**3. Results**

The electrospinning method was used to obtain nanostructured membranes from recycled PET. Di fferent flow rates were used, and the resulted samples were noted according to each flow rate. These samples, prepared at four di fferent flow rates, were further used in combination with silver nanoparticles in order to create alternative biomedical materials. A total of eight di fferent samples were obtained, four with silver nanoparticles and four as controls for in vitro and in vivo tests.

#### *3.1. Characterization of the Obtained Materials*

Silver nanoparticles obtained through a silver nitrate reduction reaction were characterized by transmission electron microscopy. Figure 1 shows that the size of the obtained particles was in the nanoscale range, varying between 25 and 85 nm. The SAED pattern allowed the identification of the crystalline phases present in the sample, with NanoAg being the only crystalline phase.

Subsequently, NanoAg was characterized by X-ray di ffraction. Figure 2 highlights the crystallinity of the synthesized nanoparticles, with the only identified crystalline phase being NanoAg through the four di ffraction interferences characteristic of silver nanoparticles.

**Figure 1.** Transmission electron microscopy (TEM) images recorded for (**<sup>a</sup>**,**b**) silver nanoparticles (NanoAg) and selected area electron di ffraction (SAED) pattern (**c**).

**Figure 2.** X-ray diffractogram recorded for NanoAg.

Infrared spectroscopy was used to evaluate the integrity of functional groups during post-processing by electrospinning. Figure 3 reveals that the four experimental variants did not show functional group degradation, absorption band movements, or significant intensity changes. The PET characteristic absorption bands can be identified as follows: 2961 cm<sup>−</sup><sup>1</sup> characteristic of the C–H bond, and 1714 cm<sup>−</sup><sup>1</sup> characteristic of the C=O group. Also, absorption bands of C–C and C–O bonds were observed in the molecular fingerprint area.

**Figure 3.** Fourier-transform infrared (FT-IR) spectra recorded for the silver-loaded polyethylene terephthalate (PET\_X\_NanoAg) samples.

Scanning electron microscopy allowed for the identification of the nanostructured membrane morphology and the presence of silver nanoparticles on the fiber surface. Figure 4 reveals the presence of an uneven deposition of silver nanoparticles on the surface of the fibrous membrane in all experimental variants. There was a general tendency of clumping at the nodes of the fibrous network, as the nodes acted as nucleation centers for nanoparticle growth.

**Figure 4.** SEM images recorded for PET\_X\_NanoAg at various flows: (**<sup>a</sup>**,**b**) PET\_2.5\_NanoAg; (**<sup>c</sup>**,**d**) PET\_5\_NanoAg; (**<sup>e</sup>**,**f**) PET\_7.5\_NanoAg; (**g**,**h**) PET\_10\_NanoAg. Green text—dimensions for nanoAg; white text—dimensions for PET fibers.

The electrospun fibers had dimensions ranging between 60 and 250 nm, and the size of the silver particles on the surface of the fibers ranged between 8 and 50 nm. A more detailed representation of the PET\_5\_NanoAg is presented in Figure 5, highlighting the presence of silver nanoparticles agglomerating at the fiber nodes.

**Figure 5.** SEM images recorded in backscattering for PET\_5\_NanoAg.

The nanostructured membranes with modified surface obtained at a flow rate of 2.5 mL/h were also characterized by transmission electron microscopy (Figure 6), which highlighted the nanometric size of the silver particles and their dispersibility over the surface of the PET fibers.

**Figure 6.** TEM images recorded for PET\_2.5\_NanoAg.

#### *3.2. Antimicrobial Properties of the Prepared Samples*

Contamination of the environment with undesired microorganisms has negative consequences in different fields, including human health. Microorganisms can grow planktonically, although a grea<sup>t</sup> majority of them are adherent to different interfaces and surfaces. Adherent microorganisms are more difficult to remove than microorganisms developing in the planktonic state, due to their ability to form specialized multicellular communities, called biofilms, in which cells may have a different behavior compared to planktonic ones, biochemically and genetically, rendering them more resilient to different stressors. Currently, alternative methods for limiting microbial colonization of raw materials and industrial installations, as well as of biomaterials intended for medical applications, are being studied [38].

In the majority of the experimental variants (except the assays on *C. albicans*), it can be seen that the highest inhibitory activities were obtained in PET samples for which the deposition of fibers by electrospinning was achieved at a flow rate of 10 or 7.5 mL/h (*p*-values ranged from 0–0.05 for *S. aureus* and *P. aerugiosa*).

From the three tested microbial strains, the recycled PET containing NanoAg nanoparticles proved to exhibit the best inhibitory effect on the planktonic growth of *S. aureus* (*p*-value was lower than 0.001), followed by *P. aeruginosa* (*p*-value was lower than 0.05), as compared with the NanoAg free controls (Figure 7).

**Figure 7.** Graphic representation of the recorded absorbance values of *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Candida albicans* cultures, expressing the multiplication capacity of these cells after cultivation for 24 h in the presence of recycled PET\_X\_NanoAg materials. \* *p* ≤ 0.001, \*\* *p* ≤ 0.05 after the comparison of control with NanoAg-containing PET fibers obtained by applying various flow rates).

In the case of the *C. albicans* yeas<sup>t</sup> strain, the inhibitory effect of the planktonic cultures was relatively low, in contrast with the antibacterial one; surprisingly, the most obvious inhibitory effect was observed for PET\_2.5\_NanoAg, but the result was not statistically significant.

In the case of the assessment of biofilm formation capacity, the results proved to be similar with the data obtained on planktonic cultures, with few variables.

The inhibition effect of *S. aureus* biofilm development was observed at all stages of biofilm development, starting with initial adherence (up to 24 h), continuing with biofilm maturation (up to 48 h) until dispersion (when cells or cell aggregates detach from the biofilm to colonize new surfaces) (Figure 8). The anti-biofilm effect was due to the decrease of viable cells embedded in the biofilm, by 1 to 4 logs, with these data being statistically significant (*p*-values ranged from 0.001–0.05). Similar to the results obtained on planktonic cells, the PET\_7.5\_NanoAg and PET\_10\_NanoAg samples also proved to be the most efficient in biofilm inhibition. It can be observed that the anti-biofilm efficiency decreases over the course of biofilm development. This inverse relationship is seen for all four samples, although it is more evident for PET\_2.5\_NanoAg and PET\_5\_NanoAg samples (Figure 8).

**Figure 8.** Graphic representation of colony-forming units (CFU)/mL representing the number of *S. aureus* viable cells included in the monospecific biofilms developed on the surface of the materials, quantified after 24 h, 48 h, and 72 h at 37 ◦C. \* *p* ≤ 0.001, \*\* *p* ≤ 0.05 comparing control PET and NanoAg PET obtained at the same flow rate.

*P. aeruginosa* is a microorganism with multiple natural resistance mechanisms, making it an opportunistic pathogen that can colonize with maximum efficiency a grea<sup>t</sup> number of environments. Biofilms produced by this opportunistic microorganism are very difficult to eradicate with current antimicrobial substances [39]. The results obtained in this study showed that *P. aeruginosa* has a limited capacity to form biofilms on the obtained nanostructured membranes (Figure 9).

It must be noted that the PET\_7.5\_NanoAg and PET\_10\_NanoAg samples proved to be slightly more active against the biofilm formation in *P. aeruginosa*, as compared with the other tested variants. As revealed by Figure 9, the biofilm inhibition capacity of the obtained fibers was maintained relatively constant in all tested time conditions.

A poor capacity to form biofilms in the presence of the developed nanostructured membranes was observed not only for the bacterial strains, but also for the fungal *C. albicans* strain. In the case of the fungal strain, a dynamic of biofilm growth similar to that observed in case of the Gram-positive *S. aureus* strain was recorded, with an inverse relationship between the age of the biofilm and the intensity of the anti-biofilm effect. However, the efficiency of different tested samples was completely different from that obtained against the two bacterial strains, in the following order: PET\_2.5\_NanoAg > PET\_5\_NanoAg > PET\_7.5\_NanoAg > PET\_10\_NanoAg (Figure 10).

**Figure 9.** Graphic representation of CFU/mL, representing the number of *P. aeruginosa* viable cells included in the monospecific biofilms developed on the surface of the materials, quantified after 24 h, 48 h, and 72 h at 37 ◦C. \* *p* ≤ 0.001, \*\* *p* ≤ 0.05 comparing control PET and NanoAg PET obtained at the same flow rate.

**Figure 10.** Graphic representation of CFU/mL representing the number of *C. albicans* cells viable cells included in the monospecific biofilms developed on the surface of the materials, quantified after 24 h, 48 h, and 72 h at 37 ◦C. \* *p* ≤ 0.001, \*\* *p* ≤ 0.05 comparing control PET and NanoAg PET obtained at the same flow rate.

#### *3.3. In Vitro and In Vivo Biological Response*

#### 3.3.1. In Vitro Biocompatibility

The cytotoxicity of recycled PET nanostructured membranes was analyzed using human diploid cells in culture. The results obtained by applying the MTT method showed that the proliferation and activity of diploid cells in the culture underwent changes in the presence of the analyzed materials, depending on the rate of deposition of the fibers by electrospinning; additionally, the majority of cases proved that the addition of NanoAg seemed to slightly reduce the cytotoxicity of the obtained materials, as compared with PET controls. However, these results had no statistical relevance (*p*-values were higher than 0.05).

The results obtained by applying the MTT assay revealed that a high percentage of the seeded cells remained metabolically active after covering with NanoAg, suggesting a good biocompatibility of the recycled PET containing NanoAg nanoparticles in vitro (Figure 11).

**Figure 11.** Effects of PET\_X\_NanoAg on MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) specific activities in amniotic fluid stem cells (AFSC).

#### 3.3.2. In Vivo Biocompatibility and Inflammatory Response

The subcutaneous implant of the di fferent nanofiber mats showed no adverse local or systemic inflammatory e ffects. CRP is an inflammation marker, which highlights the activation of a pro-inflammatory cascade. Figure 12 shows the e ffects of PET\_X\_NanoAg biomaterials implanted subcutaneously in mice on the CRP serum level. At 24 h post-implantation, the CRP blood level was elevated for all experimental groups, followed by a gradual decrease for up to seven days. Compared to the control (*p* < 0.001), PET\_2.5\_NanoAg induced significantly lower CRP levels at all time intervals. These results sugges<sup>t</sup> that PET\_X\_NanoAg biomaterials are well tolerated by the body, and inflammation, together with other potential associated complications, which might lead to implant rejection, is avoided [40,41].

Histopathological analysis of the skin and subcutis of the control animals at days one and seven after implantation showed no significant pathological changes. Inflammatory infiltrate, necrosis, neovascularization, and fibrosis were not observed at either time point. After 24 h post-implantation, PET control samples induced significant edema at the implanting sites, which increased with the rate of fiber deposition. Assessment of inflammatory response revealed the presence of inflammatory cells, such as neutrophils, monocytes, lymphocytes, and macrophages. PET\_X\_NanoAg samples showed a decreased inflammatory reaction compared with PET-implanted samples at the same rate of electrospinning. In all implants, few eosinophils were noticed (Table 2).

**Figure 12.** The effects of PET\_X\_NanoAg subcutaneous implantation in mice on the C-reactive protein (CRP) levels at 24 h and seven days post-surgery.


**Table 2.** Tissue reactions by histometric scoring used to grade inflammation, fibrosis, necrosis, and neovascularization in the tissue surrounding subcutaneous implants.

PMN: polymorphonuclear neutrophils; MONO: monocytes; LYM: lymphocytes; EOS: eosinophils; MO: macrophages; PC: plasma cell; GC: giant cell; FC: fibrocytes, NV: neovascularization. Tissue reactions are rated from − (not present), and sp (sporadic) to ++++ (extensive).

On the seventh day post-implantation, the edema reaction persisting in high-purge-speed PET implants and a fibrous capsule of varying thickness were present in all PET-implanted tissue samples (57–76 μm). Consistent with a granulomatous reaction, mainly macrophages, plasma cells, monocytes, lymphocytes, and neutrophils were present at the interface between the mats and this capsule (Figure 13). Some of these macrophages showed marked evidence of phagocytic activity. Giant cells were observed in PET 10 and 7 mL/h samples.

**Figure 13.** Representative histological images of PET\_X\_NanoAg mats-implanted sites in mice—days one and seven post-implantation. Neutrophils (black arrowhead); monocytes (green arrowhead); eosinophils (red arrowhead); macrophages (white arrowhead); plasma cells (purple arrowhead); giant cells (blue arrowhead); lymphocytes (yellow arrowhead); \* implant (asterisk). Cells were stained with hematoxylin and eosin (H&E) stain. Scale bars = 200 and 20 μm.

The PET\_X\_NanoAg samples induced the occurrence of fibrotic capsules (35 and 40 μm) with purge speed with lower thickness as compared with PET in the same electrospinning conditions (Figure 14). Attached cells on the PET\_X\_NanoAg surface and extensive neovascularization of tissue surrounding the nanofiber mat were noticed.

**Figure 14.** Gomori's trichrome stain of PET control and PET\_X\_NanoAg-implanted tissues.

Injection or implantation of a biomaterial results in an acute inflammation response, which is most often followed by a chronic inflammatory reaction [42], characterized by the infiltration of polymorphonuclear neutrophils (PMN), macrophages, and eventually lymphocytes [43]. The inflammatory reactions toward the novel in situ PET\_X\_NanoAg materials were weak, being within the limits of a typical, normal reaction to implanted materials characterized by the accumulation of the inflammatory cells on the materials surface. Similar responses were observed in the immediate post-implant period against other implanted materials with increased biocompatibility [44,45].

Implants with prolonged stay in the host tissue generally alter the tissue wound-healing response in chronic inflammatory conditions, producing fibrous encapsulation of the foreign body, with the presence of hallmark giant cells [46]. The fibrous capsule often isolates the implanted materials from normal host tissue sites, being characterized by poor vascularization and reduced bactericidal capability, predisposing these sites to infection [43]. Results from this study showed no well-defined collagen formation around implants in the case of PET\_X\_NanoAg after seven days post-implantation, a reaction comparable with that induced by other previously reported biocompatible materials [47,48].

Immunohistochemistry staining was performed for tissue sections to analyze the inflammatory response toward the implanted nanofibers (Figure 15). An increased immunopositivity for TNF-α levels on PET-implanted tissues, as compared to those obtained for the PET\_X\_NanoAg samples, was observed at both time points.

**Figure 15.** Expression and specific distribution of tumor necrosis factor (TNF)-α at implantation site at 24 h and seven days after implantation; scale bar = 200 μm.

The progression of events in inflammation and the foreign body response require the extravasation and migration of macrophages to the implant site, which produces and releases platelet-derived growth factor (PDGF), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) [43]. In our study, the NanoAg covering of PET materials reduced TNF-α expression and consequently reduced inflammation and foreign body response at the implantation site [49,50].
