Investigation of realizing both antibacterial property and osteogenic

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Investigation of realizing both antibacterial property and osteogenic cell compatibility on titanium surface by simple electrochemical treatment Masaya Shimabukuro, Yusuke Tsutsumi, Risa Yamada, Maki Ashida, Peng Chen, Hisashi Doi, Kosuke Nozaki, Akiko Nagai, and Takao Hanawa ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01058 • Publication Date (Web): 06 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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ACS Biomaterials Science & Engineering

Investigation of realizing both antibacterial property and osteogenic cell compatibility on titanium surface by simple electrochemical treatment Masaya Shimabukuro1, Yusuke Tsutsumi2, 3, *, Risa Yamada1, Maki Ashida2, Peng Chen2, Hisashi Doi2, Kosuke Nozaki2, Akiko Nagai4 and Takao Hanawa2.

1Graduate

School of Medical and Dental Sciences, Tokyo Medical and Dental

University, Bunkyo, Tokyo, Japan

2Institute

of Biomaterials and Bioengineering, Tokyo Medical and Dental University,

Chiyoda, Tokyo, Japan

3Graduate

School of Engineering, The University of Tokyo, Bunyko, Tokyo, Japan

4Department

of Anatomy, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi,

Japan

ACS Paragon Plus Environment

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*Corresponding Author ([email protected])

KEYWORDS: Biofilm, Silver, Titanium, Micro-arc oxidation, Anodic spark deposition, Plasma electrolytic oxidation, Antibacterial property, Osteoblast-like cell, Cytotoxicity, Calcification.

Abstract

Recently, the problem of infection on implanted devices caused by the formation of biofilms has been recognized. Surface treatment to prevent the initial stages of bacterial adhesion and subsequent bacterial growth is the only possible solution against such infection. In this study, simple electrochemical treatment was used for introducing silver, an antibiotic agent, on the titanium surface. A porous oxide layer containing small amounts of silver was formed on the metal of the substrate. This was done by micro-arc oxidation using the electrolyte silver nitrate. The porous oxide layer was almost amorphous with a small fraction of anatase phase. The samples prepared using the


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electrolyte containing 0.04 mM or a higher concentration of silver nitrate showed an excellent antibacterial effect against both E. coli and S. aureus. However, the proliferation of osteoblast-like cells in the samples was not affected when a concentration of 0.5 mM or lower was used. Moreover, samples containing silver showed no-harmful effects on the process of bone differentiation. Furthermore, the calcification process of the cells on the samples treated with and without silver were much promoted than that on untreated Ti. Thus, we found that it is possible to use this optimum concentration of silver to realize the conflicting biofunctions; its antibacterial property and osteogenic cell compatibility.

1. Introduction

Titanium (Ti) and its alloys are widely used in medical devices owing to their excellent mechanical properties and biocompatibility. With regards to implant devices, accelerated and strong adhesion between the implant surface and the surrounding bone is desired to achieve a shorter healing period and immediate loading 1-4. It is impossible to achieve any biofunctions on metal surface only by processing and following thermal treatments.


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Surface treatment of Ti is a successfully technique applied to enhance the osteogenic cell compatibility. Many researches have been reported that both dry processes 5-16 and wet processes

17-33

were found to be effective to promote a biological reaction on

bone/material interface to enhance osteogenic cell compatibility. Micro-arc oxidation is a conventional wet process, based on electrochemical reactions in a specific electrolyte under high-voltage. The MAO treatment has already been utilized as a surface treatment in dental implants, which enables immediate loading after surgery

34-37.

However,

problems that are related to infection due to the formation of biofilms on implant surfaces have been recognized as a major cause of failure in implant surgeries

38-41.

It is almost

impossible to eliminate a grown biofilm formed on a device implanted into the body. The only alternative is removing the contaminated device from the patients’ system to avoid any subsequent undesirable biological reaction such as an infectious disease. Therefore, it is essential to inhibit the formation of biofilms. This can be done by preventing the initial stages of the process, such as bacterial adhesion and subsequent bacterial growth. It has been reported that silver (Ag) shows an antibacterial property against almost all bacterial species

42-48.

Ag can be used as an antibacterial agent to inhibit both bacterial adhesion


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and bacterial growth. However, the cytotoxic effects of Ag have also been reported 49-54. For a novel antibacterial implant devices, not only should they have sufficient antibacterial properties, but also they should not causing any harmful effect to the surrounding living tissue. An antibacterial surface that can kill bacteria, without harming the osteogenic cell is required. In other words, the surface of an ideal implant device contacting the bone must achieve both competing biocompatibilities; antibacterial property and osteogenic cell compatibility. Therefore, in this study, the authors have focused on MAO as a prospective surface treatment. MAO can incorporate both calcium (Ca) and phosphorous (P) into the resulting oxide layer

55, 56.

For the treatment, the mentioned elements are

originally present in the electrolyte in their ionic states. In other words, incorporation of the selected element into the oxide layer formed by the MAO treatment is possible only when the element is dissolved in the electrolyte. This technique is expected to incorporate the desirable element into the resulting oxide layer. Previous researchers (Won-Hoon Song et al. 57 and A. Cochis et al. 58) reported that they could incorporate Ag into the oxide layer along with Ca and P using the MAO treatment. A. Cochis et al. 58 reported that the MAO-treated Ti surface with Ag ion effectively inhibited the growth of multi-drug


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resistance (MDR) bacteria. Y. Tsutsumi et al. 59 reported that both the bioactivity and the antibacterial property was retained by incorporating Ag into the oxide layer of MAOtreated Ti alloy. Moreover, Won-Hoon Song et al.

57

reported the possibility for realizing

both the biosafety and the antibacterial property of the Ag-incorporated surface with a specific treatment condition. Xiaojing He et al. 45 reported that MAO samples containing both Ag and Strontium could expedite osseointegration with antibacterial property. Thus, the study of incorporating Ag into Ti surface can be expected to develop a novel antibacterial implants without any harmful effect against osteogenic cells. However, the relationships among the presence of Ag in the modified surface, the Ag-ion release behavior, and biological effects on both bacteria and osteogenic cells are still unveil. Therefore, the aim of this study was to find the optimum ranges of Ag in the MAO treated surface and Ag ion release rate to realize multi-biofunctionalization; antibacterial property and osteogenic cell compatibility without any cytotoxicity. In other words, the effect of Ag on cytotoxicity, initial attachment, proliferation, and bone differentiation of an osteoblast like cell line was studied. In addition, antibacterial properties of the samples were also evaluated using both gram-negative and -positive facultative anaerobic bacteria.


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2. Materials and Methods

2.1.

Sample preparation

The two titanium disks with diameters 8 mm and 25 mm were obtained by mechanically cutting a rod of commercially pure grade 2 titanium. The surfaces of the disks were mechanically grinded using #150, #320, #600, and #800 grid SiC abrasive papers. This was followed by ultrasonication using acetone and ethanol. The disks were then kept in an auto-dry desiccator until the next step of further use. The Ti disk was fixed onto the polytetrafluoroethylene holder with an o-ring. The area in contact with the electrolyte was 39 mm2 (7.0 mm in diameter) or 398 mm2 (22.5 mm in diameter). Details of the working electrode are described

22.

A Type 304 stainless steel plate was used as a counter

electrode. The base composition of the electrolyte for the MAO treatment was 100 mM calcium glycerophosphate and 150 mM calcium acetate. In this study, 0 to 10 mM silver nitrate (AgNO3) was added to the base electrolyte. After pouring the electrolyte into the electrochemical cell, both the electrodes were connected to a DC power supply (PL-650-


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0.1, Matsusada Precision Inc., Shiga, Japan), and a positive voltage with a constant current density of 251 A m-2, was applied for 10 min. Thus, a major part of the Ti disk was MAO-treated, with an annular untreated area of 0.5 mm from the edge. All the surface characterization details described below were performed within the MAO-treated area.

2.2.

Surface characterization and Ag ion release evaluation

Scanning electron microscope with energy dispersive X-ray spectrometry (SEM/EDS, S-3400NX, Hitachi High-Technologies Corp., Tokyo, Japan) was used to observe the surface morphology of each sample. X-ray photoelectron spectroscopy (XPS, JPS9010MC, JEOL) with Mg K line (1253.6 eV, 10 kV, detection angle: 90) was used to investigate the concentration of Ag in the oxide layer. The concentration of Ag of each sample was calculated according to a method described in previous study

22.

X-ray

diffraction (XRD, BRUKER D8 DISCOVER, Bruker AXS K.K Cu K radiation, 40 kV) was used to study the crystal structure of each sample. Inductively coupled plasma-atomic emission spectrometry (ICP-AES, ICPS-7000 ver. 2, Shimadzu Corp., Kyoto, Japan) was used to investigate the amounts of Ag ion released. To determine the amount of Ag ion


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released from the surface, the MAO-treated Ti in the electrolyte with various concentration of AgNO3 was incubated in 5 mL of physiological saline (0.9% NaCl). They were fixed onto a polyethylene container to allow the release of Ag ion from the surface of the sample. These were maintained at 310 K in a humidified chamber under constant shaking (100 rpm). Every 7th day, the pooled solution was transferred into fresh physiological saline.

2.3.

Antibacterial property evaluation

The antibacterial property tests were conducted in accordance with ISO 22196:2007. To examine the antibacterial effect of MAO-treated Ti with various concentration of Ag, we used Staphylococcus aureus (NBRC122135) and Escherichia coli (NBRC3972). The experiment was approved by Pathogenic Organisms Safety Management Committee of Tokyo Medical and Dental University (22012-025 c, 22,016-060). S. aureus and E. coli are used in antibacterial property tests as representative gram-positive and gramnegative bacteria, respectively, as described in ISO 22196. S. aureus is a gram-positive cocci frequently found in the nose, respiratory tract, and on the skin. However, E. coli is a gram-negative rod-shaped bacterium commonly found in the gut. Each bacterial strain


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was cultured according to its specific requirements. S. aureus and E. coli were cultured on tryptic soy broth (TryptoSoya Broth, Nissui, Japan) and Luria-Bertani (LB) broth (LBMedium, MP Biomedicals, CA, USA), respectively, for 24 h at 37°C under ambient conditions. The optical densities of the bacterial suspensions were measured at 600 nm using an ultraviolet-visible (UV–vis) spectrometer (V-550, JASCO, Tokyo, Japan) to obtain concentrations of 0.4-3.0 × 108 colony-forming unit (CFU) mL-1. Prior to the antibacterial property tests, all samples were sterilized with 70% ethanol, washed with distilled water, and dried. Bacterial suspensions were dropped on all samples, which were subsequently covered with a sterilized plastic film and incubated at 37°C for 24 h (n=3). After collecting bacteria from the incubated samples, the obtained suspensions were diluted, pipetted onto nutrient agar plates, and incubated overnight at 37°C. The number of viable bacteria was determined by counting the number of colonies formed on the petri plates.

2.4.

In vitro osteogenic cell compatibility test

2.4.1. Cell culture


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As described in our previous work 6145, MC3T3-E1 cells (RIKEN BioResource Center, Ibaraki, Japan) were maintained in a cell culture medium, which was an alpha modification of Eagle’s minimum essential medium (α-MEM; GIBCO, CA, USA), supplemented with 10% fetal bovine serum (FBS; GIBCO, NY, USA), 100 U mL−1 penicillin, 100 mg mL−1 streptomycin, and 0.25 mg mL−1 amphotericin B (GIBCO, NY, USA). In this study, cells were plated on the sterilized specimens at an approximate initial density of 10,000 cells cm−2. As a control, cells were also seeded on MAO-treated Ti without Ag. The cells were incubated at 37°C in a fully humidified atmosphere containing 5% CO2 in air. For induction of osteogenic differentiation, a cell culture medium supplemented with 2 mM β-glycerophosphate (Calbiochem, Darmstadt, Germany) and 50 mg mL−1 L-ascorbic acid was used(Wako Pure Chemical Industries, Osaka, Japan) when 100% confluence was observed on the specimens. This medium used for induction was named as, ‘differentiation-inducting medium.’ The differentiation-inducting medium was changed every 3 days. All metallic disks were sterilized in 70% ethanol for 20 min and thoroughly rinsed with deionized (DI) water before the in vitro test.


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2.4.2. Cellular proliferation

Number of attached cells on the sample surface on day 1, 2, and 3 was counted using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). The attached cells on tissue culture polystyrene (TCPS) were harvested by treating it with Trypsin/EDTA and then resuspended it into the cell culture medium. Trypan blue (Trypan Blue Stain 0.4%, Gibco, Tokyo, Japan) was used to count the attached cells on TCPS. Then, these cell suspensions were diluted progressively and were counted on a hematocytomater, from the result, the standard line calibrating the cell number and the light absorbance was finally obtained. These cell suspensions were transferred into a 96 well microplate, in which the cell counting assay was carried out. A reaction reagent was added to each sample, according to the manufacturer’s instructions, and the reaction was continued for 4 h at 37°C. The absorbance of the samples was measured at 450 nm using a microplate reader (ChroMate® Microplate Reader, Awareness Technology, USA). A reference wavelength of 630 nm was used.

2.4.3. Osteogenic differentiation quantification


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Alkaline phosphatase (ALP) activity was determined to evaluate the osteogenic differentiation of MC3T3-E1 cells cultured on each sample. After the indicated time, cells were washed twice with ice-cold PBS and the cells on each sample were disrupted by sonication (UD100, Tomy Seiko, Japan). Sonication was performed on ice. The ALP activity was determined by LabAssay™ ALP (Wako Pure Chemical Industries) method. According to the instructions, the absorbance was measured at 405 nm using the ChroMate® Microplate Reader (Awareness Technology).

2.4.4. Calcification analysis

The calcification of MC3T3-E1 cells on each sample was evaluated by the calcified deposits demonstrated by color change after alizarin red S staining. The medium was removed and the samples containing the cells were rinsed with PBS solution thrice. The cells were then fixed with 4% formalin for 1 h. After an hour, they were rinsed with ultrapure water thrice. Each sample was stained with 1% alizarin red S solution (adjusted to pH 4.2 with ammonium hydroxide) at room temperature for 30 min. After removing the alizarin red S solution, the cells were repeatedly rinsed with ultrapure water. When the


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samples were fully dry, the surface of each sample was observed using an optical microscope (OLYMPUS SZX12, Olympus).

3. Results

3.1.

Surface characterization and Ag ion release evaluation

Figure 1 shows the SEM images of MAO-treated Ti surfaces in the electrolytes with various Ag ion concentrations. The oxide layer morphology of MAO-treated samples with 0 and 5 mM Ag ions was almost similar. In contrast, a porous oxide layer was not formed after a treatment in 10 mM Ag ions because the applied voltage during the treatment had not been fully raised. Figure 2 shows the atomic percentage of Ag in the oxide layer calculated from XPS measurements. The small amount of Ag was incorporated into the oxide layer on Ti surface. Moreover, the atomic percentage of Ag increased with a rise in Ag ion concentration for MAO treatment.


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Figure 1 SEM images of each sample surface. a) MAO-treated sample with 0 mM Ag ions, b) MAO-treated sample with 5 mM Ag ions, c) MAO-treated sample with 10 mM Ag ions. Scale bar was 10 m.

Figure 2 The atomic percentage of Ag in the oxide layer calculated from XPS measurements


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Figure 3 XRD spectra of each sample. a) Oxide layer with Ti substrate, b) Agincluded oxide layer with Ti substrate, c) Ag-included oxide layer without Ti substrate.

Figure 3 shows the XRD spectra of oxide layer with and without the Ti substrate formed by MAO treatment using 0 or 2.5 mM Ag ions-containing electrolyte. From the results of the sample with Ti substrate, the peaks originating from α-Ti and anatase type TiO2 were detected. However, the XRD spectrum of the oxide layer without Ti substrate showed a typical halo pattern indicating low crystallinity with a slight peak of anatase type TiO2.


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Figure 4 Ag ion concentration of 0.9mass% NaCl solution with immersion of the MAO treated Ti with various Ag ion concentration for every one week.

Figure 4 shows the amount of Ag ion released from the oxide layer into the physiological saline measured by ICP-AES. The amounts of Ag ion released during each unit period showed an increasing trend with immersion time. Moreover, the amount of Ag ion increased with a rise in Ag ion concentration for MAO treatment. It was confirmed that the release of Ag-ion continued for at least 28 days, which was the maximum experimental period employed in this study.

3.2.

Evaluation of antibacterial properties


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Figure 5 shows the results of the antibacterial evaluation using E. coli. E. coli grew on the untreated Ti surface. Moreover, E. coli proliferated even more on the MAO-treated sample than on the untreated Ti. In contrast, it drastically decreased with increasing concentrations of Ag ion in the electrolyte. No bacterial colonies were observed on the surface of MAO-treated Ti samples in the electrolyte with 0.05 mM or higher Ag concentrations. Figure 6 shows the results of the antibacterial evaluation using S. aureus. The proliferation of S. aureus as well as E. coli increased on untreated Ti surface. Moreover, the proliferation of S. aureus was much more on the MAO-treated sample than untreated Ti. The bacterial colonies formed were less on the surface of the MAO-treated Ti samples in the electrolyte with 0.05 mM or higher Ag concentrations. However, this inhibitory effect on S. aureus was weaker than that on E. coli.


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Figure 5 Results of antibacterial evaluations using E. coli. Data are shown as the mean ± SD. *: P

Investigation of realizing both antibacterial property and osteogenic

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