| Despite advances in surgical techniques and the inherent capacity of the peripheral nervous system (PNS) to regenerate, the outlook for a full functional recovery following nerve injury remains dismal. To regain full function, the growing portion of the regenerating axon, the growth cone, must correctly navigate through the injury site to the innervation target. Due to the disruption in the PNS environment by the destruction of the natural guidance tubes and the formation of scar tissue, growth cone migration is often misguided. Schwann cells, a population of support cells, closely associated with the growing axons have been shown to influence growth cone migration. Our hypothesis states that Schwann cell orientation can guide neurite outgrowth. To provide a means of testing this hypothesis, this work focused on the development of an in vitro model to investigate neuronal-Schwann cell interactions; specifically, neurite outgrowth was examined in the presence of an oriented Schwann cell monolayer.; Micropatterned substrates containing alternating laminin-rich and BSA-rich stripes were used to align Schwann cells and were characterized both physically and chemically to identify features present responsible for directing Schwann cell alignment. Using both static images and time-lapse microscopy, Schwann cell alignment and morphological response to the laminin micropattern was quantified, providing information necessary to create an oriented Schwann cell monolayer. Finally, neurons were grown on oriented Schwann cell substrates and neurite outgrowth was found to be directed by the underlying oriented cellular substrate. Co-cultures were investigated at higher magnification providing a detailed look of neurites tracking between and around Schwann cell nuclei, indicative of a contact guidance mechanism, in addition to tracking over nuclei indicating a potential receptor-ligand interaction.; Investigation of neurite outgrowth in response to Schwann cell orientation provided insight into the synergistic effects of the combined factors (Schwann cells and orientation) on neurite outgrowth. In addition, the continued systematic investigation of cellular interactions will not only elucidate cellular mechanisms affecting nerve regeneration, but will also provide a rational basis for the design of novel microenvironments to successfully support and promote peripheral nerve regeneration. |
