Poster AA19 - Printability and Brain Endothelial Cell Compatibility of a Brain-ECM Inspired Bioink

Introduction: Neurological disorders such as neurodegenerative diseases and stroke are a leading cause of death and disability worldwide. Therapeutic development for neurological diseases is limited by inadequate models, which ideally should mimic the semipermeable nature of the human blood brain barrier (BBB) and interplay of different neural and immune cells in the human brain parenchyma. New 3D in-vitro models comprised of human cells and engineered biomaterials are an alternative model, which can mimic human brain anatomy, gene expression and cellular behavior better than 2D cell cultures or rodent models. Existing brain 3D in-vitro models have shortcomings such as model dimensions, substrate mechanics, and nutrient diffusion which are essential to study the intracellular interactions across the blood-brain barrier. With the advent of three-dimensional (3D) bioprinting, it is now feasible to make bio-mimetic, personalized, functional tissue constructs; with precise spatial control over depositing a printable hydrogel or bioink. Herein, we engineered a brain-inspired bioink to fabricate 3D- bioprinted neural tissue model which mimics the brain extracellular matrix composition and stiffness while enabling local delivery of encapsulated drugs.

Materials and

Methods: Materials and

Methods: The bioink was formulated using a mixture of methacrylated hyaluronic acid (HAMA), methacrylated gelatin (GelMA), polyethylene glycol diacrylate (PEGDA) and poly- (lactic-co-glycolic acid) nanoparticles (PLGA NP). HAMA was synthesized by using in-house lab protocol. GelMA and PEGDA were purchased from Millipore Sigma and PolySciences respectively. PLGA NPs were synthesized using solvent evaporation method. All the bioink components were combined in MilliQ water to form a viscous hydrogel which could be used as a precursor or incorporated with Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate (LAP) (25mg/ml) to make crosslinked hydrogel by exposing it to UV at 365nm. This brain-inspired hydrogel was optimized for application as a 3D-extrusion based printable bioink. Methacrylation of HAMA was evaluated using H1-NMR and FTIR. Additionally, the presence of all the components in the hydrogel precursor was evaluated using FTIR. Quantitative rheological assessments were conducted on various hydrogel compositions, under compression and shear stress (i.e., separate frequency and amplitude sweeps) (n = 5 technical replicates) to characterize the viscoelastic properties of the hydrogel. Cytocompatibility of the HAMA-GelMA hydrogels were assessed towards surface-seeded human brain endothelial cells (hCMEC/D3, Millipore) and evaluated by LIVE/DEAD staining (ThermoFisher).

Results, Conclusions, and Discussions: Result and

Discussion: Successful methacrylation of the HA precursor was confirmed by the presence of characteristic methacrylate peaks using NMR and FT-IR. Successful incorporation of all bioink components in cured gels was confirmed by FT-IR. Rheological characterization of the cured hydrogel revealed no significant differences in compressive modulus as the PLGA NP concentration in the hydrogel was increased linearly. The compressive modulus of the hydrogels increased linearly with the GelMA concentrations in the hydrogel (0-10 wt.%). Amplitude sweeps revealed a linear viscoelastic region (LVER) of up to 10% oscillation strain, after which the loss modulus (G’’) superseded the storage modulus (G’), indicating fluid-like viscoelastic behavior necessary for printing. Cytocompatibility characterization of the hydrogel toward surface-seeded brain endothelial cells (hCMEC/D3, Millipore) by LIVE/DEAD staining revealed 98.27% ± 1.29%, 99.71% ± 0.34%, and 99.86% ± 0.14% viability at 24, 48, and 72 hours respectively, post-seeding and revealed no statistically significant differences (One-way ANOVA) amongst the hydrogel compositions.

Conclusions: The rheological assessments helped in determining the linear viscoelastic range (LVER) of the brain-inspired bioink or hydrogel and in predicting the printing accuracy, shape fidelity and extrudability of the material. We concluded that the brain-inspired bioink whilst maintaining high cell viability, can be used to fabricate viable functionalized 3D bio-printed constructs which combines aspects of neuroimmunology and additive manufacturing.

Acknowledgements (Optional): Funding is gratefully acknowledged from the OUHSC-IBEST Collaborative Research Program, American Cancer Society IRG2023-1, Harold Hamm foundation, and the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number 1R35GM150970.