Methyl thiazolyl tetrazolium (mtt)

Cells were seeded in 48-well plates at density of 5 × 10³ cells per well. For cell viability assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Glentham Life Sciences, Corsham, Wiltshire, UK) was added to the cells after 24 h at a final concentration of 0.1 mg/ml and cells were incubated for 4 h at 37°C. After 4 h, medium including MTT was removed and DMSO was added. The absorbance at 575 nm was measured with Epoch™ Microplate Spectrophotometer (BioTek, Winooski, VT, USA). For colony formation assay, cells were seeded in the same way and stained with 0.01% crystal violet after MeOH fixation. Colonies were counted with Image J software.

3T3 cells were seeded in 96-well plates at a 30% confluency (6000 cells per well) and allowed to adhere for 24 h. Polymers were purified with Sephadex G25 columns (PD10 desalting column), nanoparticles were suspended in DDW while the free drug was dissolved in DMSO. Untreated cells (control) were used to establish 100% viability. The effect on cell viability was measured with MTT (5 mg/mL, Glentham Life Sciences, Corsham, UK).

Adipose-derived mesenchymal stem cells (ADMSC, ATCC-PCS-500-011) at 5 × 10³ were seeded onto the scaffolds in the 96-well plates, as in the standard cell seeding procedure, after 4 h ultraviolet (UV) light sterilization. At the same time, monolayer cell cultures were incubated with the same number of cells in 150 μL as a control. The cell-scaffold constructs and monolayer cultures were incubated at 37 °C, 5% CO₂ for 1 and 3 days in a humidified incubator (NuAire). The toxic effect of scaffolds was checked on days 1 and 3. To investigate cytotoxicity at a given time point, the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (Glentham Life Sciences) cytotoxicity assay method was used according to the manufacturer’s protocol. The absorbance value of the cytotoxicity test was measured at 570 nm wavelength (690 nm as Ref. value) in an ELISA reader (Enspire, Perkin Elmer, Waltham, MA, USA). The assay was studied 3 times, and the average of the results was considered the final result.