Synergy ht detector

An MTT test ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) was used to indirectly analyze the cytotoxicity of the different scaffolds. Rounded scaffold (d = 11 mm) were immersed in 5 ml of FBS-free culture medium and incubated at 37°C. Thermanox^® (TMX) discs (Nunc^®, Thermofisher) were used as negative control. Aliquots of the supernatant medium were taken at 1, 2, 7, 14 and 21 days under sterile conditions and replaced with fresh medium. The samples were kept frozen until use. hObs and hACs were seeded separately in a 96-well plate at 9 × 10⁴ cells/ml of density and incubated for 24 h in complete medium. Then, the medium was substituted by the extracts and incubated at 37°C. After 24 h the medium was replaced by a solution of MTT (0.5 mg/ml) in warm FBS-free medium and the cells were incubated at 37°C for 3–4 h. Mediua containing MTT were removed and DMSO was added to dissolve the formazan crystals formed in the cells. The absorbance of the medium was measured with a Biotek Synergy HT detector at 570 nm and a reference wavelength of 630 nm. Cell viability was calculated as: $CellViability\left(\%\right)=100\times \left(O{D}_S-O{D}_B\right)/\left(O{D}_C-O{D}_B\right)$ where $O{D}_S$ , $O{D}_B$ and $O{D}_C$ are the optical density (OD) of formazan production for the sample, blank and control, respectively.