Multimaterial chaotic printing of reinforced and prevascularized hydrogel filaments: Fabrication of mechanical robust constructs for long-term muscle tissue culture

Engineering vascularization in hydrogel constructs remains a significant challenge in tissue engineering. Prevascularized hydrogels, engineered with void channels, enhance cell viability but often lack the mechanical stability needed for long-term culture, which is crucial for proper tissue maturation. In this study, we introduce chaotic bioprinting—a chaos-enabled biofabrication strategy—to produce mechanically robust hydrogel prevascularized filaments (with inner void channels) suited for extended culture. Utilizing a Kenics Static Mixer (KSM) printhead with various inlets (4 or 8), we developed fibers with intercalated layers of a myoblast-laden gelatin methacryloyl (GelMA)-alginate bioink, a sacrificial material for channel formation, and a reinforcing alginate scaffold. By optimizing ink ratios, we maximized cell-laden compartments while reinforcing the fiber structure and embedding microchannels for efficient mass and gas transport. Mechanical testing and degradation analysis reveal that optimized fibers achieve sufficient resistance (elastic modulus = 12.8 kPa) to withstand extended periods of cell culture up to 21 days. Additionally, C2C12 myoblasts cultured within these prevascularized and reinforced hydrogel filaments maintained high cell viability (>90%) for more than 21 days and demonstrated superior cell proliferation, spreading, and alignment throughout the filament volume compared to solid fibers (reinforced but without inner void channels). Sacrificial layers created void microchannels, enhancing mass and gas transport, while the reinforcing layers provided structural integrity. Multimaterial chaotic printing enabled the fabrication of mechanically stable, functional constructs with compartmentalized architectures, facilitating extended culture and tissue maturation. Our results demonstrate the potential of this method for engineering thick tissues, including skeletal muscle, and highlight its versatility for various regenerative medicine applications, advancing biofabrication towards thicker and mature tissues.