*2.10. Cell Culture Studies*

Human osteosarcoma cells (HOS) were purchased from the European Collection of Authenticated Cell Cultures (catalogue number 87070202). Cultured cells were grown in RPMI medium supplemented with 10% *v*/*v* fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine as a monolayer in T75 cell culture flasks in a humidified atmosphere (5% CO2 incubator; 95% air) at 37 ◦C. Cells were sub-cultured at approximately 80% confluency twice-weekly using a standard trypsin-EDTA protocol. All cell cultures were confirmed as being free from Mycoplasma sp. contamination using the EZ-PCR mycoplasma detection kit according to the manufacturer's instructions (Biological Industries USA, Cromwell, CT, USA). All cells were cultured up to passage 20 before being discarded. Before cell culture, coated surfaces were placed flat with the coated surface facing upwards into a 6-well cell culture dish. Surfaces were sterilised with 2 mL of 70% *v*/*v* ethanol for 30 min. Subsequently, surfaces were washed with sterile phosphate-buffered saline (PBS, 3 × 2 mL). HOS cells (100,000) were added and left to attach for 4 h. The medium was changed and cells were incubated for 5 and 7 days before being prepared for analysis by electron microscopy.
