Live dead

The possible cytotoxicity of the synthesized material was assessed using Live/Dead (Sigma Aldrich, St. Louis, MO, USA) staining and AlamarBlue (Invitrogen, Carlsbad, CA, USA) and MTT (Sigma Aldrich, St. Louis, MO, USA) assays. To reveal live and dead cells, we seeded scaffolds with the previously described design [48 (link),49 (link),50 (link)] with 1 × 10⁵ cells (per scaffold) and stained them for 72 h with calcein-AM (live, green) and propidium iodide (dead, red) (Sigma Aldrich, St. Louis, MO, USA); cell nuclei were additionally stained with Hoechst 33342 (blue). For AlamarBlue and MTT assays, we inoculated 5 × 10³ cells to each well in a 96-well plate and added a matrix extract or sodium dodecyl sulfate (positive control, in seven dilutions) in 24 h. The matrix extract was prepared by dipping its part with the surface area of 6 cm² into 1 mL DMEM/F12 supplemented with 5% FBS and 1% penicillin-streptomycin and placing for 24 h in a thermostat (ISO 10993-12:2012). The AlamarBlue and MTT assays were performed in accordance with the manufacturer’s instructions.

The material cytotoxicity was assessed using Live/Dead (Sigma Aldrich, Steinheim am Albuch, Germany) staining and AlamarBlue (Invitrogen, Carlsbad, CA, USA) and PicoGreen (Invitrogen, Carlsbad, CA, USA) assays. We used mesenchymal stromal cells (MSC) derived from the gingiva. The cells were cultured as described elsewhere [29 (link)] and monitored using a phase contrast microscope. A scaffold was inoculated with 50,000 cells and stained with calcein-AM (green, live cells) and propidium iodide (red, dead cells) in 5 days. Then it was analyzed using an LSM 880 laser scanning confocal microscope equipped with an AiryScan module and GaAsP detector (Carl Zeiss, Jena, Germany). For colorimetric assays, the material extract was prepared as previously described [30 ] and used to prepare serial dilutions. The sample solution or sodium dodecyl sulfate (SDS, positive control) were added to the cells cultured in a 96-well plate for 24 h at a temperature of 37 °C.