## 3 Results and Discussion
### Antimicrobial Activity of AgNP Evaluated in an Ex Vivo Skin Model  
AgNP have been proposed to be an excellent antimicrobial agent against bacteria both in vitro and in vivo, and scientific achievements in this area have been extensively reviewed [50]. However, studies on the effects of AgNP on the skin and their penetration have only been conducted in vitro, mainly using skin cell lines [51]. In the present work, the antimicrobial effect of the developed AgNP after topical application was compared with that of other solutions used in the disinfection of tissues and surfaces and with skin irritant solutions in an ex vivo model using full-thickness pig skin [24] initially developed to study skin bacterial translocation. PBS was used as a control in the experiments. A schematic representation of the procedure is presented in Figure 2 (Methods Section). Figure 8 shows the aspect of the skin incubated at 30 \({}^{\circ}\)C for 14 days from which, at defined time points, swabs were cultured on TSA plates. Figure 6: The evaluation of AgNP cytocompatibility in mammalian cell lines. The effect of increasing concentrations of AgNP on the viability of HaCaT and THP-1 cells after 48 h of incubation. The percentage of cell viability was calculated relative to the control using a colorimetric assay. Data are expressed as the mean \(\pm\) SD of 3 independent measurements. Figure 7: The microscopic appearance of bacterial growth in the presence of AgNP measured by the broth microdilution assay. Note that the concentration of AgNP decreases from left to right. The dashed line indicates the MIC (\(\mu\)g/mL). The B, C, and D lines correspond to replicates. Figure 8: The antimicrobial activity of AgNP in an ex vivo porcine skin model. **(a)** Images of the skin portions on day 0, day 7, and day 14 after the sterilization procedure. **(b)** An analysis of skin tissue viability after the sterilization procedure compared with the no-treatment control (PBS). The viability analysis data are plotted as mean \(\pm\) SD (\(n=5\)). Statistical analysis: * \(p<0.001\) vs. PBS. **(c)** Microbial growth on TSA agar from swabs of the porcine skin portions, where, in each image, the following treatments are indicated: top row, from left to right: 1—PBS for 40 min immersion; 2—70% EtOH for  
40 min immersion; 3--100% EtOH for 40 min immersion; 4--AgNP for 40 min immersion; bottom row, from left to right: 5--extract for 40 min immersion; 6--5% SDS for 40 min immersion; 7--AgNP for 30 min + 70% EtOH for 10 min immersion; and 8--AgNP for 30 min + 100% EtOH for 10 min immersion. (**d**) Microphotographs of H&E-stained skin slices after different experimental treatments: A--PBS for 40 min immersion; B--70% EtOH for 40 min immersion; C--100% EtOH for 40 min immersion; D--AgNP for 40 min immersion; E--extract for 40 min immersion; F--5% SDS for 40 min immersion; G--AgNP for 30 min + 70% EtOH for 10 min immersion; and H--AgNP for 30 min + 100% EtOH for 10 min immersion. Scale bars = 500 \(\upmu\)m.  
In Figure 8a, the images from day 0, taken just after the soaking procedures, show a black color on the skin specimens treated with the AgNP solution, and this is more pronounced on the sample treated only with AgNP. On day 7, the skin treated with the extract already had colonies that could be seen with the naked eye. This rapid microorganism growth is explained by the high concentration of TE phytochemicals. All skin samples were darker, but ethanol-treated samples present a more preserved aspect. Over the experimental period, skin decay is more apparent. The skin portions submerged in 70% EtOH and 100% EtOH had less decay. The skin portion soaked in AgNP looks similar to the one with 5% SDS. The skin portions submerged in the extract, AgNP + 70% EtOH, and AgNP + 100% EtOH look like the control skin. Chemical disinfectants can alter the skin structure and affect the viability of skin cells. Using the MTT assay, the viability of the cells was evaluated to understand which condition had the least impact on the skin cells (Figure 8b).