A 3D bioprinted in vitro contusion injury platform for mechanobiological studies of spinal cord injury.

Spinal cord injury (SCI) models are crucial for understanding injury mechanisms and evaluating potential therapeutic strategies. However, most in vitro SCI models rely on chemical or scalpel-induced damage and do not adequately reproduce the controlled mechanical loading associated with traumatic injuries. This study aimed to develop and optimise a reproducible injury protocol for application to 3D bioprinted cell-laden hydrogel constructs and to evaluate the resulting cellular responses. A custom mechanical loading system was used to apply compressive injury to 3D bioprinted gelatin methacryloyl (GelMA) cylindrical constructs. Three loading scenarios incorporating different displacement depths (20%, 40%, and 80% of construct height) and impact velocities (100, 1000, and 3000 mm/s) were investigated. System performance was assessed by comparing prescribed and measured displacement profiles. C6 astrocyte-like cells and NG108-15 neuronal cells encapsulated within GelMA constructs were subsequently used to examine biological responses to injury. Among the protocols tested, Injury Scenario 3, which incorporated an additional low-velocity ramp prior to peak compression, achieved the closest agreement between prescribed and measured displacements and substantially reduced displacement errors. Application of this protocol produced injury severity-dependent cellular responses, including increased GFAP expression and metabolic activity in astrocyte-like cells and reduced neuronal viability. These findings demonstrate a reproducible approach for applying controlled mechanical injury to 3D bioprinted constructs for mechanobiological studies in vitro.