Metal TiO2 Nanotube Layers for the Treatment of Dental Implant

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Biological and Medical Applications of Materials and Interfaces

Metal TiO2 nanotube layers for the treatment of dental implant infections Agata Roguska, Anna Belcarz, Justyna Zalewska, Marcin Holdynski, Mariusz Andrzejczuk, Marcin Pisarek, and Grazyna Ginalska ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04045 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 3, 2018

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ACS Applied Materials & Interfaces

Metal TiO2 nanotube layers for the treatment of dental implant infections Agata Roguska,*,† Anna Belcarz,‡ Justyna Zalewska,‡ Marcin Hołdyński,† Mariusz Andrzejczuk,§ Marcin Pisarek,† and Grazyna Ginalska‡ †

Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland

‡

Chair and Department of Biochemistry and Biotechnology, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland

§

Faculty of Materials Science and Engineering, Warsaw University of Technology, Woloska 141, 02-507 Warsaw, Poland

KEYWORDS: TiO2 nanotubes; Ag and Zn nanoparticles; Candida species; Staphylococcus mutans; antimicrobial activity; dental infections

ACS Paragon Plus Environment

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ABSTRACT: Titanium oxide nanotubes layers with silver and zinc nanoparticles are attracting increasing attention in the design of bone and dental implants, due to their antimicrobial potential and their ability to control host cell adhesion, growth and differentiation. However, recent reports indicate that the etiology of dental infections is more complex than has been previously considered. Therefore, the antimicrobial potential of dental implants should be evaluated against at least several different microorganisms cooperating in human mouth colonization. In this study, Ag and Zn nanoparticles incorporated into titanium oxide nanotubular layers were studied with regard to how they affect Candida albicans, Candida parapsilosis and Streptococcus mutans. Layers of titanium oxide nanotubes with an average diameter of 110 nm were fabricated by electrochemical anodization, annealed at 650°C, and modified with approx. 5 wt.% Ag or Zn nanoparticles. The surfaces were examined with the SEM-EDAX, STEM and XPS techniques, and subjected to evaluation of microbial-killing and microbial adhesion-inhibiting potency. In 1.5 h-long adhesion test, the samples were found more effective towards yeast strains than for S. mutans. In a release-killing test, the microorganisms were almost completely eliminated by the samples, either within 3 h of contact (for S. mutans) or 24 h of contact (for both yeast strains). Although further improvement is advisable, it seems that Ag and Zn nanoparticles incorporated into TiO2 nanotubular surfaces provide a powerful tool for reducing the incidence of bone implant infections. Their high bidirectional activity (against both Candida species and Streptococcus mutans) makes the layers tested particularly promising for the design of dental implants.


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INTRODUCTION Because of their advantageous mechanical properties, high corrosion resistance and biocompatibility, titanium and its alloys are the most commonly used materials in bone and dental implants1. However, the long-term performance of titanium implants depends on the stability of the connection between the implant and the surrounding bone tissue, and on the effectiveness of the agents used to reduce inflammation caused by the presence of a foreign body in the living organism. Unfortunately, difficulties related to implant integration still exist (e.g. loosening failures), as do perioperative problems caused by, for example, bacterial infections2. Recent studies have focused on minimizing these effects in bone and dental implants. Methods of functionalizing the surface of Ti and its alloys have been developed that yield layers having appropriate morphology, structure, chemical composition and wettability3. Nanotubular layers of titanium oxide (TiO2 NT) obtained by anodic oxidation in a specific, optimized electrolyte mimic the porous structure of bone4, and can be formed directly on the surface of a metal or alloy. The anodizing process provides precise control over the diameter of the as-formed nanotubes and the distance between them. Furthermore, hydroxyapatite layers or antimicrobial agents that enhance biocompatibility (e.g. Ag and ZnO nanoparticles) may be deposited on nanotubular titanium oxide (IV) surfaces, thus significantly increasing their range of application5,6. For dental implants, modifying titanium surfaces with metal nanoparticles (NPs) seems to be particularly important in view of the spread of antibiotic-resistant microbial strains and the risk of infection-related implant failure. Ag NPs have already been reported to inhibit microbial growth, with lower toxicity towards Eucaryota than ionic silver7. For dental implants, modifying titanium surfaces with metal-based nanoparticles (NPs) seems to be particularly important in view of the spread of antibiotic-resistant microbial strains and the risk of infection-related


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implant failure. Ag NPs have already been reported to inhibit microbial growth, with lower toxicity towards Eucaryota than ionic silver7.According to available literature, the mechanism of antibacterial action of metal and metal oxide NPs is similar. In detail, Ag NPs act by a multi-side mechanism based mainly on (i) direct interaction with cell wall resulting in the extensive damage of cell wall structure, (ii) the release of Ag+ ions, which bind to bacterial and fungal DNA (thus inhibiting cell multiplication) and attach to amino acid residues in proteins, and (iii) the generation of reactive oxygen species (ROS), which inhibit a number of oxidative enzymes, thus inducing apoptosis via the mitochondrial pathway8–10. Regarding the antibacterial mechanism of Zn NPs, it is presumably comparable to ZnO NPs, and includes direct contact of the nanoparticles with the cell walls (destruction of cell integrity), the release of antimicrobial ions (Zn2+ ions), and ROS formation11. Moreover, microbial resistance to metal ions has, so far, been rarely reported8. However, to our knowledge the metallic Zn NPs haven’t been investigated before in terms of antibacterial action This makes Ag and Zn-based nanoparticles ideal candidates as powerful antimicrobial agents in dental titanium implants. An examination of the antimicrobial activity of titanium implants modified with metal nanoparticles should not be limited to one only selected bacterial strain, as is often encountered in the literature. This is particularly important for dental implants, because there is a wide range of mouth-colonizing pathogens. These include Streptococcus mutans, a facultatively anaerobic gram-positive bacterial (procaryotic) strain, which is a part of the “normal flora” and one of the main contributors to dental caries12. S. mutans generates (by lactic acid production from sucrose) and tolerates an acidic pH in the mouth environment, thus causing the demineralization of tooth enamel leading to tooth decay13. This strain, apart from its cariogenic properties, also shows a high biofilm-forming capacity, both on smooth and rough titanium surfaces, and the ability to


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induce titanium corrosion (due to its acidogenic abilities), thus jeopardizing implant survival14,15. Infections caused by strains of Candida are another serious - and underestimated - problem. Candida, eukaryotic single-celled microorganisms from fungus kingdom, ranks fourth among the most common organisms causing nosocomial bloodstream infections16. In some reports, the mortality rate in a group of patients with catheter-related candidemia was nearly 40%17. Among the Candida species, C. albicans is the predominant cause of fungal infections18, although it is a part of the normal microflora in the human digestive tract. It transforms into a pathogen if there is an attenuation of the host defense system. C. parapsilosis is frequently cited as the second most commonly isolated fungal strain19–21. Yeast cells present in the oral cavity have a high tendency to adhere to dentures or dental implants, in almost the same manner as to oral tissues22,23. When Candida cells colonize the surface of a bioprosthetic device, a biofilm develops on the implant surface. The treatment of such infections is difficult because biofilmassociated yeast cells exhibit almost total resistance to antifungal drugs24–26, and may develop drug resistance very quickly (within 6 hours), as shown for fluconazole27. Often, the only effective therapeutic procedure is to remove the infected implant. Interestingly, Candida species form tight associations with some specific oral bacterial species. It was been suggested that C. albicans and S. mutans coexist in biofilms, causing cariogenic development28. Streptococci provide the adhesion sites for C. albicans cells and excrete lactate, the carbon source for yeast cells; those cells in turn lower the oxygen tension to the level preferred by streptococci29. Therefore, due to this close cooperation between streptococci and Candida strains, new antimicrobial dental materials should be able to combat both these types of microorganisms. It should be noted that positive response of both S. mutans and Candida strains to new antimicrobially active therapeutic systems is not obvious because prokaryotic and eukaryotic


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cells differ in numerous aspects, including the susceptibility to antimicriobial agents (e.g. antibiotics). Streptococcus mutans and Candida species have been reported to be successfully eliminated by silver and zinc-based nanoparticles7,30–34. However, the effectiveness of metallic Zn NPs in limiting the bacterial colonization on biomaterial surface has not been studied before. In addition, to our knowledge, one only report is available on the antimicrobial activity of TiO2 nanotubular layers adorned with ZnO nanoparticles against these microbial species, precisely for S. mutans (but not Candida species35. As mentioned, evaluation of antimicrobial efficacy of new therapeutic systems designed for dental implantations against both these microorganisms is of particular importance, due to their cooperative development in oral cavity. Moreover, TiO2 nanotubular layers deserve particular attention, not only because they can serve as a specific reservoir for metal nanoparticles deposition but also because of the antimicrobial activity exhibited by TiO2 NT themselves, as shown in our previous studies6,36. This makes TiO2 NT surfaces highly promising in the design of antimicrobial dental devices. Therefore, this study aims to fill the gap in the evaluation of antimicrobial efficacy of metal NPs/TiO2 NT surfaces against the representatives of prokaryotic and eukaryotic pathogens responsible for dental implants failure. It presents a synthesis of Ag and Zn NPs decorated TiO2 nanotubular layers, characterizes them, and evaluates their antimicrobial activity against both Streptococcus mutans and Candida species.

EXPERIMENTAL SECTION TiO2 nanotubes fabrication. TiO2 NT nanotubular layers were fabricated by the electrochemical anodization of Ti samples (Ti foil; 0.25 mm-thick; 99.5% purity; impurities


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content: O – 0.18%, C – 0.08%, N – 0.03%, Fe – 0.2%, H – 0.015%; Alfa Aesar, USA) in an optimized electrolyte: a glycerol/water mixture (volume ratio 50:50) with 0.27 M NH4F at a constant voltage of 25 V37. After anodization, the samples were rinsed with DI water and dried in air. Subsequently, thermal annealing in air was performed at 650°C for 3 h in order to transform the TiO2 NT structure from amorphous to crystalline (a mixture of anatase and rutile) and to obtain mechanically stable nanotubes well integrated with the Ti substrate. Deposition of metal nanoparticles. The titania nanostructures were covered with Ag or Zn by the DC magnetron sputtering technique using a Leica EM MED020 apparatus. The average amount of metal deposited per cm2 was strictly controlled by quartz microbalance in situ measurements. Certainly, both the true average amount and local amount of the metal deposits may vary substantially due to the highly-developed specific surface area of the nanotube arrays and the resulting non-uniform distribution of the metal deposits. The configuration of the setup was perpendicular to the surface of the sample. Characterization. The surface morphology and chemical composition of the samples were observed/examined with the use of a scanning electron microscope (SEM, an FEI Nova NanoSEM 450 equipped with an EDAX Energy Dispersive Spectroscopy Detector and GENESIS software). For typical imaging low-energy electron detectors, an Everhart-Thornley detector (ETD) and a Through-The-Lens (TLD) detector were used, in the low and high resolution modes, respectively. For highlighting the nanoparticles, a Concentric Backscatter (CBS) detector was used. All modes were performed in the same configuration, at a primary beam energy of 10 kV. For the nanoparticles size analysis, ImageJ software was used38. Based on the data obtained (NPs areas), by expressing the nanoparticles as circles of equivalent area, their equivalent diameters were calculated. The chemical states of individual elements were verified


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by X-ray photoelectron spectroscopy (XPS) using a PHI 5000 VersaProbeTM (ULVAC-PHI) spectrometer with micro-focused, monochromatic AlKα radiation. The microstructure was characterized using a Hitachi-HD2700 STEM (Hitachi High Technologies Corporation, Tokyo, Japan) equipped with a Cs corrector and operated at 200 kV. The Bright Field and High Angle Annular Dark Field STEM (BF-STEM and HAADF-STEM) modes were used for microstructure imaging. Thin specimens for the microscopy observations were prepared on a Hitachi NB5000 focused ion beam (FIB) system. Samples were prepared as cross-sections of the oxide layers deposited with nanoparticles. A carbon protection layer was deposited to avoid destruction of the nanoparticles during FIB machining. After FIB preparation, the lamellas were finally thinned using low-energy argon ion milling on a Gentle Mill (Technoorg Linda Ltd.). A special 3D tomography Hitachi holder with a 360° tilt range was used to acquire the tilt series 2D images using an HAADF-STEM detector. A needle-shaped specimen for electron topography was prepared using the FIB system. Visualization of the reconstructed 3D datasets was performed using Avizo software. Antimicrobial properties evaluation. In the experiments, the reference strains were used: Candida parapsilosis ATCC 22019 (from the American Type Culture Collection), Candida albicans NCPF 3179 (equivalent to ATCC 10231; from the Polish National Collection of Pathogenic Fungi) and Streptococcus mutans PMC 2502 (from the Polish Microbiological Collection, Institute of Immunology and Experimental Therapy in Wroclaw, Poland). All of the strains were maintained as stocks in sterile microbanks (Technical Service Consultants Limited, UK) at -20°C. Prior to use, the yeast strains were transferred onto a fresh Yeast Malt Agar medium (YMA) and grown for 24 h at 37°C. The bacteria strains were transferred onto a fresh Brain Heart Infusion Agar medium (BHIA) and grown for 48 h at 37°C. Subsequently, the yeast


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strains and bacteria were transferred into liquid media (Yeast Malt Broth (YMB) and Brain Heart Infusion Broth (BHIB), respectively) and cultured at 37°C for 24 h (Candida strains) or 48 h (S. mutans). The microorganism suspensions were then diluted to the appropriate densities directly before the experiment. All culture media were provided by Biomaxima, Poland. Sample preparation. The samples were defatted and cleaned in a series of acetone (15 min, twice), chloroform (15 min, twice) and ethanol (15 min, twice) and placed in 24-well plates (Costar, Corning Inc., USA), individually for each of the antimicrobial tests described below. The plates containing the cleaned samples were then sterilized by the ethylene oxide method in a paper/plastic peel pouch (1 h at 55°C, followed by 20 h aeration). Release-killing antimicrobial activity evaluation. The antifungal and antibacterial activity of the samples was estimated as described in the standard method JIS Z 2801:200039. The survivability of the yeast and bacterial cells was evaluated in triplicate, after 3 h and 24 h incubation at 37°C, with individual controls for each incubation period. The results were calculated from 3 individual experiments as means ± SD using the Microsoft Office Excel 2007 program. Contact-killing antimicrobial activity estimation (yeast and bacteria adhesion test). 1 ml of a sterile yeast and bacteria cells suspension (1.0 x 108 cells/ml) in YM and BHI broths, respectively, standardized to the McFarland Equivalence Standards using a PhoenixSpec nephelometer (Becton Dickinson, USA), was added to each well of the plate containing the tested samples. After incubation of the samples for 1.5 h at 37°C under stable conditions, the non-adhered yeast and bacteria cells were gently washed away using 0.9 % NaCl (3 times, 50 ml). The adhered yeast cells were detected on the sample surfaces after chitin staining with Calcofluor White Stain (Sigma-Aldrich, USA). Streptococcus mutans adhered cells were


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detected using Biotium (Biotium Inc., USA), which makes it possible to differentiate between live and dead/dying bacterial cells. The visualization and counting of the cells was carried out by fluorescence microscopy (Olympus BX41 microscope equipped with UC 12 Soft Imaging System camera) using CellSens Dimension 1.12 software (Olympus, Japan). The test was performed in triplicate. The results were calculated from 10 independently selected areas from each sample as means ± SD using the Microsoft Office Excel 2007 program. Finally, the samples with adhered microbial cells were stained as described elsewhere36 and subjected to SEM observations (FEI Nova NanoSEM 450).

RESULTS AND DISCUSSION The content of Ag and Zn used in this study was selected on a base of pilot experiment concerning the evaluation of antimicrobial activity of prepared surfaces. In pilot study, Ag and Zn NPs were deposited on TiO2 TN layers in approx. 2 wt.%, 5 wt.% and 10 wt.% content. The results showed that optimal antimicrobial activity of produced samples was observed for 5 wt.% content of both metallic NPs (data not shown). These results were in accordance with our previous observations35. Thus, samples containing 5 wt.% of Ag NPs and Zn NPs were selected for further experiments. The surface of the nanoparticle decorated nanotubular layers was first evaluated using the SEM technique. As depicted in Figures 1a and d, there was a uniform deposition of Ag and Zn NPs on the TiO2 NT, without the closure of the interior of the nanotubes or the gaps between them. Thus, spaces for the intrusion of liquids were provided; moreover, the high specific surface area of the NP-adorned TiO2 NT potentially allows an intense release of metal ions from the NPs, and effective antimicrobial action. The sizes of the nanoparticles were analyzed based on


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SEM images obtained in the CBS mode, as presented in Figures 1b and e. Plots of particle size distribution are shown in Figures 1c and f. The deposited nanoparticles are in the same size range of 1–40 nm. However, for Zn, a higher NP size was obtained. The average particle diameter for Ag and Zn NPs was 11 and 19 nm, respectively. EDX analysis revealed that the Ag content in the Ag/TiO2 NT sample was about 4.8 wt % and the Zn content in the Zn/TiO2 NT sample was about 5.9 wt % (see Table I).

Figure 1. SEM images of TiO2 NT (25 V) surface annealed at 650°C/3h with 0.01 mg/cm2 Ag deposit (a) and 0.01 mg/cm2 Zn deposit (d); CBS images of Ag/TiO2 NT (b) and Zn/TiO2 NT (e); particle size distribution of Ag NPs (c) and Zn NPs (f) deposited on the TiO2 NT surface.


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Table 1. Chemical composition of nanoparticle decorated nanotubular layers evaluated using the EDX technique Ti, wt.%

O, wt.%

Ag, wt.%

Zn, wt.%

C, wt.%

Ag/TiO2 NT

65.95

28.37

4.79

-

0.88

Zn/TiO2 NT

63.77

29.63

-

5.85

0.76

STEM observation was applied for more precisely characterize the structure of the titania nanotubes layers adorned with Ag or Zn nanoparticles. Figures 2a and b show cross-sectional views of the Ag/TiO2 NT in HAADF-STEM and BF-STEM, respectively. Ag NPs are clearly visible as bright objects due to the high atomic number of Ag in comparison with TiO2 (Figure 2a). As can be seen, the size of the Ag NPs ranges from a few to about 50 nm. Nanoparticles are located on the tops of the nanotubes and inside them in their upper parts. The BF-STEM image observations revealed that the Ag NPs are completely crystalline and can contain a few randomly oriented crystallites (Figure 2d). The high-resolution observations confirmed the crystallinity of the Ag NPs, and the interplanar spacing of 0.23 nm measured was identified as (111) atomic planes of the pure silver phase (JCPDS # 00 004 0783) (Figure 2e). A high-resolution image of the Ag NPs and TiO2 nanotube wall is presented in Figure 2f. The measured interplanar spacing of 0.19 nm is close to (200) atomic planes of the pure Ag phase, and 0.349 nm was identified as (101) atomic planes of TiO2 anatase phase. A reconstructed 3D structure is presented in Figure 2c, where the Ag NPs are shown in red. A model of the reconstructed volume was made using a segmentation procedure based on the gray-level intensities of the voxels. In this way, the two different phases were separated, and the localization of the Ag phase is clearly shown. A similar structure of TiO2 nanotubes was observed in previous work using this technique40.


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Figure 2. STEM characterization TiO2 NT with 0.01 mg/cm2 Ag deposit: (a) HAADF-STEM image of Ag/TiO2 NT layer cross-section, (b) BF-STEM image of Ag/TiO2 NT layer crosssection, (c) electron tomography visualization, (d) BF-STEM image of Ag NP, (e-f) highresolution images of Ag NP.

The STEM observation results for Zn/TiO2 NT are presented in Figure 3. In this case, the contrast between the nanoparticles and nanotubes in the HAADF-STEM image is relatively weak - a result of the smaller difference between the mean atomic numbers of Zn and TiO2 (Figure 3a). However, small particles close to the top of the nanotubes can be seen. In the BF-STEM images, Zn NPs can also be found (Figure 3b). The visualization of three-dimensional structure using electron tomography revealed relatively large agglomerates of Zn nanocrystals spread over the


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nanotubes. The higher magnification clearly shows the presence of nano-sized Zn crystallites on the tops of the nanotubes. The different orientation of the nanocrystals results in various intensities in the BF images (Figure 3d). Zn nanocrystals are also visible in the HAADF-STEM image as bright clusters on the tops of the nanotubes (Figure 3e). The high-resolution observations confirmed the crystallinity of the Zn nanoparticles. The measured interplanar spacing of 0.247 nm was identified as (002) atomic planes of the pure zinc phase (PDF# 00– 004–0831) (Figure 3f). A 3D reconstruction is presented in Figure 3c.

Figure 3. STEM characterization TiO2 with 0.01 mg/cm2 Zn deposit: (a) HAADF-STEM image of Zn/TiO2 NT layer cross-section, (b) BF-STEM image of Ag/TiO2 NT layer cross-section, (c) electron tomography visualization, (d) BF-STEM image of Zn NP, (e) HAADF-STEM image of Zn NP, (f) high-resolution images of Zn NP.


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The chemical state of the elements in the Ag NPs or Zn NPs decorated titania nanotubular layers was analyzed using X-ray photoelectron spectroscopy (XPS). The survey spectrum (Figure 4) clearly indicates three major sets of signals from O 1s and Ti 2p for both samples, and Ag 3d for the Ag/TiO2 NT and Zn 2p for Zn/TiO2 NT samples, respectively. No trace of any impurity was observed, except for a small amount of adventitious carbon (C 1s). A detailed XPS analysis confirmed the presence of metallic Zn on the TiO2 nanotube surface: Zn 2p doublet peaks located at 1022 eV (Zn 2p3/2) and 1045 eV (Zn 2p1/2). Similar results were obtained for the Ag NPs decorated nanotubes. The XPS spectrum confined to the Ag window gave the binding energies of Ag 3d doublet peaks located at 368.0 eV (Ag 3d5/2) and 374.0 eV (Ag 3d3/2), which implies that Ag appears at the Ag/TiO2 NT surface as metallic silver.


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Figure 4. Survey XPS spectrum of TiO2 NT (25 V) sample with Ag deposit of 0.01 mg/cm2 (a) and Zn deposit of 0.01 mg/cm2 (b). The insets show high-resolution XPS spectra of the Ag3d and Zn 2p regions, respectively.

The samples produced were then tested for antimicrobial activity in two tests: a release-killing assay (samples incubated in a microbial cell suspension for a defined period of time followed by counting the number of surviving cells), and a contact-killing assay (test of microbial cell adhesion). The release-killing assay measures the survivability of microbial cells after contact with substances released from the samples tested. The results obtained indicate that the survivability of C. albicans cells after 3 h of incubation with Ag and Zn NPs-adorned nanotubular surfaces was relatively high: 92% and 80% control, respectively (Figure 5). However, after a prolonged incubation time (24 h) almost all of the C. albicans cells died (survivability less than 1%) (Figure 5). C. parapsilosis cells were more vulnerable to Ag and Zn NPs. Their survival rate after 3 h of incubation dropped to 46% and 37% control, respectively, with almost complete mortality after 24 h of incubation (Figure 6). S. mutans exhibited much higher susceptibility to tested samples than did the two yeast strains. These bacteria died almost completely (survivability under 0.2% control) after just 3 h of contact with the Ag and Zn NPs-adorned nanotubular surfaces, with total mortality after 24 h of contact (Figure 7). This result is similar to that reported by Liu et al.35 for TiO2 NT decorated with ZnO nanoparticles. Therefore, both Ag and Zn NPs are effective as antimicrobial agents deposited on TiO2 nanotubular layers produced of Ti metal substrate. One should note that Ti, and more distinctly, the TiO2 NT surfaces themselves exhibited some microbial-killing activity. This was observed in particular for C. albicans and S.


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mutans after 24 h of incubation: cell survivability dropped to 62% and 0% control, respectively (Figures 5 and Figure 7). Interestingly, C. parapsilosis reacted to the presence of TiO2 NT in a slightly different way. This species showed decreased survivability (46% control) after 3 h of incubation, but increased survivability (120% control) after 24 h (Figure 6). It is not surprising that TiO2 nanostructures (such as nanotubular layers) exhibit some microbial-killing properties because, as reported elsewhere, TiO2 induces the formation of ROS even without photooxidation41. Similarly, due to the presence of an ultrathin native TiO2 layer that forms naturally after Ti exposure to atmospheric oxygen42, Ti samples may also display some antimicrobial potency. The specific surface area of such a native TiO2 layer on the surface of the Ti samples is of course smaller than that of the TiO2 NT; therefore, its antimicrobial activity is also lower. These results are also in agreement with our previous study on the reaction of four bacterial strains to Ag NPs-adorned surfaces6. That report suggested that TiO2 nanotubular layers exhibited significant antimicrobial potential, even without further modification with antimicrobial agents. However, this does not explain the significant increase in the survivability of C. parapsilosis after 24 h of incubation on Ti and TiO2 NT surfaces (Figure 6). It seems possible that the Ti substrate itself released certain ions (present in the Ti matrix in trace amounts) into the incubation medium that favored propagation of the yeast. The main impurity detected in Ti foil used as a substrate for synthesis of studied surfaces is iron (0.2%). Thus, the foil is similar to other Ti substrates used for the synthesis of TiO2 nanotubes, obtained from Sigma-Aldrich, Advent Materials, Chempur or Goodfellow43. The content of ferric ions in Ti substrate is of particular importance because metallic impurities could be potentially embedded in various forms in the TiO2 nanotubular layer grown by anodization and thus they could influence biological properties of these surfaces44. Iron is an essential nutrient, also for yeasts,


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because iron cofactors activate enzymes which are involved in most of the major metabolic processes in the cell, is used for synthesis of heme and iron-sulfur clusters, is required for oxygen delivery, the oxidation of acetyl CoA via the Krebs cycle, generation of ATP, biosynthesis of DNA, amino acids, proteins, sterols, and fatty acids45. Thus, it is possible that ferric ions released from TiO2 nanotubes or underlying Ti substrate could activate Candida cells growth. This hypothetic mechanism would also explain the initial (after 3 h) reduction in cell survivability (inhibition by ROS quickly released from the TiO2 NT surface layer) and the subsequent (after 24 h) increase (growth stimulation due to a slow release of ferric ions from the Ti substrate beneath the TiO2 NT layer). However, this phenomenon was observed only for C. parapsilosis, and so this hypothesis should be confirmed by further research.

Figure 5. C. albicans survivability after incubation of cell suspension with tested surfaces for 3 h and 24 h.


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Figure 6. C. parapsilosis survivability after incubation of cell suspension with tested surfaces for 3 h and 24 h.

Figure 7. S. mutans survivability after incubation of cell suspension with tested surfaces for 3 h and 24 h.

The contact-killing assay reflects the adhesion capability of microbial cells. The conditions of this test, in particular the sample-microorganism contact time (1.5 h), correspond to the conditions of the initial adherence step, which is crucial for dental biofilm development. The formation of an acquired pellicle (by the deposition of salivary mucins, statherin and proline-rich


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proteins) on the dental surfaces takes place within minutes; microorganisms attach to the pellicle within 1 hour, with gram-positive cocci as the first46. Moreover, the conditions of the contactkilling assay made it possible to quantify the number of bacterial cells that survived after direct contact with the tested surface (by staining live/dead cells with an appropriate dye). This assay provided more data concerning the behavior of Candida species in contact with the samples tested. Incubation of the samples with C. albicans and C. parapsilosis cell suspensions showed that 1.5 h of contact with the TiO2 NT surface did not significantly reduce the number of adhered cells in comparison with the control Ti samples (Figure 8). In fact, the number of cells adhered to the TiO2 nanotubular layers slightly increased in comparison with the titanium matrix: from 940 to 1,410 cells/mm2 for C. albicans and from 220 cells/mm2 to 270 cells/mm2 for C. parapsilosis. The same tendency to adhere to the samples was observed for S. mutans: 19,642 cells/mm2 on Ti and 31,764 cells/mm2 on the TiO2 NT samples, with a comparable percentage of live and dead cells for both surfaces (Figure 9; Table 2). This phenomenon may arise from differences in surface topography because, as is commonly known, eucaryotic and bacterial cells adhere better to rough (such as TiO2 NT layers) than smooth (such as machined titanium) surfaces. Moreover, it has been proved that both osteoblasts47 and bacteria36 show different adhesion preferences for nanotubes of different diameters.


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Table 2. Amount of S. mutans cells adhered to tested samples after 1.5 h incubation with bacterial suspension (108 cfu). Results calculated as means ± SD from 3 samples. Mean of total number of bacterial cells was taken as 100% Adhered S. mutans cells per mm2 Sample Ti

TiO2 NT

Ag/TiO2 NT

Zn/TiO2 NT

total

live

dead and dying

19 642 ± 13 873

11 362 ± 11 727

8 280 ± 6 164

(100 %)

(58 %)

(42 %)

31 765 ± 15 312

15 926 ± 11 841

15 838 ± 5 325

(100 %)

(50 %)

(50 %)

46 109 ± 20 561

25 610 ± 12 575

20 498 ± 10 858

(100 %)

(56 %)

(44 %)

18 912 ± 9 478

5 364 ± 4 672

13 548 ± 7 297

(100 %)

(28 %)

(72 %)

Different results were observed for the Ag and Zn NPs-adorned surfaces. In our experiments, a 2-6-fold reduction in the number of adhered yeast cells (both C. albicans and C. parapsilosis) was observed for these samples relative to the TiO2 NT surfaces (Figure 8). This confirmed the antifungal efficacy of metal NP-decorated nanotubular surfaces. The number of S. mutans cells adhered to the Zn/TiO2 NT samples was also lower than the number attached to the TiO2 NT surfaces, with a domination of dead cells (approx. 72%; Figure 9 and Table 2). This is in agreement with the results reported by Liu et al.35 for TiO2 nanotubular surfaces with ZnO nanoparticles. However, S. mutans adhered to the Ag/TiO2 NT samples even more abundantly (46,108 cells/mm2) than to the TiO2 NT sample (31,764 cells/mm2). Moreover, the ratio between live and dead cells was similar to that observed for the Ti and TiO2 NT surfaces (Figure 9 and


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Table 2), suggesting a lack of impact on S. mutans adhesion. It is difficult to explain this phenomenon, given the widespread opinion that Ag NPs are a powerful tool against bacterial growth and adhesion. However, some articles also suggest higher antibacterial activity of ZnO NPs than Ag NPs, as was described in experiments conducted on Vibrio cholera and enterotoxic Escherichia coli48. In our tests, the short (1.5 h) cell-surface contact time may be one possible reason for this observation; it is likely that Ag ions were not released during the 1.5 h-long experiment in an amount sufficient to inhibit the adhesion of S. mutans. To illustrate this statement, we can cite our previous experiment on TiO2 NT samples loaded with ZnO NPs (in an amount similar to that tested in this study) and Ag NPs (in an amount 4 time greater than in this work). The levels of released Zn2+ and Ag+ ions were defined after 1.5.h incubation in a liquid medium. Those levels were approx. 25-50 µg/l and 10 µg/l for the Zn2+ and Ag+ ions, respectively36. Thus, the release of Ag+ ions from dissolving NPs proceeded much slower than the release of Zn2+ ions. It is possible that the amount of Ag+ ions released was too low to inhibit the adhesion of S. mutans, but was enough to significantly inhibit the adhesion of Candida cells. The question arises as to whether this observation jeopardizes the potential of the tested as dental implants. It is undeniable that initial adherence is crucial for dental biofilm development and to combat further threats such as dental caries, periodontitis, implant infections, etc. In this respect, the increased adhesion of S. mutans to the Ag/TiO2 NT surfaces observed after 1.5 h seems problematic. However, almost all of the bacteria died after 3 h of bacteria-sample contact (Figure 7). It seems, therefore, that the period between 1.5 h and 3 h of contact between the bacteria and the Ag/TiO2 NT samples was critical in eradicating S. mutans and that the formation of a rapidly growing biofilm is unlikely to appear on such surfaces.


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SEM observations of the samples with adhered microbial cells made it possible to estimate the shape and general appearance of the microbial cells adhered to the surfaces. Despite a reduction in the number of adhered yeast cells, incubation with NPs-adorned surfaces did not inhibit the transition of C. albicans from a planktonic to a pseudohyphal cell type. This is clearly demonstrated in Figure 8, which shows Candida cells both in suspension (planktonic cells – a yeast-like form) and adhered to the surfaces tested (Ti; TiO2 NT; Ag/TiO2 NT; Zn/TiO2 NT). The pseudohyphal form precedes the shift of the cells into the true hyphal form49; thus, the C. albicans cells adhered to the surfaces were ready to form a biofilm. According to data in the literature, the transition of C. albicans cells into the hyphal form is inhibited by 0.01 mM Ag+ 33. For Zn2+ ions, C. albicans turns into the hyphal form only at a concentration of 1.4 mM33. It is possible that the critical level of released ions was not attained in the experimental conditions applied in this study. For the C. parapsilosis strain, all the adhered cells were similar to those in the control suspension (Figure 8). Based on the literature50 they exhibited the crater (yeast-like) phenotype. Probably, then, the transition of C. parapsilosis into an invasive form did not occur. For both Candida species, no particular signs of membrane detachment or destruction of cell morphology were observed (SEM images, Figure 8).


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Figure 8. Fluorescence microscopy and SEM images of C. albicans and C. parapsilosis cells in planktonic form (suspension) and adhered to tested surfaces after 1.5 h of incubation with cell suspension. Cells were stained with Calcofluor White Stain for chitin presence. The number of cells adhered to the surfaces (per 1 mm2) is presented in the top-right corners of the fluorescence images.


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Regarding the SEM observations of S. mutans, the cells were found to appear as aggregates (pairs and short chains), as commonly noted. Figure 9 shows typical images taken for the Ti, TiO2 NT and Ag/TiO2 NT samples, with bacterial cells of coccal or ellipsoid form. Interestingly, for the Ag/TiO2 NT samples, some crystals were also observed (Figure 9). The EDS technique defined their chemical composition: the crystals were composed of AgCl (data not shown). Therefore, it is probable that some dissolved Ag+ precipitated as a chloride salt. This could have decreased the bioavailability of silver ions, with the consequent antibacterial activity of the tested samples. This would explain the abundant adhesion of S. mutans cells to the Ag/TiO2 NT samples. The SEM images of the Zn/TiO2 NT surfaces did not show the presence of adhered S. mutans cells. This observation is a bit surprising, because fluorescence microscopy showed the presence of these cells on the tested samples (Figure 9). However, not only the amount but also the survivability of bacteria on these samples was significantly reduced when compared with other samples (Table 2). It seems therefore probable that repulsive forces between tested surface and bacterial cells were greater than the attractive forces in the case of this particular sample. Thus, the bacteria we likely to detach from tested surface rather than attach (weak adherence). It could be the case, then, that these weakly adhered cells became detached from the tested surfaces during the fixation procedure and SEM imaging. It deserves further consideration whether Zn NPs deposited on TiO2 NT remains in its metallic state in contact with air or/and aqueous media (during antimicrobials tests). It seems likely that Zn, owing to its nature, undergoes oxidation when exposed to air. The thickness of the native oxide layer formed spontaneously on the surface of bulk metallic Zn is about 2-3 nm51. On the


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hand it has been shown that the oxidation of Zn nanocrystals is size and orientation dependent52. In brief, the smaller particles have a lower activation energy. Our XPS and HR-TEM results suggest that the nanoparticles obtained were in metallic state. However, it is possible that the oxidation layer which might have formed on the Zn NPs is not thick enough be detected by the techniques applied or only partial. Nevertheless, when exposed to body fluid, Zn is oxidized into metal cations. Zn(OH)2 and ZnO corrosion products are likely to form on the metal surface. The dissolution of the Zn(OH)2 and ZnO surface film components will promote further dissolution of the exposed metal and constant supply of Zn2+ ions, which exhibit antimicrobial activity against various bacterial and fungal strains.


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Figure 9. Fluorescence microscopy and SEM images of S. mutans cells in planktonic form (suspension) and adhered to tested surfaces after 1.5 h of incubation with cell suspension. The cells were stained with a Biotium dye kit (live cells become green while dead and dying turn red). The number of cells adhered to the surfaces (per 1 mm2) is presented separately in Table 2 for better clarity.


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CONCLUSIONS In summary, adorning titania nanotubular layers with Ag and Zn nanoparticles distinctly enhanced their antifungal properties against C. albicans and C. parapsilosis yeast strains and their antibacterial properties against S. mutans. This approach also made it possible to reduce the adhesion of yeasts cells (as observed during 1.5 h of incubation), although in relation to S. mutans this effect can presumably be improved by further modifications. Interestingly, the surfaces obtained inhibit the growth and adhesion of both yeast and bacterial strains. This is important, because all the microorganisms tested cooperate in the colonization of tooth surfaces and are responsible for the appearance of dental caries and the failure of dental implants. Such synergistic activity is essential, for there is growing evidence that dental diseases are the result of microbial consortia that are altered due to environmental changes. Antimicrobial therapies, therefore, should take account of this pathogenic mechanism. Although further improvement is advisable, it seems that TiO2 NT surfaces adorned with Ag and Zn NPs can provide a powerful tool for reducing the incidence of infections in implants designed for bone tissue regeneration, in particular dental implants.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Author Contributions This manuscript was written collaboratively by the authors, all of whom have approved the final version of the manuscript.


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We are most thankful to D.Sc Wojciech Lisowski from the Institute of Physical Chemistry, Polish Academy of Sciences (Poland), for XPS research assistance using a high-resolution spectrometer – a PHI 5000 VersaProbeTM. The authors gratefully acknowledge the support from the Institute of Physical Chemistry PAS. Equally, author M. Andrzejczuk received funding from the National Science Centre (Poland) through the research grant UMO-2014/13/D/ST8/03224. Authors A. Belcarz, J. Zalewska and G. Ginalska received the subvention for scientific research from Ministry of Science and Higher Education of Poland for Medical University in Lublin, Poland (DS2/2017-2019). The experiments were partially developed using equipment purchased within Agreement No. POPW.01.03.00-06-010/09-00 Operational Program Development of Eastern Poland 2007–2013, Priority Axis I, Modern Economy, Operations 1.3. Innovations Promotion.

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Abstract Graphic 83x35mm (300 x 300 DPI)


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Figure 1 177x100mm (150 x 150 DPI)



Figure 2 177x115mm (150 x 150 DPI)


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Figure 3 177x120mm (150 x 150 DPI)



Figure 4 84x119mm (150 x 150 DPI)


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Figure 5 85x59mm (300 x 300 DPI)





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Figure 8 177x185mm (150 x 150 DPI)


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Figure 9 85x170mm (150 x 150 DPI)


Metal TiO2 Nanotube Layers for the Treatment of Dental Implant

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