ABSTRACT: Repairing large bone defects remains a significant clinical challenge. This study explores the development of collagen-enhanced piezoelectric microfiber barrier membranes (PLLA/ZnO@COL) for bone regeneration. The membranes' physicochemical properties, especially their piezoelectric characteristics, significantly influenced cellular behavior. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) confirmed the uniform dispersion of ZnO nanoparticles within the PLLA matrix, essential for optimal piezoelectric performance. The inclusion of collagen layers enhanced the electrical properties, cytocompatibility and osteogenic activity of the membranes. In vitro analyses, including bioinformatics, revealed that PLLA/ZnO@COL membranes upregulated key osteogenic pathways such as PI3K-AKT, MAPK, and Ras, thereby enhancing osteoblast proliferation, migration, and differentiation. Low-intensity pulsed ultrasound (LIPUS) stimulation further amplified these effects, indicating a synergistic role in enhancing osteoblast functions. In vivo studies using a rat mandibular bone defect model demonstrated that PLLA/ZnO@COL membranes, with the collagen layer facing the defect, significantly fostered new bone formation compared to control groups. Micro-CT analysis at 6 and 12weeks post-implantation showed higher bone mineral density, volume, and trabecular number in the PLLA/ZnO@COL group, indicating greater osteogenic activity. In conclusion, PLLA/ZnO@COL membranes, in conjunction with LIPUS, effectively enhance osteoblast functions and induce bone regeneration, showing potential as innovative barrier membranes in bone tissue engineering. The combination of piezoelectric properties, collagen enhancement, and LIPUS stimulation presents a novel approach to improving bone healing outcomes.
