Porosity plays a critical role in influencing the biological properties and performance of materials and devices. This study introduces hydrocolloid inks by incorporating porogens into silk fibroin (silk) protein solutions to generate porous hydrogel scaffolds. These inks exhibit robust printability, enabling the fabrication of complex geometries with hierarchical porosity, ranging from microscale porogen-templated pores (40 to 200 μm, with over 50 % ≥100 μm) to macroscale features determined by the 3D printing process (≥200 μm). Compatibility studies using human bone marrow mesenchymal stem cells (hMSCs) and murine embryonic osteoblast precursor cells (MC3T3-E1) demonstrate cell adhesion, infiltration, and proliferation both on the surface and within these hydrogels. Subcutaneous implantation in rats confirmed biocompatibility and the ability to support endogenous cell migration and proliferation by the hydrogels. In a rat femoral defect model, the microscale biomimetic structures significantly improved bone repair, outperforming control groups, including small pore-sized silk hydrogels (∼21.39 μm) and other 3D-printed constructs with a thickening agent (∼20.78 μm). These innovative multiscale silk 3D biomimetic scaffolds present a promising approach for effective bone defect repair for future clinical applications. STATEMENT OF SIGNIFICANCE: This study presents a transformative approach to bone defect repair through the development of 3D-printed silk hydrogel scaffolds with multiscale porosity. By incorporating dextran gel particles as sacrificial porogens, the silk scaffolds achieve hierarchical pore structures optimized for cell adhesion, proliferation, and migration. In vitro and in vivo results demonstrate that these scaffolds support robust cellular activity and significantly enhance bone regeneration compared to conventional designs, providing a scalable, biocompatible solution. The integration of silk's superior biological properties with advanced 3D printing methodologies underscores its potential to set new benchmarks in bone tissue engineering and regenerative medicine.
