The Study Of Graphene/single-walled Carbon Nanotubes Hybrids On Proliferation And Osteogenic Differentiation Of MSCs

Objective: To prepare and characterize the physiochemical properties of G/SWCNT hybrids.Methods: The samples were characterized by scanning electron microscopy(SEM),X-ray diffraction(XRD),X-ray photoelectron spectroscopy(XPS),transmission electron microscopy(TEM),and Raman spectrum.Results: The morphologies and structures of the as-prepared three samples were examined with SEM.From the images,G/SWCNT hybrids showed fibers and particles assembling together.The XRD showed the hump at 21°-27° appeared in the G/SWCNT hybrids,resulting from the different layer spacing between G/SWCNT hybrids.The whole XPS spectrum of G/SWCNT hybrids,graphene,and SWCNTs confirmed that the NPs were composed only of carbon and oxygen.From the TEM results,the expected three-dimensional nanostructure was clearly confirmed.Raman spectrum curve showed that the existence of large amount of SWCNTs and graphene in the hybrids.Conclusion: The three-dimensional structure of graphene/single-walled carbon nanotube hybrids has been constructed by combining one-dimensional single-walled carbon nanotubes and two-dimensional graphene.Objective: Carbon nanomaterials are increasingly significant in the biological and medical fields.Graphene/single-walled carbon nanotubes(G/SWCNT)hybrids is a novel class of 3D carbon nanomaterials.It is necessary to assess the biocompatibility of novel carbon nanomaterials prior to their use in biomedical applications.In this study,we investigate the effect of G/SWCNT hybrids on proliferation of r MSCs and the biodistribution in vivoMethods: Rat mesenchymal stem cells were isolated and cultured,and then the cells in passage 3 were used in this study.We adopted CCK-8 and LDH assays to detect the cellular viability.Reactive oxygen species,mitochondrial membrane potential,cell cycle assays,and the expression of apoptosis-related proteins were used to indicate mechanisms of cytotoxicity of G/SWCNT hybrids.To observe the biodistribution of G/SWCNT hybrids in vivo,the mice were injected through the tail vein with hybrids dispersion,and sacrificed at different time points.Major organs were then collected for further histological analysis.Results: The CCK-8 and LDH assays demonstrated that the cytotoxicity of G/SWCNT hybrids exhibits a dose-dependent behavior on r MSCs.In our conditions,the hybrids were less cytotoxic than graphene and single-walled carbon nanotubes.The results also showed the apoptosis of r MSCs induced by G/SWCNT hybrids was through the increase of intracellular reactive oxygen species,the decrease of mitochondrial membrane potential,the alternation of apoptosis-related proteins,and then triggered the ROS-mediated mitochondrial pathway.Moreover,the biodistribution of G/SWCNT hybrids in vivo was observed by histological analysis of major organs in mice,and showed that organs were neither inflammatory nor damaged.Conclusion: The cytotoxicity of G/SWCNT hybrids on r MSCs exhibits a dose-dependent behavior through the ROS-mediated mitochondrial pathway.Objective: Dynamic balance between osteogenesis and adipogenesis of MSCs is important to maintain the bone metabolism omestasis.If the osteogenic and adipogenic differentiation is imbalanced,the osteonecrosis develop.To investigate the interactions between G/SWCNT hybrids and rat mesenchymal stem cells(r MSCs),we focused on the proliferation and differentiation of r MSCs treated with G/SWCNT hybrids.Methods: The effect of G/SWCNT hybrids on the differentiation towards osteoblasts and adipocytes was evaluated.Rat mesenchymal stem cells were isolated and cultured,then the cells in Passage 3 were used in this study.ALP activity and mineralized matrix nodule formation were measured as specific markers of osteogenic differentiation of r MSCs at early and late stages.The formation of adipocytes was measured by the oil red O staining of lipid droplets.Western blot was used to analyze the expression levels of osteogenic and adipogenic differentiation specific genes and intracellular MAPK pathway studies.Results: Osteogenic differentiation,as evaluated by alkaline phosphatase(ALP)activity of MSCs proved to be,was higher after treatmented with G/SWCNT hybrids,and the mineralized matrix nodule formation was also enhanced.In addition,the expression levels of osteogenic associated genes were un-regulated,while adipocyte specific markers were down-regulated.Consistent with these results,we illustrated that the effect of G/SWCNT hybrids on the process of osteogenic differentiation of r MSCs was can be modulated by activating p38 signaling pathway.Conclusion: G/SWCNT hybrids could promote osteogenic differentiation of r MSCs to osteoblasts.p38 signaling pathway plays a positive role in enhancing osteogenic differentiation of r MSCs with respect to G/SWCNT hybrid interactions.