Effects of TiO2 nanotube layers on RAW 264.7 macrophage behaviour and bone morphogenetic protein-2 expression.

ABSTRACT

OBJECTIVES: To investigate behaviour and osteogenic cytokine expression of RAW264.7 macrophages grown on TiO2 nanotube layers. MATERIALS AND METHODS: The murine macrophage cell line RAW 264.7 was cultured on TiO2 nanotubes of varying diameter; macrophage morphology was examined using scanning electron microscopy. Cell adhesion and viability were assessed with the aid of the MTT method and BMP-2 and TGF-ß gene expression were examined by RT-PCR analysis. Levels of BMP-2, TGF-ß1 and ICAM-1 proteins secreted into the supernatant were measured by ELISA assay. RESULTS: Macrophages cultured on nanotube layers had spread out morphology, the largest (120 nm) nanotube layer eliciting an elongation by 24 h. Macrophages adhered significantly less to 120 nm TiO2 nanotubes than to control discs at 4 h after application; after 24 h incubation, macrophages were sufficiently viable (P < 0.05) on 30 and 70 nm nanotube layers. Increasing nanotube diameter led to increased BMP-2 protein secretion and increased BMP-2 mRNA expression. CONCLUSION: These results demonstrate that nanoscale topography of TiO2 nanotube layers can affect macrophage morphology, adhesion, viability and BMP-2 expression. Macrophages grown on layers of large nanotubes had the highest potential to enhance bone formation during bone healing.