Review Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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Peripheral Nerve Conduit: Materials and Structures Shadi Houshyar,*,† Amitava Bhattacharyya,‡ and Robert Shanks§ †
School of Engineering, RMIT University, Melbourne, Victoria 3000, Australia Nanoscience and Technology, Department of Electronics and Communication, PSG College of Technology, Coimbatore − 641004, India § School of Science, RMIT University, Melbourne, Victoria 3000, Australia
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‡
ABSTRACT: Peripheral nerve injuries (PNIs) are the most common injury types to affect the nervous system. Restoration of nerve function after PNI is a challenging medical issue. Extended gaps in transected peripheral nerves are only repaired using autologous nerve grafting. This technique, however, in which nerve tissue is harvested from a donor site and grafted onto a recipient site in the same body, has many limitations and disadvantages. Recent studies have revealed artificial nerve conduits as a promising alternative technique to substitute autologous nerves. This Review summarizes different types of artificial nerve grafts used to repair peripheral nerve injuries. These include synthetic and natural polymers with biological factors. Then, desirable properties of nerve guides are discussed based on their functionality and effectiveness. In the final part of this Review, fabrication methods and commercially available nerve guides are described. KEYWORDS: Peripheral nerve injuries, nerve regeneration, nerve guide conduit, polymeric conduit structures
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INTRODUCTION Peripheral nerve injuries (PNIs) are the most common types of injuries affecting the nervous system.1 Twenty million people in the United States suffer from peripheral nerve injuries, and there are over 300,000 cases per year in Europe.2,3 These injuries are a result of trauma, disease, or surgical procedures, resulting in permanent muscle movement impairment with little or no normal sensations in the affected area.3−9 The nervous system monitors and controls every organ and system in the body. It consists of two parts: central nervous system (CNS) and peripheral nervous system (PNS).2 The CNS consists of spinal cord and brain, while the PNS includes the rest of the nerves coming from the spinal cord and brain. The CNS is responsible for transferring electrical impulses to the PNS, and the PNS processes the impulses by transferring information and signals to and from the CNS.4,10 The PNS consists of thousands of individual nerve fibers or axons (myelinated or nonmyelinated axons by Schwann cells) that can reach more than one meter in length.10−12After transection of a PN, a degeneration process, called Wallerian or Anterograde, happens,3,13 Figure 1(a,b). In this process, newly generated axons are produced and supported by the local and produced cytoskeletal protein, toward the distal stump.3,5,13 © XXXX American Chemical Society
Meanwhile, Schwann cells accelerate degeneration of the distal nerve stump, Figure 1b, resulting in morphological changes in the gap called bands of Burger.4,10,12 This means Schwann cells align to direct in-growing axons toward the distal stump, Figure 1c. If the regenerated axons reach the distal stump, within a “favorable time period”, restoration of nerve function is possible,3,4,11,14 Figure 1d. Otherwise chronic Schwann cell denervation happens, resulting in slowed growth of axons. Therefore, for effective nerve regeneration, newly growing axons and Schwann cells in the distal stump should be in contact in a “favorable time period”.3,11,15
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CURRENT TREATMENT FOR PNIS Current treatment for gaps < 3 cm is end-to-end connections, to immobilize a nerve; however, for a larger gap, the gold standard is an autologous graft or allograft, to avoid excessive stretch and compromising blood flow to the nerve.2−4,12,16,17 Therefore, the application of suturing is limited to short gaps Received: April 4, 2019 Accepted: June 19, 2019 Published: June 19, 2019 A
DOI: 10.1021/acschemneuro.9b00203 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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Figure 1. Wallerian or Anterograde process after PNIs: (a) transection of peripheral nerve, (b) local and produced cytoskeletal protein support newly generated axons, (c) Schwann cells align to direct in-growing axons toward the distal stump (bands of Burger), and (d) regenerated nerve.
Figure 2. Parameters for the development of a successful nerve conduit.
and average axon density of the donor and recipient nerves match.20,25,26 The benefits of using autologous grafts includes nontoxicity, good biocompatibility, and provision of support for the extension of Schwann and other support cells.4,5,18 A major disadvantage, in addition to the need for multiple surgeries, is poor functional recovery at the donor site and donor morbidity.4,5,18,19,27 As reported by Winter and Schmidt, only 80% of patients recover functionality after a nerve repair procedure by autologous graft technique.28 A reliable alternative would be an artificial nerve guide to resolve these issues. An ideal nerve guide should have an internal structure that mimics an autograft and provide enough support and mechanical strength, while being flexible, porous, and with suitable degradation time, and at the same time not delivering pressure on the regenerated nerve,2,3,14 Figure 2. In their review paper, Chiono and Tonda-Turo mention that topographic, chemotactic, and hepatotactic cues to help increase
with some form of bridging graft essential for larger gaps to act as a physical guidance for directing the axonal sprout and reducing the scar tissue formation in the injured site.14,18 Autologous grafts or allografts undergo Wallerian degeneration, explained in the previous section, resulting in a rich source of neurotrophic factors to support axonal growth and nerve regenerations.10,19,20 Several types of biological tubular grafts have been used by researchers to cover this issue,21 such as decalcified inside out veins, blood vessels, arteries, xenografts, muscle fibers, bone conduits, and allogenic and autologous nerve grafts.21,22 However, there are concerns about using them due to limited availability and formation of neuroma resulting in pain sensation and donor site morbidity.6,18,23,24 An autologous nerve graft is harvested from elsewhere in the body (most commonly dissected from the patient’s lower legs),4,5,24 and the best results are achieved when the diameter B
DOI: 10.1021/acschemneuro.9b00203 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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Figure 3. Diagram showing an artificial nerve conduit as an alternative for nerve autograft and grafting process.
formation and ingrowth of unwanted tissue into the gap, and then it slowly degrades.21,34 The degradation rate should be sufficiently slow for the conduit to retain its shape and strength throughout the period of regeneration. The conduit should degrade more quickly near the proximal nerve end and slowly at the distal end,2,3,9,21 Figure 2. Incorporated drugs and trophic factors can be released as the conduit degrades. The growth of myelinated and unmyelinated axons across the gap can be enhanced more in this method compared with a conventional tube by controlling releasing compounds.5,21,35−37 The evaluation of nerve conduit biological performance has been done through in vivo and in vitro study. Cell viability, adhesion, and proliferation of glial cells have been investigated in response to the first stage of the regeneration process. To investigate the performance of a nerve conduit for nerve regeneration, a wide range of assays have been developed by independent laboratories, which leads to comparison difficulties. However, the degree of nerve regeneration has been evaluated by histological analysis along with functional recovery.18,38 In summary, a nerve guide should be biocompatible, have enough strength and flexibility without pressurizing the nerve, have low immunogenicity with good cell adhesion in the required direction, and have an appropriate degradation time, with nontoxic degradation products,2,14,29 Figure 4. This can be achieved by combination of various materials and structures, additives, and biological materials. Some of the biodegradable materials, natural and synthetic polymers,
axonal growth and minimize end-organ atrophy are main critical aspects of the nerve conduit which results in functional nerve recovery.18
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SYNTHETIC NERVE GRAFT Alternative techniques such as artificial nerve guides have been developed to reconstruct the transacted nerve ends and eliminate the above-mentioned problems. Artificial nerve grafts are made of synthetic polymers that have many advantages over the traditional autologous nerve grafts. It is possible to tailor their properties to serve similar application.2,4,14,19 Synthetic polymers can be formed into various shapes, including knitted structures, sponges, solid or porous tubes, meshes, and foams, to be similar to the shape of nerve tissue.29 These artificial nerve guides would be placed between two ends of a damaged nerve, distal and proximal, to allow the nerve to regenerate across a well-defined gap. This protocol prevents nerve dislocation or rotation,10,29−33 Figure 3. The nerve conduit should be nontoxic, minimally immunogenic, and uncomplicated to manufacture. Furthermore, it should guide regenerative nerve direction, isolate the regenerated axon from scar tissue, and protect the regenerated nerve against surrounding compression.18 The designed nerve conduit can be in the form of a single non/porous hollow tube with/out fillers or a multichannel nerve conduit to mimic the natural compartment structure of nerves.18 A silicone elastomer tube was one of the initial tubes that were used for synthetic nerve guides, due to the flexibility and biocompatibility of polydimethylsiloxanes (PDMS).10,30 Moreover, transparency is another advantage for in vivo models as it facilitates complete 3D examination of the regeneration process. “Follow up” surgery must be done to remove nondegradable nerve conduit because it causes immunological reactions, leading to constricting the nerve and hampering nerve regeneration.2,3,9 A biodegradable nerve conduit is an excellent substitute with the greatest potential, and it is preferred by researchers. The degradable nerve conduit is designed to degrade after guiding the axonal regeneration toward the distal nerve stumps. Furthermore, a conduit prevents neuroma
Figure 4. Nerve conduit and different forms of luminal fillers. C
DOI: 10.1021/acschemneuro.9b00203 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience Table 1. Some Examples of Nerve Guide Materials materials synthetic biostable polymer poly(ethylene-co-vinyl acetate) (EVA)59
poly(tetrafluoroethylene) (PTFE)60
expanded poly(tetrafluoroethylene) (ePTFE)2 silicone elastomers (SE)62
Millipore (cellulose filter)2 PTFE-electric60 polyethylene (PE)5 polysulfone (PS)2 poly(vinylidene fluoride) (PVDF)64 silicone rubber (PDMS)39 polyethylene (PE)14
polyacrylonitrile (PAN) and its copolymers61 poly(vinyl chloride) (PVC)61 polyurethane63 polypyrrole (PPy)65 poly(acrylonitrile-co-vinyl chloride) (PAN−PVC)61
poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (PHEMA-MMA)66 synthetic degradable polymer poly(glycolic acid) (PGA)67 poly(lactic acid) (PLA)41 poly(ε-caprolactone) (PCL)45 63 poly(hydroxyethyl polyurethane (PU) PCL-acrylate (PCLA)69 methacrylate) PGA-PLA blends70 poly(lactic acid−glycolic acid) (PLGA) poly(ethylene glycol) (PEG)70 methacrylated dextran (Dex-MA) copolymerized with aminoethyl methacrylate (AEMA)
PCL-fumarate (PCLF)68 poly(D,L-lactide-cocaprolactone) (PDLLC)70 collagen-terpolymer poly(lactide-co-glycolide-cocaprolactone) (PLGC) PCL-co-PDO (PCD)
poly(propylene fumarate-coPCL) (PPFPCL) poly(glyceryl-sebacate) (PGS)
polydioxanone (PDO)
poly(organo) phosphazine
poly(glycolide-co-caprolactone) (PGC)
poly(3-hydroxybutyrate-co-3hydroxyhexanoate) (PHBHHx) metals stainless steel72 natural polymers hyaluronic acid derivatives57 agarose14 alginate42 chitosan75 main biological materials glial cells (GD)
poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-poly(Llactide-co-D,L-lactide)-PLGA [PHBV-P(L-D,L)LA-PLGA] blend
poly(lactide-co-ethylene glycol-comethacrylate) (PLLA-PEG-MA) polyphosphoester (PPE)
poly(3-hydroxybutyrate) (PHB)71 poly(trimethylene carbonateε-caprolactone) (PTMCCL)
laminin67 collagen60 artery−vein3 spider silk fiber2
collagen−gelatin29 Matrigel2,74 gelatin54
decalcified bone73 polyamide74 Matrigel9 silk fibroin51
stem cells14
brain-derived neurotrophic factor (BDNF)29 neural progenitor cells9 neurotrophin (NT3)69 fibroblast growth factor (FGF)78 olfactory ensheathing cells80,81
tantalum2
Schwann cells76 astrocytes77 neural stem cells77 glial growth factor (GGF) biomimetic materials5 nerve growth factor (NGF)78 79 synergism neurotrophic factors5 widely used fabrication methods injection molding38 mandrel coating2 82 extrusion electrospinning80 78 fused deposition soft lithography39
dip-coating,77 centrifuge casting wet phase inversion2 microbraiding of filaments79
film rolling and sealing82 immersion precipitation82
sively used for nerve guides.22,43 Several nerve guides made of cross-linked collagen type I have been approved by the U.S. Food and Drugs Administration (FDA) and are commercially available,44 Table 3. Generally, dip-coating and injection molding are the two preferred fabrication methods for collagen; however, recently, many researchers have an interest in wet spinning and electrospinning collagen.45 The collagen fibers produced can be wound to form a tubular shape or they can be used as a longitudinally aligned luminal filler.30,31 In one study, collagen sponge was made with interconnected pores and then cross-linked using microwave irradiation. This sponge exhibited a high tensile modulus. Blended collagen−chitosan is an alternative biocompatible material for nerve guide application.46 In another study, the nerve conduit was fabricated with silk fibroin−collagen and then was cocultured with Schwann cells and adipose-derived stem cells (ADSC). In vivo results showed improvement in the regenerative microenvironment and acceleration of nerve regeneration.47 Choi et al. fabricated a biodegradable nerve conduit made with collagen-based materials and filled it with NGF, BDNF, and
biological materials, and fabrication techniques are listed in Table 1. Natural Polymers. A well-known natural polymer is chitosan, known for its biocompatibility and nontoxicity. In one study, multichannel cross-linked chitosan with a complex structure was formed through soft lithography on a PDMS mold.39 Chitosan was injected into a stainless-steel mold and cross-linked with formaldehyde. A freeze-drying or lyophilization method has been investigated by researchers, and poly(glycolic acid) (PGA) fibers have been added to the empty space inside the nerve conduit, PGA.40 They improved the mechanical properties of the nerve conduit by injecting chitosan on mounted poly(lactic acid-co-glycolic acid) (PLGA) and then dried the material, or dip-coated a chitosan tube with poly(lactic acid) (PLA).41 Another study confirmed development of a hydrogel nerve guide consisting of alginate and chitosan formed in acetic acid solution.42 Collagen is another natural biocompatible polymer, which has been used for nerve guides for many years. Despite its low mechanical properties, cross-linked collagen has been extenD
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materials degrade through hydrolyzing their ester groups in the polymer chain, which can be synthesized to degrade rapidly or slowly depending upon structure. Blending these materials with a natural polymer has been reported in the literature.2,4,72 Blending PCL with collagen-based beads or chitosan has been reported to provide strength and flexibility to natural polymers while retaining biocompatibility with natural polymers.45 Copolymer of PCL with PLA, PEG, and plasma polymerized using ring opening polymerization have been reported to provide more strength and faster degradation time in comparison with pure PCL.14,17,24,29,89,90 Cross-linked PCL and copolymers such as PCLA or PCLF68,69 have been studied as nerve conduits to provide more strength to the conduit. These cross-linked polymers can be used for hard and soft tissue implants due to their broad range of mechanical and thermal properties. Melded and photo-cross-linked, single or multichannel PPF-PCL and PCLF nerve conduits provide biocompatibility and axonal growth guides in the rat sciatic nerve.91−93 Qian et al. reported a PCL nerve conduit loaded with polydopamine (PDA), arginylglycylaspartic acid (RGD), and graphene, through a multilayer 3D fabrication technique. In vivo and in vitro results showed that the nerve conduit promoted axonal regrowth and improved neural expression.94 Based on this finding, a graphene-based nerve conduit will have a great potential for repairing PNI in clinical applications.94 Another study by Shah et al. investigated a spiral structured PCL nerve guide with multichannels and aligned inner nanofibers of PCL.95 They fabricated the inner aligned nanofibers through an electrospinning technique and then incorporated collagen into multichannel inside the tube. The newly developed nerve conduit in this study presented promising data in nerve regeneration within a relatively short recovery time.95 Quigley et al.65 reported knitted PLA nerve conduits coated with electrospun PLA (to control the pore size) and filled with PLGA fibers in a neurotrophic enriched alginate hydrogel. These conduits were tested in rat sciatic nerves, and they showed promising results for the presence of myelinated axons inside the lumen and in the distal stump, while preventing selfmutilation and overgrooming. Another widely available biopolymer is PHB with adjustable mechanical properties, depending on copolymerization method or additives.68 It is synthesized via micro-organisms. Generally, dip-coating and electrospinning are the main fabrication techniques for PHB. PHB was blended with PLLA and PLGA to produce porous films using solvent casting, then filled with electrospun PHBV-PLGA aligned fibrous mat.70,96 Zhu et al. investigated a PEGDA nerve conduit fabricated through a 3D-bioplotting technique. They confirmed directional sciatic nerve regeneration in vivo with motor and functional sensory recovery.97 Biodegradable polyurethane (PU), consisting of soft and hard segments, is a suitable candidate for nerve conduits due to its flexibility and other mechanical properties. In situ extrusion, dip-coating, and solvent casting are the main fabrication techniques for PU.63 Mostly, flexibility is provided by the soft segment while the hard segment delivers mechanical support. In this case, the soft segment degrades quickly and the hard segment degrades over a longer period of time. The 3D porous structure of nerve conduits of PU with PEG and PCL segments can be fabricated through rapid prototyping methods.98,99 Poly(glyceryl sebacate) (PGS) with calcium titanate has been used for nerve conduit fabrication. The developed nerve guide
laminin; this resulted in significant improvement in motor and sensory nerve function.48 Rajaram investigated an alginate−hyaluronic acid scaffold for nerve regeneration using biofabrication techniques including 3D bioplotting, reactive printing, and wet spining. He incorporated living Schwann cells into the conduit, and his in vivo study confirmed the possibility of implanting a conduit for nerve repair along with promising results.49 Stevens investigated a nerve conduit made of anionic polysaccharide gellan gum (GG) and incorporated encapsulated neural cells.50 Fibroin, the protein constituent of silk, is an excellent candidate for nerve conduit application due to its high biocompatibility, slow degradation, and suitable mechanical properties.51 Electrospun fibers and film were produced from pure silk, but they were crushed and fractured easily under pressure inside the body; therefore, a combination of silk with other materials would be a potential solution.52 Oriented silk filament has been used for inside the nerve conduit and filler.32 A nanofiber formed through wet spinning or electrospinning methods by blending silk with other polymers such as polyolefin (PO), PLGA, and poly(vinyl acetate) (PVA) showed promising results.2 Li et al. investigated waterinsoluble silk fibroin (SF) for the nerve conduit, through a freeze-drying technique. The results showed promising results for nerve regeneration and the possibility of controlling the degradation rate through a controlled degumming procedure.53 Gelatin is a biodegradable natural polymer that is watersoluble, but does not provide sufficient strength for nerve conduit application. Cross-linked gelatin, after preparation through injection molding or dip-coating, is a potential candidate.54 The gelatin cross-linking degree is an important factor for this material as it determines degradation time.55 Another natural polymer is hyaluronan (HA) with unique properties including nonadhesive character, support of axonal growth, and nonimmunogenic properties, but even crosslinked HA is too weak to handle.56 A fabricated knitted nerve conduit has been reported using an esterified HA derivative.57 The knitted conduit was coated with the same polymer to enhance its strength, but it degraded too fast and caused massive ingrowth of cells and fibrous tissue formation, which were unsuitable for this application, resulting in delayed nerve regeneration and formation.57 Lackington et al. developed a gene-activated nerve conduit by incorporating polyethylenimine-plasmid DNA nanoparticles with the pDNA encoding glial derived neurotrophic factor (GDNF). In vitro results showed promising results for future nerve conduit application.58 In summary, natural polymers are suitable candidates for nerve conduits, while blending them with biodegradable synthetic materials provides more strength and flexibility, with decreasing or tailoring their degeneration time to match the rate of axonal growth along the nerve guide. Synthetic Polymers. Among biodegradable synthetic polymers, some are more widely used in vivo due to their excellent biocompatibility and mechanical properties. Polyesters including PLA, PGA, PLGA, PCLA, and PCL, Table 1, and their copolymers are the most frequently used materials for tissue engineering application.2,13,14,83 These materials are thermoplastic and can be fabricated into any form and shape through coating,84 extruding,85 molding,39 printing,86 and immersion precipitation. Nerve conduits can be fabricated through braiding,87 knitting,65 electrospinning,88 and weaving,2 which provide more potential for fabrication methods. These E
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In 2013, Martin et al. developed a wireless electrical stimulator that could be implanted into the injured site and used to produce an alternating electric field to align sensory neurons. In this study, electrodes were printed on PCL using a biofabrication technique, and were implanted to deliver the electrical field using inductively powered electrical stimulation from an external, wireless source. In vitro studies showed that alternating electric field influenced neural growth at the cellular level and the neurons grew between two electrodes, where the neurons were faced with a barrier on either side.117 Recently, researchers at Washington University developed a new implantable wireless device, monophase electrical impulses, to produce electrical stimulation on demand. Koo et al. demonstrated that electrical stimulation resulted in enhanced neuro-regeneration and accelerated functional recovery in a shorter period of time. This technology overcame the limitation of existing surgical approaches to deliver electrical stimulation. A loop antenna with a dual-coil configuration, electrodes made of Mg (∼50 μm) and PLGA, was used for fabrication of the device.33 Biological Factor. An nerve conduit inserted between proximal and distal nerve stumps formed a cable filled with cell-and-matrix containing materials to support axons and to extend across the gap,4,6,11,15,79 Figure 2. Materials produced in the gap were supplied by damaged nerve stumps. A cable can be designed to support and direct the longitudinal growth of neural cells (nerve cable) and enhance regeneration of axons.29 An artificial nerve conduit with a tube structure has been investigated.29,118 In such studies, regeneration failed for long nerve gaps due to exceeding regenerative capabilities of the damaged nerve stumps.118,119 When this gap is long, fibroblast and Schwann cells do not migrate through the gap to provide a suitable environment inside the conduit for axonal growth.5,10,119−121 Therefore, the lack of effective biocompatibility and bioactivity is one of the key disadvantages of most empty artificial nerve guides suitable for long gaps. A filled tube with glial cells, growth factors, cells, or fibers such as Schwann cells, laminin, collagen, and stem cells enhances the regeneration potential of the tubulation method, which is necessary for optimal axon immigration.20,24,122−124 Tubes are preloaded with appropriate biological factors (growth factors) to create an appropriate environment inside a conduit to improve migration of non-neuronal cells and support axonal growth; this, however, does not result in significant improvement of nerve repair rate and its overall functionality.31,76,120,125−127 It is difficult to find one specific biological factor with an appropriate concentration to accelerate nerve regeneration and repair; however, a mixture of several biological factors with suitable concentration can provide a favorable environment for fiber growth from a proximal stump.36 In the case of a large gap (15−20 cm), tubes were filled with an appropriate combination of growth factors and exogenous matrix precursors, resulting in better axonal regeneration in comparison with an empty tube.35,36,79,128 Harvesting Schwann cells is difficult; addition of various growth factors such as neurotrophic−neurotrophic-3, nerve growth factor (NGF),77,79,81 brain derived neutrophic factor (BDNF),29,123,129 and ciliary neutrophic factor (CNTF) is more attractive.4,81,130,131 However, addition of these growth factors directly to a system has been less attractive due to unpredictable release and leakage into surrounding tissue. Important biological factors are listed in Table 1. Stem cell
showed good cell adhesion and proliferation with improved axonal growth and extension.100 Singh et al. investigated the use of photocurable PGS for nerve repair. They developed a nerve guide through stereolithography, and both in vitro and in vivo results indicated regeneration of axons through the nerve guide into the distal stump. This is a new technique for making a personalized nerve conduit.101 Electro-conductive polymers are another class of synthetic polymers that are capable of enhancing nerve regeneration through electrical stimulation. This class of material is called “intelligent” materials, with great potential for tissue engineering. Conductive polymers such as polytetrafluoroethylene and piezoelectric polymers including poly(vinylidene fluoride) and poly(vinylidene fluoride-co-trifluoroethylene) have been investigated, and the results revealed neural regeneration enhancement in mice.102−106 Research has been undertaken on conducting materials and electromagnetic field activated materials, both in vivo and in vitro, to confirm the influence of electrical conductivity and stimulation on enhancement of axonal regrowth and nerve regeneration.21,29 However, improvement is required in biocompatibility and physical properties of these conductive materials. There are several techniques that have been used to improve these properties such as surface texturizing, addition of charged side groups, or suitable proteins or peptides.8,105,107−109 Polypyrrole (PPy) is a polycationic electrical conductor that is able to incorporate anionic biomolecules (dopants) to improve biocompatibility. As mentioned in one study, films were fabricated using electrochemical deposition, and then negatively charged molecules were added as dopants to the films to make them stable, noncytotoxic, and biocompatible with a high charge density and wettability.110,111 This suggests that PPy can be synthesized and fabricated for specific applications through the selection of suitable dopants.111 Another benefit is that surface properties of both sides of PPy films that can be reversibly changed with an applied electrical potential to make them suitable for many biological applications.103,104,107,112 Investigations have been performed on doping PPy with a broad range of materials such as polymeric or biologically active anions and buffer salts,111 to make the PPy more attractive for future applications in tissue engineering due to its unique properties such as conductivity, antigenicity, and nontoxicity.102,103,105,111−113 Sun et al. investigated a polypyrrole-coated PLCL-SF nerve conduit for nerve repair. It was shown that nerve regeneration was close to that with autograft with good functional recovery.114 Carbon nanotubes (CNTs) and nanofibers (CNFs) have been studied for guided nerve growth due to their excellent electrical and mechanical properties.8 CNFs mimic the structure of tissue and proteins. It was reported by Gopinathan et al. that CNFs were more effective than CNTs and graphene in PCL based nanocomposite scaffolds for PC12 nerve cell proliferation.16 Much research has been performed on nerve guides to achieve successful clinical outcomes in nerve repair procedures; however further improvements in fabrication methods, material selection, and characteristics are still necessary. To achieve this future target, research should emphasize development of biohybrid nerve conduits115,116 to be able to imitate structure and properties of autologous and autograft. The effects of electrical stimulation on nerve regeneration and axonal growth have been investigated widely and broadly. F
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fabricated a 3D nerve conduit through electrospinning PCLPLA with aligned fibers along the inside of the channels.90 The rat sciatic nerve study showed that it acted as vehicle for cotransplanted cells; however, it required further study. Generally, the porosity and permeability of these tubes can be tailored for controlling entry of nutrients while preventing unwanted tissue permeation into the nerve conduit,88 Figure 5.
transplantation is another adjacent therapy to the conventional strategy for PN regeneration, due to stem cells’ ability to respond to the need for regenerating tissue and secreting appropriate pro-regenerative factors.132 In this method, stem cells can be injected directly to the injury site to increase the local concentration and improve tissue regeneration. Although the method of delivery, type of cells, and cell numbers should be optimized, clinical trials (Clinicaltrials.gov; NCT01739023 and NCT01909154) have been started in the CNS. Sun et al. reported a conduit with stem cells encapsulated within the central part. The developed nerve guide showed significant enhancement in function and axonal myelination compared to standard cell lumen injection, which could be a new approach for cell application in peripheral nerve repair.133 Another strategy to improve nerve regeneration is modification of cell surfaces to interact better with the nerve conduit leading to better nerve regeneration.132 In addition to the biological factors, incorporating nanofibers, nanoparticles, and synthetic polymer filler would provide more benefit to fiber growth and proper nerve regeneration.30,88 Generally, aligned fibers are inserted into a nerve guide to provide higher cross-sectional area for supporting myelinated axons. This interface can change the direction of fibers growing and cable of nerve that benefit nerve regeneration and sensory recovery.2,3,134 These materials create more interaction between the nerve guide and biological factors, resulting in better adhesion. Torabinejad et al.135 reported very promising recovery results that indicate a hybrid implant is the future of nerve implants.24
Figure 5. Schematic of different techniques for nerve guide fabrication: 3D bioplotting, 3D printing, electrospinning, braiding, and knitting.
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Injection molding is a widespread technique for fabricating nerve guides from tough and flexible polymers. In this procedure, polymer, with high thermal stability and relatively low melting temperature, was melted and injected into an appropriate mold, usually a tubular form, to form the required shape for nerve conduits.2,39 Generally, polymer with high molecular weight would be used to provide sufficient mechanical strength for nerve conduits, due to multidirectional polymer chain alignment during the injection molding process.13 However, for natural polymer or polymer with low molecular weight, cross-linking is essential. Freeze-drying is another common method for nerve guide fabrication especially with natural polymers, due to unidirectional polymer chain alignment during a freeze-drying technique. One of the conventional techniques to process thermoplastic polymers is melt extrusion. Generally, a single screw extruder is used and rods or fibers are extruded through a nozzle, die, or spinneret. The diameter of the rod or fibers can be varied depending on the size of the die holes and drawing process. Rapid prototyping methods, including three-dimensional printing, inkjet printing, fused deposition modeling, and threedimensional bioplotting techniques, are techniques for nerve conduit fabrication81,83 with an advantage of guide architectures and an ability to incorporate biological components.86,88 The 3D printing technique is capable of fabricating a conduit with a uniform channel diameter in nanodesign which was limited by the conventional fabrication techniques. Tailormade nerve conduits in various shapes and dimensions based on patient requirements can be produced precisely using this technique with the help of medical imaging to fulfill clinical needs.132 Microextrusion is a 3D printing technique that been used to print silicon nerve guides. For example, a complex nerve guide structure was fabricated by Johnson et al.139 that showed promising results. A 3D printed PEGDA nerve guide with complex structure and tunable mechanical properties was
NERVE GUIDE FABRICATION Varied fabrication techniques have been utilized to fabricate appropriate materials into 3D nerve guides. Fabrication techniques will be different depending on the type of materials and their characteristics, including whether they are a synthetic or natural polymer. Some of the widely used fabrication techniques, including dip-molding,39 inkjet printing,136 freezedrying,121 solvent casting-salt leaching,16,82 electrospinning,88 knitting,65 bioprinting, and microbraiding,87 were studied to develop nerve guides. Other techniques such as wet phase inversion,113 fused deposition,137 and soft lithography39 can also be used. The dip-molding technique involves dipping and removal of a mandrel into a polymer solution, with subsequent evaporation of the solvent resulting in a solid tube.2,39 In one of the studies discussed, a bioresorbable tube made using this method revealed a well organized nerve trunk, with a normal pattern of axon and the formation of a connective sheath.2,13 The knitting and microbraiding techniques refer to interweaving of separate yarns with a cross-way overlapping design.65,87 The few studies that have been conducted on braided and knitted nerve guides have reported formation of a fibrin matrix 1 week after implantation. This technique provides a highly flexible and porous structure.65,74,87 The electrospinning technique is a randomly or longitudinally aligned nanofiber production process using an electric field.4,45,89 Collected polymer nanofibers are rolled to prepare tubular nerve conduits.1 For example, recently, Quan et al. developed a 3D helix flexible nerve guide through electrospinning of PCL and implanted it into the rat sciatic nerve, which showed less tension during operation and easy postoperative rehabilitation.138 In another study, Frost et al. G
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rabbit
empty
empty or with autograft bone
Corglass, solid glass tube (CRG-wrap) Bioactive glass
H
neurotrophic factors (NGF) or PGA filament
acellular sciatic nerve ECM
chitosan chitosan
chitosan−PGA graft
collagen hollow fiber
fabrication techniques
30 10
dog, sciatic nerve rat
solvent casting mold porcine dermis was decellularized to create a tube
solvent casting mold
−
rat
electrospinning, hydrogel
dip-coating, fiber spinning, and winding technique
electrospun
photolithography and wet etching electrospinning and braiding
immersion-precipitation phase inversion using casting process and dipping technique and casting mold, leaching Neurolac nonwoven constructs
stereolithography
using a combined solvent casting, extrusion, and leaching or dip-coating process using a 5% w/v copolymer in chloroform solution
GEM NeuroTube woven PGA mesh tube
one-dimensional (1D) A1−A12O3 nanostructures were synthesized by the chemical vapor deposition (CVD) of [BuOAlH2]2
glass was melted in a platinum crucible for 3 h at 1360 °C; after casting in a preheated graphite mold, it was crushed and remelted for homogenization; glass was crushed again and sieved to a fraction of 630−800 μm PGA woven mesh tubes coated with collagen solution and dried at room temperature
commercial silicone nerve melting BLF-1700 at 1250 and 1350 °C for 45 min; Bioglass 45S5 monlit rod of 10 mm was immersed in liquid bioglass commercial CRG-wrap
commercial silicone tube, filled with PBS in silicone graft group (SIL) and with HGF in the HGF-SIL group commercial silicone tube
hollow commercial chitin conduit manufactured by Medovent GmbH extrusion process followed by distinctive washing and hydrolysis steps
5
6
10
10 12
10
10−12
3
10
10
15
30−50
30−50
10 5
30−50
10
gap (mm)
2 15
rat rat
rat
rat
rat rat
empty-wireless electrical stimulator empty
empty
rat
empty
rat
rat, sciatic nerve
empty
empty
Wistar rat
Schwann cells combined with a collagen matrix (Vitrogen), collagen alone, empty silicone tube empty rat, facial nerve
neurons isolated from dorsal root ganglia (DRG) rat
empty
mesenchymal bone marrow stem cells (BMSC)
mice
rat sciatic nerve rat sciatic nerve
collagen sponge and sponge tube
RADA16-1, RADA 16RGD, and RADA 16IKVAV fibroblast growth factor and empty PBS
PLLA
polycaprolactone (PCL) PCL
poly(glyceryl sebacate methacrylate) (PGS) poly(lactic-co-glycolic acid) (PLGA) PLGA and caprolactone−Neurolac PLGA PDMS-Mg PLGA
poly(glycolic acid) (PGA)−GEM NeuroTube poly(lactic acid) (PLA)
poly(glycolic acid) (PGA)−collagen aluminum based Al− Al2O3 nanowires
sheep
empty Bioglass fibers
silicone Silastic conduit
human
empty
silicone
rat sciatic nerve
animal model
hepatocyte growth factor (HGF)
luminal filling
silicone
conduit polymer
Table 2. Review of Materials Used in Vivo as Peripheral Nerve Grafts ref
149
Zhang et al.159,160 Gonzalez-Perez et al.;161 Simoes et al28 Wang et al.;75 Hu et al.;163 Jiao et al.162 Wang et al.40 Choi et al. 201948
Vleggeert-Lankamp et al.157 Wu et al.158
Koo et al.33 Bini et al.;155 Zhou et al.156 Quan et al.138
Chang and Hsu,;75,152 Yu et al.153 Luis et al.154
Singh et al.101
Evans et al.;82,150 Jansen et al.151
Costa et al.73
Veith et al.
Ito et al.148
Lindfors et al.147
Jeans et al.96
Lundborg et al.;142,143 Puente-Alonso et al.62 Urabe et al. 144 Bunting et al.145,146
Mohammadi et al.141
ACS Chemical Neuroscience Review
DOI: 10.1021/acschemneuro.9b00203 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
empty FGF-1 dispersed in collagen matrix silk fibrin fiber/Schwann cells Schwann cells and adipose-derived stem cells NGC−silk fibroin filaments or Fibres of Spidrex coated with hyaluronic acid PGA filament coated with laminin empty gelatin methacrylate hydrogel with NGF or glial cell line-derived neurotrophic factor (GDNF) saline solution empty aligned PCL nanofiber and collagen type I/ III with and without stromal cell
collagen PHEMA-MMA silk fibroin−chitosan silk fibrin−collagen silk fibroin
PCL-PLLA
PLGA-PCL PLGA PCL
PGA−collagen PEGDA−GelMA PLGA/PCL- silicon
acellular skeletal muscle matrix or empty
luminal filling
collagen filament or tube
conduit polymer
Table 2. continued
nerve nerve nerve nerve
rat
rat sciatic nerve Lewis rat Sprague−Dawley rat
Beagle dog rat rat
Lewis rat rat sciatic rat sciatic rat sciatic rat sciatic
rat sciatic nerve
animal model
10
10 5 15
80 6 10
Electrospinning
electrospinning immersion-based method solvent evaporation-salt leaching and electrospinning
woven, dip-coating 3D bioprinting 3D bioplotting
wall coated with fibers and tubes dip-coating liquid−liquid centrifugal casting cast molding cast molding hollow porous cylindrical sheath fabricated by solvent casting, dip-coating and freeze-drying
30 5 10 10 10 10
fabrication techniques solvent casting mold
10
gap (mm) ref
Frost et al.90
Panseri et al.168 Daly et al.166 Shah et al.95
Daly et al.166 Belkas et al.128 Gu et al.167 Xu et al.47 Yang et al.;51 Huang et al.32 Mastsumoto et al.67 Zhu et al.97 Johnson et al.139
Alluin et al.;43 Okamoto et al.;165 Yoshii et al.164
ACS Chemical Neuroscience Review
I
DOI: 10.1021/acschemneuro.9b00203 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Review
ACS Chemical Neuroscience fabricated by Zhu et al.97 The developed nerve guide had the capability of guiding the directional regeneration of the sciatic nerve along with motor and sensor recovery of function during in vivo testing.97 Recently the use of bioplotting, a 3D biofabrication technique, has shown promise in the fabrication of nerve guides, especially with hydrogel inside the conduit. In this type of fabrication, living cells would be mixed with biomaterials, which should be handled in aqueous and physiologically mild conditions, to create more complicated tissue scaffolds. Good organization of the cellular components within the nerve guide mimicking a real situation is the main advantage of this technique over seeding cells on a prefabricated nerve guide. Rajaram fabricated a nerve guide containing alginate-HA hydrogels and Schwann cells, which showed enhanced axonal regeneration without inducing an immune response.49 In another study, Stevens used a bioplotting technique to fabricate a nerve guide by encapsulating primary mouse cortical neurons and glia within a multilayered RGD-GG hydrogel. The hydrogel was encapsulated within the fibers which enhanced differentiation of PC12 cells and aligned neural outgrowths.50 Another study by Ning et al. revealed the application of a 3D bioplotting nerve guide with living Schwann cells. In this study, they incorporated Schwann cells into the composite hydrogels of alginate, fibrin, HA, and RGD peptide, which showed a great potential for supporting peripheral nerve regeneration.140 In recent years much research has been carried out in the area of bioplotting nerve guides, which brought hopes and promise for developing a next generation of nerve guide. A diagram illustrating these processing techniques for scaffold preparation is provided in Figure 3. Some of the current techniques used for fabrication of artificial nerve conduit are listed in Table 2. Other techniques, including solvent casting and wet extrusion, have been used to form nerve guides.82 In some instances, the combined use of multiple techniques has been reported.21,29,109,169 After implantation of nerve guides, the coating functions as a medium for adhesion, proliferation, passage, and function of neural cells.169 Autologous, “gold standard”, means a complex structure that supports axonal regrowth and nerve regeneration; therefore, it is essential that the artificial nerve conduit is fabricated to have a structure close to autologous to be able to provide support for nerve regeneration and sensory recovery.13,169
nerve function recovery. Long-term complications often result in repeat surgery to remove the conduit.4,18,62,170,171 The successful alternative is biodegradable nerve guides that provide initial structural support and space for regenerating axons. These guides degrade over time, avoiding the need for repeat surgery to remove the device.3,13 The growth of axons (myelinated and unmyelinated) across the gap may be enhanced when compared with the use of conventional tubes, as the biodegradable conduits allow better nutrition supply to the regenerating nerve.172 This type of conduit provides a better structure for the initial matrix and subsequent regenerated nerve. The flexibility of the conduit increases with degradation, resulting in less damage to the maturing regenerated nerve.170,172 Details of some of the materials used for nerve guides are summarized in Table 2. A nerve guide may be formed to actively stimulate nerve regeneration. Many factors including polymer composition, surface texture, longitudinal orientation of microchannels, porosity, and electrical stimulation play important roles during axonal outgrowth.4,5,19,21,35,128,173,174 Some of the important aspects of biodegradable nerve guides are listed in the following sections. Degradation. One of the main factors in a nerve conduit is degradation rate. An ideal nerve guide would degrade at a controlled rate in accordance with nerve regeneration, slow enough to maintain its shape and strength throughout the period of regeneration and cause no inflammatory response.13,29 If a guide degrades too quickly, scar tissue may be formed together with a low level of mechanical support. If it degrades too slowly, it might compress the regenerated nerve, thereby causing damage.2,3,13 Furthermore, the guide should degrade faster closer to the proximal nerve-end and resorb slowly at the distal-end, increasingly so over long distances.10,19,85,126 Porosity. The nerve guide should display high porosity (>80%) to provide sufficient permeability for the passage of nutrients from the outside to the inside of the conduit. This will support nerve regeneration and simultaneously inhibit infiltration of unwanted tissue.170 Testing has found that nerve guides with large pores (
