Abstract
Hyaluronic acid (HA) is an abundant extracellular matrix (ECM) component in soft tissues throughout the body and has found wide adoption in tissue engineering. This study focuses on the optimization of methacrylated HA (MeHA) for three-dimensional (3D) bioprinting to create in vitro test beds that incorporate regeneration-promoting growth factors in neural repair processes. To evaluate MeHA as a potential bioink, rheological studies were performed with PC-12 cells to demonstrate shear thinning properties maintained when printing with and without cells. Next, an extrusion-based Cellink BIO X 3D printer was used to bioprint various MeHA solutions combined with collagen-I to determine which formulation was the most optimal for creating 3D features. Results indicated that MeHA (10 mg/mL) with collagen-I (3 mg/mL) was most suitable. As Schwann cells (SCs) are a critical component of neural repair and regeneration, SC adhesion assessment via integrin β1 immunostaining indicated that the bioink candidate adequately supported SC adhesion and migration when compared to Col-I, a highly cell-adhesive ECM component. MeHA/collagen-I bioink was adapted for neural specific applications by printing with the neural growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). These test beds were conducive for SC infiltration and presented differential migration responses. Finally, a two-chamber in vitro test bed design was created to study competitive biochemical cues. Dorsal root ganglia were seeded in test beds and demonstrated directional neurite extension (measured by β-III tubulin and GAP43 immunostaining) in response to NGF and GDNF. Overall, the selected MeHA/collagen-I bioink was bioprintable, improved cell viability compared to molded controls, and was conducive for cell adhesion, growth factor sequestration, and neural cell infiltration. MeHA is a suitable bioink candidate for extrusion-based bioprinting and will be useful in future development of spatially complex test beds to advance in vitro models as an alternative to common in vivo tests for neural repair applications.
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