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

Abstract

This study investigated whether hydroxyapatite (HAp)-mineralized graphene film could support osteogenic differentiation of human adipose-derived, stromal cell (hASCs) in vitro. Graphene was produced by a chemical vapor deposition (CVD) method and the physical and chemical characteristics of the graphene film, which was functionalized with hydroxyapatite 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 graphene 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, (I2D /IG ratios 2.22) with maximal density of HAp-graphene layer. In vitro-cell proliferation, viability and adhesion of hASCs after being cultured on HAp-mineralized, graphene-coated glass (HAp/GR) with the optimized GR_UVO treatment (5 min) demonstrated a significant increment of proliferation (1.560.1 vs. 1 to 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 hydroxyapatite-mineralized graphene film prepared by CVD method and optimal ultraviolet treatment promoted osteogenic differentiation of hASCs, which BMP/Smad signaling was involved.