Consecutive Hybrid Bioprinting of Microfiber‐Reinforced Living Muscle Constructs with Highly‐Aligned Cellular Organizations

Abstract Replicating the highly‐organized extracellular matrix microfibrillar networks and directional cellular organization of native skeletal muscles is essential for engineering functional muscle constructs. Here, we propose a consecutive hybrid bioprinting (CHB) strategy to fabricate living composite constructs with polymeric microfibers, sacrificial gelatin and cell‐laden fibrin hydrogels by combining electrohydrodynamic (EHD) printing and extrusion‐based bioprinting, which enables the engineering of mechanically‐matched and highly‐aligned porous muscle constructs. The bioprinted hydrogel components provide a smooth and dynamically‐rising conductive surface for stable EHD printing of well‐organized microfibers with centimeter height, which conversely provides mechanical support to ensure the structural integrity of the resultant composite constructs. Upon removal of the sacrificial hydrogel, the porous composite constructs maintain their original shape, and native muscle‐like mechanical properties can be achieved by modulating the microfiber configurations. Notably, these microfibrous structures facilitate cell‐induced anisotropic remodeling of fibrin filaments, resulting in cross‐sectional contraction to form highly‐aligned myoblast bundles along the bioprinting trajectory. This enables the CHB of circumferentially or layer‐specifically aligned cellular constructs. The aligned myoblast constructs can be differentiated into multinucleated myotubes with enhanced muscle‐specific protein and gene expression. This CHB strategy provides a promising platform to directly engineer living composite constructs with native anisotropic mechanical properties and cellular organizations.