nerve guidance conduit

8/7/2019 nerve guidance conduit

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Superstructure

The superstructure of a conduit or scaffold is important for simulating in vivo conditionsfor 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 useof thermo-responsive hydrogels, longitudinally oriented channels, longitudinally orientedfibers, stretch-grown axons, and nanofibrous scaffolds.Thermo-responsive hydrogels

In traumatic brain injury (TBI), a series of damaging events is initiated that lead to celldeath and overall dysfunction, which cause the formation of an irregularly-shaped lesioncavity.[8] The resulting cavity causes many problems for tissue-engineered scaffoldsbecause invasive implantation is required, and often the scaffold does not conform to thecavity shape. In order to get around these difficulties, thermo-responsive hydrogels havebeen engineered to undergo solution-gelation (sol-gel) transitions, which are caused by

differences in room and physiological temperatures, to facilitate implantation through insitu gelation and conformation to cavity shape caused, allowing them to be injected in aminimally invasively manner.[8]

Methylcellulose (MC) is a material with well-defined sol-gel transitions in the optimalrange of temperatures. MC gelation occurs because of an increase in intra- and inter-molecular hydrophobic interactions as the temperature increases.[8] The sol-gel transitionis governed by the lower critical solution temperature (LCST), which is the temperatureat which the elastic modulus equals the viscous modulus. The LCST must not exceedphysiological temperature (37 °C) if the scaffold is to gel upon implantation, creating aminimally invasive delivery. Following implantation into a TBI lesion cavity or peripheral nerve guidance conduit, MC elicits a minimal inflammatory response.[8] It isalso very important for minimally invasive delivery that the MC solution has a viscosityat temperatures below its LCST, which allows it to be injected through a small gaugeneedle for implantation in in vivo applications.[8] MC has been successfully used as adelivery agent for intra-optical and oral pharmaceutical therapies.[8] Some disadvantagesof 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 inneural 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 withglycerophosphate (GP) salt.[9] This solution experiences gelation at temperatures above37 °C. Gelation of chitosan/GP is rather slow, taking half an hour to initially set and 9more hours to completely stabilize. Gel strength varies from 67 to 1572 Pa depending onthe concentration of chitosan; the lower end of this range approaches the stiffness of braintissue. Chitosan/GP has shown success in vitro, but the addition of polylysine is neededto 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.[9]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 straightalong the scaffold. In a scaffold with microtubular channel architecture, regeneratingaxons are able to extend through open longitudinal channels as they would normallyextend through endoneurial tubes of peripheral nerves.[10] Additionally, the channelsincrease the surface area available for cell contact. The channels are usually created byinserting 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 isremoved 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.[10] In their 2006study, Wang et al. threaded acupuncture needles through a hollow chitosan tube, wherethey are held in place by fixing, on either end, patches created using CAD. A chitosansolution 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 approximatelycircular with slight irregularities; all channels were aligned with the inner diameter of theouter tube wall. It was confirmed by micro-CT imaging that the channels went throughthe 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 isnecessary 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 wereable 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.[10]

lyophilizing and wire-heating process is another method of creating longitudinallyoriented channels, developed by Huang et al. (2005).[11] A chitosan and acetic acidsolution 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 theyhave a high resistance level. Temperature-controlled lyophilizers were used to sublimatethe 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 thewire used. The scaffolds were neutralized to a physiological pH value using a base, whichhad dramatic effects on the porous structure.[11] Weaker bases kept the porous structureuniform, but stronger base made it uncontrollable. The technique used here can beslightly modified to accommodate other polymers and solvents.[11]



Another way to create longitudinally oriented channels is to create a conduit from onepolymer with embedded longitudinally oriented fibers from another polymer; thenselectively dissolve the fibers to form longitudinally oriented channels. Polycaprolactone(PCL) fibers were embedded in a (Hydroxyethyl)methacrylate (HEMA) scaffold. PCLwas 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 HEMAwas used for the main conduit material and acetone was used to selectively dissolve thepolymer fibers. Extruded PCL fibers were inserted into a glass tube and the HEMAsolution was injected. The number of channels created was consistent from batch to batchand the variations in fiber diameter could be reduced by creating a more controlled PCLfiber extrusion system.[12] The channels formed were confirmed to be continuous andhomogeneous by examination of porosity variations. This process is safe, reproducibleand has controllable dimensions.[12] 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 dissolvedby acetone with sonication to create channels. It was found that HEMA in mixture with

1% AEMA created the strongest gels.[13] When compared to scaffolds without channels,the addition of 82-132 channels can provide an approximately 6-9 fold increase in surfacearea, which may be advantageous for regeneration studies that depend on contact-mediated cues.[13]

Itoh et al. (2003) developed a scaffold consisting of a single large longitudinally orientedchannel was created using chitosan tendons from crabs.[14] Tendons were harvestedfrom crabs (Macrocheira kaempferi) and repeatedly washed with sodium hydroxidesolution to remove proteins and to deacetylate the tendon chitin, which subsequentlybecame 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 thecircular-shaped and triangular-shaped tubes, it was found that the triangular tubes hadimproved mechanical strength, held their shape better, and increased the surface areaavailable.[14] While this is an effective method for creating a single channel, it does notprovide as much surface area for cellular growth as the multi-channel scaffolds.Longitudinally oriented fibers

In addition to longitudinally oriented channels, longitudinally oriented fibers can also beadded to a conduit to provide regenerating axons with guidance for longitudinally-directed growth. Studies conducted by Newman et al. (2006) and Cai et al. (2005)showed that adding filaments to a scaffold promotes inner contact guidance and increasespermeability for better nutrient and waste exchange such that the scaffold has superior nerve repair performance over non-permeable conduits that lack filaments.[15][16]

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 slideand sandwiching a collagen-TERP solution between it and another glass slide; spacersbetween 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 theother three fibers, neurites extended in fine web-like conformations. The neurites showedno directional growth on the carbon and Kevlar fibers, but they grew along the nylon-6fibers 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 growingalong the surface. All fiber gels except Kevlar showed a significant increase in neuriteextension compared to non-fiber gels. There was no difference in the neurite extensionbetween the non-conductive and the conductive fibers.[15]

In their 2005 study, Cai et al. added Poly (L-lactic acid) (PLLA) microfilaments tohollow poly(lactic acid) (PLA) and silicon tubes. The microfiber guidance characteristicswere inversely related to the fiber diameter with smaller diameters promoting better longitudinally oriented cell migration and axonal regeneration. The microfibers alsopromoted myelination during peripheral nerve repair.[16]Stretch-grown axons

Mature axon tracts has been demonstrated to experience growth when mechanicallystretched at the central portion of the axon cylinder.[17] Such mechanical stretch wasapplied by a custom axon stretch-growth bioreactor composed of four main components:custom-designed axon expansion chamber, linear motion table, stepper motor andcontroller.[17] The nerve tissue culture is placed within the expansion chamber with aport for gas exchange and a removable stretching frame, which is able to separate twogroups of somas (neuron cell bodies) and thus stretch their axons.[17] Collagen gel wasused to promote the growth of larger stretch-grown axon tracts that were visible to theunaided eye. There are two reasons for the growth enhancement due to the collagencoating: 1) the culture became hydrophobic after the collagen dried which permitted adenser concentration of neurons to grow, and 2) the collagen coating created anunobstructed coating across the two elongation substrates.[17] Examination by scanningelectron microscope and TEM showed no signs of axon thinning due to stretch, and thecytoskeleton appeared to be normal and intact. The stretch-grown axon tracts werecultured on a biocompatible membrane, which could be directly formed into a cylindricalstructure for transplantation, eliminating the need to transfer axons to a scaffold after growth was complete. The stretch-grown axons were able to grow at an unprecedentedrate of 1 cm/day after only 8 days of acclimation, which is much greater than the 1mm/day maximal growth rate as measured for growth cone extension. The rate of 1mm/day is also the average transport speed for structural elements such asneurofilaments.[17]Nanofibers scaffolds

Research on nanoscale fibers attempts to mimic the structure of collagen in theextracellular matrix by creating fibers that approach the nanoscale diameter of naturalcollagen bundles.[7] Three distinct methods for forming nanofibrous scaffolds are self-assembly, phase separation and electrospinning. However, there are many other methodsfor forming nanofibrous scaffolds.



Self-assembly of nanofibrous scaffolds is able to occur only when the fibers themselvesare engineered for self-assembly. One common way to drive the self-assembly of scaffoldfibers is to use amphiphilic peptides so that in water the hydrophobic moiety drives theself-assembly.[7] Carefully calculated engineering of the amphiphilic peptides allows for precise control over the self-assembled matrix. Self-assembly is able to create both

ordered and unordered topographies. Phillips et al. (2005) developed and tested in vitroand in vivo a self-aligned collagen-Schwann cell matrix, which allowed DRG neuriteextension alignment in vitro. Collagen gels have been used extensively as substrates for three-dimensional tissue culture. Cells are able to form integrin-mediated attachmentswith collagen, which initiates cytoskeleton assembly and cell motility. As cells movealong the collagen fibers they generate forces that contract the gel. When the collagenfibers are tethered at both ends, cell-generated forces create uniaxial strain, causing thecells and collagen fibers to align. The advantages of this matrix are its simplicity andspeed of preparation.[2] Soluble plasma fibronectin can also self-assemble into stableinsoluble fibers when put under direct mechanical shearing within a viscous solution.Phillips et al. (2004) investigated a new method of shear aggregation that causes an

improved aggregation.[18] The mechanical shearing was created by dragging out a 0.2 mlbolus to 3 cm with forceps; fibronectin aggregates into insoluble fibers at the rapidlymoving interface in an ultrafiltration cell. The proposed mechanism for this fiber aggregation is protein extension and elongation under mechanical shear force, whichleads to lateral packing and protein aggregation of fibers. Phillips et al. showed thatmechanical shear produced by stretching a high viscosity fibronectin gel causessubstantial changes in its structure and that when applied through uniaxial extension, aviscous fibronectin gel forms oriented fibrous fibronectin aggregates; additionally, thefibrous aggregates have a decreased solubility and can support the various cell types invitro.[18]

Phase separation allows for three-dimensional sub-micrometre fiber scaffolds to becreated without the use of specialized equipment. The five steps involved in phaseseparation are polymer dissolution, phase separation and gelation, solvent extraction fromthe gel, freezing and freeze drying in water.[7] The final product is a continuous fiber network. Phase separation can be modified to fit many different applications, and porestructure can be varied by using different solvents, which can change the entire processfrom liquid-liquid to solid-liquid. Porosity and fiber diameter can also be modified byvarying the initial concentration of the polymer; a higher initial concentration leads toless pores and larger fiber diameters. This technique can be used to create networks of fibers with diameters reaching type I collagen fiber diameters. The fibrous network created is randomly oriented and so far work has not been done to attempt to organize thefibers. Phase separation is a widely used technique for creating highly porous nanofibrousscaffolds with ease.[7]

Electrospinning creates nanofibers by electrically charging a droplet of polymer melt or solution and suspending it from a capillary. Then, an electric field is applied at one end of the capillary until the charge exceeds the surface tension, creating a polymer jet thatelongates and thins. Electrically charged polymers are left behind as the solventevaporates from the jets and are collected on a grounded surface. Fibers have been spun



with diameters ranging from less than 3 nm to over 1 µm. The process is affected bysystem parameters such as polymer molecular weight and solution properties and byprocess parameters such as flow rate, distance between the collector and the capillary,and motion of the collector.[19] Electrospinning forms fibers with controllable diametersand alignment. The fibrous network created is unordered and contains a high surface-to-

volume ratio as a result of a high porosity; a large network surface area is ideal for growth and transport of wastes and nutrients in neural tissue engineering.[7] The twofeatures of electrospun scaffolds that are advantageous for neural tissue engineering arethe morphology and architecture, which closely mimics the ECM, and the pores, whichare the correct range of sizes that allows nutrient exchange but prevents in growth of glialscar tissue (around 10 µm).[20] Random electrospun PLLA scaffolds have beendemonstrated to have increased cell adhesion, which may be due to an increased surfaceroughness.[20] Electrospun fiber networks can also be ordered and used to presentalignment cues to cells; this is advantageous because large scale three-dimensionalaligned scaffolds cannot be created easily using macrofabrication techniques.[7] In astudy conducted by Yang et al. (2005), aligned and random electrospun poly (L-lactic

acid) (PLLA) microfibrous and nanofibrous scaffolds were created, characterized, andcompared. Fiber diameters were directly proportional to the initial polymer concentrationused for eletrospinning; the average diameter of aligned fibers was smaller than that of random fibers under identical processing conditions. It was shown that neural stem cellselongated parallel to the aligned electrospun fibers.[19] The aligned nanofibers had alonger average neurite length compared to aligned microfibers, random microfibers, andrandom nanofibers. In addition, more cells differentiated on aligned nanofibers thanaligned microfibers.[19] Thus, aligned nanofibers are more beneficial than nonalignedfibers and microfibers for promoting nerve regeneration.Microstructure and nanostructure

Microstructure and nanostructure, along with superstructure are three main levels of scaffold structure that deserve consideration when creating scaffold topography.[7] Whilethe superstructure refers to the overall shape of the scaffold, the microstructure refers tothe cellular level structure of the surface and the nanostructure refers to the subcellular level structure of the surface. Microstructure and nanostructure have been the maininterest of recent research but there are yet to be many established methods for modifyingthe nanoscale structure.[7] There has been a recent change in interest from microscale tonanoscale motivated by the numerous nanoscale structures of the ECM. Both chemicaland physical cues can be used to direct cell growth.Physical cues

Physical cues are formed by creating an ordered surface structure at the level of themicrostructure and nanostructure. Physical cues alone have been shown to have asignificant effect on cellular organization in culture; this property of physical guidance isknown as contact guidance.[7] There are numerous methods for forming physicaltopographies; they can be divided into those that create ordered topographies and thosethat create unordered topographies.



Ordered topographies are defined as patterns that are organized and geometricallyprecise.[7] Though there are many methods for creating ordered topographies, they areusually time-consuming, requiring skill and experience and the use of expensiveequipment.[7]

Photolithography involves exposing a light source to a photoresist-coated silicon wafer; amask with the desired pattern is place between the light source and the wafer, therebyselectively allowing light to filter through and create the pattern on the photoresist.Further development of the wafer brings out the pattern in the photoresist.Photolithography performed in the near-UV is often viewed as the standard for fabricating topographies on the micro-scale.[7] However, because the lower limit for sizeis a function of the wavelength, this method cannot be used to create nanoscale features.[7] In their 2005 study, Mahoney et al. created organized arrays of polyimide channels(11 µm in height and 20-60 µm in width) were created on a glass substrate byphotolithography.[21] Polyimide was used because it adheres to glass well, is chemicallystable in aqueous solution, and is biocompatible. It is hypothesized that the

microchannels limited the range of angles that cytoskeletal elements within the neuritegrowth cones could accumulate, assemble, and orient.[21] There was a significantdecrease in the number of neurites emerging from the soma; however, there was lessdecrease as the range of angles over which the neurites emerged was increased. Also, theneurites were on average two times longer when the neurons were cultured on themicrochannels versus the controls on a flat surface; this could be due to a more efficientalignment of filaments.[21]

In electron beam lithography (EBL), an electron-sensitive resist is exposed to a beam of high-energy electrons. There is the choice of a positive or negative type resist; however,lower feature resolution can be obtained with negative resists.[22] Patterns are created byprogramming the beam of electrons for the exact path to follow along the surface of thematerial. Resolution is affected by other factors such as electron scattering in the resistand backscattering from the substrate. EBL can create single surface features on the order of 3-5 nm. If multiple features are required over a large surface area, as is the case intissue engineering, the resolution drops and features can only be created as small as 30-40nm, and the resist development begins to weigh more heavily on pattern formation.[22]To prevent dissolution of the resist, ultrasonic agitation can be used to overcomeintermolecular forces. In addition, isopropyl alcohol (IPA) helps develop high-densityarrays. EBL can become a quicker and less costly process by replicating nanometer patters in polymeric materials; the replication process has been demonstrated withpolycaprolactone (PCL) using hot embossing and solvent casting.[7] In a study conductedby Gomez et al. (2007), microchannels 1 and 2 µm wide and 400 and 800 nm deepcreated by EBL on PDMS were shown to enhance axon formation of hippocampal cellsin culture more so than immobilized chemical cues.[22]

X-ray lithography is another method for forming ordered patterns that can be used toinvestigate the role that topography plays in promoting neuritogenesis. The mask parameters determine the pattern periodicity, but ridge width and depth are determined bythe etching conditions. In a study, ridges were created with periods ranging from 400



through 4000 nm, widths ranging from 70 through 1900 nm, and a groove depth of 600nm; developing neurites demonstrated contact guidance with features as small as 70 nmand greater than 90% of the neurites were within 10 degrees of parallel alignment withthe ridges and grooves.[23] There was not a significant difference in orientation withrespect to the feature sizes used. The number of neurites per cell was constrained by the

ridges and grooves, producing bipolar rather than branching phenotypes.[23]

Unordered topographies are generally created by processes that occur spontaneouslyduring other processing; the patterns are random in orientation and organization withimprecise or no control over feature geometry.[7] The advantage to creating unorderedtopographies over ordered is that the processes are often less time consuming, lessexpensive, and do not require great skill and experience. Unordered topographies can becreated by polymer demixing, colloidal lithography and chemical etching.

In polymer demixing, polymer blends experience spontaneous phase separation; it oftenoccurs during conditions such as spin casting onto silicon wafers. Features that can be

created by this method include nanoscale pits, islands, and ribbons, which can becontrolled to an extent by adjusting the polymer ratio and concentration to change thefeature shape and size, respectively.[7] There is not much control in the horizontaldirection, though the vertical direction of the features can be precisely controlled.Because the pattern is very unordered horizontally, this method can only be used to studycell interactions with specific height nanotopographies.[7]

Colloidal lithography is inexpensive and can be used to create surfaces with controlledheights and diameters. Nanocolliods are used as an etch mask by spreading them alongthe material surface, and then ion beam bombardment or film evaporation is used to etchaway around the nanocolliods, creating nanocolumns and nanopits, respectively. Thefinal surface structure can be controlled by varying the area covered by colloids and thecolloid size. The area covered by the colloids can be changed by modifying the ionicstrength of the colloid solution. This technique is able to create large patterned surfaceareas, which is necessary for tissue engineering applications.[7]

Chemical etching involves soaking the material surface in an etchant such as hydrofluoricacid (HF) or sodium hydroxide (NaOH) until the surface is etched away to a desiredroughness as created by pits and protrusions on the nanometer scale.[7] Longer etch timeslead to rougher surfaces (i.e., smaller surface pits and protrusions). Structures withspecific geometry or organization cannot be created by this rudimentary method becauseat best it can be considered a surface treatment for changing the surface roughness. Thesignificant advantages of this method are ease of use and low cost for creating a surfacewith nanotopographies. Silicon wafers were etched using HF, and it was demonstratedthat cell adhesion was enhanced only in a specified range of roughness (20-50 nm).[7]Chemical cues

In addition to creating topography with physical cues, it can be created with chemicalcues by selectively depositing polymer solution in patterns on the surface of a substrate.There are different methods for depositing the chemical cues. Two methods for



dispensing chemical solutions include stripe patterning and piezoelectricmicrodispensing.

Stripe-patterned polymer films can be formed on solid substrates by casting dilutedpolymer solution. This method is relatively easy, inexpensive, and has no restriction on

the scaffold materials that can be used. The procedure involves horizontally overlappingglass plates while keeping them vertically separated by a narrow gap filled with apolymer solution. The upper plate is moved at a constant velocity between 60 to 100µm/s.[24] A thin liquid film of solution is continuously formed at the edge of the slidingglass following evaporation of the solvent. Stripe patterns prepared at speeds of 60, 70,and 100 µm/s created width and groove spacings of 2.2 and 6.1 µm, 3.6 and 8.4 µm, and4.3 and 12.7 µm, respectively; the range of heights for the ridges was 50-100 nm.[24]Tsuruma, Tanaka et al. demonstrated that embryonic neural cells cultured on film coatedwith poly-L-lysine attached and elongated parallel to poly(ε-caprolactone)/chloroformsolution (1g/L) stripes with narrow pattern width and spacing (width: 2.2 µm, spacing:6.1 µm).[24] However, the neurons grew across the axis of the patterns with wide width

and spacing (width: 4.3 µm, spacing: 12.7 µm). On average, the neurons on the stripe-patterned films had less neurites per cell and longer neurites compared to the neurons onnon-patterned films. Thus, the stripe pattern parameters are able to determine the growthdirection, the length of neurites, and the number of neurites per cell.[24]

Microdispensing was used to create micropatterns on polystyrene culture dishes bydispensing droplets of adhesive laminin and non-adhesive bovine serum albumin (BSA)solutions.[25] The microdispenser is a piezoelectric element attached to a push-bar on topof a channel etched in silicon, which has one inlet at each end and a nozzle in the middle.The piezoelectric element expands when voltage is applied, causing liquid to bedispensed through the nozzle. The microdispenser is moved using a computer-controlledx-y table. The micropattern resolution depends on many factors: dispensed liquidviscosity, drop pitch (the distance between the centre of two adjacent droplets in a line or array), and the substrate.[25] With increasing viscosity the lines become thinner, but if the liquid viscosity is too high the liquid cannot be expelled. Heating the solution createsmore uniform protein lines. Although some droplet overlap is necessary to createcontinuous lines, uneven evaporation may cause uneven protein concentration along thelines; this can be prevented through smoother evaporation by modifying the dispensedsolution properties.

For patterns containing 0.5 mg/ml laminin, a higher proportion of neurites grew on themicrodispensed lines than between the lines.[25] On 10mg/ml and 1mg/ml BSA proteinpatterns and fatty-acid free BSA protein patterns a significant number of neurites avoidedthe protein lines and grew between the lines. Thus, the fatty-acid-containing BSA lineswere just as non-permissive for neurite growth as lines containing BSA with fatty acids.Because microdispensing does not require direct contact with the substrate surfaces, thistechnique can utilitze surfaces with delicate micro- or nanotopology that could bedestroyed by contact. It is possible to vary the amount of protein deposited by dispensingmore or less droplets. An advantage of microdispensing is that patterns can be created



quickly in 5-10 minutes. Because the piezoelectric microdispenser does not requireheating, heat-sensitive proteins and fluids as well as living cells can be dispensed.[25]Scaffold material

The selection of the scaffold material is perhaps the most important decision to be made.

It must be biocompatible and biodegradable; in addition, it must be able to incorporateany physical, chemical, or biological cues desired, which in the case of some chemicalcues means that it must have a site available for chemically linking peptides and other molecules. The scaffold materials chosen for nerve guidance conduits are almost alwayshydrogels. The hydrogel may be composed of either biological or synthetic polymers.Both biological and synthetic polymers have their strengths and weaknesses. It isimportant to note that the conduit material can cause inadequate recovery when (1)degradation and resorption rates do not match the tissue formation rate, (2) the stress-strain properties do not compare well to those of neural tissue, (3) when degradingswelling occurs, causing significant deformation, (4) a large inflammatory response iselicited, or (5) the material has low permeability.[26]

HydrogelHydrogels are a class of biomaterials that are chemically or physically cross-linkedwater-soluble polymers. They can be either degradable or non-degradable as determinedby their chemistry, but degradable is more desirable whenever possible. There has beengreat interest in hydrogels for tissue engineering purposes, because they generally possesshigh biocompatibility, mechanical properties similar to soft tissue, and the ability to beinjected as a liquid which gels.[4] When hydrogels are physically cross-linked they mustrely on phase separation for gelation; the phase separation is temperature-dependent andreversible.[4] Some other advantages of hydrogels are that they use only non-toxicaqueous solvents, allow infusion of nutrients and exit of waste products, and allow cellsto assemble spontaneously.[27] Hydrogels have low interfacial tension, meaning cells caneasily migrate across the tissue-implant boundary.[9] However, with hydrogels it isdifficult to form a broad range of mechanical properties or structures with controlled poresize.[4]Synthetic polymer

A synthetic polymer may be non-degradable or degradable. For the purpose of neuraltissue engineering degradable materials are preferred whenever possible, because long-term effects such as inflammation and scar could severely damage nerve function. Thedegradation rate is dependent on the molecular weight of the polymer, its crystallinity,and the ratio of glycolic acid to lactic acid subunits.[4] Because of a methyl group, lacticacid is more hydrophobic than glycolic acid causing its hydrolysis to be slower.[4]Synthetic polymers have more wieldy mechanical properties and degradation rates thatcan be controlled over a wide range, and they eliminate the concern for immunogenicity.[4] There are many different synthetic polymers currently being used in neural tissueengineering. However, the drawbacks of many of these polymers include a lack of biocompatibility and bioactivity, which prevents these polymers from promoting cellattachment, proliferation, and differentiation.[28] Synthetic conduits have only beenclinically successful for the repair of very short nerve lesion gaps less than 1-2 cm.[29]



Furthermore, nerve regeneration with these conduits has yet to reach the level of functional recovery seen with nerve autografts.[26]Collagen-terpolymer

Collagen is a major component of the extracellular matrix, and it is found in the

supporting tissues of peripheral nerves. A terpolymer (TERP) was synthesized by freeradical copolymerization of its three monomers and cross-linked with collagen, creating ahybrid biological-synthetic hydrogel scaffold.[15] The terpolymer is based onpoly(NIPAAM), which is known to be a cell friendly polymer. TERP is used both as across-linker to increase hydrogel robustness and as a site for grafting of bioactivepeptides or growth factors, by reacting some of its acryloxysuccinimide groups with the – NH2 groups on the peptides or growth factors.[15] Because the collagen-terpolymer (collagen-TERP) hydrogel lacks a bioactive component, a study attached to it a commoncell adhesion peptide found in laminin (YIGSR) in order to enhance its cell adhesionproperties.[15]Poly (lactic-co-glycolic acid) family

The polymers in the PLGA family include poly (lactic acid) (PLA), poly (glycolic acid)(PGA), and their copolymer poly (lactic-co-glycolic acid) (PLGA). All three polymershave been approved by the Food and Drug Administration for employment in variousdevices. These polymers are brittle and they do not have regions for permissible for chemical modification; in addition, they degrade by bulk rather than by surface, which isnot a smooth and ideal degradation process.[4] In an attempt to overcome the lack of functionalities, free amines have been incorporated into their structures from whichpeptides can be tethered to control cell attachment and behavior.[4][edit] Methacrylated dextran (Dex-MA) copolymerized with aminoethyl methacrylate(AEMA)

Dextran is a polysaccharide derived from bacteria; it is usually produced by enzymesfrom certain strains of leuconostoc or Streptococcus. It consists of α-1,6-linked D-glucopyranose residues. Cross-linked dextran hydrogel beads have been widely used aslow protein-binding matrices for column chromatography applications and for microcarrier cell culture technology.[30] However, it has not been until recently thatdextran hydrogels have been investigated in biomaterials applications and specifically asdrug delivery vehicles. An advantage of using dextran in biomaterials applicationsinclude its resistance to protein adsorption and cell-adhesion, which allows specific celladhesion to be determined by deliberately attached peptides from ECM components.[30]AEMA was copolymerized with Dex-MA in order to introduce primary amine groups toprovide a site for attachment of ECM-derived peptides to promote cell adhesion. Thepeptides can be immobilized using sulfo-SMMC coupling chemistry and cysteine-terminated peptides. Copolymerization of Dex-MA with AEMA allowed themacroporous geometry of the scaffolds to be preserved in addition to promoting cellular interactions.[30]



Poly(glycerol sebacate) (PGS)

A novel biodegradable, tough elastomer has been developed from poly(glycerol sebacate)(PGS) for use in creation of a nerve guidance conduit.[26] PGS was originally developedfor soft tissue engineering purposes to specifically mimic ECM mechanical properties. It

is considered an elastomer because it is able to recover from deformation in mechanicallydynamic environments and to effectively distribute stress evenly throughout regeneratingtissues in the form of microstresses. PGS is synthesized by a polycondensation reactionof glycerol and sebacic acid, which can be melt processed or solvent processed into thedesired shape. PGS has a Young's modulus of 0.28 MPa and an ultimate tensile strengthgreater than 0.5 MPa.[26] Peripheral nerve has a Young's modulus of approximately 0.45MPa, which is very close to that of PGS. Additionally, PGS experiences surfacedegradation, accompanied by losses in linear mass and strength during resorption.[26]Following implantation, the degradation half-life was determined to be 21 days; completedegradation occurred at day 60.[26] PGS experiences minimal water absorption duringdegradation and does not have detectable swelling; swelling can cause distortion, which

narrows the tubular lumen and can impede regeneration. It is advantageous that thedegradation time of PGS can be varied by changing the degree of crosslinking and theratio of sebacic acid to glycerol.[26] In a study by Sundback et al. (2005), implanted PGSand PLGA conduits had similar early tissue responses; however, PLGA inflammatoryresponses spiked later, while PGS inflammatory responses continued to decreases.[26]Polyethylene glycol hydrogel

Polyethylene glycol (PEG) hydrogels are biocompatible and proven to be tolerated inmany tissue types, including the CNS. Mahoney and Anseth formed PEG hydrogels byphotopolymerizing methacrylate groups covalently linked to degradable PEG macromers.Hydrogel degradation was monitored over time by measuring mechanical strength(compressive modulus) and average mesh size from swelling ratio data.[31] Initially, thepolymer chains were highly cross-linked, but as degradation proceeded, ester bonds werehydrolyzed, allowing the gel to swell; the compressive modulus decreased as the meshsize increased until the hydrogel was completely dissolved. It was demonstrated thatneural precursor cells were able to be photoencapsulated and cultured on the PEG gelswith minimal cell death. Because the mesh size is initially small, the hydrogel blocksinflammatory and other inhibitory signals from surrounding tissue. As the mesh sizeincreases, the hydrogel is able to serve as a scaffold for axon regeneration.[31]Biological polymers

There are advantages to using biological polymers over synthetic polymers. They arevery likely to have good biocompatibility and be easily degraded, because they arealready present in nature in some form. However, there are also several disadvantages.They have unwieldy mechanical properties and degradation rates that cannot becontrolled over a wide range. In addition, there is always the possibility that naturally-derived materials may cause an immune response or contain microbes.[4] In theproduction of naturally-derived materials there will also be batch-to-batch variation inlarge-scale isolation procedures that cannot be controlled.[16] Some other problemsplaguing natural polymers are their inability to support growth across long lesion gaps



due to the possibility of collapse, scar formation, and early re-absorption.[16] Despite allthese disadvantages, some of which can be overcome, biological polymers still prove tobe the optimal choice in many situations.Polysialic acid (PSA)

Polysialic acid (PSA) is a relatively new biocompatible and bioresorbable material for artificial nerve conduits. It is a homopolymer of α2,8-linked sialic acid residues and adynamically regulated posttranslational modification of the neural cell adhesion molecule(NCAM). Recent studies have demonstrated that polysialylated NCAM (polySia-NCAM)promotes regeneration in the motor system.[32] PSA shows stability under cell cultureconditions and allows for induced degradation by enzymes. It has also been discoveredrecently that PSA is involved in steering processes like neuritogenesis, axonal pathfinding, and neuroblast migration.[32] Animals with PSA genetically knocked outexpress a lethal phenotype which has unsuccessful path finding; nerves connecting thetwo brain hemispheres were aberrant or missing.[32] Thus PSA is vital for proper nervous system development.

Collagen Type I/IIICollagen is the major component of the extracellular matrix and has been widely used innerve regeneration and repair. Due to its smooth microgeometry and permeability,collagen gels are able to allow diffusion of molecules through them. Collagen resorptionrates are able to be controlled by crosslinking collagen with polypoxy compounds.[6]Additionally, collagen type I/III scaffolds have demonstrated good biocompatibility andare able to promote Schwann cell proliferation. However, collagen conduits filled withSchwann cells used to bridge nerve gaps in rats have shown surprisingly unsuccessfulnerve regeneration compared to nerve autografts.[6] This is because biocompatibility isnot the only factor necessary for successful nerve regeneration; other parameters such asinner diameter, inner microtopography, porosity, wall thickness, and Schwann cellseeding density will need to be examined in future studies in order to improve the resultsobtained by these collagen I/III gels.[6]Spider silk fiber

Spider silk fibers are shown to promote cellular adhesion, proliferation, and vitality.Allmeling, Jokuszies et al. showed that Schwann cells attach quickly and firmly to thesilk fibers, growing in a bipolar shape; proliferation and survival rates were normal on thesilk fibers.[33]

They used spider silk fibers to create a nerve conduit with Schwann cells andacellularized xenogenic veins. The Schwann cells formed columns along the silk fibers ina short amount of time, and the columns were similar to bands of Bungner that grow invivo after PNS injury.[33] Spider silk has not been used in tissue engineering until nowbecause of the predatory nature of spiders and the low yield of silk from individualspiders. It has been discovered that the species Nephila clavipes produces silk that is lessimmunogenic than silkworm silk; it has a tensile strength of 4 x 109 N/m, which is sixtimes the breaking strength of steel.[33] Because spider silk is proteolytically degraded,there is not a shift in pH from the physiological pH during degradation. Other advantages



of spider silk include its resistance to fungal and bacterial decomposiiton for weeks andthe fact that it does not swell. Also, the silk's structure promotes cell adhesion andmigration. However, silk harvest is still a tedious task and the exact composition variesamong species and even among individuals of the same species depending on diet andenvironment. There have been attempts to synthetically manufacture spider silk. Further

studies are needed to test the feasibility of using a spider silk nerve conduit in vitro and invivo.[33]Silkworm silk fibroin

In addition to spiders, silkworms are another source of silk. Protein from Bombyx morisilkworms is a core of fibroin protein surrounded by sericin, which is a family of glue-like proteins. Fibroin has been characterized as a heavy chain with a repeatedhydrophobic and crystallizable sequence: Gly-Ala-Gly-Ala-Gly-X (X stands for Ser or Tyr). The surrounding sericin is more hydrophilic due to many polar residues, but it doesstill have some hydrophobic β-sheet portions. Silks have been long been used as suturesdue to their high mechanical strength and flexibility as well as permeability to water and

oxygen. In addition, silk fibroin can be easily manipulated and sterilized. However, silk use halted when undesirable immunological reactions were reported. Recently, it hasbeen discovered that the cause of the immunological problems lies solely with thesurrounding sericin.[34] Since this discovery, silk with the sericin removed has been usedin many pharmaceutical and biomedical applications. Because it is necessary to removethe sericin from around the fibroin before the silk can be used, an efficient procedureneeds to be developed for its removal, which is known as degumming. One degrummingmethod uses boiling aqueous Na¬2CO3 solution, which removes the sericin withoutdamaging the fibroin. Yang, Chen et al. demonstrated that the silk fibroin and silk fibroinextract fluid show good biocompatibility with Schwann cells, with no cytotoxic effectson proliferation.[34]Chitosan

Chitosan and chitin belong to a family of biopolymers composed of β(1-4)-linked N-acetyl-D-glucosamine and D-glucosamine subunits.[35] Chitosan is formed by alkalineN-deacetylation of chitin, which is the second most abundant natural polymer after cellulose.[14] Chitosan is a biodegradable polysaccharide that has been useful in manybiomedical applications such as a chelating agent, drug carrier, membrane, and water treatment additive.[11] Chitosan is soluble in dilute aqueous solutions, but precipitatesinto a gel at a neutral pH.[11] It does not support neural cell attachment and proliferationwell, but can be enhanced by ECM-derived peptide attachment. Chitosan also containsweak mechanical properties, which are more challenging to overcome.[9]

Degree of acetylation (DA) for soluble chitosan ranges from 0% to 60%, depending onprocessing conditions.[35] A study was conducted to characterize how varying DAaffects the properties of chitosan. Varying DA was obtained using acetic anhydride or alkaline hydrolysis. It was found that decreasing acetylation created an increase incompressive strength.[35] Biodegradation was examined by use of lysozyme, which isknown to be mainly responsible for degrading chitosan in vivo by hydrolyzing itsglycosidic bonds and is released by phagocytic cells after nerve injury. The results reveal



that there was an accelerated mass loss with intermediate DAs, compared with high andlow DAs over the time period studied.[35] When DRG cells were grown on the N-acetylated chitosan, cell viability decreased with increasing DA. Also, chitosan has anincreasing charge density with decreasing DA, which is responsible for greater celladhesion.[35] Thus, controlling the DA of chitosan is important for regulating the

degradation time. This knowledge could help in the development of a nerve guidanceconduit from chitosan.Aragonite

Aragonite scaffolds have recently been shown to support the growth of neurons from rathippocampi. Shany et al. (2006) proved that aragonite matrices can support the growth of astrocytic networks in vitro and in vivo. Thus, aragonite scaffolds may be useful for nerve tissue repair and regeneration. It is hypothesized that aragonite-derived Ca2+ isessential for promoting cell adherence and cell-cell contact. This is probably carried outthrough the help of Ca2+-dependent adhesion molecules such as cadherins.[36]Aragonite crystalline matrices have many advantages over hydrogels. They have larger

pores, which allows for better cell growth, and the material is bioactive as a result of releasing Ca2+, which promotes cell adhesion and survival. In addition, the aragonitematrices have higher mechanical strength than hydrogels, allowing them to withstandmore pressure when pressed into an injured tissue.[36]Alginate

Alginate is a polysaccharide that readily forms chains; it can be cross-linked at itscarboxylic groups with multivalent cations such as Cu2+, Ca2+, or Al3+ to form a moremechanically stable hydrogel.[37] Calcium alginates form polymers that are bothbiocompatible and non-immunogenic and have been used in tissue engineeringapplications. However, they are unable to support longitudinally oriented growth, whichis necessary for reconnection of the proximal end with its target. In order to overcomethis problem, anisotropic capillary hydrogels (ACH) have been developed. They arecreated by superimposing aqueous solutions of sodium alginate with aqueous solutions of multivalent cations in layers.[37] After formation, the electrolyte ions diffuse into thepolymer solution layers, and a dissipative convective process causes the ions toprecipitate, creating capillaries. The dissipative convective process results the oppositionof diffusion gradients and friction between the polyelectrolyte chains.[37] The capillarywalls are lined with the precipitated metal alginate, while the lumen is filled with theextruded water.

Prang et al. (2006) assessed the capacity of ACH gels to promote directed axonalregrowth in the injured mammalian CNS. The multivalent ions used to create thealginate-based ACH gels were copper ions, whose diffusion into the sodium alginatelayers created hexagonally structured anisotropic capillary gels.[37] After precipitation,the entire gel was traversed by longitudinally oriented capillaries. The ACH scaffoldspromoted adult NPC survival and highly oriented axon regeneration.[37] This is the firstinstance of using alginates to produce anisotropic structured capillary gels. Future studiesare need to study the long-term physical stability of the ACH scaffolds, because CNSaxon regeneration can take many months; however, in addition to being able to provide



long-term support the scaffolds must also be degradable. Of all the biological andsynthetic biopolymers investigated by Prang et al. (2006), only agarose-based gels wereable to compare with the linear regeneration caused by ACH scaffolds. Future studieswill also need to investigate whether the ACH scaffolds allow for reinnervation of thetarget in vivo after a spinal cord injury.[37]

Hyaluronic acid hydrogel

Hyaluronic acid (HA) is a widely used biomaterial as a result of its excellentbiocompatibility and its physiologic function diversity. It is abundant in the extracellular matrix (ECM) where it binds large glycosaminoglycans (GAGs) and proteoglycansthrough specific HA-protein interactions. HA also binds cell surface receptors such asCD44, which results in the activation of intracellular signaling cascades that regulate celladhesion and motility and promote proliferation and differentiation.[38] HA is alsoknown to support angiogenesis because its degradation products stimulate endothelial cellproliferation and migration. Thus, HA plays a pivotal role in maintaining the normalprocesses necessary for tissue survival. Unmodified HA has been used in clinical

applications such as ocular surgery, wound healing, and plastic surgery.[38] HA can becrosslinked to form hydrogels. HA hydrogels that were either unmodified or modifiedwith laminin were implanted into an adult central nervous system lesion and tested for their ability to induce neural tissue formation in a study by Hou et al.. They demonstratedthe ability to support cell ingrowth and angiogenesis, in addition to inhibiting glial scar formation. Also, the HA hydrogels modified with laminin were able to promote neuriteextension.[38] These results support HA gels as a promising biomaterial for a nerveguidance conduit.Cellular therapies

In addition to scaffold material and physical cues, biological cues can also beincorporated into a bioartificial nerve conduit in the form of cells. In the nervous systemthere are many different cell types that help support the growth and maintenance of neurons. These cells are collectively termed glial cells. Glial cells have been investigatedin an attempt to understand the mechanisms behind their abilities to promote axonregeneration. Three types of glial cells are discussed: Schwann cells, astrocytes, andolfactory ensheathing cells. In addition to glial cells, stem cells also have potential benefitfor repair and regeneration because many are able to differentiate into neurons or glialcells. This article briefly discusses the use of adult, transdifferentiated mesenchymal,ectomesenchymal, neural and neural progenitor stem cells.Glial cells

Glial cells are necessary for supporting the growth and maintenance of neurons in theperipheral and central nervous system. Most glial cells are specific to either the peripheralor central nervous system. Schwann cells are located in the peripheral nervous systemwhere they myelinate the axons of neurons. Astrocytes are specific to the central nervoussystem; they provide nutrients, physical support, and insulation for neurons. They alsoform the blood brain barrier. Olfactory ensheathing cells, however, cross the CNS-PNSboundary, because they guide olfactory receptor neurons from the PNS to the CNS.



Schwann cells

Schwann cells (SC) are crucial to peripheral nerve regeneration; they play both structuraland functional roles. Schwann cells are responsible for taking part in both Walleriandegeneration and bands of Bungner. When a peripheral nerve is damaged, Schwann cells

alter their morphology, behavior and proliferation to become involved in Walleriandegeneration and Bungner bands.[34] In Wallerian degeneration, Schwann cells grow inordered columns along the endoneurial tube, creating a band of Bungner (boB) thatprotects and preserves the endoneurial channel. Additionally, they release neurotrophicfactors that enhance regrowth in conjunction with macrophages. There are somedisadvantages to using Schwann cells in neural tissue engineering; for example, it isdifficult to selectively isolate Schwann cells and they show poor proliferation onceisolated. One way to overcome this difficulty is to artificially induce other cells such asstem cells into SC-like phenotypes.[39]

Eguchi et al. (2003) have investigated the use of magnetic fields in order to align

Schwann cells. They used a horizontal type superconducting magnet, which produces an8 T field at its center. Within 60 hours of exposure, Schwann cells aligned parallel to thefield; during the same interval, Schwann cells not exposed oriented in a random fashion.It is hypothesized that differences in magnetic field susceptibility of membranecomponents and cytoskeletal elements may cause the magnetic orientation.[40] Collagenfibers were also exposed to the magnetic field, and within 2 hours, they alignedperpendicular to the magnetic field, while collagen fibers formed a random meshwork pattern without magnetic field exposure. When cultured on the collagen fibers, Schwanncells aligned along the magnetically oriented collagen after two hours of 8-T magneticfield exposure. In contrast, the Schwann cells randomly oriented on the collagen fiberswithout magnetic field exposure. Thus, culture on collagen fibers allowed Schwann cellsto be oriented perpendicular to the magnetic field and oriented much quicker.[40]

These findings may be useful for aligning Schwann cells in a nervous system injury topromote the formation of bands of Bungner, which are crucial for maintaining theendoneurial tube that guides the regrowing axons back to their targets. It is nearlyimpossible to align Schwann cells by external physical techniques; thus, the discovery of an alternative technique for alignment is significant. However, the technique developedstill has its disadvantages, namely that it takes a considerable amount of energy to sustainthe magnetic field for extended periods.

Studies have been conducted in attempts to enhance the migratory ability of Schwanncells. Schwann cell migration is regulated by integrins with ECM molecules such asfibronectin and laminin. In addition, neural cell adhesion molecule (NCAM) is known toenhance Schwann cell motility in vitro.[41] NCAM is a glycoprotein that is expressed onaxonal and Schwann cell membranes. Polysialic acid (PSA) is synthesized on NCAM bypolysialyltransferase (PST) and sialyltransferase X (STX).[41] During the developmentof the CNS, PSA expression on NCAM is upregulated until postnatal stages. However, inthe adult brain PSA is found only in regions with high plasticity. PSA expression doesnot occur on Schwann cells.



Lavdas et al. (2006) investigated whether sustained expression of PSA on Schwann cellsenhances their migration. Schwann cells were tranduced with a retroviral vector encodingSTX in order to induce PSA expression. PSA-expressing Schwann cells did obtainenhanced motility as demonstrated in a gap bridging assay and after grafting in postnatal

forebrain slice cultures.[41] PSA expression did not alter molecular and morphologicaldifferentiation. The PSA-expressing Schwann cells were able to myelinate CNS axons incerebellar slices, which is not normally possible in vivo. It is hopeful that these PSA-expressing Schwann cells will be able to migrate throughout the CNS without loss of myelinating abilities and may become useful for regeneration and myelination of axons inthe central nervous system.[41][edit] Astrocytes

Astrocytes are glial cells that are abundant in the central nervous system. They are crucialfor the metabolic and trophic support of neurons; additionally, astrocytes provide ionbuffering and neurotransmitter clearance. Growing axons are guided by cues created by

astrocytes; thus, astrocytes can regulate neurite pathfinding and subsequently, patterningin the developing brain.[36] The glial scar that forms post-injury in the central nervoussystem is formed by astrocytes and fibroblasts; it is the most significant obstacle for regeneration. The glial scar consists of hypertrophied astrocytes, connective tissue, andECM. Two goals of neural tissue engineering are to understand astrocyte function and todevelop control over astrocytic growth. Studies by Shany et al. (2006) have demonstratedthat astrocyte survival rates are increased on 3D aragonite matrices compared toconventional 2D cell cultures. The ability of cell processes to stretch out across curvesand pores allows for the formation of multiple cell layers with complex 3Dconfigurations.

The three distinct ways by which the cells acquired a 3D shape are:[36]

1. adhering to surface and following the 3D contour 2. stretching some processes between 2 curvatures3. extending processes in 3D within cell layers when located within multilayer tissue

In conventional cell culture, growth is restricted to one plane, causing monolayer formation with most cells contacting the surface; however, the 3D curvature of thearagonite surface allows multiple layers to develop and for astrocytes far apart to contacteach other. It is important to promote process formation similar to 3D in vivo conditions,because astrocytic process morphology is essential in guiding directionality of regenerating axons.[36] The aragonite topography provides a high surface area to volumeratio and lacks edges, which leads to a reduction of the culture edge effect.[36]Crystalline matrices such as the aragonite mentioned here are allow for the promotion of a complex 3D tissue formation that approaches in vivo conditions.Olfactory ensheathing cells

The mammalian primary olfactory system has retained the ability to continuouslyregenerate during adulthood.[42] Olfactory receptor neurons have an average lifespan of



6-8 weeks and therefore, must be replaced by cells differentiated from stem cells forminga layer at the epithelium's base. The new olfactory receptor neurons must project their axons through the CNS to an olfactory bulb in order to be functional. Axonal growth isguided by the glial composition and cytoarchitecture of the olfactory bulb in addition tothe presence of olfactory ensheathing cells (OECs).[42] It is postulated that OECs

originate in the olfactory placode, suggesting a different developmental origin than other similarly related nervous system microglia. Another interesting concept is that OECs arefound in both the peripheral and central nervous system portions of the primary olfactorysystem, that is, the olfactory epithelium and bulb.[42] OECs are similar to Schwann cellsin that they provide an upregulation of low-affinity NGF receptor p75 following injury;however, unlike Schwann cells they produce lower levels of neurotrophins. Severalstudies have shown evidence of OECs being able to support regeneration of lesionedaxons, but these results are often unable to be reproduced.[42]Stem cells

Stem cells are characterized by their ability to self-renew for a prolonged time and still

maintain the ability to differentiate along one or more cell lineages. Stem cells may beunipotent, multipotent, or pluripotent, meaning they can differentiate into one, multiple,or all cell types, respectively.[43] Pluripotent stem cells can become cells derived fromany of the three embryonic germ layers.[43] Stem cells have the advantage over glialcells because they are able to proliferate more easily in culture. However, it remainsdifficult to preferentially differentiate these cells into varied cell types in an orderedmanner.[4] Another difficulty with stem cells is the lack of a well-defined definition of stem cells beyond hematopoietic stem cells (HSCs). Each stem cell 'type' has more thanone method for identifying, isolating, and expanding the cells; this has caused muchconfusion because all stem cells of a 'type' (neural, mesenchymal, retinal) do notnecessarily behave in the same manner under identical conditions.Adult stem cells

Adult stem cells are not able to proliferate and differentiate as effectively in vitro as theyare able to in vivo. Adult stem cells can come from many different tissue locations, but itis difficult to isolate them because they are defined by behavior and not surface markers.A method has yet to be developed for clearly distinguishing between stem cells and thedifferentiated cells surrounding them. However, surface markers can still be used to acertain extent to remove most of the unwanted differentiated cells. Stem cell plasticity isthe ability to differentiate across embryonic germ line boundaries. Though, the presenceof plasticity has been hotly contested. Some claim that plasticity is caused byheterogeneity among the cells or cell fusion events. Currently, cells can be differentiatedacross cell lines with yields ranging from 10% to 90% depending on techniques used.[43]More studies need to be done in order to standardize the yield with transdifferentiation.Transdifferentiation of multipotent stem cells is a potential means for obtaining stem cellsthat are not available or not easily obtained in the adult.[4]Mesenchymal stem cells

Mesenchymal stem cells are adult stem cells that are located in the bone marrow; they areable to differentiate into lineages of mesodermal origin. Some examples of tissue they



form are bone, cartilage, fat, and tendon. MSCs are obtained by aspiration of bonemarrow. Many factors promote the growth of MSCs including: platelet-derived growthfactor, epidermal growth factor β, and insulin-like growth factor-1. In addition to their normal differentiation paths, MSCs can be transdifferentiated along nonmesenchymallineages such as astrocytes, neurons, and PNS myelinating cells. MSCs are potentially

useful for nerve regeneration strategies because:[44]

1. their use is not an ethical concern2. no immunosuppression is needed3. they are an abundant and accessible resource4. they tolerate genetic manipulations

Keilhoff et al. (2006) performed a study comparing the nerve regeneration capacity of non-differentiated and transdifferentiated MSCs to Schwann cells in devitalized musclegrafts bridging a 2-cm gap in the rat sciatic nerve. All cells were autologous. Thetransdiffereniated MSCs were cultured in a mixture of factors in order to promote

Schwann cell-like cell formation. The undifferentiated MSCs demonstrated noregenerative capacity, while the transdifferentiated MSCs showed some regenerativecapacity, though it did not reach the capacity of the Schwann cells.[44]Ectomesenchymal stem cells (EMSCs)

The difficulty of isolating Schwann cells and subsequently inducing proliferation is alarge obstacle. A solution is to selectively induce cells such as ectomesenchymal stemcells (EMSCs) into Schwann cell-like phenotypes. EMSCs are neural crest cells thatmigrate from the cranical neural crest into the first branchial arch during earlydevelopment of the peripheral nervous system.[39] EMSCs are multipotent and possess aself-renewing capacity. They can be thought of as Schwann progenitor cells because theyare associated with dorsal root ganglion and motor nerve development. EMSCdifferentiation appears to be regulated by intrinsic genetic programs and extracellular signals in the surrounding environment.[39] Schwann cells are the source for bothneurotropic and neurotrophic factors essential for regenerating nerves and a scaffold for guiding growth. Nie, Zhang et al. conducted a study investigating the benefits of culturing EMSCs within PLGA conduits. Adding foskolin and BPE to an EMSC culturecaused the formation of elongated cell processes, which is common to Schwann cells invitro.[39] Thus, foskolin and BPF may induce differentiation into Schwann cell-likephenotypes. BPE contains the cytokines GDNF, basic fibroblast growth factor andplatelet-derived growth factor, which cause differentiation and proliferation of glial andSchwann cells by activating MAP kinases. When implanted into the PLGA conduits, theEMSCs maintained long-term survival and promoted peripheral nerve regenerationacross a 10 mm gap, which usually demonstrates little to no regeneration. Myelinatedaxons were present within the grafts and basal laminae were formed within the myelin.These observations suggest that EMSCs may promote myelination of regenerated nervefibers within the conduit.Neural progenitor cells



Inserting neurons into a bioartificial nerve conduit seems like the most obvious methodfor replacing damaged nerves; however, neurons are unable to proliferate and they areoften short-lived in culture. Thus, neural progenitor cells are more promising candidatesfor replacing damaged and degenerated neurons because they are self-renewing, whichallows for the in vitro production of many cells with minimal donor material.[27] In order

to confirm that the new neurons formed from neural progenitor cells are a part of afunctional network, the presence of synapse formation is required. A study by Ma,Fitzgerald et al. is the first demonstration of murine neural stem and progenitor cell-derived functional synapse and neuronal network formation on a 3D collagen matrix. Theneural progenitor cells expanded and spontaneously differentiated into excitable neuronsand formed synapses; furthermore, they retained the ability to differenitate into the threeneural tissue lineages.[27] It was also demonstrated that not only active synaptic vesiclerecycling occurred, but also that excitatory and inhibitory connections capable of generating action potentials spontaneously were formed.[27] Thus, neural progenitor cells are a viable and relatively unlimited source for creating functional neurons.Neural stem cells

Neural stem cells (NSCs) have the capability to self-renew and to differentiate intoneuronal and glial lineages. Many culture methods have been developed for directingNSC differentiation; however, the creation of biomaterials for directing NSCdifferentiation is seen as a more clinically relevant and usable technology.[citationneeded] One approach to develop a biomaterial for directing NSC differentiation is tocombine extracellular matrix (ECM) components and growth factors. A very recent studyby Nakajima, Ishimuro et al. examined the effects of different molecular pairs consistingof a growth factor and an ECM component on the differentiation of NSCs into astrocytesand neuronal cells. The ECM components investigated were laminin-1 and fibronectin,which are natural ECM components, and ProNectin F plus (Pro-F) and ProNectin L (Pro-L), which are artificial ECM components, and poly(ethyleneimine) (PEI). Theneurotrophic factors used were epidermal growth factor (EGF), fibroblast growth factor-2(FGF-2), nerve growth factor (NGF), neurotrophin-3 (NT-3), and ciliary neurotrophicfactor (CNTF). The pair combinations were immobilized onto matrix cell arrays, onwhich the NSCs were cultured. After 2 days in culture, the cells were stained withantibodies against nestin, β-tubulin III, and GFAP, which are markers for NSCs, neuronalcells, and astrocytes, respectively.[45] The results provide valuable information onadvantageous combinations of ECM components and growth factors as a practicalmethod for developing a biomaterial for directing differentiation of NSCs.[45]Neurotrophic factors

Currently, neurotrophic factors are being intensely studied for use in bioartificial nerveconduits because they are necessary in vivo for directing axon growth and regeneration.In studies, neurotrophic factors are normally used in conjunction with other techniquessuch as biological and physical cues created by the addition of cells and specifictopographies. The neurotrophic factors may or may not be immobilized to the scaffoldstructure, though immobilization is preferred because it allows for the creation of permanent, controllable gradients. In some cases, such as neural drug delivery systems,they are loosely immobilized such that they can be selectively released at specified times



and in specified amounts. Drug delivery is the next step beyond the basic addition of growth factors to nerve guidance conduits.Biomimetic materials

Many biomaterials used for nerve guidance conduits are biomimetic materials.

Biomimetic materials are materials that have been design such that they elicit specifiedcellular responses mediated by interactions with scaffold-tethered peptides from ECMproteins; essentially, the incorporation of cell-binding peptides into biomaterials viachemical or physical modification.[46]Synergism

Synergism often occurs when two elements are combined; it is an interaction betweentwo elements that causes an effect greater than the combined effects of each elementseparately. Synergism is evident in the combining of scaffold material and topographywith cellular therapies, neurotrophic factors, and biomimetic materials. Investigation of synergism is the next step after individual techniques have proven to be successful by

themselves. The combinations of these different factors need to be carefully studied inorder to optimize synergistic effects.Optimizing neurotrophic factor combinations

It was hypothesized that interactions between neurotrophic factors could alter the optimalconcentrations of each factor. While cell survival and phenotype maintenance areimportant, the emphasis of evaluation was on neurite extension. A combination of NGF,glial cell-line derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) was presented to Dorsal root ganglion cultures in vitro. One factor from eachneurotrophic family was used.[47] It was determined that there is not a difference inindividual optimal concentration and combinatorial optimal concentration; however,around day 5 or 6 the neurites ceased extension and began to degrade. This washypothesized to be due to lack of a critical nutrient or of proper gradients; previousstudies have shown that growth factors are able to optimize neurite extension best whenpresented in gradients.[47] Future studies on neurotrophic factor combinations will needto include gradients.Combination of neural cell adhesion molecules and GFD-5

Cell adhesion molecules (CAMs) and neurotrophic factors embedded together intobiocompatible matrices is a relatively new concept being investigated.[48] CAMs of theimmunoglobulin superfamily (IgSF), which includes L1/NgCAM and neurofascin, areparticularly promising, because they are expressed in the developing nervous system onneurons or Schwann cells. They are known to serve as guidance cues and mediateneuronal differentiation. Neurotrophic factors such as NGF and growth differentiationfactor 5 (GDF-5), however, are well established as promoters of regeneration in vivo. Arecent study by Niere, Brown et al. investigated the synergistic effects of combining L1and neurofascin with NGF and GDF-5 on DRG neurons in culture; this combinationenhanced neurite outgrowth. Further enhancement was demonstrated by combining L1and neurofascin into an artificial fusion protein, which improves efficiency since factors



are not delivered individually.[48] Not only can different cues be used, but they may evenbe fused into a single 'new' cue.

Topography in synergy with chemical and biological cues

The effect of presenting multiple stimuli types such as chemical, physical, and biologicalcues on neural progenitor cell differentiation has not been explored. A study wasconducted in which three different stimuli were presented to adult rat hippocampalprogenitor cells (AHPCs): postnatal rat type-1 astrocytes (biological), laminin (chemical),and micropatterned substrate (physical).[49] Over 75% of the AHPCs aligned within 20°of the grooves compared to random growth on the non-patterned substrates.[49] WhenAHPCs were grown on micropatterned substrates with astrocytes, outgrowth wasinfluenced by the astrocytes that had aligned with the grooves; namely, the AHPCsextended processes along the astrocytic cytoskeletal filaments. However, the alignmentwas not as significant as that seen by the AHPCs in culture alone with the micropatternedsubstrate. In order to assess the different phenotypes expressed as a result of

differentiation, the cells were stained with antibodies for class III β-tubulin (TuJI),receptor interacting protein (RIP), and glial fibrillary acidic protein (GFAP), which aremarkers for early neurons, oligodendrocytes, and astrocytes, respectively. The greatestamount of differentiation was seen with AHPCs cultured on patterned substrates withastrocytes.[49]

nerve guidance conduit

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