Electrospun nanofibers: Formation, characterization, and evaluation for nerve tissue engineering applications

The effects of fiber alignment and surface chemistry, including the covalent attachment and physical adsorption of the extracellular matrix (ECM) proteins laminin and collagen, on the neurite outgrowth of neuron-like PC12 cells were examined. Neuron-like PC12 cells responded to fiber orientation, and were successfully contact-guided by aligned electrospun nanofibers. In addition, fibers with attached protein, either physically adsorbed or covalently attached, improved neurite outgrowth lengths. Furthermore, aligning the fibers and attaching the ECM protein laminin, in particular, significantly improved neurite outgrowth over randomly oriented fibers with laminin.;Since this research suggested that protein concentration on the fibers was the dominant driving force for improved neurite outgrowth, the effect of protein concentration, incorporated onto the surface of the nanofibers, on neurite outgrowth was examined. Two ways to control protein concentration on the fibers were explored---the variation of the fiber-protein reaction time and the variation of the protein soaking solution concentration. In addition, analytical methods to quantify the concentration of protein, as well as the protein coverage, on the surface of the fibers were developed. Although most of the fiber mats had multilayer protein coverage, and hence physically adsorbed proteins which could potentially mean a loss in bioactivity, the neuron-like PC12 cell neurites responded in a dose-dependent manner with increased neurite lengths on scaffolds with higher protein concentrations. The work was extended further by forming protein gradients on the fiber mats in hopes of locally directing neurite outgrowth and orientation. Fiber mats with both linear gradients (continuous change in protein concentration) and step gradients (six regions of uniform protein coverage, with protein concentration increasing from region to region) were fabricated and analyzed. The step gradients formed in the aligned fiber direction showed the most promise for use in cell culture assays.;While surface chemistry and topography are important, porosity of the scaffold is also critical to control cellular infiltration and tissue formation. To enhance the porosity of our electrospun nanofiber scaffolds and improve the infiltration of cells, two methods were explored to control porosity. In the first method, the scaffold polymer polycaprolactone was co-electrospun with a sacrificial polymer polyethylene oxide, which was removed after the bi-component fiber mat was formed. In doing so, the void space was increased. In the second method, the spinning solution concentration of polycaprolactone was varied to control fiber diameter and porosity. The second method proved to be more effective at improving the cellular infiltration of PC12 cells. Two orders of magnitude range of fiber diameters were achieved, and nearly full infiltration of PC12 cells was observed for the mats with the highest porosity. The pore sizes of these mats were on the order of the size of the cell bodies (approximately 6-10 microm).;Although the majority of this work focuses on using conventional electrospinning to generate solid-core fibers, core-shell fibers, have many applications in tissue engineering, among other fields. We explored an efficient way to generate these fibers from an emulsion solution using a conventional electrospinning apparatus. We characterized the fibers using an atomic force microscope (AFM) elastic modulus mapping technique, along with AFM phase imaging, angle-resolved x-ray photoelectron spectroscopy and thermal gravimetric analysis, to determine the chemical and molar composition of the core and shell layers. This work presents novel analytical techniques for the characterization of core-shell nanofibers in order to better predict and understand their material properties. (Abstract shortened by UMI.)...