Precise Cell Stratification in Alginate/Gelatin Methacrylate Based 3D Construct Using Coaxial Chaotic Bioprinting

Three-dimensional (3D) bioprinting has shown great promise for fabricating constructs with complex architecture; however, achieving precise microscale biomimetic multicellular organization over a large tissue volume remains a challenge. In this study, a coaxial-chaotic 3D bioprinting platform that combines coaxial extrusion with static mixers to enable controlled cell stratification was developed. A hybrid bioink, composed of 2% sodium alginate and 5% gelatin methacrylate (GelMA) as the core and a crosslinking shell of calcium chloride and 2% GelMA, was employed to establish stable layered patterns. The static mixers modulated bioink flow and generated layered intrafibrillar stratification by repeatedly splitting, stretching, and folding laminar streams, producing predictable chaotic stratification. Under controlled printing conditions, the chaotically patterned bioink was deposited layer-by-layer to build 3D constructs. Printing parameters, including bioink and crosslinker compositions, printing speeds, and flow rates, were optimized to achieve high shape fidelity and consistent internal architecture. This approach effectively fabricated both acellular and cellular stratified 3D constructs. In addition, L929 mouse fibroblasts within the constructs exhibited well-defined and stable layered arrangements, along with sustained viability and high proliferation over 7 days of culture. These results demonstrate that the coaxial-chaotic bioprinting provides an effective strategy for the rapid fabrication of large, multicellular, and stratified 3D tissue constructs, with broad potential in tissue engineering and regenerative medicine.