*4.7. Biomineralization Assay*

As previously mentioned, the PLA/ZrO2 of 0.5 g nanocomposite fiber membrane scaffold demonstrated the best results in the biocompatibility assays. Therefore, it was used for biomineralization assays. Briefly, hFOB 1.19 cells were seeded onto the PLA fiber scaffold and PLA/ZrO2 of 0.5 g scaffold at a density of 1 × 10<sup>4</sup> cell/mL and left to adhere overnight. Then, a group of scaffolds (PLA and PLA/ZrO2) were incubated with mineralizing media (complete medium, 50 μg/mL of ascorbic acid, 10 mM glycerol-2- phosphate, and 10−<sup>7</sup> M of dexamethasone) and, together with the control group (PLA and PLA/ZrO2 scaffolds), were maintained in standard media without any osteogenic factors for 3 and 21 days of culture. After a time, PLA and PLA/ZrO2 composite fiber membrane scaffolds were washed three times with PBS. Then, cells were fixed with 4% formaldehyde for 1 h, washed five times carefully with distilled water, and then stained with a saturated solution (40 mM) of Alizarin Red S pH 4.2 for 30 min at room temperature. After several washes with distilled water to remove the dye excess, scaffolds were examined under an optical microscope.

For the quantitative analysis of ARS staining, the fiber membrane scaffolds were transferred to a 1.5 mL microcentrifuge tube, and ARS was extracted with 10% acetic acid, incubated for 30 min, and then heated at 85 ◦C for 10 min. Samples were then cooled for 5 min on ice and centrifuged at 20,000 rpm for 15 min; the reaction was stopped with 10% ammonium hydroxide. Finally, the dye solution sample was read at 405 nm in the spectrophotometer. The concentration of ARS was determined by correlating the absorbance of the experimental samples with a standard known curve of ARS dye concentrations. The experiments were repeated twice, and three fiber scaffolds were used in each experiment. Moreover, the mineral-like tissue deposited onto the PLA and PLA/ZrO2 composite fiber membrane scaffold at 3 and 21 days of culture was analyzed by FTIR.
