Soft tissue reconstruction in the nervous system is sensitive to the mechanical and chemical cues of the growth microenvironment. Many technologies have been designed to study these stimuli and their effect on the regional extracellular matrix (ECM). Because of the hard-to-achieve nature and costliness of these technologies, biologists are usually reluctant to employ them to study cellular behaviors. In addition, the complexity of the nervous system, particularly in cases of nerve repair and reconstruction, necessitates the development of facile high-throughput investigational tools.

The objective for this proposal is to develop a series of novel tools to manipulate neuronal cell-cell and cell-ECM responses to varying nervous system microenvironment stimuli in a 3-D in vitro model. In Specific Aim 1 a structurally tunable hydrogel is synthesized to investigate the effect of mechanical stimuli on nerve regeneration. We developed a novel interpenetrating network of polymers (IPN) of photocrosslinkable hyaluronic acid and Puramatrix components to promote cellular growth and allow adjustment of mechanical properties by varying the degree of methacrylation. The results demonstrated that neurite outgrowth in 3-D was significantly higher in the more compliant and less interconnected environment.

In Specific Aim 2 we analyzed the effect of different light wavelengths on cultured neurons in a 3-D dual hydrogel model. We performed cellular studies including neurite growth, neurite viability and DNA fragmentation. These studies agree that utilizing visible light is more practical for cellular encapsulation in photopolymerization applications as it is less damaging to the cells in our studies than UVA light. However, visible light still causes damages to our cells. These data also confirm that increasing the irradiation dosage through raising the exposure time will result in more cellular damage and DNA fragmentation.

We then utilized the optimized system from Specific Aim 3 to examine myelination events in co-cultures of dorsal root ganglion (DRG) with Schwann cells. Here, we demonstrated that this co-culture setting provided us with aligned, highly fasciculated neuronal growth with myelin sheath nicely wrapped along them. We also used two culture systems, and also studied the influence of collagen on neuronal growth and myelination. We also studied the influence of collagen and ascorbic acid (AA) exposure duration on neuronal growth and myelination. We demonstrated that the longer exposure to AA along with the presence of collagen in the system would lead to higher neurite growth and more myelination.