*2.4. Physico-Chemical Characterization*

**Infrared spectroscopy.** Infrared (IR) spectra were obtained using a Nicolet iN10 MX Fourier-transform (FT)-IR microscope (Thermo Fischer Scientific, Waltham, MA, USA) with a liquid nitrogen-cooled mercury cadmium telluride (MCT) detector with a measurement range of 4000–700 cm<sup>−</sup>1. Spectra collection was performed in reflection mode at a resolution of 4 cm<sup>−</sup>1. For each spectrum, 32 scans were co-added and converted to absorbance using the OmincPicta software (Version 1, Thermo Fischer Scientific, Waltham, MA, USA).

**X-ray di**ff**raction.** Grazing incidence X-ray diffraction (GIXRD) was performed with a Panalytical Empyrean diffractometer (PANalytical, Almelo, The Netherlands), using CuK radiation (1.541874 A) equipped with a 2× Ge (2 2 0) hybrid monochromator for Cu and a parallel plate collimator on the PIXcel3D. Scanning was performed on the 2θ axis in the range of 5–80◦, with an incidence angle of 0.5◦, a step size of 0.04◦, and a time step of 3 s.

**Scanning electron microscopy.** In order to investigate the morphology and size of the nanostructured membranes, images produced by recording the resultant secondary electron beam with an energy of 30 keV of the samples were taken with a scanning electron microscope purchased from FEI (Hillsboro, OR, USA).

**Transmission electron microscopy (TEM). Electron di**ff**raction on selected area.** In order to obtain transmission electron microscopy images, the samples were placed on a carbon-coated copper grid at room temperature. The TEM images were acquired using a high-resolution TecnaiTM G2 F30 S-TWIN transmission microscope equipped with selected area electron diffraction (SAED), purchased from FEI (Hillsboro, OR, USA). This microscope operates in transmission mode at a voltage of 300 kV, while the point and line resolution are guaranteed with values of 2 Å and 1 Å, respectively. The SAED analysis for silver nanoparticles was performed in a light field using the TecnaiTM G2 F30 S-TWIN high-resolution electronic microscope equipped with SAED, purchased from FEI (Hillsboro, OR, USA).

#### *2.5. In Vitro Biocompatibility*

## 2.5.1. Cell Line

Human amniotic fluid stem cells (AFSC) were used to evaluate the biocompatibility of the prepared samples. The AFSC cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin, streptomycin/neomycin). All cells were maintained at 37 ◦C in a humidified incubator with 5% CO2.

#### 2.5.2. MTT (3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) Assay

The evaluation of cell viability was performed by measuring the degree of reduction of a tetrazolium salt solution, MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), to insoluble purple formazan crystals by viable cells. In this purpose, the AFSC cells were seeded in 96-well plates, with a density of 3000 cells/well under different experimental conditions. Subsequently, a volume of 10 μL of 12 mM MTT was added and the cells were incubated at 37 ◦C for 4 h. A volume of 100 μL of SDS–HCl solution was further added, vigorously pipetted to solubilize the formazan crystals, incubated for 1 h, and read at 570 nm (TECAN spectrophotometer, Männedorf, Switzerland).

#### *2.6. In Vitro Antibacterial Tests*

#### 2.6.1. Growth of Planktonic (Floating) Microorganisms in the Presence of Material

To test the effect of the obtained material on the growth of microorganisms in liquid medium (planktonic cultures), the obtained material was cut into 1-cm<sup>2</sup> samples and sterilized by exposure to ultraviolet (UV) radiation for 30 min on each side. One fragment of the sterile material was individually deposited in a well of a six-well sterile plate. Over the deposited materials, 2 mL of liquid medium and then 20 μL of 0.5 McFarland microbial suspension (bacteria—*Staphylococcus aureus* American Type Culture Collection (ATCC) 25923 and *Pseudomonas aeruginosa* ATCC 27853) or 1McFarland (yeast—*Candida albicans* ATCC 10231) prepared in sterile physiological water, 0.9% NaCl salt, was added to the wells. The as-prepared six-well plates were incubated at 37 ◦C for 24 h. At the end of the incubation time, 200 μL of the obtained microbial suspension was transferred to 96 sterile plates, and the turbidity of the microbial cultures (absorbance) was spectrophotometrically measured at 600 nm.

#### 2.6.2. Evaluation of Adhesion and Biofilm Formation

To test the e ffect of fibrillated materials on microbial adhesion and biofilm production, the sterile material samples treated as described above were washed with sterile saline water (SSW), and the medium was changed to allow the microbial cells—adhered onto the surface of the material samples in the first 24 h of incubation—to continue biofilm development and maturation for another 24, 48, and 72 h. After the end of each incubation period, the colonized sample was washed with AFS to remove non-adherent microorganisms and deposited in a sterile tube in 1 mL of SSW. The tube was vigorously vortexed for 30 s and sonicated for 10 s to harvest the cells from the biofilm. The obtained cell suspension was diluted, and various ten-fold serial dilutions were seeded on solid culture media plates in triplicate, to obtain and quantify the number of viable cells, expressed in colony-forming units (CFU)/mL.

#### *2.7. In Vivo Biocompatibility Evaluation*

#### 2.7.1. Animals and Ethics

CD1 mice were housed in controlled-airflow cabinets with 12-h light cycles and constant temperature and humidity conditions. Animal experiments were performed in accordance with the guidelines of the Vasile Goldis Western University of Arad and approved by the Ethical Committee.

#### 2.7.2. Experimental Design and Surgical Procedures

The mice were randomly assigned to 18 groups (*<sup>n</sup>*=5): control, PET\_2.5\_ctrl, PET\_5\_ctrl, PET\_7.5\_ctrl, PET\_10\_ctrl, PET\_2.5\_NanoAg, PET\_5\_NanoAg, PET\_7.5\_NanoAg, and PET\_10\_NanoAg, for one day and seven days.

Before the experiment, the material samples (1 cm2) were sterilized under UV light for 30 min (both faces) and implanted into a subcutaneous pocket in the dorsum of the animals. For the surgical procedure, the animals were anesthetized by intraperitoneal injection of xylazine/ketamine.

Animals were allowed to recover from anesthesia, housed in individual cages, and observed daily for evidence of wound complications, such as redness, infection, edema, abscess, hematoma, encapsulation, or skin dehiscence.

On days two and seven post-surgery, the animals were euthanatized by anesthetic overdose and the implanted materials, together with the surrounding tissues, were explanted and collected for analysis. Blood was sampled by cardiac puncture to assess acute inflammatory markers, in order to be able to exclude systemic inflammation.
