**1. Introduction**

The last decades witnessed an unprecedented demand for innovative bio-functional materials, capable of not only preventing failure, but also prolonging the life time of orthopaedic/dental implants and bone grafting sca ffolds. Silica-based bioactive glasses (SBG) represent a group of reactive biomaterials that possess outstanding features (e.g., bioactivity, osteoproduction, angiogenesis), which are crucial for assuring an excellent interfacial bonding between the host bone tissue and implants [1–7]. The continuous e ffervescence in the SBG realm, associated with its unique physicalchemical and biological functional properties [2,5,7], is certified by a progressive yearly increase in the number of publications [5].

Most of the reported SBG compositions investigated so far were inspired by the 45S5 silicate glass (mol.%: SiO2—46.1, Na2O—24.4, CaO—26.9, P2O5—2.6), developed by Hench and his co-workers [8]. Over 30 years since its first implantation in 1985, 45S5 bioactive glass has been implanted into 1.5 million patients worldwide under various forms and trade names (e.g., Perioglas ®, NovaBone ®, Biogran ®, NovaMin ®, TeraSphere ®) [2,5]. Due to their high indices of bioactivity, 45S5-based formulations have been proposed as coating materials for the bio-functionalisation of various metallic titanium-based implants [7,9], to enhance their bone-bonding ability. However, the disparity of the coe fficients of thermal expansion (CTE) of 45S5 (i.e., ~14–17 × 10−<sup>6</sup> ◦C−1) and titanium-based (i.e., ~8.5–9.6 × 10−<sup>6</sup> ◦C−1) materials cannot be circumvented with ease [10], and represents a hazard for the long-term mechanical performance of the implant coating. Furthermore, because of the burst release of high concentrations of sodium and overall excessive bio-reactivity [5,11], 45S5 bioactive glass could generate highly alkaline pH environments, which, in the absence of preconditioning procedures could lead to less than favourable biological outcomes [5,12]. More recently, alkali-free SBGs such as FastOs ®BG (mol.%: SiO2—38.49, CaO—36.07, P2O5—5.61, MgO—19.24, CaF2—0.59) have been introduced and acknowledged for their notable performances such as reduced CTEs mismatch, chemical durability, high biomineralization capacity and excellent cytocompatibility [5,9,13].

The possibility to provide new biological functionalities to SBG materials by the inclusion of therapeutic ions within their chemical structure has changed the paradigm in this field, opening new avenues in healthcare and tissue regeneration. Various biologically active elements (e.g., boron, cobalt, magnesium, strontium, zinc) commonly present in the human body have been incorporated in SBGs, as medicinal micronutrients to promote bone regeneration [2,3,5,14,15]. These active ions could influence to some extent the structural properties (e.g., morphology, crystallinity, solubility) of these glasses and their biological responses (e.g., degradability, stem cells di fferentiation and proliferation, osteogenesis, angiogenesis) [5,16–18]. Upon the dissolution of glasses in the physiological environment, these ions are released and induce additional therapeutic e ffects.

A growing issue in biomedicine is the increased number of implant failures associated to microbial infection. Thus, it is of paramount importance to provide antibacterial properties to the implant by the addition of specific antimicrobial agents. In this context, various studies have reported the benefit of silver and zinc added as antimicrobial agents in SBGs [5,15,17]. More recently, gallium (Ga) and copper (Cu) have emerged as a potent new generation of antibacterial ions that may be useful in treating and preventing localized infections [19–29]. Valappil et al. [19] reported on the positive bactericidal effects of gallium-substituted phosphate-based glasses (1–5 mol.% Ga2O3) against *Escherichia coli*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, methicillin-resistant *S. aureus* and *Clostridium di*ffi*cile*. The bactericidal activity due to Ga3+ ions was found e ffective for a Ga2O3 concentration as low as 1 mol.% [19]. The potential antimicrobial activity of gallium against *E. coli* and *S. aureus* [22], as well as *P. aeruginosa* [23], has been further confirmed in Ga-substituted SBGs. In relation to CuO-substituted SBGs, recent studies have also demonstrated the potency of Cu to eradicate the *E. coli* [24,25,28], *P. aeruginosa* [28], *S. aureus* [21,25,27], *Salmonella enterica* [28] and *Staphylococcus epidermidis* [25] bacterial strains. Moreover, Cu-substituted SBG-derived glass-ceramic was acknowledged for its excellent antimicrobial e fficiency (≥99.9% reduction) against *P. aeruginosa*, *E. coli*, *Klebsiella aerogenes* and *S. aureus* [29]. Furthermore, Cu seems to have the ability to be a fungicide as shown in both

silica- [26] and apatites-based materials [16]. The association of both Cu and Ga, as co-dopants in SBGs, can thereby be an attractive way to significantly boost and extend the antimicrobial range of such biomaterials. Furthermore, Cu and Ga have other biological benefits, strengthening their potential in healthcare. For example, Cu containing SBGs were reported to encourage the proliferation and di fferentiation of cells towards the osteogenic phenotype and inhibit osteoclast activity [14,30,31]. A similar behaviour has been reported for Ga ions [23,32,33].

Such results advocate for the investigation of the coupled e ffects of Cu and Ga in alkali-free SBG implant coatings. The benefits that could emerge from the future implementation of co-substituted SBG implant coatings, possessing both e ffective therapeutic response and adequate mechanical behaviour is nowadays a subject of intense research activity.

In this context, radio-frequency magnetron sputtering (RF-MS), a prominent physical vapour deposition technique, has been selected for the fabrication of implant coatings, due to its proficiency to synthesise high purity uniform films, and the potential to scale-up a refined process to industrial levels [5]. To the best of our knowledge, no studies have been performed ye<sup>t</sup> on RF-MS synthesised SBG films containing Cu and/or Ga. As aforementioned, the poor interfacial bonding strength of typical SBG films may constitute their Achilles' heel in terms of their clinical applicability. The engineering of SBG film compositions by tuning the network formers/network modifiers ratio could be a strategy to follow when targeting mechanically performant SBG films. Previous attempts have shown that SBG RF-MS layers with a silica content in the range of ~55–60 mol.% presented excellent pull-o ff bonding strength values, exceeding 85 MPa [34], thus, being compliant to both recommendations of ISO 13779-2:2018 (minimum value of 15 MPa) [35] and FDA—STP1196:1994 draft guidance (minimum value of 50.8 MPa) [36] for implant coatings.

In this work, results concerning thin silica-based implant-type coatings with double addition of potentially antimicrobial Cu and Ga agents are presented for the first time, in terms of preliminary mechanical and in vitro biological behaviour. The study could be of importance for an accelerated development of SBG-based implant coatings with excellent mechanical properties, good cell viability and proliferation and high antimicrobial e fficiency.

### **2. Materials and Methods**

### *2.1. Synthesis of Source Cathode Target Materials*

FastOs ®BG (mol.%: SiO2—38.49, CaO—36.07, P2O5—5.61, MgO—19.24, CaF2—0.59) and Cu and Ga co-substituted FastOs ®BG (mol.%: SiO2—38.49, CaO—34.07, P2O5—5.61, MgO—16.24, CuO—2.00, Ga2O3—3.00, CaF2—0.59) glasses were prepared by melt-quenching, following the protocol reported elsewhere [37,38]. The glass powders with fine granularity were obtained in a high-speed agate ball mill. The mean particle size of the SBG powders was of ~20–40 μm, as inferred from the particle size distribution curves obtained by the dynamic light scattering method (Beckman Coulter, model LS 230, Brea, CA, USA) by applying the Fraunhofer optical di ffraction model.

Around 25 g of each SBG powder were mildly pressed (5 kgf) at room-temperature (RT) into copper dishes (diameter 110 mm, depth 3 mm) to obtain a magnetron cathode target. The benefits of using such type of targets (i.e., prevention of risk of target cracking and better control over the deposited coating composition) instead of classical ones, melt-poured into moulds, have been advocated in a previous work [39].

### *2.2. Deposition by RF-MS of Double-Layered Glass Coatings*

SBG coatings were deposited onto 10 mm × 10 mm titanium (Mateck GmbH, Jülich, Germany) and silicon (Medapteh, Magurele, Romania) mirror polished substrates with a Vacma UVN-75-R1-type deposition system (Kazan, Soviet Union), equipped with planar magnetron cathodes (110 mm in diameter) and a RF generator of 1.78 MHz. The layer thicknesses were adjusted based on the deposition rates previously determined by spectroscopic ellipsometry.

The FastOs ®BG and Cu and Ga substituted FastOs ®BG layers with a thickness of ~600 nm were intentionally enriched in silica by applying the RF-MS protocol defined in Reference [40], with fused silica plates (Präzisions Glas & Optik GmbH, Iserlohn, Germany) positioned on the racetrack (annular zone of maximum sputtering erosion). All depositions were carried out at an argon pressure of 0.4 Pa and a target-to-substrate distance of 35 mm.

### *2.3. Physico-Chemical Characterization*

The films' thickness was determined by spectroscopic ellipsometry (SE) using a Woollam apparatus (Lincoln, NE, USA) equipped with a HS-190 monochromator. The measurements were made at 65◦, 70◦ and 75◦ incidence angles, with a resolution of 3◦, in the 400–1700 nm spectral range. The SBG films were modelled using a Cauchy dispersion relation [41,42].

As their morphology was extremely smooth, the topological features of the SBG films were explored by atomic force microscopy (AFM), using an NT-MDT NTEGRA NanoLaboratory Probe system (Moscow, Russia). AFM images were recorded in non-contact mode on 5 × 5 μm<sup>2</sup> areas.

The elemental compositions of the source SBG materials and derived RF-MS coatings were estimated by energy dispersion X-ray spectroscopy (EDXS) using the EDAX Inc. (Mahwah, NJ, USA) micro-probe attached to a scanning electron microscope, operated at an acceleration voltage of 10 kV. The measurements were made on at least four randomly chosen sample regions with areas of 250 × 250 μm2. Only the elemental (in at.%) concentrations of Si, Ca, P, Mg, Cu and Ga were inferred and will be further represented as arithmetic means ± standard deviations, since the quantification of lighter elements (i.e., oxygen and fluorine) by EDXS is prone to large errors [43].

The chemical state of SBG elements was analysed by X-ray photoelectron spectroscopy (XPS) using a Specs GmbH Multimethod System (Berlin, Germany). The XPS system is equipped with a Phoibos 150 hemispherical energy analyser with a multi-element two-stage transfer lens and a nine channeltron detector array. The photo-emission studies were carried out at a pressure of ~10−<sup>7</sup> Pa, using a XR-50M Al K α (1486.7 eV) source coupled to a FOCUS500 single-crystal quartz monochromator. The X-ray source was set at 300 W. A pass energy of 20 eV was used for the high-resolution core level spectral recordings. An ultimate resolution of 0.44 eV (given as full width at half maximum of the Ag 3d5/2 peak recorded with a pass energy of 20 eV, using monochromated Al K α radiation, for a clean silver film) can be attained. The sample neutralization during the measurements was achieved by using a flood gun, working at acceleration energy of 1 eV and an emission current of 0.1 mA. In order to remove surface contaminants due to environmental/adventitious carbonaceous species, an in situ preliminary argon ions etching (using a Specs IQE11/35 ion gun) session was performed for 5 min, at an energy of 3 keV and a pressure of 1 × 10−<sup>3</sup> Pa. The background was subtracted using the Shirley method. The fitting of spectra was performed with a dedicated software Spectral Data Processor using Voigt functions, version 3.0 (XPS International, Mountain View, CA, USA).

The structure of the SBG films was investigated by Fourier transform infrared (FTIR) spectroscopy measurements with a Perkin Elmer Spectrum BX II (Waltham, MA, USA) spectrophotometer. The FTIR spectroscopy investigations were performed in transmission mode, on films deposited onto infrared transparent Si substrates. The spectra were acquired in the wave numbers range of 4000–400 cm–1, at a resolution of 4 cm<sup>−</sup>1, and represent the average of 32 individual scans.

The surface free energy of the SBG films was determined based on static contact angle measurements (performed at least in triplicate), using a Drop Shape Analysis system (model DSA 100) from Krüss GmbH (Hamburg, Germany) and two standard solutions, one polar (i.e., water) and one dispersive (i.e., diiodomethane). The measurements were performed at RT, with the two solutions being precisely poured in droplets on the sample surface via a needle attached to a syringe pump controlled by the DSA3 ® software (Hamburg, Germany). The volume of the droplet (i.e., 1 μL) and the distance from the droplet to the sample was kept constant throughout the experiments. The contact angles were estimated, and the surface energy was calculated using the Owens–Wendt method [44].

The bonding strength of the films to the Ti substrate was estimated by the pull-o ff test method, using a DFD ®Instruments PAThandy adhesion tester (Kristiansand, Norway), with a maximum force of 1 kN, and applying the testing procedure described in References [45,46], conducted in accordance with the ISO 4624:2016 [47] and ASTM D4541—17:2017 [48] standards.

The nano-mechanical properties were evaluated by nano-indentation tests using an NHT-2 CSM Instruments/Anton Paar GmbH (Peseux, Switzerland), equipped with a Berkovich diamond nano-indenter. The Oliver–Pharr method [49] was used to determine the modulus of elasticity (E) and hardness (H) of films from the load-displacement curves. The penetration depth was chosen in such a manner to minimize as much as possible the influence of the substrate on the results.

The scratch and wear resistances were evaluated with a dedicated MicroScratch Tester from CSM Instruments (Peseux, Switzerland), equipped with a diamond Rockwell conical indenter. The scratch test involves the passage (with a progressive applied load) of the Rockwell indenter, on the surface of the sample to result in a scratch. The load applied on the conical indenter increases progressively producing elastic and plastic deformations of the layer-substrate systems, which are becoming more and more pronounced. Conventionally, three critical load values are detected: (i) the load which causes the first deterioration of the cohesion of the layer (first cracks) (Lc1); (ii) the load leading to the partial removal of the coating (partial delamination) (Lc2); and (iii) the applied load that causes the removal of at least 50% of the deposited layer (Lc3) [50,51]. The device is equipped with sensors that measure and record the acoustic emissions during the scratch process, the penetration depth and the friction force. The adhesion to the substrate, as well as the cohesion of the SBG layers were evaluated according to the European standard EN1071-3/2005: "Advanced technical ceramics—Methods of test for ceramic coatings—Part 3: Determination of adhesion and other mechanical failure modes by a scratch test" [52] and the "Scratch test atlas of failure modes" guide [53]. The abrasive wear behaviour of the SBG films was determined using the same MicroScratch Tester, by using a heat-treated steel tip (Rockwell conical geometry) (HV = 1000) to employ a friction torque. While the sample is in translation motion, the frictional forces that occur between the sample and the tip are measured using a Linear Variable Di fferential Transformer sensor (CSM Instruments, Peseux, Switzerland). Five incremental load values (i.e., 1, 2, 3, 4 and 5 N) were used, with 10 passages per load at a travel speed of 4 mm/min. The wear track section areas for each sample and each applied load were assessed by transversal scanning with the same MicroScratch tester, using a 0.03 N applied load. The wear coe fficient was calculated on the basis of the wear volume loss at the maximum used load (5 N), as function of the total test length.

### *2.4. In Vitro Preliminary Biological Testing*

Prior to each in vitro test all samples were sterilized by applying a dry-heat procedure performed at 180 ◦C for 1 h [54,55].

### 2.4.1. Antibacterial Tests

The antimicrobial activity against the *Staphylococcus aureus* (ATCC ® 6538) bacterial strain was performed according to the ISO 22196:2011 standard [56]. Briefly, suspensions of *S. aureus* in nutritional broth (Sanimed International Impex SRL, Bucharest, Romania) were prepared at a concentration of 10<sup>6</sup> CFU/mL. 50 μL of nutrient broth containing 5 × 10<sup>3</sup> viable CFUs were deposited on the surface of the specimens. Then, an inert sterile plastic film was placed over the samples and the plates were inserted into the incubator. After 24 h, 3 mL of nutrient broth was added to each well and the samples were completely scraped with a dedicated sterile rubber scrubber to detach any bacterial cells that could be encapsulated in the biofilm. 10-serial dilutions were prepared for each studied situation, and from each sample dilution two simple agar plates were seeded with 1 mL of the inoculum. After 24 h of incubation, the colonies on the plates were counted. The number of viable CFUs developed after 24 h in the presence of control and SBG coatings were calculated with the formula: 3 × dilution factor × (average number of colonies on plates).
