Gaseous liquid nitrogen-assisted cryo-printing strategies for challenging volumetric anisotropic tissues fabrication

Engineering physiologically relevant anisotropic tissues remains a major challenge due to limitations in replicating native structural cues across complex geometries. Here, we developed a cryogenic extrusion bioprinting platform by precisely guiding ice crystal growth to achieve centimeter-scale anisotropic constructs, even with ultrasoft, low-viscosity inks (2% weight/volume GelMA). This approach enables the fabrication of anatomically matched, patient-specific constructs with tunable anisotropy, including bone graft substitutes and osteoporotic disease models. To overcome the height constraints of cryo-printing, we further developed cryo-assembly and LEGO-assembly strategies that allow integration of anisotropic units while maintaining continuous internal alignment. In vivo studies across wound healing, cranial defect, and femoral defect models validated that these anisotropic cues drive spatially guided cell migration, extracellular matrix (ECM) remodeling, and tissue site-specific regeneration, as evidenced by the ECM deposition. Our platform offers a scalable, clinically adaptable solution for engineering structurally and functionally relevant tissues, redefining the boundaries of anisotropic soft tissue fabrication.