The nanofibers were produced by electrospinning and the setup was similar to what we used in previous studies.[11 , 18 (link)–21 ] Poly(ε-caprolactone) (PCL) (Mw=65,000 g/mol; Sigma-Aldrich, St. Louis, MO) was dissolved in a solvent mixture consisting of dichloromethane (DCM) and N, N-dimethylformamide (DMF) (Fisher Chemical, Waltham, MA) with a ratio 8:2 (v/v) at a concentration 20% (w/v). Polymer solution was pumped at a flow rate of 0.2 mL/h using a syringe pump. A DC high voltage of 12 kV was applied between the nozzle (a 22-gauge needle) and a grounded collector. Different collectors were employed to generate different types of nanofiber assemblies. Random nanofibers were directly collected using cover glass slips. A stainless steel frame (with an open void of 2 cm × 5 cm) was used as the collector. Subsequently, the aligned nanofibers were easily transferred to the cover glass slips by lifting off the fibers. Samples containing both random and aligned fibers next to each other were obtained by using two metal frames separated by an air gap. Fibers were deposited in the random and aligned form on the metal part and across the air gap, respectively.
The electrospun PCL nanofibers were coated with laminin (Millipore, Temecular, CA) as the following. The electrospun fibers were immersed in a 0.1% poly-L-lysine (PLL) (Sigma-Aldrich) solution for 1 h at room temperature, followed by washing with phosphate buffered saline (PBS) buffer (Invitrogen) three times. Subsequently, the nanofiber sample was immersed in a laminin solution (26 μL 50 μg/mL laminin solution diluted with 5 mL PBS buffer) at 4 °C overnight. Prior to DRG seeding, the nanofiber scaffold was rinsed with PBS buffer three times.
PEG (Mw=8,000 g/mol, Sigma-Aldrich) coating on the polystyrene substrate was completed by physical adsorption. Briefly, the small piece of polystyrene substrate fabricated by cutting Petri dishes was immersed in a 1% PEG solution overnight, followed by washing with water three times.
The morphologies and structures of various fiber assemblies were characterized by scanning electron microscopy (SEM) (200 NanoLab, FEI, Oregon). To avoid charging, the polymer fiber samples were coated with platinum using a sputter coater for 40 sec in vacuum at a current intensity of 40 mA after the sample had been fixed on a metallic stud with double-sided conductive tape. The accelerating voltage was 15 kV for the imaging process.
FFT analysis was performed by utilizing the FFT function of the Scion Image processing software. The detailed information on measuring fiber alignment by FFT can be found in an excellent review article.38 (link) The spatial information presented by an image can be processed into a mathematically defined frequency domain using 2D FFT function. The frequency domain maps the rate where pixel intensities vary in the spatial domain. Pixel intensities and the intensity distraibution of the resulting image correspond to the directional content of the original image and the results of the FFT yields frequencies orthogonal to those in the original image.15 , 17 (link), 38 (link)–40
The electrospun PCL nanofibers were coated with laminin (Millipore, Temecular, CA) as the following. The electrospun fibers were immersed in a 0.1% poly-L-lysine (PLL) (Sigma-Aldrich) solution for 1 h at room temperature, followed by washing with phosphate buffered saline (PBS) buffer (Invitrogen) three times. Subsequently, the nanofiber sample was immersed in a laminin solution (26 μL 50 μg/mL laminin solution diluted with 5 mL PBS buffer) at 4 °C overnight. Prior to DRG seeding, the nanofiber scaffold was rinsed with PBS buffer three times.
PEG (Mw=8,000 g/mol, Sigma-Aldrich) coating on the polystyrene substrate was completed by physical adsorption. Briefly, the small piece of polystyrene substrate fabricated by cutting Petri dishes was immersed in a 1% PEG solution overnight, followed by washing with water three times.
The morphologies and structures of various fiber assemblies were characterized by scanning electron microscopy (SEM) (200 NanoLab, FEI, Oregon). To avoid charging, the polymer fiber samples were coated with platinum using a sputter coater for 40 sec in vacuum at a current intensity of 40 mA after the sample had been fixed on a metallic stud with double-sided conductive tape. The accelerating voltage was 15 kV for the imaging process.
FFT analysis was performed by utilizing the FFT function of the Scion Image processing software. The detailed information on measuring fiber alignment by FFT can be found in an excellent review article.38 (link) The spatial information presented by an image can be processed into a mathematically defined frequency domain using 2D FFT function. The frequency domain maps the rate where pixel intensities vary in the spatial domain. Pixel intensities and the intensity distraibution of the resulting image correspond to the directional content of the original image and the results of the FFT yields frequencies orthogonal to those in the original image.15 , 17 (link), 38 (link)–40
