Investigation on the antimicrobial properties of cerium‐doped bioactive glasses

Abstract Cerium‐doped bioactive glasses (Ce‐BGs) are implant materials that present high biocompatibility, modulate the levels of reactive oxygen species, and exert antimicrobial activity. The potential of BGs, 45S5, and K50S derived glasses doped with CeO2 (1.2, 3.6, and 5.3 mol%) to inhibit the growth of pathogen microbes was thoroughly investigated according to the ISO 22196:2011 method properly adapted. A significant reduction of the E. coli charge was detected in all glasses, including the BGs without cerium. The evolution of pH of the medium not inoculated following the immersion of the Ce‐BGs was monitored. The presence of cerium did not affect markedly the pH trend, which increased rapidly for both compositions. The change of pH was strongly mitigated by the presence of 200 mM phosphate buffer pH 7.0 (PB) in the medium. In media buffered by PB, the growth of E. coli, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, and C. albicans was not affected by the presence of BGs doped or not with cerium, suggesting that the antibacterial activity of Ce‐BGs is linked to the increase of environmental pH rather than to specific ion effects. However, Ce‐BGs resulted promising biomaterials that associate low toxicity to normal cells to a considerable antimicrobial effect, albeit the latter is not directly associated with the presence of cerium.


| INTRODUCTION
Bioactive glasses (BGs) are implant materials that can be used for biomedical applications, such as dentistry, orthopedics, and maxillofacial surgery. BGs present high biocompatibility and can effectively promote bone and soft tissue regeneration. 1 Phospho-silica-based 45S5 Bioglass ®2,3 (abbreviated as 45S5) and silica-based Kokubo glass (abbreviated as K50S) 4 are among the earliest developed and best characterized BGs and show comparable bioactivity. The properties of BGs can be improved by doping with therapeutic inorganic ions (TII) 5 ; the addition of cerium to 45S5 (H series) and to K50S (K series) allowed us to obtain novel cerium-containing bioactive glasses (Ce-BGs) with improved cytocompatibility and antioxidant properties. [6][7][8][9][10][11] Cerium is the first element in the lanthanide group, and it is the only lanthanide stable in the tetravalent state. The easy exchange between Ce 3+ $ Ce 4+ oxidation states underlies its catalytic activity as a scavenger of reactive oxygen species (ROS), and thus its antioxidative properties that protect osteoblasts from oxidative stress. 12 Furthermore, Ce-BGs are nontoxic to the cells and enhance the osteoblastic differentiation, the mineralization of primary osteoblasts, and the production of collagen. 13 Our studies on H and K cerium doped series show that the presence of cerium enhances proliferation and vitality of osteocyte-like cells. 7,8,14 We have also examined the structural role of cerium in the BGs; in the K series, cerium is coordinated by non-bridging oxygens (NBOs), whereas in the H series, the NBOs around cerium ions belong to orthophosphate groups. The latter groups stabilize the Ce 3+ ions subtracting them from the interconversion process between Ce 3+ and Ce 4+ ; this could explain the higher catalase mimetic activity of the K with respect to the H series. 10 Importantly, the efficacy of a BG in inducing bone regeneration requires the prevention of bacterial adhesion and proliferation that can occur on the implant surface. 15 Antibacterial properties of BGs can be induced or improved by the addition of metal ions with bactericidal effects. BGs doped with silver, copper, zinc, and gallium are considered potential candidates as antibacterial agents. [16][17][18][19][20][21][22][23][24] Cerium salts (oxide, nitrate, chloride, etc.) were among the first agents used against bacterial species, with evidence of some antibacterial activity dating back to 1947. 25 Cerium ions bind rapidly to E. coli cells, interfering with respiration and other metabolic functions. 26 Cerium nitrate significantly reduced the biofilm metabolic activity of C. albicans. 27 The inhibitory activity of CeO 2 on microbial growth was studied in planktonic cultures and biofilms enumerating the colony-forming units, 18 by the agar diffusion method 28 or by turbidity measurement. 28 The antiseptic effect of cerium oxide nanoparticles (CeNPs) is still To this aim, we synthesized BGs, 45S5, and K50S derived glasses, doped with increasing amounts of CeO 2 (1.2, 3.6, and 5.3 mol%). These BGs have been previously synthesized and thoroughly characterized within our research group. [6][7][8][9][10][11]14,[39][40][41] We then monitored the effect of Ce-BGs on the pH evolution over time of the medium to assess the role of pH in the antibacterial and antifungal action of the BGs.

| Cerium-doped bioactive glasses
The parent glasses are 45S5 and K50S and the molar composition of the studied BGs (hereafter named H0, H1.2, H3.6, H5.3, H series, K0, K1.2, K3.6, K5.3, K series) are presented in Table 1. The samples were prepared as reported 6 by the melting method and used in the form of slices. 7 Prior to testing, glass slices of appropriate size were smoothed by lapping.

| Antimicrobial activity tests
The   The presence of cerium did not affect the extent of growth for any microbe ( Table 2), even at the highest amount (5.3 mol%).
These results confirm on E. coli the antimicrobial activity of ceriumdoped 45S5 and K50S already demonstrated against S. aureus, a frequent cause of osteomyelitis, 43 and against other pathogens, such as Gramnegative bacteria, commonly involved in bone infection. 44 This notwithstanding, our data strongly suggest that the addition of cerium does not confer a specific antibacterial activity to the BGs investigated.
The effect of pH buffering on the antimicrobial activity of 45S5 and K50S, doped or not with cerium, strongly suggests that hindrance of microbial viability is rather a pH-related phenomenon. Indeed, bacterial growth inhibition was observed also in the absence of cerium, while buffering the pH near the physiological value eliminated the glass inhibitory effect even in the presence of increasing cerium amount. Interestingly, Allan et al. 28 already highlighted that 45S5 exhibited an intrinsic antibacterial activity, tested against oral pathogenic bacteria, that was clearly associated with the increase of pH.
The increase of pH of a BG is associated with glass degradation, dissolution, and a spontaneous formation of an apatitic layer. 2 In vivo, a continuous fluid flow clears the glass dissolution products, thus minimizing changes in the pH. However, alkaline biodegradable materials, when implanted, generate a microenvironmental pH, which is higher than the normal physiological value, reaching up to 9.2. 45 In the same study, pH dropped to pH 7.7 1 week after implantation, but residual material is expected to influence pH even 9 weeks post-surgery. The release of alkaline ions drives the nucleation of the apatitic material by raising the local pH, modulates osteoclast cells bone reconstruction, and likely affects bacterial propagation. 45 In our study, the antibacterial effect seems to be ascribable mainly to the formation of Our results are in accord with some previous literature: when the antibacterial activity of Ce-BGs was investigated by a zone inhibition method, growth hindrance of E. coli and S. aureus was similar in Ce-BGs and in the control. 23 Also, in all Ce-BGs studied, the antimicrobial activity seemed mainly due to the composition of native glass, which generated a fast pH increase in the surrounding solution, determining a strong antimicrobial effect, regardless of the addition of cerium. Similar antimicrobial activity against Gram-positive bacteria, but not against Gram-negative, was registered by phosphate glass fibers, supplemented or not with cerium, 46 confirming some intrinsic antimicrobial activity of these BGs not ascribable to cerium doping. In summary, the ability of these Ce-BGs to hamper the growth of pathogens remains, and it is generally recognized in vivo, albeit it cannot be directly ascribed to the presence of cerium ions.

| CONCLUSIONS
Cerium-doped bioactive glasses are promising biomaterials that present low toxicity to normal cells, modulate reactive oxygen species levels, and were confirmed to exert a considerable antibacterial effect. This notwithstanding, this effect is not directly associated with the presence of cerium, at least up to a 5.3 mol% content. We have previously shown that higher cerium amounts in the BG composition lead to the formation of ceramic and not vitreous material.
An increase in cerium content is then not a viable strategy to achieve cerium-based materials with both antioxidant and antibacterial properties. To this end, we postulate that alternative approaches such as doping with additional TII or functionalization with drugs should be considered.

CONFLICTS OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
All experimental data and discussed results of this technical note are available.
T A B L E 2 Charge [Log(CFU/cm 2 )] of microorganisms on the glass surface after 24 hr incubation at 30 C. Inocula were resuspended in the proper medium containing 200 mM PB (pH 7.0). All the Ce-BGs were tested and compared with controls. The reported data are means ± standard deviations of three independent experiments. No significant difference between the H and K series and among controls and Ce-BGs with different cerium amounts were observed (test-t and ANOVA, p > .05)