Assessment of TiO₂ nanoparticle cytotoxicity by real-time impedance-based monitoring

Titanium (Ti) and its alloys are commonly used as implant materials. As a result of abrasion, micron- and nano-sized particles can be released into the tissues surrounding Ti-based implants. Previous studies have shown that nanoparticles (NPs) are more biologically active and cytotoxic than micron-sized particles.  Objectives: The effect of titanium dioxide (TiO2) NPs on cellular proliferation, viability, morphology, spreading, and detachment from substrate was investigated.  Methods: An impedance-based assay was chosen due to its advantages of being label-free and monitoring the cells in real-time. Trypan Blue exclusion and ultrahigh resolution imaging (URI) were also employed.  Fibroblasts seeded in microelectrode-embedded E-plates were exposed to 0.05, 0.5, and 5 mg/L of nano-TiO2 for up to 120 h. The particles used (Sigma–Aldrich, Norway) had a diameter < 100 nm and a mixed anatase /rutile crystallographic form.  An alternative excitation signal (20 mV control voltage amplitude) was applied at 10, 25, and 50 kHz to the microelectrodes in the E-plates. Cells act as insulators and their attachment to the electrode surfaces increases the impedance.  Continuous impedance measurements were made with an xCELLigence system (ACEA Inc, USA). The experiments were run in duplicate and repeated at least three times. Unexposed cells served as controls. Results: Both the impedance-based monitoring and the Trypan blue assay showed an overall decrease of proliferation and viability of the cells exposed to TiO₂ NPs. URI microscopy revealed a functional dependence of apoptosis on the nano-TiO₂ concentration. Conclusion: Overall, impedance-based results were in accordance with those from the Trypan Blue exclusion and URI and indicated a significant cytotoxic effect at 5 mg/L of TiO₂NPs.

The study was supported by funding from UH-nett Vest, the Department of Clinical Dentistry, Faculty of Medicine and Dentistry in Bergen, and the Regional Development Program Hordaland-Thüringen.