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 fibers (f-GFs), uniformly embedded in an alginate/gelatin hydrogel matrix (f-ALG/Gel). Upon micro-extrusion bioprinting, shear-induced f-GF alignment enabled the fabrication of microfilament-based scaffolds with intrinsic anisotropy. The resulting constructs exhibited high shape fidelity, 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 on C2C12 differentiation and maturation. In conclusion, this work proposed 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 modelling and the engineering of cultured meat.