Synergistic Osteogenesis of Biocompatible Reduced Graphene Oxide with Methyl Vanillate in BMSCs

Methyl vanillate (MV), a recently characterized small molecule, can promote the Wnt/β-catenin signaling pathway and induce osteoblast differentiation both in vitro and in vivo. On the other hand, graphene-based materials have been introduced into the field of biomedical sciences in the past decade, and graphene oxide (GO), which serves as an efficient nanocarrier for drug delivery, has attracted great attention for its biomedical applications in tissue engineering. This study aimed to develop a biocompatible gelatin-reduced graphene oxide (GOG) for MV delivery so as to realize the effective osteogenesis for bone repair. First, GOG was prepared, and its morphology as well as properties were then characterized using scanning electron microscope (SEM), transmission electron microscopy (TEM), atomic force microscope (AFM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and thermal gravimetric analysis (TGA), respectively. In addition, the endocytosis of GOG in bone marrow stromal cells (BMSCs) was also investigated with the treatment of Rhodamine 6G (R6G)-labeled GOG. Our results found that GOG could be easily absorbed by cells and was distributed in both nucleus and cytoplasm, thus suggesting the favorable biocompatibility of GOG. Moreover, the effect of MV on osteogenesis was also tested, the results of which indicated that MV could promote BMSC osteogenesis in a concentration-dependent manner, and significant enhancement could be achieved at the concentration of 1 μg/mL. In addition, the complex containing different concentrations of GOG and an optimal concentration of MV was used to investigate the synergistic effect between GOG and MV on pro-osteogenesis. The results revealed that the weight ratio of MV/GOG of 1:1000 could attain remarkably enhanced osteoinduction in BMSCs, as evaluated by alkaline phosphatase (ALP) assay, alizarin red S (ARS) staining, immunofluorescence staining, and gene expression of related osteogenic markers. Taken together, these data had provided strong evidence that the complex of MV and GOG could induce osteogenesis, which was promising for bone tissue engineering.