The antibacterial W-containing microarc oxidation coating on Ti6Al4V

Surface & Coatings Technology 374 (2019) 242–252

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The antibacterial W-containing microarc oxidation coating on Ti6Al4V a

a

a

a

Tong Zhou , Jie Liu , Xinwen Zhang , Bin Shen , Jinlong Yang

b,⁎

c

, Wenbin Hu , Lei Liu

a,d,⁎⁎

T

a

State Key Laboratory of Metal Matrix Composites, School of Material Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China School of Material Science and Engineering, Tianjin University, Tianjin 300072, China d Collaborative Innovation Center for Advanced Ship and Deep-Sea Exploration, Shanghai Jiao Tong University, Shanghai 200240, China b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Microarc oxidation Titanium alloy Sodium tungstate Antibacterial property Reactive oxygen species

The antibacterial properties of the microarc oxidation coating have gained wide attentions, and W-containing surface has been verified to be notably bactericidal. Herein, in this work, doping Na2WO4 into the electrolyte to prepare the antibacterial W-containing microarc oxidation coating is proposed for the first time. No obvious crystal phase related to hexavalent W exists in the coating, and sodium tungstate may transform into a trace amount of tungsten oxides and amorphous substances. The sodium tungstate plays a crucial role in the antibacterial capability of the coating, the W-containing coating exhibits conspicuous bactericidal performance against both planktonic and adherent Escherichia coli and Staphylococcus aureus. The antibacterial mechanism of the W-containing coating is first revealed to be linked to the extracellular and intracellular reactive oxygen species (ROS). The W-containing dissolved matters spontaneously trigger the formation of the extracellular ROS, which directly destroy the bacterial cytoderm and cytomembrane. Meanwhile, the nanoscale W-containing dissolved matters penetrate the cell wall, and stimulate the generation of intracellular ROS which interact with the cytoplasmic components. Ultimately, the bacteria die from the cooperative effects of the extracellular and intracellular ROS.

1. Introduction Microarc oxidation (MAO), also named plasma electrolyte oxidation (PEO), is an innovative surface treatment technique due to its low cost, high productivity and environmental friendliness [1], and mainly applied on the valve metals (Al, Mg and Ti, etc.) and their alloys to fabricate an oxide coating [2]. The MAO process, which evolves from the conventional anodic oxidation, is frequently carried out in the solution containing silicate, aluminate and phosphate. The MAO coating prepared with specific electrolyte exhibits excellent biocompatibility [3,4], wear and corrosion resistance [5–8]. The MAO treatment involves the dielectric breakdown of the barrier layer, and plasma discharge occurs at the interface between the electrolyte and oxide film under the high potential in the aqueous solution [9]. Vast volumes of gases, noises of plasma discharge, numerous sparks and arcs on the surface of substrate are produced along with the process [10]. Ultimately, a porous and volcano-like oxide coating is formed on the substrate [11]. The properties of the MAO coating are dependent of the output mode of the power supply referred to the unipolar and bipolar pulse under the

potentiostatic or galvanostatic condition [12,13], electrical parameters of the output waveform [14], such as potential [15], current density [16], duty ratio [17] and frequency [18], and the compositions of the electrolyte [19,20]. Titanium and its alloys have been extensively used in the field of aerospace engineering, marine industry and biomedical materials due to their good mechanical stability, anticorrosion properties and biocompatibility [21–23]. Ti6Al4V is one of the most commonly used titanium alloys, and has been widely applied in the MAO process to fabricate a ceramic coating with bioactivity, abrasive and corrosion resistance [24–26]. In recent years, the antibacterial properties of the MAO coating on pure titanium and its alloys have gained wide attentions. As the orthopedic implants, the MAO coating on titanium and its alloys is bound to overcome the problems caused by microbial infection. At present, doping the antiseptics into the MAO coating is the most common way to solve the biocidal trouble. Zinc (Zn), silver (Ag) and copper (Cu) are the most commonly used inorganic bactericidal additives in the electrolyte during the MAO process [27–29]. Lan Zhang et al. [30] have added Ag nanoparticles and zinc acetate into the

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Corresponding author. Correspondence to: L. Liu, State Key Laboratory of Metal Matrix Composites, School of Material Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China. E-mail addresses: [email protected] (J. Yang), [email protected] (L. Liu). ⁎⁎

https://doi.org/10.1016/j.surfcoat.2019.05.089 Received 13 March 2019; Received in revised form 6 May 2019; Accepted 31 May 2019 Available online 03 June 2019 0257-8972/ © 2019 Published by Elsevier B.V.

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performances against planktonic and adherent Gram-negative E. coli and Gram-positive S. aureus bacteria were evaluated by means of spread plate method and fluorescence staining. Moreover, the leached metal ions were measured after soaking the coating. Meanwhile, the morphology and fluorescent staining of the adherent bacteria was observed to further verify the notable antibacterial efficiency of the W-containing MAO coating. The possible antibacterial mechanism of the coating was also accessed by means of the detection of reactive oxygen species (ROS). This is the first work about fabricating the antibacterial MAO coating by doping the sodium tungstate into the electrolyte, and the antibacterial mechanism of the W-containing coating is also revealed.

solution to prepare a Zn and Ag co-doped antimicrobial TiO2 MAO coating, the coating significantly inhibits the adhesion of Staphylococcus aureus (S. aureus) and reduces the amounts of planktonic bacteria in the culture medium. In addition, organic antimicrobial agents also can be employed on the MAO coating [31–33]. For instance, the cationic germicidal peptide LL-37 has been applied in the antibacterial MAO coating on commercial bare titanium disk, the coating presents significant antibacterial activity against both Gram-positive S. aureus and Gram-negative Escherichia coli (E. coli) bacteria [34]. On the other hand, the antibacterial MAO coating on titanium and its alloys also can be introduced to the marine antifouling field [35,36]. The formation of biofouling is generally acknowledged to be the following process: the surface of the underwater components is mainly dominated by organic biomolecular first, then followed by the adherent bacteria to form a matrix membrane. Subsequently, vicinal algae and invertebrates tightly settle on the bacterial film [37]. A mass of marine microbes, invertebrates and plants adhere to the underwater surface, leading to increase the navigational resistance, accelerate the corrosion damage and reduce service life of the components [38]. Up to now, the most efficient way to prevent the formation of the microbial biofilm is to implement the marine antifouling paints. Likewise, Ag, Cu2O and ZnO nanoparticles are the most frequently used antifouling additives due to their excellent antibacterial abilities [39–41]. Here, it should be noted that the bacterial colonization caused by the adherent bacteria is a necessary condition for subsequent settlement of algae and invertebrates, and it is scarcely possible to remove the biofilm of algae and invertebrates under natural field conditions once formed on the surface of the components. Therefore, preventing the bacterial adhesion is a prerequisite for restricting the formation of biofouling. Sodium tungstate has been used for addressing the related problem caused by bacteria. Wenhan Zhu et al. [42] have found that tungstatemediated microbiota editing reduces the severity of intestinal inflammation in mouse models of colitis, and the dysbiotic expansion of Enterobacteriaceae during gut inflammation can be prevented by sodium tungstate treatment. Additionally, the W-containing coating also possesses notable antibacterial performance. E. M. Cazalini et al. [43] have tested the antimicrobial and antifouling properties of polypropylene meshes coated with metal-DLC thin film, tungsten-DLC film inhibits the growth of Candida albicans, E. coli, Enterococcus faecalis and Pseudomonas aeruginosa strains and significantly reduces the formation of biofilm. Meanwhile, the carbonitride coating doped with W deposited on AISI 316 L stainless steel also exhibits marked antibacterial performance, the CNx-W coating obviously inhibits the formation of bacterial colonies of S. aureus, and the antibacterial ability of the coating appears to be related to hydrophilicity [44]. In view of the current research status, most of the antibacterial MAO coatings are fabricated by doping the typical inorganic bactericidal agents (Ag, Zn and Cu) into the electrolyte, and some researchers have published relevant works about adding sodium tungstate into the electrolyte for photocatalysis, anticorrosion and abrasive properties of the MAO coating [45–47], but the antibacterial properties of the Wcontaining MAO coating are never considered. Additionally, despite the W-containing coating has been verified to be antibacterial, the antibacterial mechanism is not given in relevant articles. Based on the aforementioned facts, the introduction of sodium tungstate into the MAO coating may be an effective way to achieve the antibacterial characteristics. So far, no articles have been published about the addition of Na2WO4 into the electrolyte to fabricate the antibacterial MAO coating. Therefore, we propose that the MAO coating prepared with sodium tungstate may present notable antibacterial performance, and the W-containing MAO coating could be used in the field of antibacterial application. In this work, the basic Na2SiO3-(NaPO3)6-NaAlO2 solution was used for MAO treatment, and the effects of sodium tungstate on the microstructure, phase structure and chemical composition of the MAO coating on Ti6Al4V were investigated. Specifically, the antimicrobial

2. Materials and methods 2.1. Preparation of the specimen Ti6Al4V plate with a dimension of 50 mm × 50 mm × 3 mm was used as the substrate. Prior to MAO treatment, the Ti6Al4V plates were polished using 150#, 360#, 800# and 1500# Al2O3 waterproof abrasive papers. Then, all the samples were ultrasonically rinsed with acetone, ethanol and deionized water for 5 min, respectively. Subsequently, these plates were thoroughly dried in oven at 40 °C. 2.2. Microarc oxidation process The basic electrolyte consisted of 4 g/L Na2SiO3.9H2O, 10 g/L (NaPO3)6 and 10 g/L NaAlO2, and 4 g/L Na2WO4.2H2O was added into the basic solution. All the aforesaid analytically pure reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The samples fabricated in the basic electrolyte without and with sodium tungstate dihydrate were labeled as MAO-0W and MAO-4W. A bipolar pulse power source system was applied for MAO process, the Ti6Al4V substrate was connected to the anode, and a 316L stainless steel container was set as the cathode. The process was performed under a potentiostatic mode at 600 Hz for 20 min. The positive and negative pulse voltage were +500 V and −60 V, and the duty ratios of positive and negative pulse were 20% and 10%, respectively. During the process, the solution was stirred by the circulatory system to guarantee the uniformity, and the cooling system was opened to keep the temperature of the solution below 40 °C. After the treatment, the samples were cut into pieces with sizes of 10 mm × 10 mm × 3 mm, next ultrasonically washed with acetone, ethanol and deionized water for 3 min, then dried in air. 2.3. Characterization of MAO coating The surface and cross-sectional morphologies and compositions of the MAO coatings were studied by scanning electron microscopy (SEM, Hitachi S-4800) equipped with energy dispersive spectrometer (EDS, Horiba EMAX). Phase structures of the MAO coatings were identified by X-ray diffractometer (XRD, Rigaku Ultima IV) with Cu Kα (λ = 0.15405 nm) radiation at 30 kV and 40 mA. XRD patterns were obtained in the range from 20° to 80° with 5°/min scanning rate. The chemical state of the elements in the MAO-4W coating was analyzed by X-ray photoelectron spectroscopy (XPS, Axis Ultra DLD) with Al Kα (1486.6 eV) X-ray source. The measured data were calibrated with respect to the C 1s (hydrocarbon CeC, CeH) binding energy of 285.0 eV. The amounts of Al and W released from the MAO coating in the phosphate buffer saline solution (PBS, Aladdin, Moby (Shanghai) Biotechnology Co., LTD) were detected by inductively coupled plasma optical emission spectroscopy (ICP-OES, Thermo-iCAP7600). The samples were immersed in 5 mL PBS and kept at 37 °C for 7 days. The liquid was collected on day 1, 2, 4 and 7, and the PBS solution was refreshed at every time point. The antimicrobial performances of the MAO coatings and Ti6Al4V were evaluated with Gram-negative E. coli and Gram-positive S. aureus 243

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the great bonding strength of the coating. However, more tiny holes were observed from the MAO-0W in Fig. 1a, and the diameter of porosities on the MAO-4W was slightly larger. In addition, the thickness of MAO-4W was larger than that of MAO-0W. These phenomena may be attributed to the addition of sodium tungstate in the basic electrolyte. Based on the theory in the literature [48], the breakdown potential decreased with the concentration of conductive ions in the solution in potentiostatic mode. When the sodium tungstate was added into the basic electrolyte, more electrolytic ions participated in the oxidation process, leading to the easier breakdown of the barrier layer and the enhancement of plasma discharge. Thus, larger holes were left on the surface, and the growth of the MAO coating was accelerated. Therefore, a thicker coating with larger cavity was obtained with the addition of sodium tungstate.

bacteria. The samples and PBS solution were sterilized in an autoclave at 121 °C for 30 min. The bacterial suspension in the sterile liquid LuriaBertani (LB) medium was incubated on a shaker with 150 rpm at 37 °C for 12 h. Then, the bacterial suspension was diluted to the concentration of 1.0 × 105 colony forming units (CFU)/mL. All the disinfectant samples were placed in individual well in a 24-well plate, and 500 μL diluted bacterial suspension was added into the well. Next, the 24-well plate was placed in an incubator at 37 °C for 24 h. Afterwards, the samples were washed with 1 mL sterile PBS solution to remove the unattached bacteria and medium. The plate counting method was applied to assess the antimicrobial properties of the MAO coating. As for the planktonic bacteria, after taking out the samples, the bacterial suspension in each well was diluted to an appropriate concentration, and 200 μL diluted bacterial suspension containing the planktonic bacteria was spread on the LB agar plate. After incubation at 37 °C for 24 h, the number of the bacterial colonies was counted. On the other hand, the sample washed by PBS solution was put in a disinfectant tube with 1 mL sterile PBS solution, then the tube was placed in an ultrasonic scrubber for 10 min to shock off the adherent bacteria on the surface of the MAO coating. The PBS solution containing the adherent bacteria was also diluted to a suitable concentration, and 200 μL diluted bacterial suspension containing the adherent bacteria was incubated on the LB agar plate at 37 °C for 24 h, then the number of the bacterial colonies was determined. The antibacterial rate (AA) of the MAO coatings for planktonic and adherent bacteria was evaluated with the following formula: AA = (N0eNi)/N0 × 100%, N0 and Ni represent the number of bacterial colonies on the LB agar plate corresponding to the Ti6Al4V and MAO coatings, respectively. The antibacterial rate was expressed as mean and standard deviations. Statistical analysis of the data was implemented analysis of variance (One Way ANOVA), and the statistical significance was defined as p 0.01. In order to observe the morphology of the adherent bacteria on the surface of the samples, after incubation and washed with disinfectant PBS solution, the samples were immersed in 1 mL 2.5 wt% glutaraldehyde solution at 4 °C for 30 min, then dehydrated in a series of ethanol/distilled water solutions (30, 50, 70, 90 and 100% v/v) for 10 min, and dried for SEM observation. Confocal laser scanning microscopy (Leica, TCS SP8 STED 3X) was also utilized to assess the viability of adherent bacteria. After removing the unattached bacteria, the samples were strained with 1 mL 0.01 wt% acridine orange/ethidium bromide mixed solution in dark for 15 min. Subsequently, the samples were gently washed with the sterile PBS solution for fluorescence observation. The potential antibacterial mechanism was supposed to be the generation of extracellular and intracellular ROS. The extracellular ROS trapped by the dimethylpyridine nitrogen oxide (DMPO) were directly measured by the electron paramagnetic resonance spectrometer (EPR, Bruker EMX-8). In addition, 500 μL 2,7-dichlorodihydrofluorescein diacetate (DCFHDA, Beyotime Biotechnology Co., LTD) with concentration of 10 μM was added in the aforementioned process of bacterial culture to indirectly detect the intracellular ROS. The fluorescence intensity of the aqueous medium was measured at 488 nm excitation wavelength with fluorescence spectrophotometer (Hitachi, F7000) after 24 h incubation.

3.2. Composition analysis The elemental contents of the surface were detected by EDS and shown in Table 1. The MAO coatings were mainly composed of O, Ti and Al, and additional W existed in the MAO-4W along with doping the sodium tungstate into the basic electrolyte. Obviously, Ti was derived from the substrate, while O, Si and P came from the electrolyte. The weight percentage of Al in the MAO coating was much higher than that in substrate, indicating that NaAlO2 participated in the plasma discharge to form the coating. The phase structures of the MAO coatings were investigated by XRD and illustrated in Fig. 2. The MAO coatings principally consisted of aluminium titanate (Al2TiO5), rutile titania (TiO2) and alumina (Al2O3), which suggested that the addition of sodium tungstate had negligible effects on the phase compositions of the MAO coating. Furthermore, an obvious broad peak like a steamed bun in the range of 20° to 40° corroborated the amorphous substances in the MAO coating, Si and P should exist in the amorphous phase due to the missing crystal referred to them in the XRD patterns. On the other hand, despite the content of W in the MAO-4W coating was 7.48 wt%, no phases related to W were detected in Fig. 2. Therefore, the content of formed crystal phase related to W may be too few to reach the threshold of detection for the equipment. Perhaps W also entered the coating to form the amorphous matters to some extent. Due to the negligible difference of XPS spectra between MAO-0W and MAO-4W, Fig. 3 just exhibited the chemical states of the elements in the MAO-4W coating, and the XPS results of MAO-0W were presented in Fig. S1. The full spectrum in Fig. 3a showed the existence of O, Al, Si, P, Ti and W, which was consistent with the EDS results. The highresolution spectra of Al 2p, Si 2p, P 2p, Ti 2p, W 4f and O 1s were depicted in Fig. 3b–g. The Al 2p 3/2 peak at 75.37 eV in Fig. 3b was related to AleO bond in Al2O3 and Al2TiO5. The sole Si 2p peak in Fig. 3c was linked to the SieO bond in silicate. Fig. 3d showed the P 2p core level corresponding to the PeO bond in orthophosphates, the two distinct P 2p and P 2p 3/2 peaks located at the binding energy of 134.96 eV and 133.14 eV. The two peaks at 465.59 eV and 459.87 eV in the spectrum of Ti 2p were assigned to the Ti 2p 1/2 and Ti 2p 3/2 in TiO2 and Al2TiO5. The binding energies of W 4f 5/2 and W 4f 7/2 peaks were 38.74 eV and 36.79 eV, indicating that W still exhibited the same hexavalent chemical state as sodium tungstate and might exist as WO3 or tungstate. The profile of O 1s in Fig. 3g was decomposed into six peaks, the peak located at the binding energy of 534.42 eV belonged to the OeH bond oxygen. Since the coating was fabricated in aqueous solution, some crystal water can be trapped in the coating during the MAO process. The peaks at 533.55 eV and 533.30 eV corresponded to the OeAl bond (Al2O3 and Al2TiO5) and OeTi bond (TiO2 and Al2TiO5), respectively. Moreover, the peak of OeW bond at the binding energy of 532.67 eV was obvious, which might be ascribed to the comparatively high mass fraction of W on the surface according to the EDS in Table 1. Besides, the two small peaks located at 532.00 eV and 531.71 eV were determined as the OeSi and OeP bond, proving the existence of silicate

3. Results and discussion 3.1. Surface and cross-sectional morphology The surface and cross-sectional morphologies of the MAO coatings were shown in Fig. 1. A porous and craterlike morphology was presented on the surface of both MAO-0W and MAO-4W, and numerous cavities with several micrometers dispersedly distributed on the surface. According to the cross section of the MAO coating, a typical porous and uneven microstructure was exhibited, and no distinct cracks were found at the interface between the substrate and coating, implying 244

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Fig. 1. Surface and cross-sectional morphology of the coatings: (a, b) MAO-0W, (c, d) MAO-4W.

the surface of the coating gradually dissolved in the PBS solution. As for the MAO-4W coating, many obvious cavities existed in the area marked with red ellipses in Fig. 4b. Especially, the regions around the volcano hole on the original coating were much smooth in Fig. 1c, while the corresponding areas near the crater were coarse and concave (marked with red arrows at the right side in Fig. 4b), and these representative places cannot be found on the original coating. These topographic features verified that the soluble products fell off the surface of the coating and left abundant tiny spots. The cumulative concentration of Al and W with different soaking times was presented in Fig. 5. The leached metal ions infused into the solution at a decreasing rate, and the dissolution rate of Al was larger than that of W. The only difference between the MAO-0W and MAO-4W was the released W ions. Thus, it can be inferred that the dissolved products from the MAO-4W coating contained W.

Table 1 The elemental contents (wt%) on the surface of the MAO coatings prepared in the electrolyte with and without sodium tungstate. Samples

O

Si

Al

P

Ti

W

MAO-0W MAO-4W

40.40 36.06

5.24 4.79

24.67 21.81

7.58 7.32

22.11 22.54

– 7.48

3.4. Antibacterial activity of MAO coating The pictures of the bacterial colonies on the LB agar plate were taken, and the antimicrobial efficiency of the MAO coatings against planktonic and adherent E. coli and S. aureus was evaluated from the number of the bacterial colonies. The quantitative antibacterial rate for planktonic and adherent bacteria was displayed in Fig. 6. Figs. 7 and 8 showed the macrophotographs of the planktonic and adherent E. coli and S. aureus bacterial colonies on the LB agar plate, respectively. Fig. 6a exhibited the antibacterial rate of the MAO coatings and substrate against planktonic E. coli and S. aureus. It was found that the MAO-4 W obviously inhibited the growth of the planktonic E. coli and S. aureus, and the antibacterial rate of the MAO-4W reached 99%. While the MAO-0W just restricted the viability of the planktonic E. coli and S. aureus to a minuscule degree. Additionally, its antibacterial rate fiercely fluctuated in repeated trials, which further suggested that the bactericidal effect of the MAO-0W was unstable. These results can be verified by the macrophotographs of the planktonic E. coli and S. aureus bacterial colonies on the LB agar plate in Fig. 7. The plate was covered with large numbers of bacterial colonies corresponding to the bacterial suspension of E. coli and S. aureus on substrate in Fig. 7a and d,

Fig. 2. XRD patterns of the MAO coatings and substrate.

and phosphate radicals. The deconvoluted results of O 1s were well consistent with that of the other elements.

3.3. Metal ions release The release of metal ions from the MAO coating was measured after immersion in PBS solution for 1, 2, 4 and 7 days. Fig. 4 showed the surface morphology of the MAO coating after immersion for 7 days. The porous and craterlike topography of the MAO coatings almost remained unchanged. Whereas, vast fine pits were found on the soaked surface in Fig. 4a, which was distinctly different from the Fig. 1a, unveiling that 245

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Fig. 3. XPS spectra of MAO-4W coating: (a) full spectrum, (b) Al(2p), (c) Si(2p), (d) P(2p), (e) Ti(2p), (f) W(4f) and (g) O(1s).

appeared on the LB agar plate in Fig. 7b and e when the bacterial suspension was cultured with MAO-0W. Consequently, it can be reasonably concluded that the addition of sodium tungstate is the crucial factor to endow the MAO coating with the notable antimicrobial effects

demonstrating that the substrate possessed the poor antimicrobial activity against planktonic bacteria. In contrast, only several colonies appeared on the plate in Fig. 7c and f after incubating the planktonic bacteria with MAO-4W. Additionally, many bacterial colonies still 246

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Fig. 4. Surface morphology of the MAO coatings after immersion in PBS solution for 7 days: (a) MAO-0W, (b) MAO-4W.

bacteria in Fig. 6a. Because of the small quantities of the colonies for adherent bacteria in control group (Ti6Al4V), the high antibacterial rate of MAO-0W against adherent bacteria was extremely unreasonable in Fig. 6b. Thereby, the antibacterial properties of the MAO-0W coating should be evaluated mainly by antibacterial rate for planktonic bacteria. No bacterial colonies of the bacteria attaching on the surface of MAO-4W were found on the plate, which also elucidated the excellent antibiotic performance of MAO-4W. Combined with the two kinds of antibacterial rates, the MAO-4W coating was indeed superior to the MAO-0W coating. Based on the above results, the MAO-4W showed the excellent antibacterial activity against planktonic and adherent E. coli and S. aureus bacteria, which was closely related to the sodium tungstate. The sodium tungstate promoted the antibacterial properties of the MAO coating, and the tungsten and its correlative substances in the MAO coating played a pivotal role in disturbing the metabolism of both the adherent and planktonic E. coli and S. aureus bacteria. The attached bacteria on the surface of Ti6Al4V, MAO-0W and MAO-4W were further visualized by SEM observation. The morphology of the adherent E. coli and S. aureus bacteria on the surface of the samples were shown in Fig. 9. After incubation for 24 h, bacterial membrane of the adherent E. coli and S. aureus on the surface of the substrate still maintained integrated, and bacterial cells of E. coli exhibited undamaged rod shape in Fig. 9a, while the adherent S. aureus bacteria kept the spherical shape in Fig. 9d, indicating that the substrate presented limited antibacterial effects on the E. coli and S. aureus bacteria. The surface profile of the adherent bacteria was slightly altered after contacting with MAO-0W, and the adherent bacteria were marked with red arrows in Fig. 9. The attached E. coli still held rod-like and smooth morphology in Fig. 9b, and the shape of the adherent S. aureus bacteria was almost unchanged in Fig. 9e despite some tiny dimples existed on the surface, elucidating that the cytoderms of the adherent bacteria were slightly affected by MAO-0W. In the case of adhesive E. coli on MAO-4W, the bacteria were completely decomposed as shown in Fig. 9c, the cell fragments were left in the vicinity of the

Fig. 5. The cumulative concentration of the metal ions Al and W released from the MAO coatings.

against planktonic E. coli and S. aureus bacteria. As for the adherent E. coli and S. aureus, the MAO-4W still presented the most efficient bactericidal properties against adherent bacteria, and its antibacterial rate was > 99% in Fig. 6b. The formed colonies of the adherent E. coli and S. aureus on LB agar plate were shown in Fig. 8, on the whole, the sequences of the numbers of bacterial colonies in Fig. 8 conformed to the results in Fig. 7. The number of the bacterial colonies cultured with substrate was just a few dozens in Fig. 8a and d, which was much lower than that in Fig. 7a and d, suggesting that the population of adherent bacteria on the surface of substrate was greatly less than that of planktonic bacteria. It also meant that most of the bacteria suspended in the aqueous LB medium, and only trace amounts of bacteria adhered to the surface of samples. Whereas, the MAO-0W displayed a relatively high bacteriostatic action for adherent E. coli and S. aureus compared with its antibacterial rate against the planktonic

Fig. 6. The antimicrobial rate of the MAO coatings and Ti6Al4V against (a) planktonic and (b) adherent bacteria (⁎⁎p < 0.01). 247

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Fig. 7. Photos of culture plate for planktonic bacteria: (a) Ti6Al4V, (b) MAO-0W and (c) MAO-4W against E. coli; (d) Ti6Al4V, (e) MAO-0W and (f) MAO-4W against S. aureus.

attached bacteria was characterized by fluorescent images and presented in Fig. 10. It was clearly showed that the substrate displayed the maximum amounts of the adherent bacteria, and all the bacteria exhibited green fluorescence, implying that they were live on the surface of matrix. Thus, the Ti6Al4V was insufficient to restrain the bacterial activity. Many fluorescent points were still observed on the surface of MAO-0W coating in Fig. 10b and e compared with the substrate. Whereas, few green points and some orange fluorescent dots were found, which unveiled that the cell wall of the partial bacteria was damaged to some extent. Meanwhile, the flaxen dots derived from superimposed effect of the green and orange fluorescence also existed on the surface, this was owing to the mixed dyeing of the acridine orange and ethidium bromide. Furthermore, only several scattered fluorescent points lied on the Fig. 10c, and almost all of the bacteria exhibited faint yellow in Fig. 10f, manifesting that the MAO-4W presented pronounced

bacteria and marked with red ellipses. Despite the globular form was still kept by the adherent S. aureus bacteria on the surface of MAO-4W in Fig. 9f, many irregular fine particles adhered to the surface of the S. aureus bacteria, these granules might come from the exfoliated Wcontaining dissolved matters, which was in accordance with the morphology after immersion in PBS solution in Fig. 4b. Furthermore, the contractive cell wall of the adherent S. aureus bacteria was displayed in the image inserted in Fig. 9f, suggesting that the bacterial cell wall was obviously destructive, and the MAO-4W coating had significant influences on the adherent bacteria. Hence, it can be concluded that the coating prepared with addition of sodium tungstate exhibited the notable antibacterial performance against the attached E. coli and S. aureus bacteria. To further illustrate the antibacterial properties of the MAO coatings against the adherent E. coli and S. aureus bacteria, the viability of the

Fig. 8. Photos of culture plate for adherent bacteria: (a) Ti6Al4V, (b) MAO-0W and (c) MAO-4W against E. coli; (d) Ti6Al4V, (e) MAO-0W and (f) MAO-4W against S. aureus. 248

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Fig. 9. Morphology of the adherent E. coli (a–c) and S. aureus (d–f) on Ti6Al4V (a, d), MAO-0W (b, e) and MAO-4W (c, f).

Fig. 10. Fluorescence images of the adherent E. coli (a–c) and S. aureus (d–f) on Ti6Al4V (a, d), MAO-0W (b, e) and MAO-4W (c, f).

antibacterial activity. These results of fluorescent staining were strongly consistent with that of the spread plate method and morphology of the attached bacteria. Based on the above results, it can be concluded that doping the

sodium tungstate into the electrolyte strikingly enhances the antibacterial activity of the MAO coating against planktonic and adherent E. coli and S. aureus bacteria. The MAO-4W contained no obvious antibacterial crystal phases according to the XRD patterns. Despite the

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intensity between the MAO-0W and Ti6Al4V was almost same. Whereas, the difference among the intensity of DMPO-OH∙ adduct for all samples was small in Fig. 12b. These results demonstrated the existence of the extracellular ROS, and the superoxide radicals played a more dominant role in antibacterial process for MAO-4W. Combined with the previous results, the MAO-4W can simultaneously cause the formation of extracellular and intracellular ROS in medium, and the Wcontaining coating presented the optimum antibacterial effects. The potential antibacterial mechanism was schematized in Fig. 13. The W-containing coating gradually dissolved into the solution, and the exfoliated dissolved matters contained W and amorphous substances. The exfoliated W-containing dissolved substances adhered to the bacterial cell wall and induced the formation of the extracellular and intracellular ROS with high oxidation potential. The extracellular ROS harmed the biomolecules (e.g. lipopolysaccharide, phospholipid and lipoprotein) of the outer membrane and facilitated the deleterious change in peptidoglycan membrane of the Gram-negative E. coli. As for the Gram-positive S. aureus, because of the structural differences in cell wall between the Gram-negative and Gram-positive bacteria, the extracellular ROS directly attacked the peptidoglycan membrane and interacted with the teichoic acid. Consequently, the cell wall was destroyed, further leading to the rupture of the cytomembrane. While parts of nanoscale W-containing dissolved matters penetrated the cell membrane and directly caused the oxidation of the intercellular components by virtue of the formation of intracellular ROS. The decomposition of the cytoplasmic constitutes (e.g. enzymes, DNA and RNA) gave rise to the loss of bacterial respiratory activity and cell death.

existence of TiO2, the rutile TiO2 presented the poor antibacterial performance in consideration of incubation in dark condition [49–51], thus it can be reasonably deduced that the antibacterial properties of the MAO-4W coating were related to the W and amorphous phase. After soaking in the PBS solution for 7 days, many corrosion spots were left on the surface of the MAO coating, and the results of the ICP-AES confirmed that the exfoliated dissolved matters contained the W element. Both the macrophotographs of the agar plates and fluorescent images of the adherent bacteria corroborated the antibacterial properties of the MAO coating were enhanced with the addition of sodium tungstate into the electrolyte. Moreover, the difference of the released ions between the MAO-0W and MAO-4W was W. Therefore, the antibacterial activity of the MAO-4W derived from the sodium tungstate in the electrolyte, and must be linked to the amorphous substances and the tungsten. Interestingly, in consideration of the released W of MAO-4W coating after immersion in PBS solution, we also cultured the E. coli and S. aureus bacteria with the 0.2 g/L sodium tungstate aqueous solution, and its concentration was 60 times higher than that of the released W in PBS solution. However, there were no notable differences in numbers of the bacterial colonies between the agar plates (see Fig. S2), the activity of the bacteria was almost unaffected by sodium tungstate aqueous solution, indicating that sodium tungstate displayed no antibacterial properties. Hence, the antimicrobial capability of the particles cannot come from tungstate radicals, and the sodium tungstate must undergo certain transition into the coating during the MAO process. In fact, the sodium tungstate is very likely to exist in the coating by means of tungsten trioxide, and the literature have reported that sodium tungstate enters the coating by transforming into tungsten oxide during the MAO process [52,53]. Additionally, the oxygen vacancies in the WO3 may be formed under the plasma discharge during the MAO process, and the WO3 turns into the similar structure phase (e.g. W18O49) [54]. Besides, we have calculated the contents of the crystal WO3 phase in the MAO coating in our previous paper [55], the results verify that the volume fraction of the WO3 is extremely close to the detection limit. Hence, the diffraction signal of WO3 is difficult to distinguish and disappeared in XRD pattern. The potential antibacterial mechanism of MAO-4W was assumed to be linked to the generation of ROS, such as hydroxyl radicals, superoxide radicals and hydrogen peroxides [56,57]. The DCFHDA and DMPO were used to verify the existence of the intracellular and extracellular ROS caused by the W-containing dissolved matters. The fluorescence intensity of the bacterial liquid after cultured with DCFHDA was shown in Fig. 11. The intensity of bacterial liquids incubated with the Ti6Al4V and MAO-0W was almost identical, while the bacterial liquid cultured with MAO-4W exhibited the higher fluorescence intensity. Therefore, the MAO-4W coating triggered the largest content of intracellular ROS in both E. coli and S. aureus. The EPR spectra of the DMPO-ROS adducts were shown in Fig. 12. The signal of DMPO-O2∙ adduct for MAO-4W was highest in Fig. 12a, and the

4. Conclusions The antibacterial MAO coating was fabricated in the basic Na2SiO3(NaPO3)6-NaAlO2 electrolyte doped with sodium tungstate. The crystalline phase compositions of the MAO coating were almost independent of the addition of sodium tungstate. The chemical state of tungsten presented a hexavalent state in the coating, and W may exist in amorphous substances and trace amounts of tungsten oxides. Doping Na2WO4 into the electrolyte to prepare the antibacterial W-containing microarc oxidation coating was verified to be feasible for the first time, and the antibacterial properties of the coating was mainly dependent on the sodium tungstate. The W-containing MAO coating significantly inhibited the adhesion of bacteria and reduced the quantities of planktonic bacteria in culture medium. The potential antibacterial mechanism of the W-containing coating was initially unveiled. The generation of the extracellular and intracellular ROS was induced by the exfoliated W-containing dissolved matters, the extracellular ROS with high oxidation potential interacted with the cell wall and membrane of the bacteria, causing the decomposition of the cytoderm and cytomembrane. Meanwhile, the nanoscale W-containing dissolved matters penetrated the bacterial cytoplasm, and triggered the formation

Fig. 11. Fluorescence intensity of the E. coli (a) and S. aureus (b) bacterial suspension incubated with DCFHDA. 250

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Fig. 12. EPR spectra of DMPO-ROS adducts: (a) DMPO-O2∙ and (b) DMPO-OH∙.

Fig. 13. Schematic illustration of potential antibacterial mechanism of MAO-4W coating.

of the intracellular ROS which directly harmed the intracellular macromolecules. Ultimately, the synergistic effects of the extracellular and intracellular ROS led to the death of the planktonic and adherent bacteria.

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