Abstract
Aligned collagen microstructure is essential for the mechanical and biological function of anisotropic tissues. However, conventional engineering methods often fail to achieve consistent and tunable fiber alignment within complex geometries. In this study, we developed a step-ladder printing (SLP) approach by incorporating successive segments of channels of variable widths into a custom barrel design, combining controlled extensional flows with 3D bioprinting to enhance collagen fiber alignment. The results revealed that constructs 3D-printed via SLP demonstrated improved anisotropy of collagen fibers and narrower fiber angle distributions compared to both extrusion-based bioprinting with a conventional straight nozzle and drop casting methods. Furthermore, SLP effectively guided the directionality of seeded cells, aligning them consistently with underlying collagen fibers. To exemplify the utility of SLP, we built corneal constructs, achieving high transparency and shape fidelity, and articular cartilage constructs, showing mechanical properties within the range of native tissue and supported extracellular matrix production. These results suggest that the SLP approach offers a strategy for fabricating complex anisotropic tissues with integrated fiber alignment and cellular guidance.