**3. Discussion**

Tissue engineering (TE) is a multidisciplinary area of research aimed at repairing, replacing, or regenerating tissues or organs to restore impaired function due to defects and aging. Nanotechnology for TE application focuses on imitating the size and role of the extracellular matrix (ECM) with a biomimetic characteristic for improving the migration, proliferation, and differentiation of cells [28,29]. This ECM biomimetic related to bone tissue could be obtained by synthesizing a scaffold with a combination of different biomaterials as polymers and bioceramic, via several manufacturing methods that allow the combining of its properties for translation to the new composite phase, to improve its functionality [5–10,30]. Thus, in this study, we used hydrothermal synthesis and air-jet spinning (AJS) technology to engineer a multifunctional composite scaffold as a platform for bone tissue engineering applications.

First, we used hydrothermal synthesis, defined as a homogeneous reaction in the presence of aqueous solvents under temperature and pressure conditions to dissolve and recrystallize insoluble materials under normal conditions, to obtain the fiber's ceramic phase composite scaffold. Our XRD and SEM images show that hydrothermal synthesis allows a homogeneous thermally stable crystalline nanoceramic of ZrO2 to be obtained. Furthermore, the nanoceramic showed agglomerates of crystallites, of nearly uniform size, between 10–80 nm on average for the particle diameter, with nanometric dimensions considering that the grains are spheres, and the results showed that nanocrystals coincide with the monoclinic and cubic structural phases specific to ZrO2; the results are consistent with previous studies reported [31–33].

For bone application, nanocomposite fiber biomaterials are a relatively new class of materials that combine a homogeneous size of the crystalline structure with biopolymeric matrix structure, that enables the improvement of the fibrillar design nanocomposite [34,35]. In this regard, we explore the use of AJS to design a polymeric fibrillar matrix of 10% wt of PLA, doped with two different concentrations (0.1 and 0.5 g) of the nanoceramic of ZrO2. As demonstrated by our results, the SEM images of the PLA/ZrO2 nanocomposite had a random orientation of the fiber morphology with a submicron/nanometer range between 100 nm up to 1 μm in diameter, with an average size of 395 nm; by FTIR, it was possible to detect differences between the intensity peak of 0.5 and 0.1 g of ZrO2 that could be related to the mass fraction of the nanoceramic for 0.1 g (~0.0099 g) and for 0.5 g (~0.047 g) in the composite. This difference in the peak intensity of samples showed that, with increased concentrations of nanoceramic, the corresponding interaction of the Zr-O bonds with the characteristic bands of the functional groups of PLA (carbonyl group, esters CH and CH3) at 758 cm<sup>−</sup><sup>1</sup> could be detected; furthermore, this different concentration improves the anisotropic properties of the scaffold that could be related to the adhesion between the interface of the fiber matrix and the nanoceramic particles, in concordance with previous studies reported [25–27,36,37].

In bone tissue engineering, the goal of a nanocomposite is intended to provide a suitable surface for supporting the tissue regeneration process, and therefore requires osteoconduction or osteoinduction properties [35]. Our study found that the doped polymer matrix with the nanoceramic of ZrO2 could be considered adequate to the bone because the result showed appropriate surface property cues and physicochemical stability that have been defined as important parameters for influencing cell cytocompatibility and osteogenesis [37–40]. In our study, we analyzed the biocompatibility of the PLA/ZrO2 composite fiber scaffold by cell adhesion and viability response of human osteoblasts cells hFOB 1.19. In the adhesion assay and viability evaluation, we found that the composite scaffold of PLA doped with 0.5 g of the nanoceramic had higher cell attachment and proliferation of cells. This cellular behavior could be influenced by the optimal surface topography, the physicochemical properties of the material, the nanometric surface features, and the critical size of the doped nanophase that plays a role in mediating the cell behavior response [14,41].

Moreover, the physical, chemical, and surface topography property cues are critical parameters that induce cellular recognition signals. Our results showed that hFOB cells attached well to the fiber composite, covering long area extensions with very spreading, elongated, and lamellae morphology. Thus, the morphology of the topographic cues (spherical nanostructure) of the nanocomposite of PLA related to the presence of ZrO2 demonstrated an excellent surface that stimulated cell attachment and proliferation and supported the extracellular matrix deposit by the osteoblast cells, as shown by alizarin red staining. This is an important effect of the nanoscale because AJS allows the synthesis of a more suitable scaffold with the physical integration of nanoceramic, conferring a homogenous distribution for cell binding. Furthermore, these could be improved by the adsorption of biomolecules by the media that can activate signaling pathways that change the cell behavior, recreating a microenvironment similar to the native tissue, indicating the recognition or sense of the surface by the osteoblast cells, favorably inducing a good cell-material interaction and differentiation [13].

In our study, we evaluated the potential of the 0.5 g of PLA/ZrO2 fiber nanocomposite in promoting the mineral deposit of the ECM of the hFOB by a differentiation assay. Our analysis by alizarin red staining (ARS) and FTIR showed that osteoblasts could deposit a mineral-like tissue over the nanocomposite surface after 21 days of culture. This indicates the importance of the chemical components at the nanoscale, and the submicronsized topographical cues that could modulate the cell osteoblasts behavior. Moreover, the chemical component related to the insertion of the ZrO2 nanoceramic onto the fiber polymer matrix may confer a possible cue role on the bioactivity related to the osteogenesis potential that influences the proliferation and differentiation. The mineral deposit evaluated by ARS and FTIR results indicate that topographical properties of the nanocomposite were related

to the size and morphology of the fiber, the porosity, but also to the osteogenic stimuli (inductive factors) presence on the cell culture media, together performing a specific role supporting osteoblast cell differentiation. Thus, our results are in agreemen<sup>t</sup> with different studies that reported that zirconia nanofibers or ZrO2 composites may have pro-osteogenic properties, have good mechanical biocompatibility, and improve the metabolism of bone precursor cells. Furthermore, they could also be considered as a self-regulation material that possesses an excellent osteoinductive capacity that could modify the deposition of ECM by bone cells during differentiation or in response to the surface material, because the osteoblast cell functions could be substrate-sensitive, either with respect to stiffness, roughness, or to the size of the composition of the material [16,17,42–46].

## **4. Materials and Methods**

## *4.1. Synthesis of Nanoceramic of Zirconium Oxide*

Zirconium oxide nanoceramic was synthesized via the hydrothermal technique. The volume ratio of zirconium tetrachloride (ZrCl4) and sodium hydroxide (NaOH) was 3:1. The obtained solution was mixed with pH 14 and let to settle for 20 h, until a substitution reaction happened between ZrCl4 and NaOH, obtaining sodium chloride (NaCl) and zirconium oxide (ZrO2) as products. After that, the reaction product was removed, washed five times with deionized water, then the clean ZrO2 was loaded into a Teflon vessel for autoclaving. Afterward, the autoclave was placed in a furnace (Barnstead model 1500), heated, and maintained at 200 ◦C for 2 h, then cooled to room temperature. The powder obtained was crushed on an agate mortar. The characterization of the ceramic was made by X-ray diffraction analysis and scanning electron microscopy (SEM).

The crystal size was calculated by the equation of Scherrer, where d is the mean size of the particle, *K* is the Scherrer constant, *λ* is the wavelength, *β* is the peak at half the maximum intensity (FWHM), and *θ* is the Bragg angle (in degrees). The following equation is applied only for crystals with sizes in the range of 1–100 nm.

$$d = \frac{K\lambda}{\beta \cos \theta}$$

## *4.2. Polymer Fiber Composite Fabrication of PLA/ZrO2*

Fibrous composite spun scaffolds were fabricated via the AJS process from PLA polymeric solutions of 10% wt. First, polymeric solutions of 10% wt of PLA were prepared: PLA pellets (C3H6O3; MW 192,000 from Nature Works, Minnetonka, MN, USA) were dissolved in chloroform (CHCl3 from J. T. Baker) and stirred for 20 h. After that, acetone was added, and the solution was stirred for 30 min to obtain a homogeneous solution. The volume ratio of chloroform/acetone was 3:1. Then, the polymeric solution was prepared with two different amounts of nanoceramic of ZrO2 (0.1 and 0.5 g) for composite scaffolds. During their preparation, solutions were incubated in an ultrasonic bath for 1 h to guarantee the homogenization of nanoparticles onto the polymeric solution. In all cases, the polymeric solution was placed in a commercially available airbrush ADIR model 699 with a 0.3 mm nozzle diameter and with a gravitational feed of the solution to synthesize the fiber membrane scaffolds. The airbrush was connected to a pressurized argon tank (CAS number 7740-37, concentration > 99%, PRAXAIR Mexico, Nuevo Leon, Mexico). For deposition of the fibers, a pressure of 30 psi with 11 cm of distance from the nozzle to the target was held constant. The fiber scaffold characterization was analyzed by a Fourier transform infrared (FTIR) spectrophotometer, employing an IRAffinity-1S (Shimadzu, Kyoto, Japan) within the 400–4000 cm<sup>−</sup><sup>1</sup> range. The morphology and structure of the fibers were observed with a field emission scanning electron microscope (FE-SEM, JSM-7800F, JEOL). Fiber diameter was measured from SEM micrographs employing image analysis software (ImageJ, National Institutes of Health, Gaithersburg, MD, USA).

## *4.3. Cell Culture*

The human fetal osteoblast cell line (hFOB 1.19, ATCC CRL-11372) was used to evaluate the cell biocompatibility response of the PLA fibers spun mat, and the PLA/ZrO2 spun composite scaffolds. Briefly, hFOB 1.19 cells were cultured in 75 cm<sup>2</sup> culture flasks, in Ham's F12/Dulbecco's modified Eagle's Media (1:1, Ham's F12-DMEM, Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS, Biosciences, Collierville, TN, USA), 2.5 mM L-glutamine, and an antibiotic solution (streptomycin 100 μg/mL and penicillin 100 U/mL, Sigma-Aldrich, St. Louis, MO, USA). The cells were incubated in a 100% humidified environment at 37 ◦C in 95% air and 5% CO2. The hFOB 1.19 cells were used between 2–6 passages of culture for all the experimental procedures.

## *4.4. Cell Adhesion*

As the first attempt to investigate how hFOB cells would interact with the PLA/ZrO2 nanocomposite fiber material, adhesion assays were performed. For this purpose, hFOB 1.19 cells were seeded at 1 × 10<sup>4</sup> cells/mL onto the composite scaffold in triplicate and were allowed to adhere under standard cell culture conditions for 4 and 24 h. After each time, nanocomposite fiber scaffolds were rinsed three times using phosphate-buffered saline (PBS) to remove non-adherent cells. Next, the adherent cells were fixed with 3.5% paraformaldehyde, and the evaluation of cell attachment was performed according to the crystal violet assay. Briefly, cells were incubated with 0.1% crystal violet solution for 15 min and then washed carefully with deionized water until clear water was obtained. Then, the dye was extracted with 0.1% of sodium dodecyl sulfate (SDS), and the optical absorption was quantified by spectrophotometry at 550 nm with an ELISA plate reader (ChroMate, Awareness Technology). Cells attached in triplicate onto a conventional tissue culture plate (TCP) were considered as 100% cell attachment of hFBO 1.19 cells, and cells seeded in triplicate onto PLA fiber membrane scaffolds were used as the control.

## *4.5. Viability Cells (MTT Assay)*

The cell viability response of hFOB 1.19 cultured onto PLA/ZrO2 composite fiber membrane scaffolds in triplicate was analyzed with the MTT assay. The assay is based on the ability of mitochondrial dehydrogenases to oxidize thiazolyl blue (MTT), a tetrazolium salt (3-[4,5-dimethylthiazolyl-2-y]-2,5-diphenyltetrazolium bromide), into an insoluble blue formazan product. Cells were seeded at 1 × 10<sup>4</sup> cell/mL density in triplicate and incubated for 3, 5, 7, 14, and 21 days of culture. After each time, PLA/ZrO2 nanocomposite fiber scaffolds were washed with PBS and incubated with fresh cultured medium containing 0.5 mg/mL of MTT for 4 h at 37 ◦C in the dark. Then, the medium was removed, and 500 μL of dimethyl sulfoxide (DMSO) was added to each well. After 60 min of slow shaking, the absorbance was read at 570 nm. Cells seeded in triplicate onto PLA fiber membrane scaffold were used as the control. During the experimental time, the culture medium was changed every two days.

## *4.6. Cell-Material Interaction*

We examine the cell morphology, the spreading patterns, and cell-material interaction of hFOB 1.19 cells cultured onto the PLA fiber scaffold and PLA/ZrO2 of 0.5 g nanocomposite fiber scaffold, because it demonstrated the best results for adhesion and viability. Briefly, hFOB 1.19 cells were cultured at a density of 1 × 10<sup>4</sup> cells/mL onto the scaffolds for 24 h in triplicate. Then, the samples were analyzed using SEM and fluorescence microscopy (AMSCOPE). For SEM analysis, after 24 h of incubation, scaffolds were washed three times with PBS, fixed with 4% formaldehyde, then dehydrated with a graded series of ethanol (25–100%) and air-dried. Next, the samples were sputter-coated with a thin layer of gold and examined by SEM. For fluorescence observation, before seeding the hFOB 1.19 cells onto the scaffolds, cells were incubated with CellTracker ™ Green (CMFDA, 5-chloromethylfluorescein diacetate) in phenol red-free medium at 37 ◦C for 30 min. Subsequently, the cells were washed with PBS and incubated for 1 h in a complete

medium. Then, hFOB 1.19 cells were trypsinized and counted to the desired cell concentration (1 × 10<sup>4</sup> cells/mL), incubated for 24 h onto the scaffolds, and examined under the microscope.
