2.3. Characterization of Biocomposite Hydrogels

The structural characteristics of the obtained hydrogels, the ionic and covalent new bonds, and also the presence of ZnONPs were analyzed using a Bruker FT-IR Spectrometer (VERTEX 70) equipped with a DLaTGS detector in the ATR operating mode (ZnSe crystal: 600–4000 cm⁻¹).

The morphological characteristics of the hydrogels were determined by scanning electron microscopy (SEM) using a Quanta 200 Scanning Electron Microscope (FEI Company, Bruno, Czech Republic). The hydrogels were analyzed in the dry state. This analysis gave us information on the porosity and homogeneity of hydrogels.

The shape and size of ZnONPs were assessed using a HITACHI-HT7700 Transmission Electron Microscope (Hitachi High-Technologies Corporation, Tokyo, Japan).

The elemental composition of the hydrogels and the presence of ZnONPs were demonstrated using a Verios G4 UC Scanning Electron Microscope from Thermo Scientific (Bruno, Czech Republic) equipped with an Octane Elect Super SDD detector (an energy-dispersive X-ray spectrometer from EDAX (Ametek, Mahwah, NJ, USA).

To provide electrical conductivity and to prevent charge buildup during exposure to the electron beam, the samples were coated with 6 nm platinum using a Leica EM ACE200 Sputter coater.

The swelling behavior of the obtained hydrogels was evaluated, using the gravimetric method, at 37 °C, in phosphate buffer solution (PBS) at pH 7.4, which mimics biological fluids [22]. The dried square-shaped hydrogel samples (1 cm side) were first weighed (W_d, mg) and then immersed in 20 mL of PBS. At predetermined times, the samples were easily removed from PBS and placed on a filter paper. The excess PBS on the surface of the hydrogels was easily absorbed with another filter paper and the swollen samples were weighed (W_st). This procedure was repeated until equilibrium was reached. The swelling degree at time t (Q_t(%)) was calculated using the following equation:

Tensile mechanical measurements were performed using a Brookfield CT3 Texture Analyzer (Texture Brookfield Engineering Laboratories Inc., Middleboro, MA, USA) with a 50N load cell, at room temperature. The Roller Cam Accessory (TA-RCA) was used for this test. The films (dry hydrogels kept in an atmosphere with 65% humidity) were cut into rectangles (length 55 mm, width 10 mm) and were caught between two clamps positioned at a distance of 30 mm. The thickness of the films was determined by taking the average of the thickness, measured at five different places with a digital Caliper and found to be around 0.5 mm in all the samples. The speed of the tensile measurements was fixed at 0.5 mm/s. The stress (σ, N/m²) was calculated from the load divided by the cross-sectional area of the undeformed sample, and the strain (ε) was determined as the clamp displacement divided by the initial distance between the two clamps. The ultimate strain and stress (from the point of failure) were determined, and the elastic modulus values were calculated from the slope of the linear climbing tract of the stress–strain plot within the fixed strain region 0.1–2%.

The in vitro cytotoxic effects of the hydrogels were assessed using human dermal fibroblasts, adult, and cell line (HDFa). HDFa cells were cultured and prepared for testing as described in our previous paper [33]. The sterilized hydrogel samples were cut with a biopsy punch to obtain discs 4 mm in diameter and incubated with the HDFa cells. After the incubation time (24 and 48 h), the cell viability was determined using the MTT assay. All procedures were performed in a laminar flow hood (Lamil Plus 13, Kartusalan Metally Oy). Each hydrogel sample was tested in triplicate in order to get the average percentage and the standard error.

The antimicrobial activities of the obtained hydrogels without and with ZnONPs were evaluated using the disk diffusion assay against 4 reference strains: S. aureus (Gram-positive bacteria), E. coli, K. pneumoniae, and P. aeruginosa (Gram-negative bacteria). Commercially available antimicrobial disks with Chapman agar for Gram-positive bacteria, and MacConkey agar for Gram-negative bacteria were used for these tests. A suspension of microorganisms (0.5 McFarland density) was inoculated on a Petri dish with the substrate. The sterilized hydrogel discs (4 mm diameter) were firstly hydrated in sterile 0.9% saline solution and then placed on the agar plate and incubated for 24 h at 37 °C. The antimicrobial activities were evaluated by measuring the diameters of the inhibition zones (din, mm) according to the Kirby–Bauer method [34]. The experiments were repeated three times. In order to evaluate the significance of their antibacterial activity against S. aureus, E. coli, and K. pneumonia strains, as compared to samples without ZnONPs, the one-way ANOVA statistical test was processed.