Printing Life: Engineering Muscle with Bioinks

Volumetric muscle loss (VML) is a clinical challenge, often resulting in irreversible functional impairments. While 3D bioprinting offers a promising strategy for muscle regeneration, replicating biomimetic structures with appropriate mechanical properties, multiple cell types, and vascularization remains difficult. Here, we demonstrate the role of decellularized extracellular matrix methacryloyl (dECM-MA) in enhancing the biofunctionality of skeletal muscle bioinks. We compared a 3 % chemically cross-linkable gelatin methacryoyl (GelMA) + 3 % dECM-MA hydrogel (G3D3) formulation to assess the effects of dECM-MA on cellular and structural outcomes. Our results showed that higher dECM-MA content significantly enhanced in vitro culture longevity, with notable cell spreading and myotube formation after 14 days. Encapsulated human umbilical vein endothelial cells (HUVECs), human skeletal muscle satellite cells (HSkMCs), and coculture of HUVECs and HSkMCs demonstrated high viability, proliferation, and differentiation, forming longitudinally aligned myofibers within the printed constructs. Quantitative analysis showed higher myotube formation efficiency in co-culture constructs compared to constructs encapsulated with HSkMCs only. Taken together, our findings highlight the crucial role of dECM-MA, and co-culture system in developing biofunctional muscle bioinks and demonstrate that higher dECM-MA concentrations and cellular cross-talk support the formation of structured, vascularized myofibers in bioprinted muscle constructs. Improved integration and regeneration in hydrogel-implanted regions were shown by in vivo implantation in a murine VML model, particularly in constructs that included the hSkMSCs-HUVEC co-culture. For the placement of the construct, fibrin gel worked well as a biological adhesive, preserving spatial stability inside the defect. These results provide a translational strategy for VML repair by highlighting the complementary effects of cellular co-culture and biomechanical tension in improving the functional maturation and regenerative potential of bioprinted muscle constructs.