Improved osteogenesis of human adipose-derived stromal cells on hydroxyapatite-mineralized graphene film

Abstract

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.