*2.5. Biomineralization Assay*

For the analysis of the biomineralization assay, we selected only the PLA/ZrO2 of 0.5 g scaffold because it demonstrated the best results for cell adhesion and cell viability. The bioactivity of hFOB cells onto the composite was analyzed by Alizarin Red S staining (ARS) after 3 and 21 days (Figure 6). The images of light microscopy showed that after three days of culture, on control media, there was almost a very weak staining onto the PLA scaffold and the PLA/ZrO2 composite fiber scaffold, in comparison with a very light signal of the red staining in both the PLA spun mat and the PLA/ZrO2 composite fiber scaffold cultured on osteogenic media. However, after 21 days of culture, on control media, redder staining onto the PLA scaffold and the PLA/ZrO2 composite fiber scaffold could be seen, in comparison with a darker red precipitate throughout the surface on both the PLA fiber scaffold and the PLA/ZrO2 composite fiber scaffold, which indicates calcium deposits (Figure 6a). Likewise, the semi-quantitative analysis of the red alizarin staining reveals that the PLA/ZrO2 composite scaffold presents a higher concentration related to the calcium precipitates at 3 and 21 days of culture than the PLA fibers in both culture media (Figure 6b).

**Figure 5.** Representative fluorescence images of the cellular morphology and SEM micrographs of the spreading pattern interaction of hFOB cells cultured onto PLA fiber scaffold (**<sup>a</sup>**,**b**) and PLA/0.5 g ZrO2 scaffolds (**<sup>c</sup>**,**d**) after 24 h of culture. Scale bar on SEM images = 100 μm.

**Figure 6.** Alizarin red staining. The hFOB cells were cultured onto the PLA and PLA/ZrO2 with the presence or absence of an osteogenic medium. (**a**) The calcified extracellular matrix deposits produced by hFOB cells onto the PLA/0.5 g ZrO2 composite scaffolds are red. The area of the calcified deposits onto the scaffolds was time-dependent and increased with the culture time. However, the calcium deposit onto the PLA/0.5 g ZrO2 scaffolds in the osteogenic medium, compared with the standard medium, was more visible, and the concentration of the alizarin was higher than in the PLA scaffolds. (**b**) The calcium (mM) concentration in PLA/0.5 g ZrO2 scaffolds after 3 and 21 days of culture in osteogenic medium and standard medium. Asterisk (\*) mean that the scaffolds showed a statistical significance (*p* < 0.05).

Moreover, the PLA scaffold and PLA/ZrO2 composite fiber scaffold were analyzed by FTIR after 3 and 21 days of culture in osteoinductive culture media and compared to the control culture. From the spectra, the peaks that are characteristic of the amide I and amide II groups could be observed at 1650 cm<sup>−</sup><sup>1</sup> and 1533 cm<sup>−</sup>1, corresponding to the extracellular collagen matrix (Figure 7).

**Figure 7.** FTIR-IR spectrum of PLA/ZrO2 scaffolds after 3 and 21 days of culture with hFOB. The PLA/ZrO2 scaffolds seeded with hFOB cells were cultured with and without the presence of the osteogenic medium. The PLA/0.5 g ZrO2 scaffold spectrum after 3 and 21 days of culture with osteogenic medium showed new peaks that are characteristic of the amide I and amide II groups at 1650 cm<sup>−</sup><sup>1</sup> and 1533 cm<sup>−</sup>1; the bands that correspond to the PLA are also present.
