Fullerene-titanium (C60/Ti) composites cause no DNA damage response in human osteoblast-like MG 63 cells

Fullerenes (C60) and fullerene-based composites are considered as promising substrates for biological cell colonization. It might be mainly due to their nanostructure, resembling the nanoarchitecture of the natural extracellular matrix. Thin films of binary C60/Ti composites with various concentrations of Ti ranging from 25% (i.e., 25 Ti atoms and 75 C60 molecules) to 70% were deposited on microscopic glass coverslips in micro-patterned form through a metallic mask, and were tested for their potential use in bone tissue engineering. It is known that fullerenes and their derivatives can cause cytotoxic injury, cell death or inhibition of cell growth. These effects are based mainly on the reactivity of fullerenes, which may weaken with time due to the oxidization and polymerization of fullerenes in an air atmosphere. We therefore tested the dependence between the age of C60/Ti composites (i.e., from one week to one year) and the level of DNA damage of human osteoblast-like MG 63 cells in cultures on these materials. The DNA damage was analyzed by immunofluorescence staining of markers of DNA damage response, such as phosphorylation of histone H2AX and focal recruitment of p53-binding protein. As positive control to markers of DNA damage response was used 7 days long treatment with 2,5 mM Thymidine. We also monitored the proliferation and morphological changes of the cells. After 7 days of cultivation, we observe no cytotoxic morphological changes, such as enlarged cells or cytosolic vacuole formation, which are signs of cell senescence, and no increased induction of cell death. In addition, there was no increased level of DNA damage response on the C60/Ti composites (FIG.1). We also found no significant differences in cell population densities and no increased level of DNA damage among various Ti concentrations (FIG.1). Moreover, there was no effect of the age of the C60/Ti composites on the cell population densities or on the DNA damage response (FIG.1). These results suggest that fullerenes in combination with Ti do not cause cytotoxic injury and this material could be used in bone tissue engineering