Scaffolds with oriented microchannels for angioneural regenerative engineering

Spinal cord injury (SCI) affects over 12,000 people annually and, due to limited therapeutic options, results in an estimated 1.3 million people living with chronic motor dysfunction or paralysis in the U.S. The medical costs associated with SCI can pose significant financial burden to patients (estimated at 2 million dollars in a lifetime-NSCICS). Finding a therapeutic strategy for repair of SCI would dramatically improve quality of life for these patients as well as ease the costs associated with chronic care.Prior studies have shown that on non-structured substrates, oriented axonal growth is very poor; axons tend to grow erratically and axon connection pathways cannot find and connect to their target neurons. Additionally spinal cord development, and presumably spinal cord regeneration requires angiogenesis for nutrient delivery as well as regulation of neuronal growth through paracrine signaling. This is supported by previous studies that showed increased micro-vessel density correlates with regeneration and functional recovery in spinal cord tissue. Based on these finding this study focuses on combining structural support for directed axonal growth with angiogenic cues for the purpose of enhancing SCI repair. It hypothesizes that the crosstalk between the vascular and the nervous systems, i.e., between cues that lead to angiogenesis and neuritogenesis is crucial for axonal regeneration across a spinal cord lesion. To test this hypothesis, a scaffold is evaluated for promoting neuritogenesis in vitro by seeding its longitudinally oriented 3D pores with endothelial cells prior to seeding with neural-crest derived PC12 cells.This study designed and tested a biodegradable, genipin cross-linked gelatin scaffold by using a controlled uniaxial freezing technique followed by lyophilization to improve mechanical properties of the scaffold and to generate longitudinal microporous channels. The scaffold had mechanical properties similar to those of the rat spinal cord with elastic moduli of (~51kPa), and it supported growth and differentiation of endothelial cells and PC12 cells. An “angioneural tissue” was generated by first seeding endothelial cells in the scaffold to generate a monolayer lining the channel walls, followed by seeding with neuronal PC12 cells a week later. In support of the hypothesis, the presence of endothelial cells facilitated the differentiation of nerve growth factor (NGF)-induced PC12 cells significantly leading to enhanced PC12 cell survival and neuritogenesis. Our results are promising for the promotion of neuritogenesis through endothelial cell-derived cues (angioneural crosstalk, i.e., concomitantly promoting ngiogenesis and neuritogenesis), which may facilitate tissue regeneration and recovery after spinal cord injury.M.S., Biomedical Engineering -- Drexel University, 201