## 3 Results
### Bacterial Tests: Microbial Viability Assay, CFU Counting and Fluorescence Microscope  
As shown in Figure 9a,b, bacterial cell viability and CFU counting was performed at three different times, 12, 24 and 48 h, respectively. About the Microbial viability assay (Figure 9a), at 12 h CS/PEO/BG showed the highest OD value (1.72 \(\pm\) 0.06), indicating the largest living bacteria number among the samples. Ti-alloy and CS/PEO had lower OD values (1.15 \(\pm\) 0.08 and 0.98 \(\pm\) 0.06) and did not present any significant difference between each other (\(p\) < 0.05). A general decrease of OD value occurred for  
Figure 8: Images collected using EDX elemental maps pf CS/PEO (**a**) and CS/PEO/BG (**b**) converted in different colors (blue for calcium, green for silicon and red for carbon) and overlaid on the SEM images in order to verify the incorporation of bioglass inside the fibers. EDX maps and SEM images were collected at 5000X magnification. Figure 7: SEM images (collected at 5000X magnification) and diameter distributions of the electrospun nanofiber coatings (CS/PEO (**a,b**) and CS/PEO/BG (**c,d**)). The diameter size distributions were obtained analyzing 200 fiber sizes on each surface. all the samples analyzed at 24 h. The bacterial growth trend of CS/PEO presented the lowest OD value (\(0.43\pm 0.02\)), as compared to OD values for Ti-alloy (\(0.51\pm 0.03\)) and CS/PEO/BG (\(0.53\pm 0.01\)), which were not significantly different (\(p<0.05\)). At 48 h of exposure, CS/PEO/BG had the lowest OD value (\(0.41\pm 0.01\)), while Ti-alloy presented a slight increase and the highest OD value (\(0.65\pm 0.02\)), whereas CS/PEO (\(0.44\pm 0.02\)) had an intermediate OD value. At this time of exposure, OD values between the tested samples were significantly different (\(p<0.05\)). Figure 9b displays the antibacterial activity performed by fiber-coated substrates evaluated by a time-lapse CFU counting assay obtained after culturing the bacteria collected from the surface. Results indicated a CFU number decreasing with the time increase on all the substrates treated. At all three times of exposure, uncoated Ti-alloy had the highest OD value compared with the two fiber-coated substrates. These latter did not show significant differences between each other at 12 h and 24 h, but at 48 h CS/PEO showed the lowest amount of CFU/ml compared to CS/PEO/BG (\(p<0.05\)). Fluorescence micrographs were obtained for all samples after first staining them with PI, CFDA, and DAPI markers, which colored dead bacteria (red), live bacteria (green), and nuclei (blue), respectively. Figure 10 illustrates images collected at 12 h of the different samples after exposure to _S. epidermidis_ (constant magnification of 4X). On CS/PEO/BG a high amount of cells, homogeneously distributed, was detected and most of them corresponded to living cells (cf. DAPI blue-stained and CFDA green-stained micrographs in Figure 10c). Differently, in images of Ti-alloy substrates (Figure 10a), bacteria were located in few spots related to both living and dead cells. Also on CS/PEO, bacteria were not homogeneously distributed and the PI-red stained images in the micrographs indicated how these areas were associated to dead bacteria (Figure 10b). Images were collected at 48 h for all the samples shown in Figure 11. For Ti-alloy substrate, a relatively high amount of bacteria was detected over the entire sample's surface (cf. DAPI blue-stained micrographs in Figure 11b). The level of living bacteria was still high, as suggested by the CFDA green-stained areas, but some PI red-stained areas associated with dead bacteria clearly indicated that lysis had occurred. Micrographs of CS/PEO (Figure 11b) presented a relatively low density of bacterial cells. They were located in few areas not homogeneously distributed on the entire surface and containing both dead and living cells. Regarding the CS/PEO/BG substrate, bacterial cell density seemed lower than on Ti-alloy (cf. DAPI blue-stained micrographs in Figure 11c), even if distributed over the entire surface.