Fibrous bioinks for the bioprinting of anisotropic scaffolds with micro-and nanoscale organization as a novel approach for in vitro skeletal muscle engineering

Replicating skeletal muscle architecture remains challenging in 3D bioprinting, as conventional bioinks lack multiscale directional cues. Herein, we propose a next- generation fibrous bioink composed of fragmented electrospun gelatin fi bers (f-GFs), uniformly embedded in an alginate/gelatin hydrogel matrix (f-ALG/Gel). Upon microextrusion bioprinting, shear-induced f-GF alignment enabled the fabrication of microfilament-based scaffolds with intrinsic anisotropy. The resulting constructs exhibited high shape fidelity, favorable viscoelastic properties, and physiologically relevant stiffness (Young’s modulus: 16.1 ± 1 .7 kPa). In vitro studies using C2C12 murine myoblasts demonstrated that the embedded f-GFs provided strong topographical guidance, enhancing cell alignment and myogenesis. After 14 days of culture, the f-ALG/Gel scaffolds supported a 2.5-fold increase in myotube fusion index and length, alongside reduced angular dispersion. These effects were achieved without the need for biochemical induction with a differentiation medium, underscoring the key role of structural cues at the micro- and nanoscale in C2C12 differentiation and maturation. In conclusion, this work proposes a scalable, cell-compatible strategy to recapitulate the hierarchical organization of skeletal muscle tissue within 3D-printed constructs. The platform holds broad potential for applications in regenerative medicine, skeletal muscle tissue modeling, and the engineering of cultured meat.