**2. Results**

## *2.1. Characterization of ZrO2 Nanoceramic*

A hydrothermal technique was used to synthesize zirconium oxide nanoparticles. This allows a nanoceramic with homogeneous semispherical morphology, with dimensions of around 25 to 80 nm, to be obtained; this could be due to the presence of agglomerates constituted by several small polycrystallites in the range of 10–20 nm in the dimension (Figure 1a). Moreover, the X-ray diffraction (XRD) pattern showed a considerable broadening of the peaks due to the nanostructure of the crystalline grains (Figure 1b). The XRD of the ZrO2 showed a crystalline behavior with two phases. Based on the XRD spectrum of the JCPDS card No. 03-065-0461, the monoclinic phase showed the peaks (1 0 1) at 30.26◦, (1 1 0) at 35.31◦, (1 1 2) at 50.28◦ and ( −2 0 2) at 62.93◦. A cubic phase was also identified,

with peaks (0 1 0) at 24◦, (1 1 1) at 28◦, (−1 1 1) at 31.5◦, (0 2 1) at 38.5◦, (1 2 1) at 41◦, (2 0 2) at 45.25◦, (−2 0 2) at 55◦, and (3 1 1) at 60.28◦, according to the JCPDS card No. 03-065-0461. Using the Debye-Scherrer formula for the line broadening fitting curve XRD program, the particle size was evaluated. The average particle diameter for the monoclinic phase was around 25–35 nm, considering that the grains were spheres. For the cubic phase, the average size was estimated at around 6–14 nm.

**Figure 1.** Characterization of zirconium oxide (ZrO2) nanoceramic. (**a**) Scanning electron microscopy images of zirconium oxide nanoparticles. ZrO2 nanoparticles with homogeneous morphology and semispherical surface; the nanoparticles tend to agglomerate; scale bar = 200 nm. (**b**) X-ray diffraction patterns of ZrO2 nanoparticles obtained with incident angles at 30.5◦, 31.7◦, 35.2◦, 50.2◦, 60.1◦, and 63.2◦.

## *2.2. Characterization of PLA/ZrO2 Nanocomposite Fiber Scaffold*

Figure 2 shows the analysis of the fiber membrane scaffolds of 10% wt of PLA and PLA/ZrO2 nanocomposite fiber scaffold by scanning electron microscopy (SEM). The PLA fiber scaffold is composed of smooth and uniform fibers with minimal bead formation and a diameter of around 400 nm with random orientation (Figure 2). The morphological analysis demonstrated that PLA/ZrO2 scaffolds have a rough surface due to the zirconium nanoceramic. The particles are observed through the fiber and sometimes as agglomerates around the interconnected strands with random orientation (Figure 2b,c). The analysis of the diameter sizes of the PLA/ZrO2 nanocomposite fiber scaffold showed that incorporating the nanoceramic (0.1 and 0.5 g) increased the fiber diameter. Moreover, the fibers were in the range of 100 to 800 nm, with an average diameter of 395 nm.

The chemical structures of PLA fiber membranes synthesized with different concentrations of the ZrO2 nanoceramic (0.1 and 0.5 g) were obtained using fourier-transform infrared spectroscopy (FTIR) spectroscopy. The results were compared to identify structural changes by incorporating the nanoceramic onto the PLA polymer matrix (Figure 3). The infrared absorbance spectra showed the typical characteristic of PLA; i.e., the absorption bands around 1750 cm<sup>−</sup><sup>1</sup> corresponding to the (C=O) ester carbonyl group; at 1445 and 1380 cm<sup>−</sup>1, corresponding to the absorbance bands of the C-H bending vibration of CH3; at 1350 cm<sup>−</sup><sup>1</sup> corresponding to the bending vibration of carbonyl CH; at 960 to 830 cm<sup>−</sup>1, corresponding to the backbone stretching and CH3 rocking; at the region of 3200 to 2800 cm<sup>−</sup>1, corresponding to the symmetric and asymmetric stretch of CH; and at 1260 cm<sup>−</sup><sup>1</sup> and 1100 cm<sup>−</sup>1, corresponding to the lactide C-O stretch. However, the typical spectral of PLA was accompanied by the absorption band peaks of the ZrO2 nanoceramic; especially, a band at 758 cm<sup>−</sup><sup>1</sup> without structural change on the PLA fiber after doping with the nanoceramic.

**Figure 2.** Scanning electron microscopy assessment of the microstructure of the polylactic acid (PLA) scaffolds loaded with different concentrations of zirconium oxide nanoparticles (ZrO2). (**a**) PLA scaffolds synthesized with the air-jet spinning technique. (**b**) PLA scaffold loaded with 0.1 g of ZrO2. (**c**) PLA scaffold loaded with 0.5 g of ZrO2. The PLA/ZrO2 scaffolds show dispersed and unsaturated ZrO2 nanoparticles along the PLA fibers, scale bar = 10 μm.

**Figure 3.** FTIR-IR spectrum of PLA/ZrO2 fiber membrane scaffolds. The absorption bands typical characteristic of PLA: around 1750 cm<sup>−</sup>1, the (C=O) ester carbonyl group; at 1445 and 1380 cm<sup>−</sup>1, the absorbance bands of C-H bending vibration of CH3; at 1350 cm<sup>−</sup>1, the bending vibration of carbonyl CH; at 960 to 830 cm<sup>−</sup>1, corresponding to the backbone stretching and CH3 rocking; at the region of 3200 to 2800 cm<sup>−</sup>1, to the symmetric and asymmetric stretch of CH; and at 1260 cm<sup>−</sup><sup>1</sup> and 1100 cm<sup>−</sup>1, corresponding to the lactide C-O stretch, with 1090*–*1189 cm<sup>−</sup><sup>1</sup> (C-O=C) and 758 cm<sup>−</sup><sup>1</sup> attributed to (Zr–O). The insert image of the absorption band of Zr-O at 758 cm<sup>−</sup><sup>1</sup> was magnified for show the differences between the PLA/ZrO2 with 0.1 g and 0.5 g composite scaffold.

## *2.3. Biocompatibility Assay*

The biocompatibility of the PLA/ZrO2 nanocomposite fiber scaffold was analyzed in in vitro cell culture to investigate the cell adhesion and cell viability of hFOB 1.19 cells. The cellular adhesion response of hFOB 1.19 cells at 4 and 24 h over the surface of the PLA/ZrO2 nanocomposite fiber scaffold are presented as the percentage of attached cells in relation to control tissue culture plates (Figure 4a).

The cell adhesion of hFOB 1.19 cells was favorable. It exceeded 100% at 4 h and ≥150% at 24 h of attachment onto the PLA/ZrO2 doped with 0.5 g of the nanoceramic with statistical differences compared to the PLA/ZrO2 doped with 0.1 g of the nanoceramic and the PLA fiber spun mat at *p* < 0.05. However, there were no statistical differences between the adhesion of hFOB 1.19 cells onto the PLA/ZrO2 doped with 0.1 g of the nanoceramic and the PLA fiber spun mat. The cell attachment for the former conditions was around 80% at 4 h and ≥120% at 24 h.

Concerning the cell viability, we performed the MTT assay to confirm that the PLA/ZrO2 nanocomposite fiber scaffold is not toxic to the cells (Figure 4b). The results are presented as the optical absorbance at 570 nm. The histogram in Figure 4b suggests that, in all scaffolds, high levels of MTT oxidation are present. However, the higher conversion rate of MTT was found in PLA/ZrO2 doped with 0.5 g of the nanoceramic from day 3, and with a constant increment until 21 days of cell culture. Furthermore, the MTT conversion rate of the PLA/0.5 g ZrO2 scaffold was followed by PLA/ZrO2 doped with 0.1 g of the nanoceramic and by the PLA fiber scaffold. Furthermore, statistical differences were found between the viability of hFOB cell culture onto the PLA/ZrO2 nanocomposite fiber scaffold and hFOB cell culture in the PLA fiber scaffold at *p* < 0.05.

**Figure 4.** The hFOB biological response, after culture onto the PLA/ZrO2 scaffolds. (**a**) The cellular adhesion response of hFOB at 4 and 24 h, evaluated with violet crystal. The cell adhesion onto the PLA/ZrO2 nanocomposite fiber scaffold surface is presented as the percentage of attached cells in relation to control tissue culture plates. (**b**) Metabolic activity was evaluated with MTT assay at 3, 5, 7, 14, and 21 days to confirm that the PLA/ZrO2 nanocomposite fiber scaffold is not toxic to the cells. The viability of the hFOB cell increased time-dependently, with the best results observed for the PLA/0.5 g of ZrO2 after 21 days of culture. Asterisk (\*) mean that scaffolds showed a statistical significance (*p* < 0.05).

## *2.4. Cell-Material Interaction*

Figure 5 showed the cell morphology and the cell spreading pattern interaction between hFOB cells onto the PLA fiber scaffold and the PLA/ZrO2 composite fiber scaffold doped with 0.5 g of the nanoceramic. The fluorescence images of the morphology showed that human osteoblasts cultured onto PLA/ZrO2 had a well-attached cell with elongated morphology and filopodia extensions (Figure 5c,d), in comparison with the less spreading and less elongated morphology shown by osteoblasts cultured onto the control PLA fiber scaffold (Figure 5a,b). Additionally, SEM micrographs showed that cells onto PLA presented a rounded cytoplasm, with few spreading cells that exhibited a flat shape (Figure 5b), compared with preferential spread on the entire surface of the PLA/ZrO2 composite fiber scaffold, wherein some points present an elongated morphology in direct contact with the nanofiber morphologies of the scaffolds due to the presence of cues exerted by zirconia nanoparticles (Figure 5d).
