*2.5. Statistical Analysis*

The statistical analysis was performed using the unpaired two-tailed Student's *t*-test, with the di fferences being considered significant when *p* < 0.05.

### **3. Results and Discussion**

### *3.1. Physico-Chemical Analyses*

### 3.1.1. AFM Morphological Examination

The AFM microscopic investigations revealed that the SBG films, regardless of their composition (i.e., FastOs ®BG—-Figure 1a or Cu&Ga-FastOs ®BG-derived—Figure 1b), had a highly similar morphology, being smooth (i.e., root-mean-square roughness of <1 nm) and compact. The films were composed of dome-shaped nano-sized particles with diameters within the ~15–20 nm narrow range, characteristic to amorphous magnetron sputtered structures.

**Figure 1.** 2D atomic force microscopy (AFM) images recorded on 5 × 5 μm<sup>2</sup> surface areas of the (**a**) FastOs®BG- and (**b**) Cu&Ga-FastOs®BG-derived films.

### 3.1.2. EDXS Compositional Analysis

The comparative elemental compositions (in at.%) of the source SBG materials and of the FastOs®BG- and Cu&Ga-FastOs®BG-derived layers, as estimated by EDXS analysis, are presented in Figure 2. The results showed that the SBG films were, as intended, successfully enriched in Si/SiO2 (~53–54 at.%/~56–57 mol.%) in both coating cases, at the expense of Ca and Mg, which recorded a concentration decrease of ~23–32% and ~18–24 at.%, respectively. The decrease (with 41–48 at.%) of the P concentration is a characteristic to RF-MS processes, and it is likely linked to the higher volatility of P2O5 (and associated with its relatively low sublimation latent heat) compared to the other oxide glass constituents [40,60]. The Cu and Ga contents of the Cu&Ga-FastOs®BG source biomaterial were well-transferred into the RF-MS films (no statistical significant differences being determined, *p* > 0.05).

**Figure 2.** Comparative elemental composition (in at.%) of the magnetron cathode targets and the deposited silica-rich films: (**a**) FastOs®BG- and (**b**) Cu&Ga-FastOs®BG-based materials. The quantification of oxygen and fluorine was not performed because of the inaccuracy of EDXS analysis method with respect to light elements (Z < 11) [43]. \* *p* < 0.05, statistically significant differences, as determined by using an unpaired two-tailed Student's *t*-test.

### 3.1.3. XPS Chemical State Examination

The high-resolution core electron XPS spectra of the Si 2p, Ca 2p, P 2p, Mg 2p, Cu 2p3/2, Ga 2p3/2 and O 1s levels of the source materials and derived RF-MS films are presented comparatively in Figure 3a–d, e–h, i–l, m–p, q–s, r–t, and u–x, respectively. The binding energy positions of the Si 2p (~101.9–102.6 eV), Ca 2p3/2 (~347.5–347.6 eV), P 2p3/2 (~133.0–133.4 eV), Mg 2p (~50.1–50.3 eV) correlated with the Auger parameter calculated as the binding energy of Mg 2p + kinetic energy of Mg KLL (~1230.0–1230.7 eV) lines (data not shown), disclosed their complete oxidation [61] in the case of both source materials and derived SBG films.

**Figure 3.** High resolution XPS spectra of the (**<sup>a</sup>**–**d**) Si 2p, (**<sup>e</sup>**–**h**) Ca 2p, (**i**–**l**) P 2p, (**<sup>m</sup>**–**p**) Mg 2p, (**q**,**<sup>s</sup>**) Cu 2p3/2, (**<sup>r</sup>**,**<sup>t</sup>**) Ga 3p3/2, (**<sup>u</sup>**–**<sup>x</sup>**) O 1s core photoelectron levels, recorded for the (**<sup>a</sup>**,**c**,**e**,**g**,**i**,**k**,**m**,**o**,**u**,**<sup>w</sup>**) FastOs®BG and (**b**,**d**,**f**,**h**,**j**,**l**,**n**,**p**,**q**–**t**,**v**,**<sup>x</sup>**) Cu&Ga-FastOs®BG (**<sup>a</sup>**,**b**,**e**,**f**,**i**,**j**,**m**,**n**,**q**,**r**,**u**,**<sup>v</sup>**) source materials and (**<sup>c</sup>**,**d**,**g**,**h**,**k**,**l**,**o**,**p**,**s**,**t**,**w**,**<sup>x</sup>**) derived films. Symbols: experimental spectrum, Solid line: fit sum. Filled area curves: spectral components.

The Ga 2p3/2 levels are situated at ~1119.3 and ~1118.9 eV for the cathode target and RF-MS film (Figure 3r,t), respectively, unveiling the 3<sup>+</sup> oxidation state of gallium [61,62]. The core electron spectra collected in the region of the Cu 2p3/2 peak indicated that the Cu is found in both 1+ (~933.4 eV) and 2+ (~935.5 eV) oxidation states in the case of the Cu&Ga-FastOs ®BG source material (Figure 3q) [61]. The presence of Cu2+ in the melt-quenched glass is also supported by the lower intensity broad peak, situated at higher binding energies and ascribed to the Cu2+ satellites [61]. In the case of the Cu&Ga-FastOs ®BG-derived RF-MS films, the Cu2+ satellites were di fficult to emphasize due to the low intensity of the peaks and consequently higher noise/signal ratio. The Cu2+ reduction to Cu+ oxidation state could be related to the lower thermodynamic driving force for the formation of CuO with respect to Cu2O, as the Gibbs free energy of oxidation is higher for the former [63]. As a matter of fact, the Gibbs free energy of formation of the Cu oxides is lower with respect to all the other glass components (i.e., SiO2, CaO, P2O5, MgO, Ga2O3) [63].

Two components were disclosed by the peak separation procedure applied to the O 1s spectra of the glass source materials and RF-MS films (Figure 3u–x). The higher energy component (positioned at ~532.3–532.8 eV) is assigned to the Si–O–Si bonds contribution (bridging oxygen bonds), whilst the lower binding energy one (centred at ~530.7–531.3 eV) is associated to the oxygen bonds with metal species (i.e., Si–O–M bonds (M = metal ions in the glass), non-bridging oxygen bonds) [64,65]. An additional third minor O 1s component, positioned at a higher binding energy (~533.8 eV), was evidenced in the case of the FastOs ®BG target material only (Figure 3u). This third component can be ascribed to the adsorbed water [58,61], and is determined by hydroscopic character of the SBG. The Si–O–Si/Si–O–M ratio was dramatically increased in the case of RF-MS coatings with respect to the source materials (Figure 3u–x), as consequence of their intentional increase in Si concentration (Figure 2). In both the source material and RF-MS derived coating cases, the incorporation of Cu and Ga into the glass structure induced a slight increase of Si–O–Si/Si–O–M ratio, which suggested an improvement of the network connectivity.

### 3.1.4. FTIR Spectroscopy Structural Investigation

When network modifiers are incorporated into SBGs, the covalent Si–O–Si bonds are broken, and non-bridging oxygen atoms (NBO) are formed. The silica-based glass network becomes disrupted through the creation of ionic bonds between NBOs and modifier cations [5]. The network connectivity is directly influencing the stability and durability of SBG in contact with the intercellular fluids [5]. The short-range order in oxide glasses is generally quantified by the Qn notation, in which *Q* represents a network-former polyhedron (in the case of SBG, [SiO4]), whilst *n* corresponds to the number of associated bridging oxygen atoms (BOs).

The FTIR spectra of the simple and Cu & Ga substituted SBG source biomaterials, and of the RF-MS derived coatings are presented together in Figure 4a–c. Both the FastOs ®BG-based source materials (Figure 4a) were characterized by the presence of large IR absorption bands with four maxima positioned at (i) ~1181 cm<sup>−</sup>1, (ii) ~1030 cm<sup>−</sup>1, (iii) ~950 cm<sup>−</sup><sup>1</sup> and (iv) 855 cm<sup>−</sup><sup>1</sup> appertaining to the asymmetric stretching (νas) vibrational modes of: (i + ii) of the Si–O–Si bonds in all the silicate tetrahedrons, TO3 (Transverse-Optical) and LO3 (Longitudinal-Optical) modes, respectively, and of Si–O bonds in (iii) Q<sup>2</sup> + Q<sup>3</sup> (with one and two NBOs) and (iv) Q<sup>0</sup> + Q<sup>1</sup> (with three and four NBOs) units [9,66–68]. The lower intensity band at ~749 cm<sup>−</sup><sup>1</sup> is assigned to the symmetrical stretching (νs) vibrations of Si–O bonds. The incorporation of Cu and Ga into the composition of the parent FastOs ®BG material induced an improvement of the glass network connectivity, as suggested by the small intensity reduction of the Q<sup>2</sup> + Q<sup>3</sup> NBO band (at ~950 cm<sup>−</sup>1), while the intensity of the Si–O–Si band (at ~1030 cm<sup>−</sup>1) remained constant (Figure 4b).

The successful increase of the network modifiers concentration (Figure 2) of SBG RF-MS coatings had as consequences the (i) disappearance of the band at ~855 cm<sup>−</sup><sup>1</sup> (associated with the νas Q<sup>0</sup> + Q<sup>1</sup> band) (Figure 4c), together with a (ii) pronounced decrease in intensity of the νas Q<sup>2</sup> + Q<sup>3</sup> band (Figure 4b).

**Figure 4.** (**a**) FTIR spectra of the (1) FastOs®BG and (2) Cu&Ga-FastOs®BG melt-quenched glass powders, and (3) FastOs®BG- and (4) Cu&Ga-FastOs®BG-derived RF-MS layers; Insets highlighting the main structural modifications occurring in the spectral regions of the (**b**) νas Q<sup>0</sup> + Q1 and (**c**) νas Q<sup>2</sup> + Q<sup>3</sup> vibration bands.

The incorporation of Cu & Ga therapeutic ions into the SBF RF-MS film structure led to minor structural changes that advocate for a slight polymerization of this type of coating with respect to the FastOs®BG-derived one (similarly to the trends experienced by the source materials). This was hinted by the minor intensity decrease and position shift of the absorption band associated with νas vibration modes of bonds in the Q<sup>3</sup> + Q<sup>2</sup> units (Figure 4b). Concurrently, the IR absorption bands generated by Si–O–Si bonds experienced a slight blue-shift (Figure 4a,b). This is in agreemen<sup>t</sup> with the afore-presented XPS results (Figure 3u–x), which indicated a higher Si–O–Si/Si–O–M ratio in the case of the Cu&Ga-FastOs®BG-based materials. Since the network former content (Si + P) is the same (~59.3%) for both type of films, and the concentration of well-known network modifiers (Ca + Mg + Cu) is higher (~40.6% vs. ~35.2%) for the FastOs®BG-derived structure (Figure 2), it is suggested that at least part of Ga ions act as network formers. The network former role of Ga is not unprecedented [23] in SBGs. However, for the time being this should be treated as merely a hypothesis, with the clarification of Ga role within the RF-MS SBG films' structure demanding further insightful analyses, which will be the focus of future systematic studies.

### 3.1.5. Surface Energy Measurements

The evolution of surface free energy (and their polar and dispersive components as nominal values and ratios) and water contact angles (CA), recorded for the bare Ti and simple and Cu-Ga doped SBG coatings are shown in Figure 5a,b. The variation of the polar and dispersive components of the surface free energy (SFE, γtot), and thus the hydrophilic/hydrophobic character, can unveil prospective biofunctional traits of a scrutinized material/construct [69,70]. The polar component (γp) is generated by the chemical bonds/interactions (e.g., dipole-dipole interactions) within the material, whilst the dispersive component (γd) is linked to the movement of electrons around atoms/molecules and temporary variation in the electron density with associated temporary dipoles [71]. SFE is known to play a major role in biocompatibility, leading to the arrangemen<sup>t</sup> of functional groups and electrical charges on the surface of the biomaterials in contact with the living environment, and thus, govern the first interactions with the intercellular fluid and the adherence of cells [70,72,73].

**Figure 5.** Evolution of the (**a**) surface free energy values (γtot), its polar (γp) and dispersive (γd) components, water contact angle average values and (**b**) ratios of the polar (γp)/dispersive (γd) components of the surface free energy, for the (1) bare Ti and (2) FastOs®BG-derived and (3) Cu&Ga-FastOs®BG-derived RF-MS coatings.

The SFE values of both the bare and FastOs®BG- and Cu&Ga-FastOs®BG-derived coated Ti specimens were found to be situated in a rather narrow range of ~37–40 mN/m (*p* > 0.05, thus, without statistical significant differences) (Figure 5a). However, the weight of the polar and dispersive components of the SFE significantly differed in the case of the silica-rich SBG coated Ti, with the polar component becoming dominant (Figure 5b). While the contact angle (CA) value with water does not change upon the incorporation of Cu & Ga into the SBG film structure (CA ≈ 62–64◦, *p* > 0.05), in the case of Ti, a CA value towards the hydrophobic domain (i.e., 84◦) was obtained. It is important to note that in the case of implant surfaces, many studies indicated that an optimal wettability range, capable of augmenting the cellular response (i.e., adhesion, proliferation and cytoskeleton organisation), is the 60–80◦ one [73–75]. Thereby, from this point of view favourable premises existed for a positive biological response in the case of the proposed silica-rich bioglass implant coating design.

### *3.2. Mechanical Performance Characterization*

The first mechanical assessment consisted in the evaluation of the bonding strength of the SBG-based coating by the pull-off test. The tensile tests yielded similar values for both simple and Cu-Ga doped SBG coatings (Figure 6a), with average bonding strength values of ~53–55 MPa (*p* > 0.05, thus, without statistically significant differences). The recorded bonding strength values are situated above the limits imposed for implant coatings by the ISO 13779–2:2018 [35] (i.e., 15 MPa), and are close to those recommended by the FDA—STP1196 draft guidance (i.e., 50.8 MPa) [36].

Further, the mechanical properties of the SBG films were evaluated by scratch, nano-indentation and wear tests.

During the scratch tests, the Lc1 load value (the first indication of cohesive failure (appearance of cracks)) was not observed, while the Lc2 (load responsible for the first delamination) and Lc3 (load at which the coating is severely delaminated from the substrate) were clearly observed on the scratch tracks. The Lc2 and Lc3 values of FastOs®BG- and Cu&Ga-FastOs®BG-derived RF-MS coatings are presented in Figure 6b. The incorporation of Cu and Ga into the silica-rich SBG RF-MS layer led to a statistically significant (*p* < 0.05) increase of ~15%, with the Lc3 having a value of 4.9 ± 0.47 N. These values are similar to those (i.e., ~5 N) required to partially delaminate bioglass coatings of similar thickness, deposited by pulsed electron deposition (PED) from a silica-rich system (mol.%: SiO2–47.2, Ca–45.6, P2O5–2.6, K2O–4.6), and superior to those (~2 N) of 600 ◦C heat-treated 45S5 PED coatings [76].

**Figure 6.** Summary of the mechanical performance of the SBG films in terms of (**a**) pull-off bonding strength (tensile), (**b**) scratch resistance, (**c**) hardness and (**d**) elastic modulus. \* *p* < 0.05, statistically significant differences, as determined by using an unpaired two-tailed Student's *t*-test.

The average hardness and elastic modulus values of the SBG coatings, determined by nano-indentation measurements, are presented in Figure 6c,d, respectively. The Cu & Ga doping moderately increased (*p* < 0.05) the hardness of the RF-MS film to a value of ~6.1 GPa (Figure 6c), whilst the elastic modulus slightly decreased (*p* < 0.05) to ~127 GPa (Figure 6d). The nano-mechanical performances were similar (E) or even higher (H) with respect to titanium (e.g., E ≈ 120 GPa, H ≈ 3.6 GPa [58,77]).

Furthermore, the wear behaviour against a steel Rockwell tip, in linear motion, was assessed using the same MicroScratch tester. The wear tracks obtained on the uncoated (Figure 7a) and FastOs®BG-coated (Figure 7b) Ti substrate were severe, regardless of the applied load size (1–5 N), with increasing widths and depths of the wear track for higher applied loads. In contrast, the Cu&Ga-FastOs®BG-derived coating showed a significantly enhanced wear behaviour (Figure 7c). For the lowest applied loads, namely 1, 2 and 3 N, some wear grooves are visible on the surface, but to a much smaller extent compared to the uncoated and FastOs®BG-coated Ti substrate. Moreover, the width of the wear tracks on the Cu&Ga-FastOs®BG coated sample is significantly smaller. This is a good indicator of an improved wear behaviour. No signs of delamination are visible for these loads. For higher applied loads, namely 4 and 5 N, coating failures (delamination) are visible on the wear tracks, after 10 reciprocating passes. These values are in good agreemen<sup>t</sup> with the ones obtained during the adhesion tests. The wear coefficients determined on the basis of the wear volume loss under a load of 5 N as a function of the total test length are presented in Figure 7d. As hinted by the optical microscopy images, the wear coefficient is significantly improved by the application of the Cu&Ga-FastOs®BG-derived coating. The so-called elastic strain to failure ratio (H/E) is a reliable indicator of wear resistance of materials, with values higher than 0.1 usually indicating a tough coating. Even though this value is not reached by the coatings presented herein, a slightly higher value of this ratio is exhibited by the Cu&Ga-FastOs®BG-derived coating, in good agreemen<sup>t</sup> with the other wear parameters (wear coefficient, wear track width). These results are quite promising at this stage of the research development, especially considering that bio-glasses are relatively soft.

**Figure 7.** Optical microscopy images of the wear tracks recorded at five incremental load values (i.e., 1, 2, 3, 4 and 5 N), with 10 passes per load at a displacement rate of 4 mm/min, in the case of the (**a**) bare and (**b**) FastOs®BG- and (**c**) Cu&Ga-FastOs®BG-coated Ti substrate. (**d**) The wear coefficient determined for the three studied specimens under the load of 5 N.

### *3.3. In Vitro Preliminary Biological Evaluation*

### 3.3.1. Antibacterial <sup>E</sup>fficacy at 24 h

Since dental implant coatings are the main targeted application, the antibacterial properties of the SBG films were preliminarily assessed against the Gram-positive *S. aureus* (ATCC® 6538) bacterial strain, both because of its presence in oral microbiota, and according to the ISO 22196:2011 recommendation [56]. 50,000 CFUs were seeded onto the samples and allowed to grow for 24 h. The silica-rich FastOs®BG did not impede the bacterial proliferation, whilst the Cu&Ga-FastOs®BG layer was found to reduce the bacterial development by 30 times with respect to the seeding CFU, and with 4 orders of magnitude with respect to the control situations (i.e., nutritive broth and bare substrate) (Figure 8). This output could be considered encouraging for combating the microbial infection at the implantation site. However, future dynamic studies need to be carried out, at several time intervals, and against a large palette of microbial strains, in order to fully probe and unveil the complete potential of such implant-type coatings.

**Figure 8.** Antibacterial activity against *S. aureus* at 24 h. The data are presented in logarithmic values of CFUs (bacterial survival). (1) seeded CFUs; (2) control = nutrient broth without sample; (3) bare and silica-rich (4) FastOs®BG and (5) Cu&Ga-FastOs®BG coated substrate. \* *p* < 0.05, statistically significant differences, as determined by using an unpaired two-tailed Student's *t*-test.

### 3.3.2. Cytocompatibility Response at 24 h

The cytocompatibility of the samples was tested on NIH/3T3 fibroblast cell cultures (ATCC® CRL-1658TM). The cell proliferation was assessed by the MTS assay (Figure 9a), while the cell death was evaluated by a LDH test. Both silica-rich FastOs®BG and Cu&Ga-FastOs®BG coatings elicited an excellent proliferation of cells, with values similar to those recorded on the standard substrate for cell cultures (i.e., polycarbonate for cell cultures) (Figure 9a). Furthermore, the Cu&Ga-FastOs®BG-derived coatings showed lower values of LDH activity with respect to the FastOs®BG-derived ones, being situated closer to the ones recorded for the polycarbonate surface for cell cultures (Figure 9b). Thereby, it is advocated that the introduction of low concentrations of Cu and Ga into the thin FastOs®BG-derived films does not alter their cytocompatibility. When cultured on the silica-rich FastOs®BG- and Cu&Ga-FastOs®BG-derived coatings, the cells retained their characteristic morphology as seen in Figure 9c,d, respectively. Actin filaments grouped in bundles spanned the cells in a usual manner, whilst the nuclei retained their normal shape and chromatin condensation pattern.

**Figure 9.** (**a**) Cell viability/proliferation assessed by MTS assay and (**b**) cytotoxicity assessed by LDH release after 24 h. (1) seeded cells; (2) biological control—polycarbonate; (3) bare and silica-rich (4) FastOs®BG and (5) Cu&Ga-FastOs®BG coated substrate. \* *p* < 0.05, statistically significant differences, as determined by using an unpaired two-tailed Student's *t*-test. Epi-fluorescence microscopy images revealing the morphology of 3T3 fibroblast cells grown on the silica-rich: (**c**) FastOs®BG and (**d**) Cu&Ga-FastOs®BG films. The actin cytoskeleton was stained with phalloidin-AlexaFluor596 (red), whilst cell nuclei were counterstained with DAPI (blue). Objective: 40×. Magnification bar: 50 μm.

When analysing the antibacterial activity and cytocompatibility responses together, it is suggested that the application of layers of Cu&Ga-FastOs®BG on metallic endo-osseous implants represents a promising conceptual solution to minimize the risk of post-surgical bacterial infection.
