3D printing- and cyclic strain-driven engineering of skeletal muscle blocks using enhanced viscoelastic extracellular matrix bioink.

Development of physiologically functional skeletal muscle constructs is vital for regenerative therapies and drug-screening applications. Herein, we present an integrated strategy that combines viscoelastic muscle-derived extracellular matrix (MdECM) bioinks with a 3D printing-assisted cyclic strain bioreactor. To enhance mechanical performance, an improved decellularization protocol incorporating isopropanol was introduced to effectively remove residual lipids. The resulting bioinks exhibited superior viscoelasticity, structural stability, and thixotropic recovery compared with conventional formulations. The viscoelastic bioinks enabled stable myoblast encapsulation and alignment within the 3D-printed pillar frames. These counterforces guided robust myotube formation, while optimized ECM concentration and cell density further promoted alignment and myogenic differentiation. Spatial regulation of myotube orientation was achieved by tuning the pillar geometry and spacing. Muscle blocks were matured in a custom-designed multi-chamber bioreactor capable of applying cyclic uniaxial strain, yielding constructs with uniaxial cellular alignment, elevated myogenic marker expression, and contractile responses confirmed by calcium imaging and electrical stimulation. This platform supports scalable production of structurally and functionally mature muscle microtissues, offering a promising model for therapeutic implantation and high-throughput drug screening targeting sarcopenia and muscle-wasting diseases.