PEO coating on Mg-Ag alloy: The incorporation and release of Ag species

Xinxin Zhng ,Yupeng Zhng ,You Lv ,Zehu Dong ,Lei Yng ,Erlin Zhng ,Teruo Hshimoto,Xiorong Zhou,?

aKey Laboratory of Material Chemistry for Energy Conversion and Storage,Ministry of Education,Hubei Key Laboratory of Material Chemistry and Service Failure,School of Chemistry and Chemical Engineering,Huazhong University of Science and Technology,Wuhan 430074,China

bHubei Engineering Research Centre for Biomaterials and Medical Protective Materials,Huazhong University of Science and Technology,Wuhan 430074,China

cKey Laboratory for Anisotropy and Texture of Materials,Education Ministry of China,School of Materials Science and Engineering,Northeastern University,Shenyang 110819,China

d Corrosion and Protection Centre,Department of Materials,The University of Manchester,Manchester M13 9PL,United Kingdom

Abstract In the present study,the distribution of Ag in the coating formed on Mg-Ag alloy by plasma electrolytic oxidation (PEO) and its ionic release kinetics when exposed to a 0.9 wt.% NaCl solution at 37 °C have been investigated.Both metallic Ag and Ag oxide particles with～5 to ～40 nm in diameters were observed in the PEO coating.Further,an Ag-enriched layer of ～20 nm in thickness at the substrate/coating interface was also observed.The PEO coating on the Mg-Ag alloy not only increases its corrosion resistance with the corrosion current density decreasing by up to 3 orders of magnitude from 8.04 × 10-3 to 4.03 × 10-6 A/cm2,but also controls the release of Ag+ to the level that is sufficient for anti-infective efficacy without causing cytotoxicity to mammal cells.

Keywords: Mg-Ag alloy;Plasma electrolytic oxidation;Ag distribution;Ag release;Corrosion resistance.

1.Introduction

Metallic materials have been employed as medical implants since the 19th century due to their load-bearing capabilities and bio-inert properties.Nowadays,a wide range of metallic materials are used as orthopaedic implants,including titanium alloys [1–3],stainless steels [4–6],CoCr alloys [7–9] and so on.However,such metallic implants are non-biodegradable,which means that a second surgery is required to remove the implant after the healing of bone tissue.The second surgery could cause further pain,increase financial burden for patients,and may also increase the possibilities of dysfunction,physical irritation and chronic inflammation[10].Hence,biodegradable metallic implants that can avoid second surgery have attracted wide attention in both scientific and industrial communities.

Magnesium (Mg) and its alloys are regarded as promising metallic implants due to their overall physicochemical properties and desired biocompatibility [10–13].Mg alloys exhibit similar elastic modulus with human bone tissue so that stress shielding effect is minimum.More importantly,Mg alloys are naturally biodegradable [14–16],i.e.Mg alloy implants can degradeinvivowithout the requirement of a second surgery.The large-scale clinical application of biodegradable Mg alloys is still faced to various restrictions [17],although a series of progress has been achieved,including but not limited to synthesis of functional biodegradable Mg alloy [18],researches on corrosion behaviour in simulated human environment [19,20] and design of Mg alloy degradation model[21].

Microbial infection associated with implants also brings significant clinical challenges [22].Hence,nowadays,it is generally required that the implanted devices should possess certain capabilities to prevent the colonization and proliferation of bacteria [23].Some researchers claimed that Mg and its alloys exhibit certain antimicrobial capabilityinvitro,which is mainly ascribed to the increased osmotic pressure and the alkalinisation of the local chemical condition associated with the rapid dissolution of metallic Mg [24].However,such rapid dissolution of Mg-based implants may not only result in premature loss of their integrity and mechanical strength,but also produce a significant amount of Mg2+ions that may exceed the threshold concentration of Mg intake allowed for human body,consequently,resulting in hypermagnesemia.Further,the buffering effect of human body fluid may effectively prevent the local alkalinisation associated with the dissolution of Mg,hence,reduce its ability to inhibit bacterial invasioninvivo[12,25].

To endow Mg with sufficient and appropriate antimicrobial capabilityinvivo,one of the viable options is to add antibacterial elements into Mg alloys [22].Although many metallic elements can effectively kill bacteria [23],considering their potential cytotoxicity,only Ag,Cu and Zn have been widely employed for alloying with implant metallic materials [26–31].Generally,Ag possesses a higher antibacterial efficacy relative to Cu and Zn,which can disable the vital proteins of bacteria by interacting with their thiol groups and,thereby,kill bacteria by damaging the integrity of their membranes[29,32].Hence,Ag has been used as an alloying element to fabricate antibacterial Mg alloys [33,34].

Previous work on Mg-Ag binary alloys suggests that Ag addition can refine grains and thus improve their mechanical properties[34].However,it was also reported that the addition of Ag results in the formation of intermetallic compounds,including Mg4Ag,Mg7Ag3and Mg54Ag17[35–37],which increase chemical non-uniformity and,thus,lead to the reduction in corrosion resistance [34,36,37].As mentioned above,the reduction of corrosion resistance of Mg-based implants may lead to the premature loss of load-bearing capability.Further,rapid dissolution of Mg-Ag alloy may also result in excessive release of Mg and Ag species,causing cytotoxicity to hinder the proliferation and differentiation of mammal cells[29,38].

To enhance the corrosion resistance of Mg and its alloys,surface modification methods [10,39–41],including conversion coating [42–44],self-assembled monolayers [45–47] and thermal spraying [48],have been explored.Amongst all sur face modification approaches,plasma electrolytic oxidation(PEO) [40,41,49,50] is regarded as an effective method to increase the corrosion resistance of Mg alloys.During PEO process,a well-adherent oxide coating is formed on Mg surface.The coating acts as a physical barrier to separate the underlying substrate from corrosive environment.However,due to the presence of porosity and cracks in PEO coatings,the external electrolyte can infiltrate through those defects to reach the underlying metallic substrate,resulting in interaction with the coating material and local dissolution of the underlying substrate,which,consequently,leads to limited release of metallic ions [51–54].Thus,for PEO-coated Mg-Ag alloys,the release of Ag+ions may be sustained,through limited dissolution of Mg-Ag alloys and leaching of ionic species from the coating if Ag+ions are incorporated into the coating.In other words,Mg-Ag alloys with PEO coatings are likely to possess suitable corrosion properties and Ag ionic release ability desired for biodegradable orthopaedic implants.

In the present work,PEO coatings are formed on Mg-2 wt.% Ag alloys.The resultant coatings are characterized with a focus on the distribution of Ag species within the coating and at the coating/alloy interface.Further,the ionic release kinetics of Mg and Ag from the PEO-coated Mg-Ag alloy are investigated.The corrosion resistance of the PEOcoated Mg-Ag alloy is also assessed.

2.Materials and methods

2.1. Materials

In the present work,a Mg-Ag alloy with 2.03 wt.%Ag was employed because of its ideal balance in antibacterial properties,biological toxicity,corrosion resistance,and mechanical properties [34,55–57].The composition of the alloy is presented in Table 1,which was determined by optical emission spectroscopy (Spectrolab M9 Kleve,Germany).To prepare the Mg-Ag alloy,high purity magnesium ingot (99.9%,Yanbang Materials,China) and silver powder (99.9%,XFJ45 and XFJ14,XFNANO) were used.In order to remove Fe impurity,Mn and Zr were added to the melt to form Fe-rich intermetallic compounds that sink to the bottom of cast,which were removed before the subsequent processes.During casting,the temperature was maintained at 700 °C in a vacuum conductive heating furnace with Ar as the shielding gas.For comparison,99.99 wt.% high purity Mg was also used in the present work.

Table 1Chemical composition of the Mg-Ag alloy.

2.2. Synthesis of PEO coatings

Mechanically polished Mg-Ag alloy and pure Mg plates were subject to plasma electrolytic oxidation (PEO) using an in-house set-up shown in Figure S1.During the PEO process,stainless steel was used as cathode with Mg/Mg-Ag substrate as anode.The aqueous electrolyte contains 15 g/L Na2SiO3·9H2O and 5 g/L KOH.The PEO process was conducted using a pulsed unipolar constant current regime with current density,duty cycle and frequency stabilized at 30 mA/cm2,10% and 100 Hz respectively.After oxidation for 600 s,the specimens were removed from the electrolyte immediately,then washed thoroughly using deionized water and finally dried in a stream of cold air.Hereafter,PEO coatings are referred to as Mg-Ag-PEO and Mg-PEO respectively.

2.3. Characterization

For microstructure characterization,the Mg-Ag alloy was first cut into 2 mm × 2 mm × 1 mm plates,which were then mechanically ground to 1200 grit and polished to a mirror-like surface finish.In order to obtain a surface condition suitable for electron backscatter diffraction (EBSD),further polishing using a suspension of silica nanoparticles of ～40 nm in diameters was conducted on the mirror-like surface to remove near-surface deformed layer introduced by coarse polishing medium used in previous polishing steps.

The resultant coatings were characterized using various techniques,including scanning electron microscopy (SEM),transmission electron microscopy (TEM),electron probe microanalysis (EPMA),X-ray diffraction (XRD) and microscratcher.The detailed description of the instruments,operation parameters and specimen preparation procedures are included in the Supporting Information to avoid superfluous materials here.

2.4. Corrosion tests

The corrosion behaviour of PEO-coated Mg-Ag alloy was evaluated using potentiodynamic polarization(PDP)as well as hydrogen evolution measurement in 0.9 wt.%NaCl solution at the temperature of 37 °C.The experiments followed standard procedures,which are described in detail in the Supporting Information.

2.5. Ionic release tests

The ionic release kinetics of Mg and Ag from both bare Mg-Ag alloy and PEO-coated Mg-Ag alloy were investigated according to ASTM G31–72 (2004).Specimens were immersed in a 0.9 wt.% NaCl solution at 37 °C for various periods of time.The backs and edges of the specimens were covered with beeswax,leaving 1 cm2area of the surface exposed to the solution.The solution volume/specimen surface ratio was 20 mL/cm2.The released dosages of Ag and Mg were analysed using inductively coupled plasma mass spectroscopy (ICP-MS).

2.6. In vitro biological performances

Plate-counting method was carried out usingStaphylococcusaureus(S.aureus) strain (ATCC 6538) to evaluate the antibacterial properties of both bare and PEO-coated Mg-Ag alloys.And MTT assay using the MC3T3-E1 subclone 4 preosteoblast cells was conducted to examine their cytocompatibilities The experimental details are recorded in the Supporting Information.For the obtained data,Student’st-test was applied to calculate the p-value.The significance was only admitted when the p-value does not exceed 0.05,which is marked by the symbol?.

3.Results

3.1. Plasma electrolytic oxidation of Mg-Ag alloy

Before PEO treatment,the microstructure of the Mg-Ag alloy was characterized (Fig.1).Cellular feature appearing brighter than the surrounding area is revealed in the backscatter electron micrograph (Fig.1(a)).Within the bright regions,micron-sized brighter spots (indicated by red dashed-line arrows) were also observed,which is confirmed in a magnified micrograph from a different region,as shown in Fig.1(b),where electron probe microanalysis (EPMA) was also performed to determine elemental distribution in the Mg-Ag alloy(Fig.1(c)).It is evident that the bright cellular feature corresponds to regions with higher Ag contents.Since the solid solubility of Ag in Mg is relatively high,up to 15 wt.%,most of Ag in the binary alloy is present in solid solution[58].However,Ag concentration in the alloy matrix is not uniform.The Mg matrix formed in early stage of solidification contains relatively low level of solute and the Mg matrix formed during late stage of non-equilibrium solidification contains relatively high level of solute,i.e.Ag tends to segregate along grain boundaries,forming the cellular structure in the alloy.Further,as shown in Fig.1(c),the micron-sized bright spots are highly enriched in Ag (indicated by red dashedline arrows),suggesting that the bright spots are eutectic Mg-Ag intermetallic compounds,presumably Mg3Ag,Mg4Ag or Mg54Ag17phases [36,37,55].EBSD analysis was conducted on the Mg-Ag alloy surface to reveal its grain structure,as shown in Fig.1(d) with the grain orientation distribution in inverse pole figure colouring,revealing equiaxed grains.Comparing Fig.1(a) and (d),similar sizes of grains and cellular pattern are noticed,confirming Ag segregation along grain boundaries.It is worth mentioning that some finer particles were also observed in the alloy,which are generally Zr-rich with a typical example shown in Figure S2.

Fig.1.(a),(b) backscatter electron micrographs of the Mg-Ag alloy surface;(c) EPMA elemental maps from the area shown in (b);(d) grain orientation distribution in inverse pole figure colouring.

The Mg-Ag alloy was then subject to plasma electrolytic oxidation (PEO) treatment.For comparison,high purity Mg(99.99 wt.%) was also treated using the same PEO procedure.As a constant current density was employed for the PEO treatment,the cell voltage response was recorded,as shown in Fig.2,revealing three stages.Initially,the cell voltage increases linearly to ～400 V,which then continues to increase at a slower rate to about 450 V before finally stabilized at about 470 V.Interestingly,the voltage-time curves for both pure Mg and Mg-Ag alloy are identical,indicating that the addition of 2.03 wt.% Ag has negligible influence on the PEO process.

Fig.2.Voltage-time curves of Mg-Ag alloy and pure Mg during the PEO process under constant current density of 30 mA/cm2 in the electrolyte consisting of 15 g/L Na2SiO3·9H2O and 5 g/L KOH.

Scanning electron microscopy (SEM) was carried out to examine the surfaces and cross sections of PEO coatings,as shown in Fig.3,revealing identical surface morphologies of the PEO coatings on pure Mg and Mg-Ag alloy.Both PEO coatings exhibit typical porous features characterized by pores of micron size (indicated by white dashed-line arrows),which are discharge channels associated with plasma discharge events during the oxidation process.The framed areas of Fig.3(a),(b) are shown in Fig.3(c),(d) at increased magnifications,exhibiting crack-free coatings.The porosities of both PEO coatings were quantified (Figure S3) using area ratios of pore area over the entire coating surface area.A minimum of 10 randomly selected areas were analysed for this porosity quantification.The porosity of Mg-Ag-PEO is approximately 9.1%,similar to that of Mg-PEO (～9.0%).Fig.3(e),(f) exhibit the framed areas of Fig.3(c),(d) at an increased magnification,revealing numerous bright spots on the surfaces of both coatings (with typical examples denoted with red dashed-line arrows).Those bright spots tend to concentrate around the pores,indicating that they may be the deposits of molten material produced by thermal transient associated with the plasma discharge.Finally,high-resolution SEM micrographs of both coatings are shown in Fig.3(g),(h),exhibiting numerous globular features of nanometre scales,indicating that the deposition of molten material contributes to the growth of the coating.

EDX analysis was then conducted to analyse the compositions of the PEO coatings on pure Mg and Mg-Ag alloy.The EDX maps in Fig.4(a),(b) exhibit uniform elemental distributions in the coatings.The detection of Si in both PEO coatings suggests the incorporation of species from the electrolyte into the coating during PEO process.Further,Ag was detected in the coating formed on Mg-Ag alloy,suggesting that Ag is also present in the PEO coating on Mg-Ag alloy.Since the PEO coatings have thickness greater than 3 μm(Fig.5),which is greater than the detection depth of EDX analysis under the selected accelerating voltage of 15 kV,the Ag yields during EDX analysis must be from the coating.Quantified chemical compositions of both PEO coatings are presented in Table 2.The values are average of ten randomly selected areas on each coating.The reduction of Ag/Mg ratio (～0.013) in PEO coating relative to the alloy substrate(～0.021) indicates lower oxidation rate of Ag or loss of Ag species to electrolyte during PEO process,which is further discussed later.

Fig.4.Scanning electron micrographs and corresponding EDX elemental maps of the PEO coatings: (a) PEO coating on pure Mg;(b) PEO coating on Mg-Ag alloy.

Fig.5.Scanning electron micrographs of the cross sections of the PEO coatings: (a),(c) PEO coating on pure Mg;(b),(d) PEO coating on Mg-Ag alloy.

Table 2Chemical compositions of PEO coatings on Mg-Ag alloy and pure Mg.

Following the characterization of PEO coating surface,cross sections of the coatings on pure Mg and Mg-Ag alloy were produced using focused ion beam (FIB).A layer of Pt was deposited on the coating surface regions where cross sections were taken to protect the underlying specimen before ion milling.Fig.5 shows the representative cross-sectional views of the PEO coatings,revealing porous features beneath coating surface and the variations in thickness across surface.From repeated examination of a number randomly selected areas,it is evident that the thickness of Mg-Ag-PEO (from～1.9 to 5.7 μm) is similar to that of Mg-PEO (from ～1.7 to 5.4 μm) although Fig.5(a),(b) display some differences in thickness since they only give local thickness of the two cross sections.The framed areas of Fig.5(a),(b) are shown in Figs.5(c),(d) at increased magnifications to reveal the details of morphological features.Both Mg-Ag-PEO and Mg-PEO display three-layered structures (as indicated by the dashed lines),a compact inner layer,a porous middle layer and an outer layer with open channels connecting electrolyte and the layers beneath.The compact inner layer of ～400 nm thickness is present immediately above the substrate.The middle layer exhibits a high population of pores with typical sizes below a micrometre,which are absent in the inner and outer layers.The outer layer exhibits open channels with diameters of 1–2 μm,consistent with the size of the pores observed on the coating surface (Fig.3).The open channels are associated with plasma discharge events during the PEO process,which connect the electrolyte and the materials beneath surface.Fig.5(c),(d) also shows the presence of intact coating/substrate interfaces in both Mg-Ag-PEO and Mg-PEO,which may lead to the desired adhesive strength between the PEO coating and the underlying metallic substrate.This is verified by the micro-scratcher test.By applying the ramping load from 0.03 N to 50 N on the PEO-coated specimen,the critical load that reflects adhesive strength could be determined by the combination of imaging and acoustic emission(AE).A typical optical micrograph of the groove formed on Mg-Ag-PEO and related curves during the scratching process are shown in Figure S4 with the critical failure point marked by a red dashed-line frame.As expected,the PEO coating exhibit an adhesive strength higher than 10 N,which is desired for the application as implants.Scrutiny of Fig.5(d)also reveals a thin layer of nanometres thickness at the coating/substrate interface with a higher brightness (indicated by red arrows) relative to the surrounding area,presumably associated with the enrichment of Ag [59–61],which will be further discussed later.

X-ray diffraction (XRD) analysis was carried out to investigate the phase components in the PEO coatings (Fig.6).For both Mg-Ag-PEO and Mg-PEO,characteristic peaks for MgO are identified,which are absent in the spectra for bare substrates that only exhibit peaks for metallic Mg.The peaks for metallic Mg are also present in the spectra for PEO-coated substrate due to the large penetration depth of X-ray relative to the thickness of the PEO coating although TF-XRD (thin film XRD,using grazing incident angle) was employed.The differences in the intensities of metallic Mg peaks at different angles in the spectra for PEO-coated specimens are due to the significant variation in coating thickness,which leads to random detection of metallic grains from the area where coating is thinner than the penetration depth of X-ray.Both PEO-coated Mg-Ag and bare Mg-Ag exhibit no Ag-related peaks,indicating that Ag mainly exists as solute atoms in the alloy matrix with a very low volume fraction of Ag-rich second phases (Fig.1).For the PEO coating,the absence of Ag-related peaks may be ascribed to the low volume fraction of Ag-rich crystals,which is confirmed by transmission electron microscopy (TEM) later.

Fig.6.XRD patterns of Mg-Ag alloy and pure Mg with and without PEO coatings.

Transmission electron microscopy was carried out to further characterize the PEO coatings,especially local phase distribution and composition.Fig.7(a) displays a high angle annular dark field (HAADF) micrograph obtained from a cross section of the PEO coating on Mg-Ag alloy,again,exhibiting a layered structure.Scrutiny of Fig.7(a) reveals nano-sized pores (indicated by red dashed-line arrows) in the inner layer,which were not resolved in SEM (Fig.5) due to their fine size.The nano-sized pores are due to the formation of oxygen gas bubbles [59,62].The framed area 1 in Fig.7(a) is shown in Fig.7(b) at an increased magnification,displaying a high population of fine bright spots with typical dimensions ranging from ～5 to ～40 nm (with typical examples indicated by red dashed-line arrows).The framed area 2 in Fig.7(a)is shown in Fig.7(c),with the corresponding EDX elemental maps shown in Fig.7(d).It is evident that the nano-sized bright spots are enriched in Ag (indicated by red dashed-line arrows).Further,a thin Ag-enriched layer is also observed at the coating/substrate interface (indicated by red solid-line arrows).Unlike Ag,the distributions of Mg,O and Si are relatively uniform across the PEO coating.

Fig.7.TEM analysis of the PEO coating on Mg-Ag alloy: (a) HAADF micrograph showing cross-sectional view of the PEO coating;(b) framed area 1 in(a) at an increased magnification;(c) framed area 2 in (a) at an increased magnification;(d) EDX maps from the area shown in (c);(e) a lattice image of the middle layer in the PEO coating;(f) the FFT pattern from the lattice image of (e);(g)-(h) lattice images of two typical Ag-rich nanoparticles in the PEO coating.

A typical high resolution TEM (HRTEM) micrograph of the middle layer in the PEO coating is displayed in Fig.7(e),clearly revealing lattice fringes of nano-crystalline regions.The corresponding fast Fourier transformation(FFT)is shown in Fig.7(f),displaying concentric rings as expected for polycrystalline materials.Further,the FFT pattern clearly contains the diffraction points for{220}MgOand{200}MgOplanes,consistent with Fig.6.In addition,diffraction points accounting for {104}Agand {101}Agplanes (PDF#41–1402) along with {23}Ag2Oand {003}Ag2Oplanes (PDF#42–0874) are also identified,indicating that both metallic Ag and Ag2O nano-crystalline particles are present in the coating.This is further confirmed by HRTEM of individual Ag-rich particles in the PEO coating,as shown in Fig.7(g) and (h).A lattice image of a Ag-rich nanoparticle of ～30 nm in diameter is displayed in Fig.7(g),revealing {11},{11},{00} and {10}planes of Ag2O (PDF#42–0874).Another Ag-rich nanoparticle with diameter of ～7 nm is shown in Fig.7(h),identified as metallic Ag (PDF#41–1402) from the spacing of {111}and {113} planes in the lattice fringe.

For comparison,PEO coating on pure Mg was also examined using TEM (Fig.8).The cross-sectional view of the PEO coating is shown in the HAADF micrograph of Fig.8(a),revealing its porous structure.The framed areas 1 and 2 in Fig.8(a) are shown in Fig.8(b) and (c) at increased magnifications,revealing a three-layered structure (indicated by the dashed lines).Within the inner layer,fine nano-sized pores associated with oxygen evolution are clearly observed,whereas micron-sized pores originated from plasma discharge are mainly present in the middle layer.Clearly,the coatings on Mg-Ag alloy and pure Mg have identical morphologies across coating thickness.The chemical composition of the PEO coating was also analysed using EDX mapping.Fig.8(d) shows elemental maps generated from the area shown in Fig.8(c),exhibiting uniform distributions of Mg,O and Si across the PEO coating.Finally,the electron diffraction of the PEO coating is shown in Fig.8(e),displaying ring-like diffraction pattern from nano-sized polycrystalline MgO.

3.2. Corrosion behaviour of PEO-coated Mg-Ag alloy

The corrosion resistance of PEO coatings were evaluated by both potentiodynamic polarization (Fig.9) and hydrogen evolution experiment (Fig.10).The potentiodynamic polarization curves shown in Fig.9 were obtained from Mg-PEO and Mg-Ag-PEO in a 0.9 wt.% NaCl solution at 37 °C,with bare Mg and Mg-Ag substrates as comparisons.The fitting results of the polarization curves based on Tafel extrapolation method are also listed in Table 3.Clearly,with PEO coatings,the ennoblement of corrosion potentials (Ecorr) for both pure Mg and Mg-Ag alloy is obvious,along with the reduction of corrosion current densities (icorr) by 2–3 orders of magnitude,indicating that PEO coatings endow the underlying substrates with enhanced corrosion resistance.It is worth noting that there are significant shifts ofEcorrin the positive direction for both coated and bare Mg-Ag alloy compared with coated and bare pure Mg,which are due to Ag-enriched layer at the coating/substrate interface for PEO-coated Mg-Ag,or Ag in solid solution for bare Mg-Ag alloy.

Fig.9.Anodic polarization curves of PEO-coated pure Mg and Mg-Ag alloy with bare Mg and Mg-Ag substrates as comparisons in a 0.9 wt.% NaCl solution at 37 °C.

Fig.10.Hydrogen evolution measurements from Mg-Ag alloy and pure Mg with and without PEO coatings immersed in 0.9 wt.%NaCl solution at 37°C,the cumulative specific hydrogen volume (specific volume: measured volume divided by exposed specimen area) versus exposure time is presented.

Table 3The fitting results of the polarization curves in Fig.9.

To further assess the corrosion resistance of Mg-PEO and Mg-Ag-PEO,hydrogen evolution was measured by immersing the specimens in a 0.9 wt.% NaCl solution at 37 °C using the set-up shown in Fig.S5.The cumulative specific hydrogen volume (specific volume: measured hydrogen volume divided by the exposed specimen area) is shown in Fig.10.As expected,the overall average hydrogen evolution rate follows the descending sequence:bare Mg-Ag(～0.43 mL cm-2h-1) >bare Mg (～0.23 mL cm-2h-1) >>Mg-Ag-PEO (～0.139 mL cm-2h-1) >Mg-PEO (～0.086 mL cm-2h-1).Clearly,Mg-Ag-PEO and Mg-PEO exhibit comparable corrosion resistance,which are significantly higher than bare Mg and Mg-Ag substrates.

3.3. Ionic release from PEO-coated Mg-Ag alloy

The ionic release kinetics of Mg and Ag from PEO-coated Mg-Ag alloy were measured in comparison with bare Mg-Ag alloy,as presented in Fig.11.The cumulative concentrations of Ag+and Mg2+released from PEO-coated Mg-Ag alloy were recorded after immersion in a 0.9 wt.% NaCl solution for 12,24,36,48,60 and 72 h,whereas,due to high dissolution rate,the immersion periods of 1,3,6,9,12,15 and 18 h were selected for bare Mg-Ag alloy.

Fig.11.Cumulative concentrations of Ag+ and Mg2+ released from PEO-coated and bare Mg-Ag alloy during immersion in 0.9 wt.% NaCl solution at 37 °C:(a) Ag+;(b) Mg2+.

The cumulative concentration of Ag+increases generally with immersion time for both PEO-coated and bare Mg-Ag alloy (Fig.11(a)).For PEO-coated Mg-Ag alloy,the change of Ag+concentration can be divided into two distinctive stages.Initially,the release of Ag+is relatively slow,increasing to～263.8 ppb/cm2after 48 h.Afterwards,its release rate increases,reaching ～457.3 ppb/cm2and ～652.2 ppb/cm2after immersion for 60 h and 72 h,respectively.The increase of Ag release rate may correspond to progressive local breakdown of PEO coating with increasing immersion time.

Compared to PEO-coated Mg-Ag alloy,Ag+release is much more rapid for bare Mg-Ag alloy.The cumulative concentration of Ag+is more than 300 ppb/cm2after only 3 h immersion,which exceeds that for Mg-Ag-PEO after 48 h immersion.With the extension of immersion time to 18 h,the cumulative concentration of Ag+for bare Mg-Ag alloy reaches ～2.98 ppm/cm2,nearly 30 times higher than that for PEO-coated alloy after 24 h immersion.Similar tendency of Mg2+release kinetics was also recorded,as shown in Fig.11(b).

3.4. In vitro biological performances of PEO-coated Mg-Ag alloy

Finally,the difference of biological performances between bare and PEO-coated Mg-Ag alloys is verified by platecounting method (Fig.12) and MTT assay (Fig.13) respectively.Ag+releases of both bare and PEO-coated Mg-Ag alloys after 12 h immersion (Fig.11) are selected to compare their antibacterial capabilities and cytocompatibilities by adding the measured Ag+amount into the corresponding culturing media.

Fig.12.Antibacterial capabilities of bare and PEO-coated Mg-Ag alloys based on plate-counting method: (a) photographs of bacterial colonies;(b)calculated antibacterial rates.

Fig.13.(a) optical densities (ODs) of culture media after 1 day’s incubation;(b) relative growth rates of PEO-coated and bare Mg-Ag alloys.

Typical photographs of bacterial colonies are shown in Fig.12(a).Clearly,compared to the control group with no Ag+added,dramatically decreased numbers of bacterial colonies could be observed for both bare and PEO-coated Mg-Ag alloys,preliminarily suggesting their desired antibacterial capabilities againstS.aureusbacteria.The calculated antibacterial rates (ARs) of bare and PEO-coated Mg-Ag alloys are shown in Fig.12(b).The AR of bare Mg-Ag alloy(～96.8%) is higher than that of PEO-coated Mg-Ag alloy(～90.5%),which is possibly due to its relatively lower Ag+release (Fig.11).Clearly,both bare and PEO-coated Mg-Ag alloys exhibit ARs higher than 90%,indicating that they both possess desired antibacterial capabilities.

MTT assay was carried out to examine the cytocompatibilities of both bare and PEO-coated Mg-Ag alloys using MC3T3-E1 cells.The optical densities(ODs)of culture media at 570 nm were recorded after 1 day’s incubation (Fig.13(a)).Since OD570value reflects the number of osteoblasts in the culture medium,the difference of cellular response between bare and PEO-coated Mg-Ag alloys could be clearly revealed.The relative growth rate (RGR) was calculated and listed in Fig.13(b).The RGR of PEO-coated Mg-Ag alloy is ～1,which indicates that the Ag+release from PEO-coated Mg-Ag alloy exhibits no negative effect on the growth of MC3T3-E1 cells.By contrast,the RGR of bare Mg-Ag alloy is much lower than 1,suggesting the cytotoxicity of bare Mg-Ag alloy to MC3T3-E1 cells.

Hence,based on Figs.12,13,it is clearly demonstrated that,with the application of PEO process to Mg-Ag alloy,the controlled Ag ionic release of PEO-coated Mg-Ag alloy exhibits desired antibacterial capability with no apparent negative effect on the growth of MC3T3-E1 cells whereas that of bare Mg-Ag alloy causes obvious cytotoxicity to mammal cells.

4.Discussion

During the PEO process,both the species in electrolyte and Ag in Mg-Ag alloy incorporated into the resultant PEO coating(Figs.4,7 and 8).It is also revealed that Ag is present in the coating in two distinctive forms of nano-sized particles(Fig.7),i.e.Ag or Ag2O.Further,Ag-enriched layer of a few nanometres thickness is present at the coating/substrate interface,on the substrate side (Figs.5 and 7).

The enrichment of alloying elements more noble than alloy matrix element during anodic oxidation of binary alloys has been studied previously [59–61].During plasma electrolytic oxidation of Mg-Ag alloy,oxidation of Mg occurs preferentially due to less negative Gibbs free energy per equivalent for the formation of silver oxide compared with that for magnesium oxide [63–67],leading to progressive accumulation of Ag in the alloy matrix immediately beneath the interface with the growing PEO coating,which results in a Ag-enriched layer of a few nanometres thickness at the coating/substrate interface.

After enriching to a critical level,Ag and Mg atoms are then co-oxidized at the coating/substrate interface,with Ag+ions being incorporated into the PEO coating in the alloy proportions [59,65,67].The positively charged Ag+and Mg2+ions migrate outward whereas the negatively charged O2-migrate inward under the high electric field during PEO process.Since the bonding energy of Mg-O (～394 kJ/mol[68]) is higher than that of Ag-O (～213 kJ/mol [68]),Ag+migrates outward faster than Mg2+.The outward migrating Ag+ions are ejected to the electrolyte when arrive the coating/electrolyte interface,consequently,leading to a lower concentration of Ag+in the PEO coating compared to the alloy(Table 2) Eq.(1).

Further,Ag+ions are bonded with O2-ions to form Ag2O in the PEO coating.Ag2O is instable at temperatures above 250 °C and may decompose into metallic Ag rapidly when temperature reaches 300 °C following reaction (1) [69]:

The temperature at the locations where plasma discharge events occur during PEO is well above 300 °C [53,54,70].Thus,Ag nanoparticles are formed in PEO coating.Therefore,both metallic Ag and Ag2O are present in the resultant PEO coating formed on Mg-Ag alloy (Fig.7).

The Ag species incorporated in the PEO coating,including both metallic Ag and Ag2O,can be released when in contact with aqueous NaCl solution.Metallic Ag is not stable in NaCl solution.Ag reacts with dissolved O2,producing Ag+ions.This is evident in the ionic release experiment by immersing Mg-Ag alloy in 0.9 wt.% NaCl solution,as shown in Fig.11.The same experiment also shows that Ag+ions are released from PEO-coated Mg-Ag alloy although the Ag+ions release rate is significantly lower than that of bare alloy.There are two sources of Ag+ions released from PEO-coated alloy,i.e.PEO coating and alloy substrate.During immersion in NaCl solution,the incorporated Ag+ions in PEO coating are released to solution when in contact with solution since the solubility of silver oxide in neutral aqueous solution is high[71];meanwhile,the metallic Ag nanoparticles in the coating are oxidised and dissolved into solution,as described above.NaCl solution can also penetrate through PEO coating at locations of porosity and other defects to directly interact with the underlying Mg-Ag alloy substrate,leading to localized corrosion of alloy substrate and the subsequent release of Ag+to the solution.However,the extent of corrosion in PEO-coated alloy is significantly less than the general corrosion that occurs across the entire surface of bare alloy.Therefore,the Ag+ions release rate from PEO-coated alloy is significantly lower than that of bare alloy,as shown in Fig.11.

The significantly reduced Ag+ions release rate from PEOcoated alloy is beneficial for medical implants.It is generally believed that both antimicrobial efficacy and cytotoxicity of Ag specie is closely associated with its ionic concentration[14,32,72].It has been reported that when the concentration of Ag+exceeds ～300 ppb,side effects could be revealed,which may damage mammal cells.On the other hand,the minimum Ag+concentration required for antimicrobial capability is approximately 0.1 ppb [73].Hence,the desired concentration of Ag+ions is within the range of ～0.1–300 ppb.For bare Mg-Ag alloy,the burst of Ag+release is obvious in the ionic release experiment,almost 30 times faster than that of PEO-coated Mg-Ag alloy (Fig.11).Potentially,such fast release may cause cytotoxicity.By contrast,the release of Ag+from PEO-coated Mg-Ag alloy is relatively slow (Fig.11).The Ag+concentrations in leaching liquid are in the range from ～60 to ～270 ppb/cm2after immersion for 48 h,which is sufficient for antibacterial activity without causing cytotoxicity.Although Ag+concentrations reached ～652.2 ppb/cm2after immersion for 72 h,it is worth noting that the ionic release experiment was performed in a still solution and cumulative concentrations were recorded,which do not reflect theinvivosituation where liquid circulation can reduce the concentration of released ions.

Thus,the PEO coating on the Mg-Ag alloy not only improves the corrosion resistance that can prevent premature loss of mechanical strength of implants,but also controls the release of Ag+to the level that is sufficient for anti-infective efficacy,but is below the level of causing cytotoxicity.

5.Conclusions

In this work,the distribution and ionic release kinetic characteristics of Ag in the PEO-coated Mg-Ag alloy have been investigated.The following conclusions can be drawn:

?Ag species are incorporated into the coating formed on Mg-Ag alloy by PEO process.Both metallic Ag and Ag2O crystalline nanoparticles are present in the resultant PEO coating.

?Due to the difference in Gibbs free energy change during oxidation of Mg and Ag,an Ag-enriched layer of a few nanometres thickness is formed at the coating/substrate interface during PEO process.

?The corrosion resistance of the PEO coating on Mg-Ag alloy is comparable to that of PEO coating on pure Mg.

?PEO-coated Mg-Ag alloy exhibits a sustained release of Ag+when exposed to 0.9 wt.% NaCl at 37 °C.

Acknowledgements

The authors wish to thank the financial support from the Hubei Provincial Natural Science Foundation of China(No.2020CFB295),the National Natural Science Foundation of China (No.52001128),the Innovative Foundation of Huazhong University of Science and Technology (No.2021JYCXJJ023) and the Innovation and Talent Recruitment Base of New Energy Chemistry and Device (No.B21003).

The authors also thank the technical supports from the Analytical and Testing Centre in Huazhong University of Science and Technology (HUST),XFNANO Company (Nanjing,China),Shiyanjia Lab (www.shiyanjia.com),the Instrumental analysis &research centre of Shanghai Yanku(www.shuyanku.com) and Experiment Centre for Advanced Manufacturing and Technology in School of Mechanical Science &Engineering of HUST.

All research data supporting this publication are directly available within this publication.

Supplementary materials

Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.jma.2021.12.006.