Objective: To explore graphene's effects on the gene expression profile of mesenchymal stem cells, and to reveal the mechanisms of graphene-guided osteogenic differentiation.
Methods: Human adipose-derived mesenchymal stem cells (hASCs) were cultured on single-layer graphene-coated titanium disks or titanium disks in proliferation medium (control) or osteoinduction medium for 7 days before RNA extraction. After library construction and RNA sequencing, identification of differentially expressed genes was performed through Limma package of R platform, with a cut-off value of log fold change (logFC) > = |1|. Pathway and Gene ontology (GO) analyses were conducted on DAVID Bioinformatics Resources 6.8 (NIAID/NIH). Network analyses were performed by the Ingenuity Pathways Analysis (IPA).
Results: Signalling pathway analysis revealed the top five pathways - cytokine-cytokine receptor interaction, neuroactive-ligand receptor interaction, calcium signalling pathway, PI3K-Akt signalling pathway and cell adhesion molecules. GO analyses demonstrated significant changes on cell adhesion, calcium signalling, and epigenetic regulation. IPA network analyses demonstrated that inflammation-related pathways were influenced by graphene, while the downstream factors of histone H3 and H4 were also altered especially under the existence of osteoinduction medium.
Conclusion: Graphene promotes osteogenic differentiation of hASCs mainly by influencing cell adhesion, cytokine-cytokine receptor interactions, inflammatory responses, and potentially influences histone H3 and H4 through epigenetic regulation.