motor and controller. the nerve tissue culture is placed within the expansion chamber with a port for gas exchange and a removable stretching frame, which is able to separate two groups of somas (neuron cell bodies) and thus stretch their axons. collagen gel was used to promote the growth of larger stretch-grown axon tracts that were visible to the unaided eye. there are two reasons for the growth enhancement due to the collagen coating: 1) the culture became hydrophobic after the collagen dried, which permitted a denser concentration of neurons to grow, and 2) the collagen coating created an unobstructed coating across the two elongation substrates. examination by scanning electron microscope and tem showed no signs of axon thinning due to stretch, and the cytoskeleton appeared to be normal and intact. the stretch-grown axon tracts were cultured on a biocompatible membrane, which could be directly formed into a cylindrical structure 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 unprecedented rate of 1 cm/day after only 8 days of acclimation, which is much greater than the 1 mm/day maximal growth rate as measured for growth cone extension. the rate of 1 mm/day is also the average transport speed for structural elements such as neurofilaments. nanofibers scaffolds research on nanoscale fibers attempts to mimic the in vivo extracellular environment in order to promote directional growth and regeneration. three distinct methods for forming nanofibrous scaffolds are self-assembly, phase separation and electrospinning. however, there are many other methods for forming nanofibrous scaffolds. self-assembly of nanofibrous scaffolds is able to occur only when the fibers themselves are engineered for self-assembly. one common way to drive the self-assembly of scaffold fibers is to use amphiphilic peptides so that in water the hydrophobic moiety drives the self-assembly. 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 vitro and in vivo a self-aligned collagen-schwann cell matrix, which allowed drg neurite extension alignment in vitro. collagen gels have been used extensively as substrates for three-dimensional tissue culture. cells are able to form integrin-mediated attachments with collagen, which initiates cytoskeleton assembly and cell motility. as cells move along the collagen fibers they generate forces that contract the gel. when the collagen fibers are tethered at both ends, cell-generated forces create uniaxial strain, causing the cells and collagen fibers to align. the advantages of this matrix are its simplicity and speed of preparation. soluble plasma fibronectin can also self-assemble into stable insoluble 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. the mechanical shearing was created by dragging out a 0.2 ml bolus to 3 cm with forceps; fibronectin aggregates into insoluble fibers at the rapidly moving interface in an ultrafiltration cell. the proposed mechanism for this fiber aggregation is protein extension and elongation under mechanical shear force, which leads to lateral packing and protein aggregation of fibers. phillips et al. showed that mechanical shear produced by stretching a high viscosity fibronectin gel causes substantial changes in its structure and that when applied through uniaxial extension, a viscous fibronectin gel forms oriented fibrous fibronectin aggregates; additionally, the fibrous aggregates have a decreased solubility and can support the various cell types in vitro. phase separation allows for three-dimensional sub-micrometre fiber scaffolds to be created without the use of specialized equipment. the five steps involved in phase separation are polymer dissolution, phase separation and gelation, solvent extraction from the gel, freezing and freeze drying in water. the final product is a continuous fiber network. phase separation can be modified to fit many different applications, and pore structure can be varied by using different solvents, which can change the entire process from liquid–liquid to solid–liquid. porosity and fiber diameter can also be modified by varying the initial concentration of the polymer; a higher initial concentration leads to less 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 the fibers. phase separation is a widely used technique for creating highly porous nanofibrous scaffolds with ease. electrospinning provides a robust platform for development of synthetic nerve guidance conduits. electrospinning can serve to create scaffolds at controlled dimensions with varying chemistry and topography. furthermore, different materials can be encapsulated within fibers including particles, growth factors, and even cells. electrospinning creates fibers 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 that elongates and thins. this polymer jet discharges as a taylor cone, leaving behind electrically charged polymers, which are collected on a grounded surface as the solvent as the solvent evaporates from the jets. fibers have been spun with diameters ranging from less than 3 nm to over 1 µm. the process is affected by system parameters such as polymer type, polymer molecular weight, and solution properties and by process parameters such as flow rate, voltage, capillary diameter, distance between the collector and the capillary, and motion of the collector. 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. the two features of electrospun scaffolds that are advantageous for neural tissue engineering are the morphology and architecture, which closely mimics the ecm, and the pores, which are the correct range of sizes that allows nutrient exchange but prevents in growth of glial scar tissue (around 10 µm). random electrospun plla scaffolds have been demonstrated to have increased cell adhesion, which may be due to an increased surface roughness. chemically modified electrospun fiber mats have also been shown to influence neural stem cell differentiation and increase cell proliferation. in the past decade, scientists have also developed numerous methods for production of aligned nanofiber scaffolds, which serve to provide additional topographic cues to cells. this is advantageous because large scale three-dimensional aligned scaffolds cannot be created easily using traditional fabrication techniques. in a study conducted by yang et al. (2005), aligned and random electrospun poly (l-lactic acid) (plla) microfibrous and nanofibrous scaffolds were created, characterized, and compared. fiber diameters were directly proportional to the initial polymer concentration used for electrospinning; the average diameter of aligned fibers was smaller than that of random fibers under identical processing conditions. it was shown that neural stem cells elongated parallel to the aligned electrospun fibers. the aligned nanofibers had a longer average neurite length compared to aligned microfibers, random microfibers, and random nanofibers. in addition, more cells differentiated on aligned nanofibers than aligned microfibers. thus, the results of this study demonstrated that aligned nanofibers may be more beneficial than nonaligned fibers or 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. while the superstructure refers to the overall shape of the scaffold, the microstructure refers to the cellular level structure of the surface and the nanostructure refers to the subcellular level structure of the surface. all three levels of structure are capable of eliciting cell responses; however, there is significant interest in the response of cells to nanoscale topography motivated by the presence of numerous nanoscale structures within the extracellular matrix. there are a growing number of methods for the manufacture of micro- and nanostructures (many originating from the semiconductor industry) allowing for the creation of various topographies with controlled size, shape, and chemistry. physical cues physical cues are formed by creating an ordered surface structure at the level of the microstructure and/or nanostructure. physical cues on the nanoscale have been shown to modulate cell adhesion, migration, orientation, contact inhibition, gene expression, and cytoskeletal formation. this allows for the direction of cell processes such as proliferation, differentiation, and spreading. there are numerous methods for the manufacture of micro- and nanoscale topographies, which can be divided into those that create ordered topographies and those that create unordered topographies. ordered topographies are defined as patterns that are organized and geometrically precise. though there are many methods for creating ordered topographies, they are usually time-consuming, requiring skill and experience and the use of expensive equipment. photolithography involves exposing a light source to a photoresist-coated silicon wafer; a mask with the desired pattern is place between the light source and the wafer, thereby selectively 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. however, because the lower limit for size is a function of the wavelength, this method cannot be used to create nanoscale features. 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 by photolithography. polyimide was used because it adheres to glass well, is chemically stable in aqueous solution, and is biocompatible. it is hypothesized that the microchannels limited the range of angles that cytoskeletal elements within the neurite growth cones could accumulate, assemble, and orient. there was a significant decrease in the number of neurites emerging from the soma; however, there was less decrease as the range of angles over which the neurites emerged was increased. also, the neurites were on average two times longer when the neurons were cultured on the microchannels versus the controls on a flat surface; this could be due to a more efficient alignment of filaments. 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. patterns are created by programming the beam of electrons for the exact path to follow along the surface of the material. resolution is affected by other factors such as electron scattering in the resist and 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 in tissue engineering, the resolution drops and features can only be created as small as 30–40 nm, and the resist development begins to weigh more heavily on pattern formation. to prevent dissolution of the resist, ultrasonic agitation can be used to overcome intermolecular forces. in addition, isopropyl alcohol (ipa) helps develop high-density arrays. ebl can become a quicker and less costly process by replicating nanometer patterns in polymeric materials; the replication process has been demonstrated with polycaprolactone (pcl) using hot embossing and solvent casting. in a study conducted by gomez et al. (2007), microchannels 1 and 2 µm wide and 400 and 800 nm deep created by ebl on pdms were shown to enhance axon formation of hippocampal cells in culture more so than immobilized chemical cues. x-ray lithography is another method for forming ordered patterns that can be used to investigate the role that topography plays in promoting neuritogenesis. the mask parameters determine the pattern periodicity, but ridge width and depth are determined by the 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 600 nm; developing neurites demonstrated contact guidance with features as small as 70 nm and greater than 90% of the neurites were within 10 degrees of parallel alignment with the ridges and grooves. there was not a significant difference in orientation with respect to the feature sizes used. the number of neurites per cell was constrained by the ridges and grooves, producing bipolar rather than branching phenotypes. unordered topographies are generally created by processes that