Sterile distilled water

MG-63, an osteosarcoma cell line (Lonza, UK) and human bone marrow derived primary mesenchymal stem cells (less than 5 passages) (Lonza, UK) were seeded in expansion media consisting of 4.5 gL⁻¹ glucose Dulbecco’s Modified Eagle’s medium (Lonza, UK) supplemented with 10% foetal bovine serum, 1% Penicillin/Streptomycin (antibiotics and antimycotics) and 1% L-glutamine in well plates at ~80% confluency and allowed to attach overnight before addition of nanoparticle suspensions. hMSCs were adherence selected and tested for stemness using standard trilineage differentiation and flow cytometry for CD markers. Aqueous suspensions of the nanoparticles were mixed with cell culture media at 10% v/v and then added to cells. For the control samples, equivalent amount of sterile distilled water (negative control) or sterile cobalt chloride solution (positive control; Sigma- Aldrich, UK) at a concentration of 500 μM were mixed with the cell culture media and added to cells. The cation/MNP concentrations were maintained constant between comparisons and cells were incubated with the additives for 72 h before being assessed.

The coating procedure was performed as described elsewhere (Dos-Santos-Pereira et al., 2018 (link); Dos-Santos-Pereira et al., 2020 (link)). For astrocyte cultures, bottom surfaces of culture flasks were coated with 1 μg/ml laminin dissolved in sterile distilled water (Sigma Aldrich). For microglial and neuronal cell cultures, we used a borate buffer solution (pH 8.3) containing 1 mg/ml polyethyleneimine (PEI; Sigma Aldrich). After at least 2 h at 37 °C, culture flasks were washed with Dulbecco’s phosphate-buffered saline (PBS) four times before applying culture medium and cell seeding.