This study investigated whether hydroxyapatite (HAp)-mineralized graphene (GR) film could support osteogenic differentiation of human adipose-derived, stromal cell (hASCs) in vitro. GR was produced by a chemical vapor deposition (CVD) method and the physical and chemical characteristics of the GR film, which was functionalized with HAp mineralization following ultraviolet-ozone (GR_UVO) treatment, were subsequently validated. Results of scanning electron microscopy, x-ray photoelectron spectroscopy and Raman spectroscopy showed GR_UVO for 5 min yielded applicable GR coverage (97.98 ± 0.85%), conversion of chemical composition ratio (29.78% C–O, 18.34% C=O and 8.49% O–C=O) and degree of oxidation, (I 2D/I G ratios 2.22) with maximal density of HAp-GR layer. In vitro-cell proliferation, viability and adhesion of hASCs after being cultured on HAp-mineralized, GR-coated glass (HAp/GR) with the optimized GR_UVO treatment (5 min) demonstrated a significant increment of proliferation (1.56 ± 0.1 vs 1–1.13 ± 0.1, p< 0.05) without changing in viability (94.83 ± 1% to 95.3 ± 1.6%, p= 0.9651) compared with the control (intact glass). There were no differences in F-actin and vinculin on day 1 (p= 0.1422 and 0.5025, respectively) and on day 4 (p= 0.3787 and 0.9208) of culture. Osteogenic differentiation of hASCs was significantly improved on the HAp/GR with increasing of osteogenesis-related genes (Runx2 and Osteocalcin). The hASCs culture with the HAp/GR glass promoted phospho-SMAD1/5/9 and SMAD4 expression with increased patterns of BMP/Smad signal-related genes, regardless of differentiation induction or not. These results demonstrated that HAp-mineralized GR film prepared by CVD method and optimal ultraviolet treatment promoted osteogenic differentiation of hASCs, which BMP/Smad signaling was involved.

Based on the concept that microenvironment and physical stimuli regulate the cell behaviors like proliferation, migration, and differentiation, this study was conducted to investigate whether nanoscale spacing by stacked graphene film affect osteogenic differentiation of human adipose-derived mesenchymal stromal cells (hADSCs). The graphene films were synthesized by a chemical vapor deposition method, followed by etching and rinsing process to fabricate single or 3-, 5-, and 7-multilayers. The height and width of wrinkles of the graphene were confirmed by SEM and atomic force microscopy (AFM), ranging from 1.5 to 12.5 nm and from 30 to 100 nm, respectively. Osteogenic differentiation was significantly (p < 0.0001) promoted as the stacking layer increased. Immunofluorescent imaging and osteogenesis-related gene expression showed which increment was saturated from three layers. The calcium deposits and expression of osteogenesis-related genes (Runt-related transcription factor 2 and Osteocalcin) were highest in the three layers. In the hADSCs cultured on the three layers, the intensity of protein expression levels of filamentous actin (F-actin) was significantly increased (p = 0.0319) and focal adhesion kinase/extracellular signal-regulated kinase signal related genes were concomitantly activated. These results demonstrated that multilayer-stacked graphene creating nanoscale spaces promotes calcium deposit and cytoskeletal integrity in hADSC-related, in vitro-osteogenesis.