Extrusion bioprinting of FibMA–fibrin semi-IPN hydrogel filaments for enhanced skeletal muscle cell alignment

Abstract We engineered a biodegradable hydrogel that can be bioprinted with C2C12 skeletal muscle cells into filamentous structures that serve as a provisional template for in vitro myofiber formation, without requiring sacrificial polymers or a secondary gelation bath. The hydrogel meets the rheological and biological requirements of direct extrusion printing and supports cellular realignment along the filament axis. In this study, we optimized the printable hydrogel formulation and investigated how C2C12 cells respond to different geometric constraints imposed by the filament microstructure, with a focus on reorientation and uniaxial alignment. To this end, we prepared a dual-crosslinked semi-interpenetrating polymer network (semi-IPN) hydrogel composed of fibrin and methacrylated fibrinogen (FibMA), enabling independent control over biological, microstructural, and viscoelastic mechanical properties. The FibMA–fibrin (FibMAtrx) semi-IPN hydrogel was successfully extruded with the cells into filaments and supported rapid cell spreading. Filament formation was enabled by fibrinogen’s rapid supramolecular chemistry upon extrusion, while covalent crosslinking of FibMA imparted mechanical stability suitable for long-term 3D culture. The fibrin-based supramolecular microstructure provided essential morphogenetic cues that promoted accelerated muscle cell spreading and organization. Filament dimensions scaled proportionally with extruder nozzle size; however, decreasing filament diameter increased the elastic modulus. Compared to bulk hydrogels, filament hydrogels promoted enhanced uniaxial cell alignment. Notably, the relationship between nozzle size and cell alignment was confounded by increased stiffness in smaller-diameter filaments, which hindered skeletal muscle cell morphogenesis. Overall, the FibMAtrx semi-IPN hydrogel represents a robust protein-based bioink for bioprinting skeletal muscle filaments, offering tunable biological and biophysical cues that support rapid morphogenesis for in vitro myogenic applications.