Parkinson’s Disease is a neurodegenerative disorder primarily affecting dopaminergic cells in the substantia nigra. The resulting degenerated axons in the nigrostriatal pathway lead to a denervated striatum, which affects circuits controlling patient movement. Implantable micro-tissue engineered neural networks (Micro-TENNs), previously developed by our lab, offer a potential alternative to the current Parkinson’s treatments. Micro-TENNs consist of a hollow hydrogel column filled with an extracellular-matrix like solution and seeded with an aggregated mass of immature neurons at one end. The hydrogel column then acts as a guide for proliferating axons, yielding unidirectional outgrowth. When fabricated with dopaminergic neurons, micro-TENNs act as Tissue Engineered Nigrostriatal Pathways (TE-NSPs) by reconnecting the substantia nigra pars compacta and striatum, theoretically restoring proper dopamine signaling required for motor regulation. Given that axonal proliferation is strongly dependent on the local neuronal micro-environment, this study aimed to optimize micro-TENN outgrowth through manipulation of the hydrogel encasement. Specifically, micro-TENN casings were fabricated from methacrylated hyaluronic acid (MeHA) with varying degrees of backbone modification (DoM) and solution concentration. As a proof of concept, we then constructed generalized micro-TENNs from embryonic rodent cortical neurons to assess the impact of casing polymeric network density on axonal outgrowth. 

The MeHA solution was synthesized by the esterification of Hyaluronic Acid and Methacrylic Anhydride (MA). Addition of the MA was calculated to control the solution’s degree of backbone modification and NMR analysis was done to estimate the DoM that was achieved. The column encasement was created by coaxially molding the synthesized MeHA solution and curing it under UV light. Cortices were isolated from E18 Sprague Dawley rat embryos and then digested with trypsin and fully dissociated under DNAse before being spun down into aggregates. An ECM-like mixture of collagen and laminin was injected into the column, and an aggregate was inserted into one end. The micro-TENN was then submerged in neurobasal media for long-term culture. 

Our findings suggest that encasements made of higher degrees of methacrylation and lower solution concentrations correlate with less axon outgrowth within the micro-TENN. This may result from higher backbone modification levels increasing the network crosslinking density within the encasement. The higher crosslinking density could then reduce the diffusion through the column walls and enforce the reliance on diffusion through the open ends of the structure. This change in diffusion balance may reduce the overall amount of nutrients available to the axons, restrict waste removal, and thus hinder axonal outgrowth through heightened metabolic stress. Future studies will characterize porosity and molecular diffusion through MeHA at varying DoM and solution concentrations. In addition, encasement. In addition, longitudinal neuronal health will be assessed via lactate dehydrogenase levels or similar nondestructive outcome measures.