Three-Dimensionally N-Doped Graphene–Hydroxyapatite/Agarose as an Osteoinductive Scaffold for Enhancing Bone Regeneration

Composite biomaterials with hierarchical structures have emerged as new approaches for bone-tissue engineering. In this study, a biomimetic, osteoconductive tricomposite scaffold made of N-doped graphene–hydroxyapatite (NG–HA) hybrids blended with an agarose (AG) matrix was prepared via a facile hydrothermal/cross-linking/freeze-drying method. The structure and composition of AG/NG–HA were examined by scanning electron microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, Fourier transform infrared, Raman spectroscopy, and thermogravimetric analysis. The as-prepared scaffolds showed hierarchical pore architecture and an organic–inorganic composition, which simulated the composition and structure of natural bone tissue. The effect of AG/NG–HA on bone mesenchymal stem cells (MSCs) osteoblast proliferation, differentiation, and mineralization was tested in vitro. The expression of osteogenic-related genes was determined by real-time polymerase chain reaction. Our results showed that the introduction of N-graphene into the hybrid scaffold significantly improved its mechanical properties, an effect that promoted the proliferation and viability of MSCs. Moreover, the scaffolds triggered selective differentiation of MSCs to osteogenic lineage while conferring good cell adhesion, enhanced alkaline phosphatase activity, and mineralization. A distal femoral condyle critical size defect in rabbits was used as a platform to confirm the effect of AG/NG–HA on bone regeneration in vivo. Our experiments show that the AG/NG–HA hybrid scaffolds provided a favorable environment for new bone formation. The results presented in this study suggest that the AG/NG–HA hybrid scaffolds have potential in bone-tissue regeneration engineering