled with fibrin matrices without PFTBA. It was found that axonal regeneration and functional recovery in the combined PFTBA group were significantly higher than those in the control group without PFTBA. Further investigations showed that the mRNA and protein levels of S-100, brain-derived neurotrophic factor and nerve growth factor were enhanced by PFTBA at 1 and 3 weeks after surgery. However, the mRNA and protein levels of vascular endothelial growth factor were in a similar range between the combined PFTBA group and the control group without PFTBA. In addition, immunohistochemical results showed that the morphological appearances of regenerated nerve and survival of SC were enhanced by PFTBA at 4 and 12 weeks after surgery. In conclusion, PFTBA-enriched nerve conduit is capable of enhancing axonal regeneration, which provides a new avenue for achieving better functional recovery in the treatment of nerve defect.     

Bioengineered nerve regeneration and muscle reinnervation

The peripheral nervous system has the intrinsic capacity to regenerate but the reinnervation of muscles is often suboptimal and results in limited recovery of function. Injuries to nerves that innervate complex organs such as the larynx are particularly difficult to treat. The many functions of the larynx have evolved through the intricate neural regulation of highly specialized laryngeal muscles. In this review, we examine the responses of nerves and muscles to injury, focusing on changes in the expression of neurotrophic factors, and highlight differences between the skeletal limb and laryngeal muscle systems. We also describe how artificial nerve conduits have become a useful tool for delivery of neurotrophic factors as therapeutic agents to promote peripheral nerve repair and might eventually be useful in the treatment of laryngeal nerve injury.     

Conductive PPY/PDLLA conduit for peripheral nerve regeneration

The significant drawbacks and lack of success associated with current methods to treat critically sized nerve defects have led to increased interest in neural tissue engineering. Conducting polymers show great promise due to their electrical properties, and in the case of polypyrrole (PPY), its cell compatibility as well. Thus, the goal of this study is to synthesize a conducting composite nerve conduit with PPY and poly(d, l-lactic acid) (PDLLA), assess its ability to support the differentiation of rat pheochromocytoma 12 (PC12) cells in vitro, and determine its ability to promote nerve regeneration in vivo. Different amounts of PPY (5%, 10%, and 15%) are used to synthesize the conduits resulting in different conductivities (5.65, 10.40, and 15.56 ms/cm, respectively). When PC12 cells are seeded on these conduits and stimulated with 100 mV for 2 h, there is a marked increase in both the percentage of neurite-bearing cells and the median neurite length as the content of PPY increased. More importantly, when the PPY/PDLLA nerve conduit was used to repair a rat sciatic nerve defect it performed similarly to the gold standard autologous graft. These promising results illustrate the potential that this PPY/PDLLA conducting composite conduit has for neural tissue engineering.