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

Abstract

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-Fe₂O₃ magnetic nanoparticles into a PLGA matrix using 3D printing technology. The biosafety and effectiveness of the scaffold was validated through <i>in vitro</i> cell assays and <i>in vivo</i> 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/Fe₂O₃ scaffolds demonstrated excellent biocompatibility and promoted both bone formation and angiogenesis <i>in vitro</i> and <i>in vivo</i>. 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/Fe₂O₃ scaffolds promote osteogenesis and angiogenesis both <i>in vitro</i> and <i>in vivo</i>. 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.