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.

三维生物打印（3D bioprinting）在制备具有复杂架构的构造体方面已展现出巨大潜力，但在大体积组织范围内实现精准的微米级仿生多细胞组织结构仍为一大挑战。本研究开发了一种融合同轴挤出与静态混合器的同轴混沌三维生物打印平台，可实现可控的细胞分层。该平台采用以2%海藻酸钠和5%甲基丙烯酸明胶（GelMA）为核心相、氯化钙与2% GelMA为交联外壳的复合生物墨水，以建立稳定的分层图案。静态混合器通过反复分割、拉伸与折叠层流，调控生物墨水流动并生成层内纤维状分层结构，从而得到可预测的混沌分层效果。在可控打印条件下，经混沌图案化的生物墨水逐层沉积以构建三维构造体。研究对打印参数（包括生物墨水与交联剂配比、打印速度及流速）进行了优化，以实现高形状保真度与一致的内部架构。本方法可高效制备无细胞及有细胞的分层三维构造体。此外，构造体内的L929小鼠成纤维细胞呈现出清晰且稳定的分层排布，且在7天的培养过程中持续保持高存活率与增殖能力。上述结果表明，同轴混沌生物打印技术为快速制备大体积、多细胞、分层三维组织构造体提供了有效策略，在组织工程与再生医学领域具有广阔应用前景。