3D printing combined with thermally induced phase separation for engineering hierarchical osteogenic PLA scaffolds

Accelerated population aging and rising incidence of bone defects have intensified the need for advanced bone regeneration strategies. While tissue-engineered scaffolds fabricated via 3D printing offer promising alternatives to conventional grafts, most techniques fail to replicate the multi-scale fibrous architecture of native bone extracellular matrix, limiting their biofunctionality. To address this, we developed a hybrid manufacturing strategy integrating low-temperature thermally induced phase separation with extrusion-based 3D printing of polylactic acid (PLA) scaffolds. By optimizing solvent ratios (THF: DMF = 3:1) and freezing temperatures (-196 °C-4 °C), we produced scaffolds with tunable micro-nano fibrous surfaces and macroporous structures. Key findings revealed that scaffolds processed at -196 °C (PLA-196) exhibited the highest porosity (pore size: 6.01 ± 2.06 μm), superior hydrophilicity, and enhanced compressive modulus. These scaffolds significantly promoted BMSC adhesion, proliferation, and osteogenic differentiation via activation of <i>Macf1</i>-mediated pro-osteogenic signaling pathways, leading to elevated expression of Runx2, Ocn, and Col-I. <i>In vivo</i>, PLA-196 scaffolds accelerated cranial defect healing, achieving 62 % bone volume fraction and robust vascularization within 8 weeks. This study demonstrates a scalable, biocompatible platform for fabricating hierarchically structured bone scaffolds that bridge the gap between structural complexity and biological efficacy, offering significant potential for clinical translation in regenerative medicine.