## 2 Materials and Methods
### Sample Characterization
Specifically, it helps assess whether the observed data is consistent with a null hypothesis, which typically states that there is no effect or no difference, and any observed effect is due to random chance [28]. Optical studies using Fourier transform infrared spectroscopy (FTIR) were conducted with a Nicolet iS50 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a deuterated triglycine sulfate (DTGS) detector, known for its high sensitivity. The FTIR analysis covered the spectral range from 1000 cm\({}^{-1}\) to 100 cm\({}^{-1}\), which is crucial for identifying the Zn-O stretching vibrations typically observed in this range. The resolution of the measurements was set at 4 cm\({}^{-1}\), ensuring detailed and accurate spectral data. Preliminary studies for the antimicrobial analysis of the efficiency of ZnO powders on the most common postoperative infections with Gram-positive, Gram-negative, and fungal microorganisms were carried out through a qualitative analysis, using an adapted diffusion method. The adapted diffusion method consisted of distributing the chemical compound in \"spots\" on the Muller-Hinton medium. The seeding of the plates was carried out in the first stage. The plates were incubated at a temperature of 37 \({}^{\circ}\)C for 24 h, after which they were left to rest at room temperature. DMSO (Dimethyl sulfoxide) was used as a standard and was tested comparatively. The bactericidal effect of ZnO was demonstrated by the appearance of an inhibition zone. The Gram-positive bacterial strain used in the preliminary tests was _Staphylococcus aureus_ 0364, while the Gram-negative bacterial strain was _Escherichia coli_ ATCC 25922. _Candidata albicans_ ATCC 10231 was used as a representative of fungal microorganisms. Primary human osteoblast cells, used to assess the interaction of proliferation, viability, and cytotoxicity with ZnO nanoparticles, were obtained from the upper part of a patient's femur. These purchased osteoblast cells were cultured in Dulbecco Modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum, DMEM sodium pyruvate, 2% glutamine, and an antibiotic mixture. The cells were incubated at 37 \({}^{\circ}\)C in a 5% CO\({}_{2}\) atmosphere. Additionally, phosphate-buffered saline (PBS) was used during the process. After 7-10 days of incubation under the specified conditions, the osteoblast cells reached a suitable state for division. Confluent cultures were treated with trypsin for 2-3 min and then centrifuged at 1500 rpm for 10 min. The cells were resuspended in a minimal volume of DMEM, counted using a Burker-Turk chamber, and evenly distributed on sterile supports that have been pre-treated with polylysine. Following this, the cells were treated with 0.05% trypsin and placed in 35/35 mm Petri dishes. The osteoblast cellswere seeded at a density of 10\({}^{5}\) cells/mL in the Petri dishes and incubated with ZnO powders for 24 h. Cell viability was determined with the MTT reduction assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium). The cells were incubated in a 5% CO\({}_{2}\) atmosphere at 37 \({}^{\circ}\)C for 4 h with MTT at a concentration of 0.1 mg/mL. The number of viable cells was directly proportional to the production of formazan. Isopropanol was added to dissolve the insoluble purple formazan into a colored solution and absorbance was measured at a wavelength of 595 nm using a TECAN spectrophotometer.