Novel Hierarchical Nitrogen-Doped Multiwalled Carbon Nanotubes/Cellulose/Nanohydroxyapatite Nanocomposite As an Osteoinductive Scaffold for Enhancing Bone Regeneration

Nanomaterials based on hybrid scaffolds have shown a high potential to promote osteointegration and bone regeneration. In this study, a novel nanocomposite scaffold was synthesized via a cross-linking/hydrothermal/freeze-drying method, resulting in layer-by-layer structures with functional and structural properties mimicking the natural bone. The hierarchical structures of the scaffold were reinforced with nitrogen-doped multiwalled carbon nanotubes (N-MWCNTs), cellulose, and nanohydroxyapatite. The N-MWCNT/Cel/nHA scaffolds were characterized and evaluated in terms of structure, morphology, biocompatibility, cellular responses, and bone repair efficiency in vivo. The resulting scaffolds showed that incorporation of 1 wt % N-MWCNTs into the hybrid scaffold with micropores (∼5 μm) significantly improved its mechanical properties, although the surface morphology of the scaffold tended to be rough and porous. Importantly, the resulting scaffolds supported in vitro cellular attachment, proliferation, viability, and mineralization of bone mesenchymal stem cells (BMSCs). On the other hand, incorporation of N-MWCNTs into the scaffold induced preferential differentiation of BMSCs to osteogenic lineage accompanied by increased alkaline phosphatase activity and expression of key osteogenic genes. Furthermore, 12 weeks after implantation, the 1%N-MWCNT/Cel/nHA porous scaffolds successfully cicatrized a distal femoral condyle critical size defect in rabbit without obvious inflammatory responses, as indicated by the results of the Micro-CT and histological analyses. In vitro and in vivo experiments confirmed that the scaffolds not only improved the interface bonding with bone tissue but also accelerated the new bone formation and regeneration by up-regulating signaling molecules that are involved in cell proliferation and differentiation. These results indicated that the novel N-MWCNT/Cel/nHA scaffold is an efficient platform for osteogenesis research and bone regeneration medicine.