J21 - Investigating Material Selection for In Vitro Modeling of TBI Associated Proteinopathies

Introduction: : Traumatic Brain Injury (TBI) is a major public health concern, attributed to millions of emergency department visits in the United States annually. Emerging evidence links repeated TBI, common among athletes and military personnel, to neurodegenerative proteinopathies such as the accumulation of tau and amyloid-beta. Accumulation of such proteins, implicated in conditions like Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE), are key biomarkers of neurodegeneration. Currently, knowledge gaps hinder the understanding of how TBI biomechanics, particularly brain strain and strain rate during head impact, contribute to these neurodegenerative proteinopathies. Our study plans to bridge these gaps by conducting in vitro tensile testing of neurons and subsequent measurements of protein aggregation. Selecting an optimal material for cell survivability represents the first step towards optimizing this in vitro model, ensuring any cellular damage is due to stretch injury rather than material incompatibility. Materials being tested include silicon elastomers, used in similar in vitro tensile tests of seeded cell cultures, and hydrogel-based cell cultures, selected for their high biocompatibility and three-dimensional structure. Once an optimal material is selected, it will be subjected to stretch injury that mimics TBI biomechanics at a cellular level, and subsequent measurements of neurodegenerative protein aggregation will be assessed. These in vitro experiments will be used to establish correlations between strain and neurodegeneration, providing valuable insights into secondary cellular processes following a TBI.

Materials and

Methods: : A comparison was conducted between four materials, including three-dimensional poly(ethylene glycol) diacrylate (PEGDA) hydrogel cultures, silicon elastomers, silicon coated with Sylgard 527, and silicon coated with Sylgard 184. Silicon constructs were 3D printed using 50A silicon resin (FormLabs), and Sylgard coatings were prepared following manufacturer instructions. Silicon resin constructs and Sylgard adhesives were selected due to their use in previous in vitro TBI models; however, these models lack justification for material selection. The use of hydrogel-based cell cultures has emerged in recent years, with cells embedded into a three-dimensional matrix closely resembling the physiological environment. PEGDA hydrogels specifically were selected for their high biocompatibility and molecular weight (6000 Da), allowing easy passage of nutrients through the hydrogel mesh. These hydrogels were prepared by mixing SH-SY5Y neuroblastoma cells cultured in DMEM/F12 media with 10% fetal bovine serum directly into a mixture of PEGDA, deionized water, and Irgacure 2959 photoinitiator prior to UV curing. Survivability of differentiated SH-SY5Y cells seeded onto these materials was assessed through various assays. Colorimetric XTT assays were used to quantify cell viability and metabolic activity over a seven-day period, providing a comparison of long-term viability of cells on each material. Results were used to calculate percent cell viability relative to an initial positive control; cells seeded directly onto a 96-well plate. Additionally, fluorescent caspase 3/7 assays are ongoing, and results will provide further insight into cell survivability and apoptosis-linked enzymatic activity. Results from both assay types will be assessed to determine the optimal material for cell survivability.

Results, Conclusions, and Discussions:: Based on the XTT assay results, PEGDA hydrogel cultures display the most consistent long-term metabolic activity, likely due to their 3D structure closely resembling neural extracellular environment. Although this material displayed a low initial viability-- around 30% -- there was a steady increase in viability until day five, and viability at the end of the seven-day period remained around 25%. In contrast, each of the silicon-based materials showed 0% viability at this seven-day time point. Additionally, SH-SY5Y cells seeded onto silicon elastomers without adhesive coatings displayed little to no viability throughout the entirety of the experiment, suggesting that for silicon elastomers, adhesive coatings are imperative for SY-SY5Y cell adhesion and survivability. Notably, elastomers coated with Sylgard 527 adhesive produced relatively high initial viability when compared to other experimental groups, however results were inconsistent over time, suggesting the need for further assays. Caspase assays are currently underway and will provide insight into apoptotic activity of seeded SH-SY5Y cells. Apoptotic activity is anticipated to be highest in the silicon elastomer group without adhesive, as this is the group exhibits the lowest metabolic activity according to XTT assays. Similarly, apoptosis activity is hypothesized to consistently be the lowest in the PEGDA hydrogel cultures, which displayed the most consistent metabolic activity over time in the XTT assays. Upon completion of both XTT and caspase assays, results will be used to inform selection of an optimized material. Follow-up studies will use this optimized material to carry out in vitro stretch experiments to help establish relationships between TBI-induced strain and proteinopathy of tau and amyloid-beta. By selecting an ideal material for both cell adhesion and survivability based on XTT and caspase assay results, we will ensure that our in vitro testing frame accurately replicates strain-induced cellular damage and protein accumulation post-injury, removing any confounding factors due to material incompatibility.

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