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This study reports a facile method for the fabrication of aligned Poly(3,4-ethylene dioxythiophene) (PEDOT) fibers and tubes based on electrospinning and oxidative chemical polymerization. Discrete PEDOT nano- and microfibers and nano- and microtubes are difficult to fabricate quickly and reproducibly. We employed poly(lactide-co-glycolide) (PLGA) polymers that were loaded with polymerizable 3,4-ethylene dioxythiophene (EDOT) monomer to create aligned nanofiber assemblies using a rotating glass mandrel during electrospinning. The EDOT monomer/PLGA polymer blends were then polymerized by exposure to an oxidative catalyst (FeCl3). PEDOT was polymerized by continuously dripping a FeCl3 solution onto the glass rod during electrospinning. The resulting PEDOT fibers were conductive, aligned and discrete. Fiber bundles could be easily produced in lengths of several centimeters. The PEDOT sheath/PLGA core fibers were immersed in chloroform to remove the PLGA and any residual EDOT resulting in hollow PEDOT tubes. This approach made it possible to easily generate large areas of aligned PEDOT fibers/tubes. The structure and properties of the aligned assemblies were measured using optical microscopy, electron microscopy, Raman spectroscopy, thermal gravimetric analysis, and DC conductivity measurements. We also demonstrated that the aligned PEDOT sheath/PLGA core fiber assemblies could be used in supporting and directing the extension of dorsal root ganglia (DRG) neurons in vitro.

Electrospinning is a technique for producing micro-to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications. Video LinkThe video component of this article can be found at https://www.jove.com/video/2389/ Protocol 1. Poly-L-lactic Acid (PLLA) Spinning Solution 1. Dissolve 0.4 g PLLA in 9 mL chloroform by stirring over low heat. 2. Add 1 mL dimethylformamide to the solution, bringing the final concentration of the solution to 4% PLLA (w/v) in chloroform:DMF 9:1 (v/v). 3. Place the solution in a polypropylene or glass syringe with a blunt 23ga metal tip. Spinning Substrate Preparation 11. Make an 8% (w/v) solution of 85:15 PLGA (poly-lactic-co-glycolic acid) in chloroform by stirring over low heat. 2. Coat clean glass cover slips in PLGA by covering the surface of each cover slip with the PLGA solution. Allow the PLGA to dry to a thin film (approx. 30 min). Electrospinning 21. Secure PLGA coated glass cover slips to collector with conductive carbon tape. For aligned fibers, the collector is a motor-driven wheel. For random fibers, the collector is a stationary plate. 2. Place syringe in pump with tip 20 cm from collector wheel. Set pump to approximately 2 mL/hr and if using a wheel, set the motor to 300-400 RPM. If possible, apply a -2 kV DC bias to the collector and +15 kV to metal tip. In the absence of access to a bipolar power supply, ground the collector. 3. Fibers will jet from the syringe tip and collect on the rotating wheel. Continue spinning until the desired density of fibers is obtained. Swipe the metal tip with a paper towel affixed to a non-conductive rod periodically to prevent clogging at the tip.

There is little remedy for the devastating effects resulting from neuronal loss caused by neural injury or neurodegenerative disease. Reconstruction of damaged neural circuitry with stem cell-derived neurons is a promising approach to repair these defects, but controlling differentiation and guiding synaptic integration with existing neurons remain significant unmet challenges. Biomaterial surfaces can present nanoscale topographical cues which influence neuronal differentiation and process outgrowth. By combining these scaffolds with additional molecular biology strategies, synergistic control over cell fate can be achieved. Here, we review recent progress in promoting neuronal fate using techniques at the interface of biomaterial science and genetic engineering. New data demonstrates that combining nanofiber topography with an induced genetic program enhances neuritogenesis in a synergistic fashion. We propose combining patterned biomaterial surface cues with prescribed genetic programs to achieve neuronal cell fates with the desired sublineage specification, neurochemical profile, targeted integration and electrophysiological properties.