Bioactive Properties of Composites Based on Silicate Glasses and Different Silver and Gold Structures

Using an ideal biomaterial to treat injured bones can accelerate the healing process and simultaneously exhibit antibacterial properties; thus protecting the patient from bacterial infections. Therefore, the aim of this work was to synthesize composites containing silicate-based bioactive glasses and different types of noble metal structures (i.e., AgI pyramids, AgIAu composites, Au nanocages, Au nanocages with added AgI). Bioactive glass was used as an osteoconductive bone substitute and Ag was used for its antibacterial character, while Au was included to accelerate the formation of new bone. To investigate the synergistic effects in these composites, two syntheses were carried out in two ways: AgIAu composites were added in either one step or AgI pyramids and Au nanocages were added separately. All composites showed good in vitro bioactivity. Transformation of AgI in bioactive glasses into Ag nanoparticles and other silver species resulted in good antibacterial behavior. It was observed that the Ag nanoparticles remained in the Au nanocages, which was also beneficial in terms of antibacterial properties. The presence of Au nanoparticles contributed to the composites achieving high cell viability. The most outstanding result was obtained by the consecutive addition of noble metals into the bioactive glasses, resulting in both a high antibacterial effect and good cell viability.


Synthesis of Silver Iodide
AgI particles were prepared via solvothermal synthesis by the addition of silver nitrate, sodium iodide, and PVP [24]. The solutions were prepared in two separate beakers. In the first beaker, silver nitrate (0.167 M) was dissolved in 20 mL of EG. In the second beaker, sodium iodide (0.077 M) and PVP (0.0036 M; calculated to units of PVP) were dissolved in 100 mL of EG and stirred for 1 h at 60 • C. After combining the contents of the two beakers, the synthesis mixture was stirred for another hour. A yellowish-green precipitate was formed in an instantaneous reaction, indicating the formation of AgI. After stirring, the suspension was crystallized at 160 • C in an autoclave (160 mL) for 2 h. The sample was then purified with 3 × ≈35 mL Milli-Q water and 1 × ≈35 mL EtOH by centrifugation at 4400 rpm for 10 min, then dried at 40 • C for 12 h.

Synthesis of Spherical Gold Nanocages
The synthesis of spherical AuNCs was based on the galvanic replacement method in which AgNPs were exchanged [26,27]. In the first part of the synthesis, a sol containing AgNPs was prepared. For this purpose, 227 mL of Milli-Q water was mixed with 33 mL (2.31 mM) of sodium citrate solution. After stirring for 30 min, 2.8 mL (83.67 mM) of silver nitrate solution was added to the mixture. After stirring for another 30 min, the AgNPs were reduced with sodium borohydride (5.25 mL, 0.55 M).
The resulting solution containing AgNPs was transferred to a reflux flask, heated to 100 • C under continuous reflux, and held at that temperature for 10 min. Then, 3.15 mL of 25.39 mM HAuCl 4 ·3H 2 O solution was added dropwise and the system was kept at 100 • C for 20 min and refluxed continuously. The solution containing AuNPs was cooled to room temperature and ≈18 g of NaCl was added to facilitate the dissolution of the resulting AgCl. Impurities were removed from the final solution by washing three times with 0.5 mM Pluronic ® F-127 polymer solution.

Synthesis of Silver Iodide with Spherical Gold Nanocages
A composite of silver iodide with spherical gold nanocages (AgIAu) was fabricated using AgI and AuNCs. First, a sol containing AuNCs was prepared and purified by washing three times with water. Second, AgI was prepared by adding 50 mg of AgI to 100 mL of distilled water under ultrasonication for 15 min. Third, the aqueous gold sol was added to the silver-containing suspension that was washed with 3 × ≈35 mL distilled water in a centrifuge at 4400 rpm for 5 min, then dried at 40 • C for 12 h. For the thermal stability analysis, the AgIAu composite was heat treated at 500 • C in air for 3 h.

Synthesis of Bioactive Glasses with Silver and Gold Content
In the sol-gel derived bioactive glass (BG) noble metals (AgI and AuNCs) were introduced in two different ways: (i) with preliminary composite mixing, i.e, with the addition of AgIAu; and (ii) consecutively added to the system. Reactants were added consecutively after 1 h intervals under continuous stirring, as described in our previous study [27,28]. Finally, the solution of colloidal AgI, AuNCs, AgIAu, AgI, and AuNCs was added and stirred for 1 h. The solutions were stored in a closed container at 37 • C until gelation was reached (~48 h). The obtained gels were aged 3 days at 37 • C, after that they were dried at 110 • C for 24 h. Material stabilization was carried out at 500 • C in air for 3 h. Prior to characterization, the samples were milled by hand in a ceramic mortar. Table 1 contains the abbreviations of the samples and details about the compositions. In the composites, the AuNCs:AgI ratio was 24:76. Glass composites are traditionally calculated in mol%. For comparison purposes, the amount of silver was expressed in at% in each sample, and the information is presented in Table 1.

Characterization of AgIAu Composites
The crystalline composition of the AgIAu composites was identified by using a Shimadzu X-ray diffractometer (XRD 6000, Kyoto, Japan), operating with CuKα radiation (λ = 1.54 Å) and a Ni filter. The diffraction patterns were recorded in the 2θ range from 10 • to 80 • with a speed of 2 • /min. The specified band for AgI and the typical localized surface plasmon resonance band for Au nanocages were identified with a Jasco-V780 spectrophotometer equipped with an ILV-724 integrating sphere (UV-Vis, Jasco, Wien, Austria) with spectra resolution of 2 mm. The morphology of the samples was determined with a FEI Technai G2 20 X-TWIN transmission electron microscope (TEM, FEI, Hillsboro, OR, USA) and a Hitachi S-4700 Type II scanning electron microscope (SEM, Hitachi, Tokyo, Japan).

Structural and Morphological Characterization of Bioactive Glass Composites
Elemental composition of the glass samples was examined using an energy dispersive X-ray spectroscope (EDX, Hitachi, Tokyo, Japan) with Röntec XFlash Detector 3001 SDD system. The morpho-structural properties of the samples were analyzed by XRD and FT-IR spectroscopy. The FT-IR absorption spectra were recorded with a Jasco 6600 FT-IR spectrometer (IR, Jasco, Tokyo, Japan) within the range of 400-4000 cm −1 and a spectra resolution of 4 cm −1 by using the KBr pellet technique.

In Vitro Bioactivity Assays
The in vitro bioactivity assays were analyzed by immersion in SBF (pH = 7.4; 37 • C) for 7 days. An incubator was used to reach 37 • C and the concentration of bioactive glasses was 10 mg·mL −1 . After 7 days the samples were filtered, rinsed with ultrapure water, and dried. The formation of a hydroxycarbonate apatite (HCA) layer on the surface of the samples was investigate by XRD, FT-IR, UV-Vis, and SEM measurements. FT-IR spectra, XRD diffractograms, and an SEM micrograph were achieved with the same equipment described above. To analyze surface changes before and after immersion in SBF, XPS spectra were recorded with a SPECS PHOIBOS 150 MCD system employing a monochromatic Al-K α source (1486.6 eV), a hemispherical analyzer, and a charge neutralization device. Samples were fixed on double-sided carbon tape, and care was taken to ensure that the sample particles covered the tape. Experiments were performed by operating the X-ray source with a power of 200 W, while the pressure in the analysis chamber was in the range of 10 −9 -10 −10 mbar. The binding energy scale was charge referenced to that of C1s at 284.6 eV. Elemental composition was determined from the survey spectra acquired at a pass energy of 60 eV. High-resolution spectra were obtained using an analyzer pass energy of 20 eV. Analysis of the data was carried out with CasaXPS software.

Cell Viability Assay
Cell viability assessment was carried out on a human epidermal keratinocyte cell line (HaCaT, Cell Line Service, Eppelheim, Germany), via the same method that we described in our previous publications [10,27]. The cells were cultured in Dulbecco's modified Eagle's medium (Lonza) supplemented with 2 mM L-glutamine, Pen/Strep 100 U/mL, and 10% FC. Then, they were incubated in a humidified incubator under 5% CO 2 atmosphere at 37 • C. The cytotoxic effects of different samples were assayed with a WST-1 dye (Merk Millipore, Burlington, MA, USA). The HaCaT cells were seeded in a 96-well plate, at a density of 1 × 10 4 cells/well. The following day, different amounts of the samples were added to the test wells and cells were placed in the incubator for an additional 24 h. All glass samples were tested at three concentrations (75 µg·mL −1 , 150 µg·mL −1 , and 300 µg·mL −1 ). Untreated cells were used as controls. At the end of the incubation period, the medium was removed from all wells, and 100 µL of fresh medium containing 10% WST-1 solution was added to each well. The cells were incubated for another 60 min at 37 • C. Empty wells with medium containing WST-1 reagent were used as blanks. Viability of HaCaT cells after 24 h of treatments was determined by measuring the absorbance at 440 nm with a microplate reader (Fluostar Omega, BMG Labtech, Ortenberg, Germany).

Microorganism and Culture Conditions
One aerobic gram-negative Pseudomonas aeruginosa (ATCC 27853) and one grampositive Staphylococcus aureus (ATCC 49444) bacterial strain were used for antibacterial activity analysis. The tested microorganisms were obtained from Food Biotechnology Laboratory, Life Sciences Institute, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania. Both bacteria were cultured on Mueller-Hinton Agar and cultures were stored at 4 • C and subcultured once a month.

Microdilution Method
A modified microdilution technique, based on the regulations of the Clinical Laboratory Standard Institute (CLSI), was used to evaluate antimicrobial activity [29]. Bacterial species were cultured overnight at 37 • C in Mueller-Hinton Broth (MHB). The bacterial cell suspensions were adjusted with a 0.9% NaCl sterile saline solution to a concentration of approximately 3 × 10 5 colony-forming units (CFU)/mL in a final volume of 100 µL per well. The inoculum was stored at +4 • C for further use. Dilutions of the inoculum were cultured on solid Mueller-Hinton (MH) for bacteria to verify the absence of contamination and check the inoculum's validity. Determinations of minimum inhibitory concentrations (MICs) were performed by a serial dilution technique using 96-well microtitre plates. The samples were transferred to the wells containing 100 µL MHB, and afterward, 10 µL of inoculum was added to all the wells. The microplates were incubated for 18 h at 37 • C. The MIC of the samples were detected following the addition of 20 µL (0.2 mg·mL −1 ) of resazurin solution to each well, and the plates were incubated for 2 h at 37 • C. A change from blue to pink indicated a reduction in resazurin and, therefore, bacterial growth. The MIC was defined as the lowest drug concentration that prevented this color change. The concentration of the samples was 10 mg·mL −1 . A control sample gentamicin solution of the same concentration was used for the tested samples.

Statistical Analysis
All data in the cell viability assay were reported as the mean ± SD of triplicate measurements. The values were analyzed by two-way analysis of variance ANOVA. Statistical significance was at p < 0.05 in all cases. Statistical values were obtained using GraphPad Prism 8.0 software. The antimicrobial analysis required no statistical validation as the method was one of the highest confidence interval methods [30].

Characterization of The Composites Containing AgI Microcrystals, Spherical Gold Nanocages, or AgIAu Nanostructures
After the synthesis of AgI microcrystals and AuNCs, their characterization was carried out to investigate their reproducibility. The results were compared with those published in our previous work [24,27]. The UV-Vis spectra, XRD patterns, and SEM micrographs of the AgI microcrystals confirmed the reproducibility of the samples as follows: (i) in the UV-Vis spectrum ( Figure 1A) of the sample the typical absorption band for AgI (415-480 nm; [31]) was identified; (ii) in Figure 1B the corresponding reflections of AgI (β-AgI-COD 00-101-1025 and γ-AgI-COD 00-901-1693) were observed; and (iii) the SEM images confirmed the pyramidal structure ( Figure 2A). It should be emphasized that AgI was semicrystalline and the two different crystal phases of AgI could not be distinguished because their reflections overlapped with each other [32]. reflection be identified but the reflection of Au (COD 00-900-8463) could also be identified. Moreover, in the TEM images small particles could be observed on the surface of the AgI microparticles ( Figure 2c). Unfortunately, the low stability of silver-based materials could also be noticed in Figure 2d as the surface of the particles changed with the energy of the electron beam, thereby their size could not be analyzed.     The UV-Vis spectra of the AuNCs showed the appropriate (localized) surface plasmon resonance band of AuNCs, located at ≈490 nm ( Figure 1A). In the TEM micrographs the presence of Au nanostructures of about 18 ± 3 nm were also observed ( Figure 2B). The first proof of successful composite mixing was obtained by UV-Vis measurements. Both the (localized) surface plasmon resonance band of AuNCs (located at 500 nm; insert graph Figure 1A) and the absorption band of AgI (between 350-470 nm) could be observed. The second proof was obtained by XRD measurements, revealing that not only could the AgI reflection be identified but the reflection of Au (COD 00-900-8463) could also be identified. Moreover, in the TEM images small particles could be observed on the surface of the AgI microparticles ( Figure 2C). Unfortunately, the low stability of silver-based materials could also be noticed in Figure 2D as the surface of the particles changed with the energy of the electron beam, thereby their size could not be analyzed.
In our previous work [27] it was shown that the gold nanocages introduced into the bioactive glasses were recrystallized into spherical gold nanoparticles by heat treatment at 500 • C. It has also been demonstrated by our group [24] that AgI microcrystals remained stable after heat treatment at 500 • C. Thermal stability of the AgIAu composite was investigated to determine possible transformations during heat treatment; thus, the composite was heat treated at 500 • C. After heat treatment, the reflection for Au and AgI were still presented, the gold became crystalline, and the AgI did not have any significant changes ( Figure 1B).

Characterization and In Vitro Bioactivity of Bioactive Glass Composites
EDX analysis was performed for elemental identification of composites. In Figure 3 the presence of all metal components can be seen. The silver and iodine values were comparable with theoretical values ( Table 2). The presence of gold was also confirmed, but due to the small amount the obtained value differed to the theoretical one.

Characterization and In Vitro Bioactivity of Bioactive Glass Composites
EDX analysis was performed for elemental identification of composites. In Figu the presence of all metal components can be seen. The silver and iodine values were parable with theoretical values ( Table 2). The presence of gold was also confirmed due to the small amount the obtained value differed to the theoretical one.    The XRD patterns (Figure 4) of gold-containing samples exhibited characteristic reflections of metallic gold (COD 00-900-8463). These signals were recorded both before and after immersion in SBF. In the BG-AgI sample an additional reflection was observed at 29.45 (2θ • ). This could be attributed to the presence of CaCO 3 (calcite) (COD 00-101-0928), which only appeared after the heat treatment of the sample. The formation of CaCO 3 could have originated from the high concentration of calcium ions and the textural parameters of the bioactive glasses [33].
After immersion in SBF for 7 days, all samples showed reflections associated with the presence of an HCA layer precipitated on the surface of the samples (COD file no. 09-0432). After soaking in SBF, AgCl (COD file no. 00-901-1666) was formed on the surface of the silver containing BG composites because of the high amount of NaCl in SBF [34]. The formation of AgCl occurred in the BG-AuNCs samples when the amount of Ag was low.
After immersion in SBF for 7 days, all samples showed reflections associa presence of an HCA layer precipitated on the surface of the samples (COD 0432). After soaking in SBF, AgCl (COD file no. 00-901-1666) was formed on of the silver containing BG composites because of the high amount of NaCl The formation of AgCl occurred in the BG-AuNCs samples when the amoun low.  Figure 5) [36]. In addition to the phosphate bands, a new band at 870 cm −1 was observed that could be ascribed to carbonate ions indicating the formation of a carbonate apatite phase.
SEM images ( Figure 6) confirmed that the BG had a porous structure before immersion in SBF. It was also ascertained that samples having a cauliflower-like structure showed good bioactivity. These structures confirmed the formation of the HCA layer on the surface of the BG composites.
The plasmon resonance band of AgNPs (≈357 nm) and gold nanoparticles (≈525 nm) could be identified in the UV-Vis spectra ( Figure 7A) of the BG composites before immersion in SBF. The plasmon resonance band of AgNPs (located at ≈394 nm) could also be identified in the BG-AuNCs composites. In these composites, a low amount of remaining silver was observed because their synthesis involves the addition of AgNPs as well. Ag + ions, with a signal around 260 nm, were visible only in the BG-AuNCs and BG-AgI+AuNCs. The similarity of these two samples was due to the fact that in both cases, AuNCs were added into the BG structure. As described before, the Ag + ions could originate from the silver that remained after the synthesis of AuNCs. The presence of Ag + ions could not be observed in the XRD patterns either because their amount was low or they were present in the amorphous phase. UV-Vis absorption bands around 290 and 250 nm of the BG samples' spectra appeared to be due to the presence of Si-O-(Si, Ca) and P-O-(P, Ca) groups, respectively [37].
After immersion in SBF for 7 days, the surface plasmon resonance of gold nanoparticles (at ≈414 nm and ≈ 425 nm) and the absorption band of Ag + ions (at around 260 nm) were detected ( Figure 7B). In addition, a band associated with the presence of AgCl was observed at 369 nm. The surface plasmon resonance signal was observed for the sample containing AuNCs. However, most probably these bands originated from the AgNPs that remained in the system during the galvanic replacement of AuNCs. For the AgI-containing samples, an additional plasmon resonance band correspond to AgNPs could be detected, presumably due to the transformation of AgI.
XPS measurements were also carried out to analyze the chemical state of the samples' surfaces and to provide the presence of the HCA layer. The proof for the formation of a HCA layer was that the atomic percentage of Si decreased after the immersion into SBF (Table 3). Another proof was the increase in the atomic percentages of Ca and P (Table 3). It is known that XPS measurements can only provide information from the surface of a sample (≈10 nm). Thus, based on the decreasing amount of Si, and the increase in Ca and P amounts, the HCA layer was formed on the surface of the composites [24]. The obtained Ca/P ratio suggested that the surface layer contained carbonates and non-stoichiometric components (decreasing from 2.3 to 1.33) [38].  SEM images (Figure 6) confirmed that the BG had a porous structure bef sion in SBF. It was also ascertained that samples having a cauliflower-lik showed good bioactivity. These structures confirmed the formation of the HC the surface of the BG composites.  The plasmon resonance band of AgNPs (≈357 nm) and gold nanoparticles (≈52 could be identified in the UV-Vis spectra (Figure 7a) of the BG composites before im sion in SBF. The plasmon resonance band of AgNPs (located at ≈394 nm) could a identified in the BG-AuNCs composites. In these composites, a low amount of rem silver was observed because their synthesis involves the addition of AgNPs as we ions, with a signal around 260 nm, were visible only in the BG-AuNCs and AgI+AuNCs. The similarity of these two samples was due to the fact that in both were detected (Figure 7b). In addition, a band associated with the presence of AgCl was observed at 369 nm. The surface plasmon resonance signal was observed for the sample containing AuNCs. However, most probably these bands originated from the AgNPs tha remained in the system during the galvanic replacement of AuNCs. For the AgI-contain ing samples, an additional plasmon resonance band correspond to AgNPs could be de tected, presumably due to the transformation of AgI. XPS measurements were also carried out to analyze the chemical state of the samples surfaces and to provide the presence of the HCA layer. The proof for the formation of a HCA layer was that the atomic percentage of Si decreased after the immersion into SBF (Table 3). Another proof was the increase in the atomic percentages of Ca and P (Table 3) It is known that XPS measurements can only provide information from the surface of a sample (≈10 nm). Thus, based on the decreasing amount of Si, and the increase in Ca and P amounts, the HCA layer was formed on the surface of the composites [24]. The obtained Ca/P ratio suggested that the surface layer contained carbonates and non-stoichiometric components (decreasing from 2.3 to 1.33) [38]. Table 3. Si, Ca, and P content of the heat-treated and immersed BG composites obtained from XPS measurements. For comparison, theoretical values were also calculated.

Sample
Elements (   High-resolution Ca2p and P2p spectra were also recorded (Figure 8), and the binding energy of 348 eV and 351.5 eV were attributed to Ca2p orbitals, while the one at 133 eV to P2p orbitals [24,38]. After immersion in SBF, both photoelectron peaks broadened and their position changed: for Ca2p, it changed from ≈348 eV to ≈349.3 eV, and from 351.5 eV to 352.64 eV; while for P2p, it changed from ≈133 eV to ≈135 eV. The broadening of the Ca2p peak could be due to the transformation of CaO into CaI 2 and Ca(IO 3 ) 2 as was proved in our previous study [24]. These transformations were not surprising since the presence of AgNPs was confirmed based on the UV-Vis spectra (Figure 7). This meant that the AgI in the BG transformed into AgNPs and other species of silver and into CaI 2 and CaIO 3 . Moreover, for the BG-AuNCs the degree of these broadenings was low and could be attributed to the formation of the apatite layer ( Figure 8).

Influence of Silver and Gold on Cell Viability and Antibacterial Activity
The toxicity of the silver content was evaluated via a cell viability assay following the viability of HaCaT cells in contact with the BG samples for 24 h. First, it needs to be mentioned that, independently of the BG composites used, and the different concentration of glasses, all samples showed cell viability greater than 100% (Figure 9). Therefore, it could be concluded that the composites were not cytotoxic.

Influence of Silver and Gold on Cell Viability and Antibacterial Activity
The toxicity of the silver content was evaluated via a cell viability assay following the viability of HaCaT cells in contact with the BG samples for 24 h. First, it needs to be mentioned that, independently of the BG composites used, and the different concentration of glasses, all samples showed cell viability greater than 100% (Figure 9). Therefore, it could be concluded that the composites were not cytotoxic. The next step of our research was to investigate the antibacterial character of the sa ples using two different bacterial strains: S. aureus (gram-positive; Figure 10, self-colo bar) and P. aeruginosa (gram-negative; Figure 10, bar with pattern). Since the gram-ne tive bacterial strains had a thinner cell wall than that of S. aureus, the outstanding antib terial character of the samples was not surprising. BG-AgI showed the highest antibac rial character, which could have originated from the transformation of AgI into AgN as confirmed by the UV-Vis spectra (Figure 7a). Reasonable antibacterial activity was served for BG-AuNCs, which could be due to the low amount of silver, as confirmed the UV-Vis spectra (Figure 7a). Composites of BG-AgIAu and BG-AgI+AuNCs show good antibacterial activity against P. aeruginosa. The composites, where the noble me based particles were added consecutively (BG-AgI+AuNCs) showed a higher resista to S. aureus, from which it was concluded that this approach was best. The silver-free gl sample has no antibacterial effect (data not shown).  The next step of our research was to investigate the antibacterial character of the samples using two different bacterial strains: S. aureus (gram-positive; Figure 10, selfcolored bar) and P. aeruginosa (gram-negative; Figure 10, bar with pattern). Since the gramnegative bacterial strains had a thinner cell wall than that of S. aureus, the outstanding antibacterial character of the samples was not surprising. BG-AgI showed the highest antibacterial character, which could have originated from the transformation of AgI into AgNPs, as confirmed by the UV-Vis spectra ( Figure 7A). Reasonable antibacterial activity was observed for BG-AuNCs, which could be due to the low amount of silver, as confirmed by the UV-Vis spectra ( Figure 7A). Composites of BG-AgIAu and BG-AgI+AuNCs showed good antibacterial activity against P. aeruginosa. The composites, where the noble metalbased particles were added consecutively (BG-AgI+AuNCs) showed a higher resistance to S. aureus, from which it was concluded that this approach was best. The silver-free glass sample has no antibacterial effect (data not shown).

Staphylococcus aureus Pseudomonas aeruginosa
Based on the results, we could conclude that the addition of Ag and Au enabled the bioactive glasses with both antibacterial activity and with the ability to enhance the viability of HaCaT cells. Furthermore, the best combination was obtained when silver and gold content were consecutively added into the BG instead of creating a composite out of them beforehand by mixing. served for BG-AuNCs, which could be due to the low amount of silver, as confirme the UV-Vis spectra (Figure 7a). Composites of BG-AgIAu and BG-AgI+AuNCs sho good antibacterial activity against P. aeruginosa. The composites, where the noble m based particles were added consecutively (BG-AgI+AuNCs) showed a higher resist to S. aureus, from which it was concluded that this approach was best. The silver-free sample has no antibacterial effect (data not shown).

Conclusions
Four different composites were prepared to investigate the synergistic effect between bioactive glass composites and two noble metals. From these four samples two were synthesized as reference materials: one containing only silver iodide and the other one containing predominantly Au nanocages. Another two samples were fabricated that contained both noble metals, either by adding them consecutively or as a AgIAu composite. In addition, noble-metal-free bioactive glass was synthetized for comparison purposes. Based on morphological and structural characterizations, bioactive glasses with amorphous structures were successfully obtained independently of the noble metal or noble-metal composites used. All composites presented excellent bioactivity and induced the proliferation of Ha-CaT cells. Composites with silver content possessed good antibacterial activity against the S. aureus and P. aeruginosa bacterial strains. The best results were achieved when silver iodide and gold nanocages were added consecutively to the bioactive glasses, resulting in versatile bioactive glasses.

Conflicts of Interest:
The authors declare no conflict of interest.