During development, neurites sample the surrounding environment for mechanical and chemical guidance cues in order to reach their desired targets. Due to the nervous system’s limited ability to regenerate after experiencing trauma, approaches from neurite development have been applied to create improved neural therapeutic and regenerative strategies. The use of guidance cues to reconnect the neural network could overcome limitations of current clinical treatments and ensure full function restoration and inhibit neuropathic pain development. This dissertation presents in vitro models that incorporate soluble gradients of chemical cues to influence neural growth at a choice point.

Axonal growth can be guided to a desired destination through the use of soluble guidance cues. These cues are usually presented as gradients in order to induce an attractive or repulsive response. In order to advance the application of our laboratory’s hydrogel choice point model, we needed to integrate soluble cues. An approach for incorporating these soluble cues was incorporating a circular reservoir patterned directly into the construct to serve as a protein source for biomolecule diffusion. The objective of Aim 1 was to determine how changes in source well position, growth restrictive border composition, and source well concentration affected the spatial and temporal concentration of gradients within the growth permissive area. Increasing the well distance caused a longer sustained release of proteins resulting in longer maintained gradients with higher concentrations. Increasing the restrictive border composition slowed the diffusion of the protein, producing gradients with lower concentrations and lasting for shorter durations. Reducing the initial well concentration lessened the overall gradient concentrations. In addition, a 2-D computational diffusion model was developed and compared to experimental results. Through the use of the computational model, we identified configurations and experimental processes that keep cells exposed to physiologically relevant concentrations of a desired chemical signal. Our results show the ability to control soluble gradient profiles within our biphasic scaffold and establish methods that inform future experiments aimed at exposing cells to concentrations found in vivo.

During development, axons frequently encounter choice points that they navigate by sensing environmental cues in order to reach their final destination. The objective of Aim 2 was to demonstrate the feasibility of growth factor induced axon guidance using a computationally informed experimental choice point model. Additionally, a biased turning neurite growth model was created as a predictive tool for evaluating newly microfabricated geometries. Our in vitro studies showed that the presence of a soluble gradient of nerve growth factor produced a more chemoattractive guidance ratio compared to conditions with no gradient. Corresponding computational simulations modified to match the choice point geometry, generated similar guidance ratio values for the gradient and no gradient conditions. Our results demonstrate an axonal guidance assay influenced by diffusible gradients at a choice point and supports the use of our computational growth model for the assessment of axonal growth patterns within newly fabricated geometries.

An issue with experimentally administering axonal guidance cues in Aim 2 was the need for multiple fillings. The use of exogenous cells that consistently secrete these cues could serve as a possible solution. The objective of Aim 3 was to demonstrate the ability to direct axon guidance using exogenous cells. In our dual phased hydrogel system, keratinocytes and dermal fibroblasts were used as they are involved in cutaneous innervation. We developed viable co-cultures of neural and dermal cells in our choice point model. Secretions from keratinocytes alone produced a more chemoattractive response compared to the control. The combination of keratinocytes and fibroblasts induced a further measured chemoattractive response. Our results show that exogenous cells can direct neurite growth and can be used as a cue source for future axonal guidance experiments.

The results establish the utility of our system for observing axonal growth under the influence of soluble guidance cues. Obtaining a better understanding of the neuriteenvironmental interaction can lead to the development of improved neural therapeutic and regenerative strategies.