Advanced 3D-printed PLA scaffolds functionalized with dual small molecules for enhanced bone regeneration: in vitro and in vivo studies

Abstract

Bone defects remain a significant clinical challenge due to limited vascularization and insufficient osteogenesis. In this study, 3D-printed PLA scaffolds functionalized with dual small molecule-loaded chitosan nanoparticles containing DIPQUO and GS4012 (DG-NP-coated scaffolds) were developed to support osteogenic and angiogenic responses. Representative physicochemical characterization confirmed successful nanoparticle coating, nanoscale particle distribution, and sustained release behavior of the incorporated small molecules. In vitro analyses demonstrated enhanced osteogenic differentiation of rat bone marrow mesenchymal stem cells cultured on DG-NP-coated scaffolds, with increased ALP activity, calcium deposition, and osteogenic gene expression. Notably, ONC expression reached a 54.19-fold increase by day 21. In vitro co-culture studies additionally demonstrated enhanced endothelial cell migration on GS4012-containing scaffold surfaces. In vivo evaluation in a critical-sized rat calvarial defect model revealed enhanced bone tissue formation and the presence of vascularized tissue regions in DG-NP-coated scaffolds compared to untreated and uncoated groups, with a bone mineral density of 757 mg HA cm⁻³ at week 8. Collectively, these findings suggest that DG-NP functionalized PLA scaffolds represent a promising bioactive platform for supporting vascularized bone regeneration in bone tissue engineering applications.