Ag nanoparticle–coated zirconia for antibacterial prosthesis

Accepted Manuscript Ag nanoparticle–coated zirconia for antibacterial prosthesis

Risa Yamada, Kosuke Nozaki, Naohiro Horiuchi, Kimihiro Yamashita, Reina Nemoto, Hiroyuki Miura, Akiko Nagai PII: DOI: Reference:

S0928-4931(17)30988-8 doi: 10.1016/j.msec.2017.04.149 MSC 7972

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

15 March 2017 21 April 2017 23 April 2017

Please cite this article as: Risa Yamada, Kosuke Nozaki, Naohiro Horiuchi, Kimihiro Yamashita, Reina Nemoto, Hiroyuki Miura, Akiko Nagai , Ag nanoparticle–coated zirconia for antibacterial prosthesis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.04.149

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ACCEPTED MANUSCRIPT Ag nanoparticle–coated zirconia for antibacterial prosthesis Risa Yamada1, Kosuke Nozaki2*, Naohiro Horiuchi3, Kimihiro Yamashita3, Reina Nemoto1, Hiroyuki Miura1, Akiko Nagai2

of Fixed Prosthodontics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University

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1Department

of Material Biofunctions, Institute of Biomaterials Bioengineering, Tokyo Medical and Dental University 3Department of Inorganic Biomaterials, Institute of Biomaterials Bioengineering, Tokyo Medical and Dental University

and and

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2Department

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Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University 1-5-45 Yushima, Bunkyo-ku, Tokyo 135-0044, Japan

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Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan

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*Corresponding author Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Tel: +81-3-5280-8087 FAX: +81-3-5280-8015 E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract Bacterial adhesion to dental materials is a major cause of caries and periodontitis, necessitating the development of compounds such as yttria-stabilized zirconia

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(YSZ) and silver nanoparticles (AgNPs), which are widely employed in medicine

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due to their high antimicrobial activity and low cytotoxicity. The main goal of this

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study is the synthesis of the broad antimicrobial activity of AgNP-coated YSZ with facile methods. The bactericidal AgNPs were immobilized on the surface of

mutans,

Escherichia

coli,

and

Aggregatibacter

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Streptococcus

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YSZ and tested for bactericidal activity against Staphylococcus aureus,

actinomycetemcomitans based on ISO 22196:2007. The loading of AgNPs was

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optimized by culturing mouse fibroblast cells on AgNP-coated YSZ with cell viability test based on ISO 10993-5. In addition, the silver release profile of

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AgNP-coated YSZ in artificial saliva was determined using an accelerated aging

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test. Antibacterial activity, and cell viability test revealed optimum performance with no cytotoxicity at a level of 32 μg/cm2. Accelerated aging test showed that the AgNP-coated surface was extremely stable, exhibiting a total silver leaching level of only 1% and confirming the effectiveness of this coating method for retaining AgNPs while exerting an antibacterial effect against oral pathogens. This finding also implies that the bactericidal action of AgNP-coated YSZ is not mediated by

ACCEPTED MANUSCRIPT the released Ag ions, but rather corresponds to contact killing.

Keywords:

dental

materials,

antibacterial

activity,

silver

nanoparticles,

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prosthesis, yttria-stabilized zirconia, cytotoxicity

emission

scanning

electron

microscopy,

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Abbreviations: Yttria-stabilized zirconia, YSZ; silver nanoparticles, AgNPs; field FE-SEM;

X-ray

photoelectron

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spectroscopy, XPS; grazing incidence X-ray diffraction, GIXRD; ultraviolet-visible,

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UV-vis; inductively coupled plasma atomic emission spectrometry, ICP-AES.

ACCEPTED MANUSCRIPT 1 Introduction In recent years, the use of yttria-stabilized zirconia (YSZ) gained popularity in dental medicine fields such as crown restoration, implant fixture, and implant

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abutment due to its superior mechanical properties and biocompatibility [1, 2].

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The long-term success of dental prosthetics is strongly dependent on dental

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biofilm formation, which causes oral pathologies such as dental caries, periodontal disease, and peri-implantitis [3, 4]. Although YSZ-based dental

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prosthetics exhibit low levels of oral bacteria accumulation, secondary caries is

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reported as one of the reasons for zirconia crown failure due to complex biofilm formation mechanisms [5]. Ideally, YSZ should possess antibacterial properties to

Currently,

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prevent the adhesion and proliferation of pathogens on a long-term basis [6, 7]. researchers

have

focused

on

imparting

antimicrobial

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properties to biomaterials by using nanoparticles of metals such as silver, copper,

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and gold [8]. Among them, silver nanoparticles (AgNPs) have been widely used in medical and pharmaceutical nanoengineering owing to their strong bactericidal effect against a broad spectrum of bacteria and low cytotoxicity, as compared to other metal NPs [9–14]. Additionally, AgNPs exhibit the advantage of a greater surface-area-to-volume ratio compared to microscale silver particles, achieving the same antibacterial effect at a lower concentration [15].

ACCEPTED MANUSCRIPT The technique of AgNP incorporation depends on the type of material [16], e.g., in the case of dental materials, AgNPs are mixed with a composite resin, dental adhesives, denture base materials, and endodontic materials, while dental

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implants and brackets are coated with AgNPs [17–20]. A number of reports

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describe methods for YSZ surface modification with Ag or AgNPs, e.g.,

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co-sintering with Ag nanopowders [21–23] and chemical-reduction-based coating [24]. Although all techniques impart sufficient antibacterial activity to YSZ-based

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materials, their high complexity and long duration necessitate the development of

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alternative processes.

Furthermore, the antibacterial effect of all materials is attributed to the

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Ag ions released from the substrate, being inactivated by biologically relevant compounds due to the reaction of Ag ions with thiol groups [25]. Recently, contact

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killing and ion-mediated killing have been proposed as two bactericidal action

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modes of AgNPs [26]. Surface-immobilized AgNPs were reported to exert a bactericidal effect via contact killing, allowing re-usability with good efficacy [26]. In this study, we present the first facile method of coating YSZ substrates with immobilized AgNPs, revealing the broad antimicrobial activity of AgNP-coated YSZ against oral bacteria. Moreover, we further optimize the biocompatibility of AgNPs and their concentration to achieve the best activity

ACCEPTED MANUSCRIPT against several bacterial species causing biomaterial-associated infections. 2 2.1

Materials and methods Sample preparation

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YSZ powder (TZ-3YSB-E, Tosoh, Tokyo, Japan) was uniaxially pressed in steel

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dies at 7.5 MPa to prepare green-phase pellets (diameter 8 mm, thickness 1.3

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mm) and sintered at 1450 C for 2 h. All samples were coated with 30 µL of 0.2, 0.5, 1.0, 2.5, and 5.0 mM AgNP dispersions prepared from a 10-mM stock solution

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(Wako, Tokyo, Japan) assuming to apply AgNP to the outer surface of dental

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prosthesis and dried at 60 C. Non-coated samples were used as controls. After drying, samples were annealed at 600 C for 30 min and ultrasonically cleaned

2.2 Characterization

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with acetone, ethanol, and water.

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The microstructures and chemical compositions of samples were characterized by

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field emission scanning electron microscopy (FE-SEM, S-4500, Hitachi, Tokyo, Japan) and X-ray photoelectron spectroscopy (XPS; JPS-9010MC, JEOL, Tokyo, Japan), respectively. The size and distribution of AgNPs were analyzed using FE-SEM image analysis software (ImageJ, National Institutes of Health, MD, USA). The amount of AgNPs on the YSZ substrate was evaluated based on the atomic density of silver calculated from the peak heights of narrow-scan XPS

ACCEPTED MANUSCRIPT spectra. Crystal structures were analyzed by grazing incidence X-ray diffraction (GIXRD; D8 Advance, Bruker, MA, USA; Cu Kα radiation) over a 2θ range of 20–65° at 40 kV and 40 mA with a grazing incidence angle of 3°. Antimicrobial test

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2.3

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The antimicrobial activity test was conducted in accordance with ISO 22196:2007

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(measurement of antibacterial activity on plastics and other non-porous surfaces, MOD). To examine the antimicrobial effect of AgNPs, we used Staphylococcus

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aureus (NBRC122135), Streptococcus mutans (MT8148), Escherichia coli

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(NBRC3972), and Aggregatibacter actinomycetemcomitans (ATCC33384). The experiment was approved by Pathogenic Organisms Safety Management

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Committee of Tokyo Medical and Dental University (22012-025 c, 22016-060).

S. aureus and E. coli are utilized in antimicrobial tests as representative

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Gram-positive and Gram-negative bacteria described in ISO 22196, respectively.

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S. aureus is a Gram-positive coccal bacterium frequently found in the nose, respiratory tract, and on the skin, and E. coli is a Gram-negative rod-shaped bacteria commonly found in gut flora. S. mutans and A. actinomycetemcomitans are utilized as representative pathogens of oral disease. The Gram-positive facultative anaerobic S. mutans is the main pathogen associated with dental caries, causing secondary caries after the installation of prosthetics [27]. The

ACCEPTED MANUSCRIPT rod-shaped Gram-negative facultative anaerobic A. actinomycetemcomitans is a representative pathogen causing aggressive dental periodontitis, chronic

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periodontitis, and peri-implantitis [28].

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Each strain was cultured according to its specific requirements. S. aureus,

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S. mutans, and E. coli were cultured on tryptic soy broth (Trypto-Soya Broth, Nissui, Japan), brain-heart infusion (Bacto Brain heart Infusion, Becton, Dickson

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and Company, MD, USA) and Luria-Bertani (LB) broth (LB-Medium, MP

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Biomedicals, CA, USA) for 24 h at 37 C in ambient atmosphere, respectively. A.

actinomycetemcomitans was anaerobically cultured on GC broth medium for 48 h

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at 37 C in 5% CO2 atmosphere (AnaeroPack CO2, Mitsubishi Gas Chemical, Tokyo, Japan). The GC broth medium was prepared as follows: 15.0 g protease

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peptone #3 (Becton, Dickson and Company), 1.0 g corn starch (Wako), 4.0 g

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dipotassium phosphate (Wako), 1.0 g monopotassium phosphate (Wako), 5.0 g sodium chloride (Wako), 10.0 g dried bovine hemoglobin (BBL Hemoglobin, Becton, Dickson and Company), and 10.0 mL IsoVitaleX Enrichment (Becton, Dickson and Company) were dissolved in 1 L deionized water. The optical densities of bacterial suspensions were measured at 600 nm to obtain concentrations of 0.4 to 3.0 × 108 colony-forming unit (CFU)/mL using an

ACCEPTED MANUSCRIPT ultraviolet-visible (UV-vis) spectrometer (V-550, JASCO, Tokyo, Japan). Prior to antibacterial tests, all samples were sterilized with 70% ethanol, washed with distilled water, and dried. Bacterial suspensions were spread on all samples,

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which were subsequently covered with sterilized plastic film and incubated at 37

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C for 24 h (n = 6). After collecting bacteria from the incubated samples, the

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obtained suspensions were diluted, pipetted onto nutrition agar plates, and incubated overnight at 37 C. The number of viable cells was determined by

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counting the number of colonies in Petri dishes, and antimicrobial activity (R)

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was determined according to ISO 22196:2007 using the following formula:

R = Ut – At (1)

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where Ut is the number of viable bacterial cells after 24-h inoculation of the untreated test piece, and At is the number of viable bacterial cells after 24-h

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inoculation of the treated test piece.

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2.4 Solubility of silver nanoparticles Artificial saliva of the following composition was prepared: 0.4 g/L NaCl, 0.4 g/L KCl, 0.795 g/L CaCl2(H2O), 0.69 g/L NaH2PO4(H2O), 0.005 g/L Na2S, and 1 g/L urea [29]. To accelerate elution from AgNPs, the pH of artificial saliva was adjusted to 2.0 by adding lactic acid. All sample disks were immersed in 1 mL of artificial saliva in sealed polypropylene tubes and kept at 37 C for 1, 7, 14, 21,

ACCEPTED MANUSCRIPT and 28 days (n = 3), with artificial saliva being reconstituted after every observation period. The amount of released silver was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES, ICPS-7000 ver.2,

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Shimadzu Corp., Kyoto, Japan).

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2.5 Cytotoxicity assay

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The cytotoxicity of the AgNP coating was investigated using mouse fibroblast strain L929 cells (ATCC CCL1) in a direct contact test according to ISO 10993-5.

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Briefly, all samples were cleaned prior to cell culturing, and L929 cells were

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grown in Eagle's minimal essential medium (E-MEM, Wako) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution inside a

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humidified incubator at 37 °C in an atmosphere of 5% CO2. YSZ and AgNP-coated samples were placed into a well plate (n = 6), and cells were seeded onto each disk

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at density of 5 × 104 cells/mL, followed by one-day incubation. Subsequently, a

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solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Dojindo Co., Ltd., Kumamoto, Japan) was added to each well, followed by 3-h incubation. After incubation, the disks were washed with phosphate buffered saline (PBS) and treated with dimethyl sulfoxide (Wako) to solubilize formazan crystals. The optical density of the resulting supernatant at 570 nm was measured using a microplate reader (Model 680, Bio-Rad, CA, USA), and the

ACCEPTED MANUSCRIPT cytotoxicity of AgNP-coated YSZ was calculated as the reduction of viability compared to uncoated YSZ. Equation is used;

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where OD570e is the mean value of the measured optical density of the 100%

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extracts of the test sample (0.2, 0.5, 1.0, 2.5, 5.0 mM Ag sample), OD 570b is the

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mean value of the measured optical density of the blanks (0 mM Ag sample). The viability <70% of the blank is judged as cytotoxic potential.

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2.6 Statistical analysis

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Statistical analysis was performed using the Mann–Whitney U test with Bonferroni corrections for particle size distribution. Results with p values below

3 Results

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3.1 Characterization

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0.05 were considered statistically significant.

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AgNPs, represented by high-brightness areas in FE-SEM images, were uniformly distributed on both grains and grain boundaries (Fig. 1) of all coated samples, with particle size distributions shown in Fig. 2. The size of AgNPs gradually increased with increasing silver dispersion liquid concentration. The following average sizes and standard deviations were obtained: 19.7 ± 14.3 nm (0.2 mM), 23.4 ± 13.5 (0.5 mM), 30.6 ± 11.2 nm (1.0 mM), 41.3 ± 18.7 nm (2.5 mM), and 46.1

ACCEPTED MANUSCRIPT ± 20.4 nm (5.0 mM). Surface crystal structure analysis was performed by GIXRD (Fig. 3), revealing peaks of tetragonal ZrO2 for all samples, while peaks of Ag were not observed.

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XPS wide-scan spectra of uncoated and AgNP-coated YSZ showed peaks of

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Zr, Y, O, Ag, and C (Fig. 4(a)). The atomic percentage of Ag, calculated from Zr 3d,

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Y 3p, O 1s, and Ag 3d peak heights, increased with increasing concentration of the silver dispersion liquid (Fig. 4(b)). Narrow-scan Ag 3d5/2 spectra were

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deconvoluted into peaks of three components: Ag at 368.2 eV [30], Ag2O at 367.7

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eV [31], and AgO at 368.2 eV [31] (Fig. 4(b)), suggesting that AgNPs mainly

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comprised Ag and Ag2O together with a small amount of AgO.

The growth inhibition curves of AgNP-coated YSZ for Gram-positive and

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Gram-negative bacteria are shown in Fig. 5. The number of viable bacteria

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declined with increasing Ag atomic ratio on the YSZ surface, as summarized in Table 1. AgNP-coated YSZ was most effective against Gram-positive S. mutans, followed by Gram-negative E. coli, Gram-positive S. aureus, and Gram-negative A.

actinomycetemcomitans. 3.3

Solubility of silver nanoparticles

The longevity of the antibacterial effect in the oral cavity was estimated by

ACCEPTED MANUSCRIPT measuring the concentration of silver eluted from the YSZ surface by low-pH artificial saliva using ICP-AES. The results are reported in Figure 6, which summarizes the one- and seven-day solubility of AgNPs. A small amount of silver

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was eluted from AgNP-coated YSZ after one day, slightly increasing after seven

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days, but no silver leaching was detected for non-coated YSZ samples. The Ag

3.4

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release at 14, 21, and 28 days were below the detection limit of ICP-AES. Cytotoxicity assay

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The viabilities of L929 cells exposed to AgNP-coated YSZ were determined by the

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MTT assay. The viability gradually decreased with increasing atomic percentage of Ag (Fig. 7). A minimum cell viability of 64.7% for 5-mM samples was considered

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as a cytotoxic effect based on the standard value described in ISO 10993-5. 4. Discussion

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The results of this study indicate that the described coating method achieved a

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homogeneous dispersion of AgNPs on the YSZ substrate. The AgNP dispersion liquid contained an organic dispersive agent, which was removed by annealing of AgNP-coated YSZ samples. Although annealing at 600 C induced a solid-state reaction between silver and YSZ, and immobilized AgNPs on the YSZ surface, AgNPs were shown to contain Ag, Ag2O, and AgO due to the reaction with oxygen in ambient atmosphere. AgNPs are known to be highly sensitive to oxygen,

ACCEPTED MANUSCRIPT producing partially oxidized AgNPs with chemisorbed Ag ions upon exposure to ambient atmosphere, in agreement with our results [32, 33]. AgNP-coated YSZ was demonstrated to exhibit broad antimicrobial

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activity against Gram-positive and Gram-negative bacteria responsible for

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biomaterial-associated infections in the fields of dentistry and orthopedics. S.

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aureus and E. coli are representative etiologic agents causing osteomyelitis after prosthetic joint infection [34] and are used for antibacterial activity testing

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according to ISO 22196:2007. S. mutans and A. actinomycetemcomitans, observed

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in the oral cavity, cause dental caries and periodontitis, respectively. All biomaterial-associated infections start with bacterial adhesion to the substrate, bacteria

growth,

colonization,

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by

and

biofilm

formation

[35].

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AgNP-coated YSZ probably inhibited bacterial cell proliferation, since the

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antibacterial activity test used in this study principally evaluated the

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contact-killing capability of antimicrobial materials. Several mechanisms have been put forward to explain the antimicrobial effect of AgNPs, which are known to penetrate bacterial cells, inducing DNA damage and enzyme inhibition. Furthermore, AgNPs generate reactive oxygen species (ROS) and induce cell membrane disruption [5]. However, the above mechanisms are based on studies of planktonic AgNP states. Since approximately 1% of the total AgNP amount was

ACCEPTED MANUSCRIPT dissolved by artificial saliva (according to ICP-AES analysis), the produced planktonic AgNPs exhibited no antimicrobial effect due to not reaching a minimum inhibitory concentration [36]. The antimicrobial activity of AgNPs fixed

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on YSZ by annealing was attributed to ROS generation followed by cell wall

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disruption, as opposed to cell penetration. The data obtained in this study for Ag

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release from AgNPs coincides with the results of other reports [26]. In this study, the antibacterial effect against A. actinomycetemcomitans

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was weak compared to the other bacterial species. A. actinomycetemcomitans was

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cultured in 5% CO2 atmosphere, i.e., at a reduced oxygen concentration, and showed a larger resistance to AgNPs than other species. The ROS-induced of

AgNPs

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capability

decreases

with

decreasing

oxygen

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contact-killing

concentration in the culture media [37]. This suggests that the antibacterial

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activity of AgNP-coated YSZ was suppressed in an anaerobic atmosphere.

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Cytotoxicity tests showed that AgNP coatings obtained using 0.2–2.5 mM dispersion liquids can be used in medical devices, because the cell viabilities of these specimens corresponded to more than 70% of the control group value. The above tests were conducted by exposure of test material extracts or direct contact between cells and material surface (ISO10993-5:2009). Since YSZ-based materials are usually used for replacing femoral heads, teeth roots, and dental

ACCEPTED MANUSCRIPT crowns, which directly contact cells, the direct contact method is suitable for assessing the cytotoxicity of AgNP coatings. Anti-pathogenic biomaterials should balance antibacterial activity and

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cytotoxicity. Our results suggest that optimized AgNP dispersion liquid

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concentrations are in range of 0.2 to 2.5 mM, corresponding to AgNP weight

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densities from 2.6 to 32 μg/cm2 and coinciding with the concentration reported by Baker et al. for anti-E. coli activity [38]. Although these concentrations were

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slightly higher than the minimal inhibitory concentration of planktonic AgNPs

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[39] due to the immobilization of AgNPs on the substrate and their contact-killing capability, AgNP-coated zirconia potentially exhibits long-term antibacterial

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effects, which is supported by the results of ICP-AES analysis that reveal a low rate of AgNP release from the zirconia substrate under the action of artificial

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5. Conclusions

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saliva.

In conclusion, antimicrobial properties were imparted to YSZ by coating with AgNPs. The antimicrobial activity of the obtained composites against S.

aureus, S. mutans, E. coli, and A. actinomycetemcomitans was found to depend on the concentration of AgNPs. In particular, excellent antimicrobial activity against E. coli was observed, whereas no cytotoxic effects on L929 cells were

ACCEPTED MANUSCRIPT detected at coating concentrations below 2.5 mM. In view of the pronounced antimicrobial properties and small toxicity of AgNPs, the biocompatible AgNP-coated YSZ can be potentially used to control dental caries and periodontal

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disease.

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Acknowledgements

The authors are grateful to Dr. Arakawa, Professor of Lifetime Oral Health Care

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Sciences, Tokyo Medical and Dental University, for his assistance with the

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antibacterial activity test. Funding

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This study was partially supported by the MIKIYA Science and Technology Foundation.

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[34] Riberio M, Monterio FJ and Ferraz MP. Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions. Biomatter. 2 (2012) 176-194. [35] Hojo. K, Nagaoka S, Oshima T and Maeda N. Bacterial Interactions in Dental Biofilm Development. J. Dent. Res. 88 (2009) 982-990.

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[36] Bondarenko O, Juqanson K, Ivask A, Kasemets K, Mortimer M and Kahru A. Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant

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test organisms and mammalian cells in vitro: a critical review. Arch. Toxicol. 87 (2013) 1181-200.

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[37] Loza K, Diendorf J, Sengstock C, Ruiz L, Gonzalez JM, Vallet M, Koller M and Epple M. The dissolution and biological effects of silver nanoparticles in biological media. J. Mater. Chem. B. 2 (2014) 1634-1643.

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[38] Baker C, Pradhan A, pakstis L, Pochan DJ and Shah SI. Synthesis and antibacterial properties of silver nanoparticles. J. Nanosci. Nanotechnol. 5 (2005)

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[39] Catalina MJ and Eric MVH. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J. Nanopart. Res. 112 (2010) 1531-1551.

Fig. 1. FE-SEM images of the surface of YSZ coated with AgNPs of different concentrations, with arrows indicating representative AgNPs. (a) uncoated YSZ, (b) 0.2 mM, (c) 0.5 mM, (d) 1.0 mM, (e) 2.5 mM, (f) 5.0 mM. Scale bar: 1.0 μm. Fig. 2. Particle size distributions for samples prepared using different AgNP concentrations: (a) 0.2 mM, (b) 0.5 mM, (c) 1 mM, (d) 2.5 mM, (e) 5.0 mM.

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Wide-scan spectra of AgNP-coated YSZ showing Zr, Y, O, Ag, and C peaks. (b)

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Atomic percentage of Ag calculated from the peak height of Zr, Y, O, and Ag. (c)

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Representative Ag 3d XPS spectrum of AgNP-coated YSZ (2.5 mM) fitted in the Ag 3d5/2 region. The solid line represents experimental data, and broken, dashed,

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and long broken lines represent the components used to fit the spectrum.

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Fig. 5. Inhibition of bacterial cell growth by AgNP-coated YSZ. Fig. 6. Ag release from AgNP-coated YSZ in low-pH (2.0) artificial saliva at 37 °C,

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reported as integrated Ag concentrations (ppb). The amount of Ag released from uncoated YSZ was below the detection limit.

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Fig. 7. Viability of L929 cells exposed to uncoated and AgNP-coated YSZ, reported

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as percentages relative to uncoated samples. The dashed line represents 70% viability, corresponding to the standard cytotoxicity level (ISO10993-5). Table 1. Relationship between AgNP concentration, antibacterial activity, and cell viability.

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ACCEPTED MANUSCRIPT Silver dispersion liquid (mM)

0.2

0.5

1.0

2.5

5.0

Atomic concentration (at%)

0.12

0.68

1.83

4.98

7.65

S.aureus

0.1

0.6

0.9

2.7

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S.mutans

0.9

1.9

3.6

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-

E.coli

0.1

0.4

0.8

6.6

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A.actinomycetemcomitans

0.3

0.5

0.6

0.9

2.6

99.5

89.3

94.7

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Cell viability (%)

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Antibacterial Activity against

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Table 1. Relationship between AgNP concentration, antibacterial activity, and cell

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viability.

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Graphical abstract

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Highlights

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• Silver nanoparticles were successfully immobilized on yttria-stabilized zirconia • The obtained composites showed antibacterial activity and low cytotoxicity • Nanoparticle loading was optimized by culturing mouse fibroblast cells on composites • Composites showed a total silver leaching level of only 1% • Bactericidal action is not du to leached Ag ions, but corresponds to contact killing