## 4 Discussion  
The development of a coating for Ti-alloy made of CS/PEO fibers functionalized with BG by electrospinning method was studied by different techniques in order to understand the topography and chemistry of the coatings. The most important aspect that emerged during pre-test characterizations was the variation of the fibers' physiochemical and morphological properties, correlated with the presence of BG. Before electrospinning, the solution containing BG presented a higher viscosity and this could be correlated with an increase in fiber diameter. Incorporating BG, the presence of rigid particles increased the shear viscosity of dilute suspensions compared to that of neat liquids. These results are consistent with previous reports, which show how an increase in viscosity arises from implementing the polymeric solution for electrospinning with bioactive powders, in turn affecting the fiber size and distributions [41, 42]. Consequently, the change in fiber diameter had an effect on wettability. In fact, substrates with CS/PEO/BG had a higher contact angle than samples without bioglass. These data also found confirmation in other works in literature in which changes in voltage led to fibers with different diameters. Analyses of contact angle showed that by increasing the size of the fibers the contact angle also increased [43]. Adding BG into the mixture usually produces a relatively rough and more hydrophilic surface [23], but in our case the use of bioglass did not bring any variation in terms of average roughness or mean roughness depth, despite the presence of small particles related to silicon and calcium as detected by EDX. The increase in roughness derived from the contribution of fiber distribution did not affect the bacterial adhesion since, as seen through the 12 h Microbial viability test, the optical density value did  
Figure 13: Fluorescence microscopy images collected on Ti-alloy (**a**), CS/PEO (**b**), and CS/PEO/BG (**c**) substrates: blue stain (osteoblast nuclei), red stain (osteepontin), and green stain (osteocalcin). Images collected at 4X. not change much between the Ti-alloy and the CS/PEO substrates. In the case of CS/PEO/BG, the high degree of optical density related to the number of living bacteria on the surface can be associated to the presence of BG. This is because during the fiber preparation process BG interacted with CS. In particular, in the FTIR spectrum there was an increase in intensity for bands related to NH bending of the amide groups and, especially, the band located at 1560 cm\({}^{-1}\) was found to be an index of interactions between BG and chitosan [23; 44]. The dissolution of chitosan in dilute acetic acid leads to the protonation of NH\({}_{2}\) groups [21; 45]. The incorporation of BG initially inhibited the effect of chitosan as visible at 12 h by WST and fluorescence microscope images. With the dissolution of BG over time, the protonated NH\({}_{3}\)\({}^{+}\) of the CS could interact through electrostatic forces with electronegative sites on the surface of the bacterial membrane, promoting changes in membrane wall permeability (osmotic imbalances), inhibiting bacterial growth, leading to leakage of intracellular electrolytes as proteins and other low molecular weight constituents, and binding the cell via protonated amino groups, which in turn led to inhibition of the microbial RNA synthesis [44; 46; 47; 48; 49; 50; 51; 52]. In fact, the antibacterial behavior of the chitosan was clearly visible on both coated surfaces at 24 and 48 h compared to Ti-alloy, according to both Microbial viability sssay and CFU counting. The tests with osteoblasts highlighted some important aspects related to the formation of a mineral and organic matrix on various surface samples. The incorporation of BG into the CS/PEO fibers led to a marked improvement compared to the CS/PEO fibers and Ti-alloy. This represented a further confirmation of how BG acts as a source of ions that can be used by osteoblasts to produce mineral (hydroxyapatite) [53].