nerve guidance conduit nerve guidance conduit a nerve guidance conduit (also referred to as an artificial nerve conduit or artificial nerve graft, as opposed to an autograft) is an artificial means of guiding axonal regrowth to facilitate nerve regeneration and is one of several clinical treatments for nerve injuries. when direct suturing of the two stumps of a severed nerve cannot be accomplished without tension, the standard clinical treatment for peripheral nerve injuries is autologous nerve grafting. due to the limited availability of donor tissue and functional recovery in autologous nerve grafting, neural tissue engineering research has focused on the development of bioartificial nerve guidance conduits as an alternative treatment, especially for large defects. similar techniques are also being explored for nerve repair in the spinal cord but nerve regeneration in the central nervous system poses a greater challenge because its axons do not regenerate appreciably in their native environment. the creation of artificial conduits is also known as entubulation because the nerve ends and intervening gap are enclosed within a tube composed of biological or synthetic materials. whether the conduit is in the form of a biologic tube, synthetic tube or tissue-engineered conduit, it should facilitate neurotropic and neurotrophic communication between the proximal and distal ends of the nerve gap, block external inhibitory factors, and provide a physical guidance for axonal regrowth. the most basic objective of a nerve guidance conduit is to combine physical, chemical, and biological cues under conditions that will foster tissue formation. materials that have been used to make biologic tubes include blood vessels and skeletal muscles, while nonabsorbable and bioabsorbable synthetic tubes have been made from silicone and polyglycolide respectively. tissue-engineered nerve guidance conduits are a combination of many elements: scaffold structure, scaffold material, cellular therapies, neurotrophic factors and biomimetic materials. the choice of which physical, chemical and biological cues to use is based on the properties of the nerve environment, which is critical in creating the most desirable environment for axon regeneration. the factors that control material selection include biocompatibility, biodegradability, mechanical integrity, controllability during nerve growth, implantation and sterilization. scaffold topography in tissue engineering, the three main levels of scaffold structure are considered to be: the superstructure, the overall shape of the scaffold; the microstructure, the cellular level structure of the surface; and the nanostructure, the subcellular level structure of the surface. superstructure the superstructure of a conduit or scaffold is important for simulating in vivo conditions for nerve tissue formation. the extracellular matrix, which is mainly responsible for directing tissue growth and formation, has a complex superstructure created by many interwoven fibrous molecules. ways of forming artificial superstructure include the use of thermo-responsive hydrogels, longitudinally oriented channels, longitudinally oriented fibers, stretch-grown axons, and nanofibrous scaffolds. thermo-responsive hydrogels in traumatic brain injury (tbi), a series of damaging events is initiated that lead to cell death and overall dysfunction, which cause the formation of an irregularly-shaped lesion cavity. the resulting cavity causes many problems for tissue-engineered scaffolds because invasive implantation is required, and often the scaffold does not conform to the cavity shape. in order to get around these difficulties, thermo-responsive hydrogels have been engineered to undergo solution-gelation (sol-gel) transitions, which are caused by differences in room and physiological temperatures, to facilitate implantation through in situ gelation and conformation to cavity shape caused, allowing them to be injected in a minimally invasively manner. methylcellulose (mc) is a material with well-defined sol-gel transitions in the optimal range of temperatures. mc gelation occurs because of an increase in intra- and inter-molecular hydrophobic interactions as the temperature increases. the sol-gel transition is governed by the lower critical solution temperature (lcst), which is the temperature at which the elastic modulus equals the viscous modulus. the lcst must not exceed physiological temperature (37 °c) if the scaffold is to gel upon implantation, creating a minimally invasive delivery. following implantation into a tbi lesion cavity or peripheral nerve guidance conduit, mc elicits a minimal inflammatory response. it is also very important for minimally invasive delivery that the mc solution has a viscosity at temperatures below its lcst, which allows it to be injected through a small gauge needle for implantation in in vivo applications. mc has been successfully used as a delivery agent for intra-optical and oral pharmaceutical therapies. some disadvantages of mc include its limited propensity for protein adsorption and neuronal cellular adhesion making it a non-bioactive hydrogel. due to these disadvantages, use of mc in neural tissue regeneration requires attaching a biologically active group onto the polymer backbone in order to enhance cell adhesion. another thermo-responsive gel is one that is formed by combining chitosan with glycerophosphate (gp) salt. this solution experiences gelation at temperatures above 37 °c. gelation of chitosan/gp is rather slow, taking half an hour to initially set and 9 more hours to completely stabilize. gel strength varies from 67 to 1572 pa depending on the concentration of chitosan; the lower end of this range approaches the stiffness of brain tissue. chitosan/gp has shown success in vitro, but the addition of polylysine is needed to enhance nerve cell attachment. polylysine was covalently bonded to chitosan in order to prevent it from diffusing away. polylysine was selected because of its positive nature and high hydrophilicity, which promotes neurite growth. neuron survival was doubled, though neurite outgrowth did not change with the added polylysine. longitudinally oriented channels longitudinally oriented channels are macroscopic structures that can be added to a conduit in order to give the regenerating axons a well-defined guide for growing straight along the scaffold. in a scaffold with microtubular channel architecture, regenerating axons are able to extend through open longitudinal channels as they would normally extend through endoneurial tubes of peripheral nerves. additionally, the channels increase the surface area available for cell contact. the channels are usually created by inserting a needle, wire, or second polymer solution within a polymer scaffold; after stabilizing the shape of the main polymer, the needle, wire, or second polymer is removed in order to form the channels. typically multiple channels are created; however, the scaffold can consist of just one large channel, which is simply one hollow tube. a molding technique was created by wang et al. for forming a nerve guidance conduit with a multi-channel inner matrix and an outer tube wall from chitosan. in their 2006 study, wang et al. threaded acupuncture needles through a hollow chitosan tube, where they are held in place by fixing, on either end, patches created using cad. a chitosan solution is then injected into the tube and solidified, after which the needles are removed, creating longitudinally oriented channels. a representative scaffold was then created for characterization with 21 channels using acupuncture needles of 400 µm in diameter. upon investigation under a microscope, the channels were found to be approximately circular with slight irregularities; all channels were aligned with the inner diameter of the outer tube wall. it was confirmed by micro-ct imaging that the channels went through the entire length of the scaffold. under water absorption, the inner and outer diameters of the scaffold became larger, but the channel diameters did not vary significantly, which is necessary for maintaining the scaffold shape that guides neurite extension. the inner structure provides an increase in compressive strength compared to a hollow tube alone, which can prevent collapse of the scaffold onto growing neurites. neuro-2a cells were able to growth on the inner matrix of the scaffold, and they oriented along the channels. although this method has only been tested on chitosan, it can be tailored to other materials. lyophilizing and wire-heating process is another method of creating longitudinally oriented channels, developed by huang et al. (2005). a chitosan and acetic acid solution was frozen around nickel-copper (ni-cu) wires in a liquid nitrogen trap; subsequently the wires were heated and removed. ni-cu wires were chosen because they have a high resistance level. temperature-controlled lyophilizers were used to sublimate the acetic acid. there was no evidence of the channels merging or splitting. after lyophilizing, scaffold dimensions shrunk causing channels to be a bit smaller than the wire used. the scaffolds were neutralized to a physiological ph value using a base, which had dramatic effects on the porous structure. weaker bases kept the porous structure uniform, but stronger base made it uncontrollable. the technique used here can be slightly modified to accommodate other polymers and solvents. another way to create longitudinally oriented channels is to create a conduit from one polymer with embedded longitudinally oriented fibers from another polymer; then selectively dissolve the fibers to form longitudinally oriented channels. polycaprolactone (pcl) fibers were embedded in a (hydroxyethyl)methacrylate (hema) scaffold. pcl was chosen over poly (lactic acid) (pla) and poly (lactic-co-glycolic acid) (plga), because it is insoluble in hema but soluble in acetone. this is important because hema was used for the main conduit material and acetone was used to selectively dissolve the polymer fibers. extruded pcl fibers were inserted into a glass tube and the hema solution was injected. the number of channels created was consistent from batch to batch and the variations in fiber diameter could be reduced by creating a more controlled pcl fiber extrusion system. the channels formed were confirmed to be continuous and homogeneous by examination of porosity variations. this process is safe, reproducible and has controllable dimensions. in a similar study conducted by yu and shoichet (2005), hema was copolymerized with aema to create a p(hema-co-amea) gel. polycaprolactone (pcl) fibers were embedded in the gel, and then selectively dissolved by acetone with sonication to create channels. it was found that hema in mixture with 1% aema created the strongest gels. when compared to scaffolds without channels, the addition of 82–132 channels can provide an approximately 6–9 fold increase in surface area, which may be advantageous for regeneration studies that depend on contact-mediated cues. itoh et al. (2003) developed a scaffold consisting of a single large longitudinally oriented channel was created using chitosan tendons from crabs. tendons were harvested from crabs (macrocheira kaempferi) and repeatedly washed with sodium hydroxide solution to remove proteins and to deacetylate the tendon chitin, which subsequently became known as tendon chitosan. a stainless steel bar with triangular-shaped cross-section (each side 2.1 mm long) was inserted into a hollow tendon chitosan tube of circular-shaped cross-section (diameter: 2 mm; length: 15 mm). when comparing the circular-shaped and triangular-shaped tubes, it was found that the triangular tubes had improved mechanical strength, held their shape better, and increased the surface area available. while this is an effective method for creating a single channel, it does not provide as much surface area for cellular growth as the multi-channel scaffolds. newman et al. (2006) inserted conductive and non-conductive fibers into a collagen-terp scaffold (collagen cross-linked with a terpolymer of poly(n-isopropylacrylamide) (pnipaam) ). the fibers were embedded by tightly wrapping them on a small glass slide and sandwiching a collagen-terp solution between it and another glass slide; spacers between the glass slides set the gel thickness to 800 µm. the conductive fibers were carbon fiber and kevlar, and the nonconductive fibers were nylon-6 and tungsten wire. neurites extend in all directions in thick bundles on the carbon fiber; however with the other three fibers, neurites extended in fine web-like conformations. the neurites showed no directional growth on the carbon and kevlar fibers, but they grew along the nylon-6 fibers and to some extent along the tungsten wire. the tungsten wire and nylon-6 fiber scaffolds had neurites grow into the gel near the fiber-gel interface in addition to growing along the surface. all fiber gels except kevlar showed a significant increase in neurite extension compared to non-fiber gels. there was no difference in the neurite extension between the non-conductive and the conductive fibers. in their 2005 study, cai et al. added poly (l-lactic acid) (plla) microfilaments to hollow poly(lactic acid) (pla) and silicon tubes. the microfiber guidance characteristics were inversely related to the fiber diameter with smaller diameters promoting better longitudinally oriented cell migration and axonal regeneration. the microfibers also promoted myelination during peripheral nerve repair. stretch-grown axons mature axon tracts has been demonstrated to experience growth when mechanically stretched at the central portion of the axon cylinder. such mechanical stretch was applied by a custom axon stretch-growth bioreactor composed of four main components: custom-designed axon expansion chamber, linear motion table, stepper