Growth Factor-Free Engineered Biphasic Scaffold for Enhanced Bone Regeneration.

Large-area bone regeneration remains a significant clinical challenge, as current grafts often mineralize only at the defect edges, leaving the core underdeveloped. This study introduces a biphasic, biomimetic scaffold integrating structural support with uniform bioactivity to address this limitation. The scaffold features a highly porous outer tube for mechanical strength and cell infiltration, paired with an electrospun nanofiber core enriched with decellularized extracellular matrix (dECM) to promote cell recruitment and mineralization. Twenty-five dECMs were derived from co-cultures of bone-healing cell types: osteoblasts (OB), chondrocytes (CH), mesenchymal stromal cells (MSCs), fibroblasts (FB), and endothelial cells (EC). Among them, OB + MSC-derived dECM showed the greatest osteogenic potential. This dECM was applied to an optimized nanofiber core (232 ± 87 nm from 5 wt% solution), with a protein content of 67.9 ± 8.3 µg/mg and DNA < 50 ng/mg. The outer tube exhibited 89.6 ± 5.8% porosity and a compressive modulus of 123 ± 6.7 MPa. After BSA coating and simulated body fluid immersion, scaffolds showed calcium phosphate deposition (0.28 ± 0.03 mmol/L Ca(2)⁺/scaffold). In a 10 mm critical-sized femoral defect in rats, scaffolds containing both CaP and OB + MSC-derived dECM significantly enhanced bone healing. Imaging and histological analyses showed a twofold increase in bone volume, mineral density, and cortical bone formation. The compressive modulus of regenerated bone was threefold higher than untreated controls and autografts. By 12 weeks, complete defect bridging and structural recovery were achieved. This biphasic scaffold design presents a promising strategy for large bone defect repair by enabling uniform tissue regeneration, combining osteoinductive cues with structural performance suited for clinical translation.