3D-printed magnetic scaffolds promote bone and vessel regeneration through CRYAB/PI3K-AKT and NF-κB pathways identified by proteomics

Iron oxide nanoparticles (IONPs)-based bone scaffolds have attracted increasing attention because of their potential to enhance osteogenesis and angiogenesis. However, the underlying mechanisms remain incompletely understood. We fabricated a biocompatible bone scaffold by incorporating γ-Fe 2 O 3 magnetic nanoparticles into a PLGA matrix using 3D printing technology. The biosafety and effectiveness of the scaffold was validated through in vitro cell assays and in vivo implantation studies. To evaluate osteogenesis and neovascularization, we employed micro-CT imaging with a vascular contrast agent. In-depth mechanistic investigations were conducted via label-free proteomic profiling and pathway enrichment analysis. The PLGA/Fe 2 O 3 scaffolds demonstrated excellent biocompatibility and promoted both bone formation and angiogenesis in vitro and in vivo . Micro-CT analysis revealed enhanced new bone and vessel formation in the presence of magnetic scaffolds. Proteomic analysis revealed that alpha-B crystallin (CRYAB) is a key regulatory protein upregulated under a static magnetic field, thereby activating the PI3K/AKT signaling cascade and promoting osteogenic differentiation. In endothelial cells, we observed the upregulation of nuclear NF-κB and HIF-1α, leading to VEGF expression and angiogenic activation. Our findings provide direct evidence that 3D-printed PLGA/Fe 2 O 3 scaffolds promote osteogenesis and angiogenesis both in vitro and in vivo . Importantly, we report for the first time that CRYAB-mediated stabilization of β-catenin plays a central role in magnetic scaffold-induced bone regeneration, offering new insights into the design of functional bone substitutes. • 3D-Printed Magnetic Scaffolds : This study demonstrates the development of biocompatible PLGA/Fe 2 O 3 scaffolds using 3D printing technology to enhance both osteogenesis and angiogenesis. • Multi-Model Evaluation : A comprehensive approach involving both in vitro and in vivo models, coupled with Micro-CT imaging , demonstrates enhanced bone formation and vascularization , showcasing the scaffolds' regenerative potential. • Mechanistic Insights via Proteomics : The novel use of label-free proteomic profiling uncovers the central role of alpha-B crystallin (CRYAB) in stabilizing β-catenin, activating the PI3K-AKT and NF-κB signaling pathways to promote bone and vessel regeneration.