New Materials and Scaffold Fabrication Method for Nerve Tissue Engineering.

Major technical challenges remain before enabling complete functional recovery after a severe nerve injury. Creating a growth permissive environment and directing the extension and myelination of surviving neurons encompass the current goals of nerve regeneration strategies. A potential solution to address these challenges is to promote neurite sprouting and guide the regenerating nerve using biomaterials. We hypothesize that neurite sprouting and extension can be enhanced by using biomaterials as a platform to present biochemical and physical cues. We present a series of biodegradable polymers with varying concentrations of acetylcholine-like motifs and a scaffold fabrication method for producing porous scaffolds containing longitudinally oriented channels. Acetylcholine is a neurotransmitter involved in neuronal processes, which regulates neurite branching, induces neurite outgrowth, and promotes the formation of synapses. Because of its various roles in neuronal activities, acetylcholine-based materials may also be useful in nerve repair. Acetylcholine-like motifs were incorporated within a polymer by the polycondensation of diglycidyl sebacate, aminoethyl acetate, and leucine ethyl ester, in a manner that permitted control over acetylcholine motif concentration. Diglycidyl sebacate was synthesized by oxidation of diallyl sebacate, while the amine-containing compounds, aminoethyl acetate and leucine ethyl ester, were liberated with sodium carbonate-sodium bicarbonate buffers. Standard analytical techniques were used to quantify acetylcholine motif concentrations, as well as to characterize molecular weights and glass transition temperatures of the polymers. The modular design of synthesizing the polymers can be used to test the effects of presenting different neurotransmitter motifs, a combination of two or more neurotransmitter motifs, or polymer backbones on neurite sprouting in future studies. Interactions between the polymers of different acetylcholine motif concentrations and neurons were characterized using rat dorsal root ganglia explants (DRG). We screened the potential application of these materials in nerve tissue engineering using the following criteria: (1) neurite sprouting, (2) neurite length, and (3) distribution of the neurite lengths. The ability of DRG to sprout neurites was influenced by the concentration of acetylcholine motifs of the polymer. All polymers permitted neurite extension and maintained the neuronal phenotype. Addition of acetylcholine receptor antagonists to DRG cultured on the polymers significantly decreased neurite sprouting, suggesting acetylcholine receptors mediate sprouting on the polymers. Future studies may examine how neurons on acetylcholine-based polymers exhibit changes in downstream signaling events and cell excitability that are associated with receptor activation. In preparation for testing the acetylcholine-based polymers in vivo, porous scaffolds with longitudinally oriented channels were fabricated using fiber templating and salt leaching. Scaffolds were made of poly(glycerol sebacate) because of this polymer's comparable mechanical properties to the peripheral nerve and its biocompatibility in nerve regeneration applications. Micro computed tomography, scanning electron microscopy, and cryo-sectioning revealed the presence of longitudinally oriented channels going along the length of the scaffold. Channel volume and average pore size of the scaffolds were controlled by the number of fibers and salt fusion time. Future studies may involve testing the effect of acetylcholine-motifs by coating polymers onto such scaffolds or assessing the effect of the scaffold's dimensional...