MgâZnâAl LDH Composite Coating on Mg ... - ACS Publications

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Bio-interactions and Biocompatibility

PEO/Mg-Zn-Al LDH composite coating on Mg alloy as a Zn/ Mg ions release platform with multifunctions: enhanced corrosion resistance, osteogenic and antibacterial activities Feng Peng, Donghui Wang, Dongdong Zhang, Bangcheng Yan, Huiliang Cao, Yuqin Qiao, and Xuanyong Liu ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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ACS Biomaterials Science & Engineering

PEO/Mg-Zn-Al LDH composite coating on Mg alloy as a Zn/Mg ions release platform with multifunctions: enhanced corrosion resistance, osteogenic and antibacterial activities Feng Peng1,2, Donghui Wang1, Dongdong Zhang1,2, Bangcheng Yan1,2, Huiliang Cao1, Yuqin Qiao1, Xuanyong Liu1,* 1

State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai

Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China 2 University

of Chinese Academy of Sciences, Beijing 100049, China

*Corresponding Author Prof. Xuanyong Liu. E-mail: [email protected]. Tel.: +86 21 52412409. Fax: +86 21 52412409.

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Abstract: The design of advanced multifunctional Mg-based bone implants with enhanced corrosion resistance, antibacterial and osteogenic activities should be brought to the forefront to fulfill the requirement of clinic. In this work, PEO/Mg-Zn-Al layered double hydroxide (LDH) composite coating on Mg alloy was development via plasma electrolytic oxidation (PEO) and hydrothermal treatment. The porous structure formed during the PEO process was filled by Mg-Zn-Al LDH. The as-prepared coating exhibited better corrosion resistance than PEO/Mg-Al LDH composite coating. In addition, the composite coating showed strong antimicrobial ability against Gram-positive Staphylococcus aureus, which was attributed to the releasing of Zn ions. When Zn content was controlled at 1.17 at% in the composite coating, rBMSCs showed long-term cytocompatibility and enhanced initial adhesion. Moreover, with the synergistic functions of Zn and Mg ions, cells on the composite coating showed a higher level of alkaline phosphatase activity and expression of osteopontin (OPN). With enhanced corrosion resistance, antibacterial and osteogenic differentiation abilities, the PEO/Mg-Zn-Al LDH composite coating exhibits a promising application in bone-related implants.

Keywords: Magnesium alloy, Mg-Zn-Al layered double hydroxide, multifunctions, bone implants


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1. Introduction Magnesium (Mg) and its alloys are widely honored as the next generation of biomedical materials for its complete degradation in vivo, favorable mechanical strength and elastic modulus close to natural bone.1-4 However, its clinical applications are hampered by its fast corrosion rate, which would lead to the accumulation of hydrogen bubbles and the violent changes of local pH value.5 Surface modification is the widest used method to improve its cytocompatibility and corrosion resistance, including electroless plating,6-7 plasma electrolytic oxidation (PEO),8-9 electrodeposition10-11, hydrothermal treatment,12-14 polymer coating15 and ion implantation16-18 et al. Only improved corrosion resistance and cytocompatibility of Mg implants are not desirable enough to meet the requirement of different clinical application, such as bone implants. The lesson from clinical applications tell us that titanium implants worked as bone-related implants, such as bone lamella, nail or bone stent et.al are poor in osseointegration and easily be infected.19-21 Moreover, though Mg substrate exhibit strong antibacterial activity in vitro,22 Hou et.al found that Mg shows dramatically reduced antibacterial property in vivo.23 Thus, Mg implants with superior osseointegration and antibacterial activities would be superior to be worked as bone-related implants. Nevertheless, many researchers only devoted to enhance the corrosion resistance of Mg via surface modification to obtain a cytocompatibility surface or antibacterial surface,24-25



and ignore the special requirement of different biomedical application. Hence, it is full of significance for us to develop coatings on Mg alloy with sufficient osseointegration and antibacterial activities for bone-related applications. Topography, bioactive metal trace elements and antibiotics are the most studied factors to regulate bone mesenchymal stem cells and bacterial behaviors. Zhang et.al fabricated a clustered nanorod structure on Ti plate using hydrogen peroxide and found that the hybrid micro/nanorod topography could enhance the initial adhesion of bone mesenchymal stem cells (BMSCs) and promote osteogenic differentiation.26 However, topography is useless to regulate bacterial behavior. Antibiotics, such as Minocycline hydrochloride and benzyl penicillin,27-28 are grafted on materials surface to kill bacteria. Nevertheless, the abuse of antibiotics would cause drug resistance problem and the emergence of superbug, which is a worldwide concerned issue.29-30 With these considerations in mind, bioactive components might be the best choice to prepare dual biological functions (osseointegration and antibacterial properties) surface. On the one hand, Mg element plays a vital role in bone metabolism and generation. Supplement of Mg ions would enhance osteoblastic cell adhesion and then improve integration of orthopedic implants.31 It is also reported that Mg ions released from Mg alloys can enhance osteogenic differentiation of human BMSCs.32 On the other hand, Zn-incorporated biomaterials can up regulate bone-related gene expression and ALP activity,33-35 and Zn ions can inhibit bone resorption via down regulating osteoclast formation and adsorbing activity.36 Meanwhile, many reports revealed that Zn ions also


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show strong antibacterial activity.33, 37-38 It is also worth noting that Yu et.al suggested that Mg and Zn would show synergistic effects on improved osteoinductivity.39 Thus, a platform with the ability to release Zn and Mg ions on Mg alloy would be promising to obtain dual biological functions. Layered double hydroxide (LDH) becomes a hot spot in the field of surface modification of Mg.40 Ye et al. grew NiAl LDH on AZ31alloy to protect the substrate.41 Guo et al. compared the corrosion of vanadate intercalated MgAl LDH and ZnAl LDH.42 However, little literature focuses on its clinic application. Our previous work revealed that PEO/Mg-Al LDH

composite coating could offer sufficient protection for Mg alloy

and cells could survive on its surface for a long time up to 14 days.43 The composite coating can work as a platform to release Mg ions, but cannot inhibit the proliferation of bacteria. Hence, if we can introduce Zn element into the composite coating and design a dual Zn/Mg ions release platform, the modified composite coating might exhibit better osteogenesis with the synergistic action of Zn and Mg ions and enhanced antibacterial ability with the help of Zn ions. PEO/Mg-Zn-Al LDH composite coatings with different Zn contents were prepared in this work, and PEO/Mg-Al LDH composite coating was used as the comparison. The corrosion resistance of the various coatings was evaluated via electrochemical and hydrogen evolution tests. The antibacterial ability of the films was evaluated by culturing Staphylococcus aureus (S. aureus). Moreover, the behaviors of rat bone marrow stem cells (rBMSCs) were systematically examined via the evaluation of cell adhesion,



proliferation, long-term viability and osteogenic differentiation. This study may provide new insight into the design of biomedical Mg-based bone implants.

2. Materials and methods 2.1 Materials preparation and characterization Commercially purchased AZ31 alloy (10 mm × 10 mm × 2 mm) was used as the substrate. Before modification, AZ31 alloy rectangle were ground with abrasive paper, and then ultrasonically cleaned in alcohol. Firstly, porous PEO coating was prepared on the substrate via PEO equipment (Southwestern Institute of Physics, China) with 0.04 M Na2SiO3·9H2O, 0.1 M KOH and 0.2 M KF·2H2O as the electrolyte. The constant current was 0.8 A, the frequency was 800 Hz and the duty cycle was 10%. The reaction was ended at 360 V. Secondly, LDH layers were formed on the porous PEO coating via hydrothermal treatment. The samples after PEO treated were placed in a Teflon-lined stainless containing 0.02 M Al(NO3)3 (pH value was adjusted to 12.8 by NaOH) and kept at 120 °C for 12 h. After that, samples were gently washed with ultrapure water and dried in ambient atmosphere. The obtained PEO/Mg-Al LDH coated samples were denoted as PEO/LDH. For PEO/Mg-Zn-Al LDH coated sample, three different Zn(NO3)2 solutions were added to the aforementioned hydrothermal solution (the final concentration of Zn2+ ions were 10 μM, 100 μM or 1000 μM), and the obtained samples were denoted as Zn-1#, Zn-2# and Zn-3#, respectively. The surface morphologies of PEO/LDH, Zn-1#, Zn-2#, and Zn-3# samples were


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observed by scanning electron microscopy (SEM; Hitachi-S3400N, Hitachi, Japan), and elemental compositions of the samples surfaces were detected by energy dispersive spectrometry (EDS; IXRF-550i, IXRF SYSTEMS, USA). Meanwhile, scanning maps of all coated samples were also detected by EDS. X-ray diffraction (XRD; D/Max, RIGAKU, Tokyo, Japan) with a Cu Kα radiation (40 kV, 30 mA) was used to analyzed samples’ phase composition.

2.2 Corrosion resistance evaluation 2.2.1

Electrochemical test CHI760C electrochemical analyzer (Shanghai, China) was used to test the

electrochemical corrosion behavior of various samples in phosphate buffer saline (PBS). The process was conducted in with a three-electrode electrochemical cell with a saturated calomel electrode (SCE) as the reference electrode, a graphite rod as the counter electrode and the sample with exposing area of 0.255 cm2 as working electrode. Samples were kept in PBS for 5 mins before the test to obtain a stable open circuit potential (OCP). The test was performed with a scanning rate of 10 mV/s. The corrosion potential (Ecorr), current density (icorr) and polarization resistance (Rp) were calculated according to Tafel extrapolation. 2.2.2

Hydrogen evolution test Four parallel samples were immersed in 400 mL 0.35 wt% NaCl at 37 °C. A 50 mL

burette, placed on the top of the samples, was used to collect the released hydrogen. The



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amount of released hydrogen was recorded each day. 2.3 Mg and Zn ions release measurement Samples were immersed in 1 mL α-MEM (Minimum Essential Medium alpha-Medium) at 37 °C for 1 day. The amounts of released Mg and Zn ions were detected by inductively coupled plasma optical emission spectrometry (ICP-OES, Varian Liberty 150, USA). 2.4 rBMSCs early adhesion process rBMSCs were used to evaluate cell behaviors on different samples. rBMSCs were purchased from Shanghai Institutes for Biological Science. Cells were cultured with α-MEM at 37 °C in a humidified atmosphere of 5% CO2. Before seeded cells, samples were sterilized by ultraviolet irradiation. In the test mentioned below, cells were seeded on the samples at a density of 5 × 104 cells/well. For osteogenic differentiation related experiments, cells at passage 3 were used. Otherwise, cells at passage 4-5 were used. Samples were seeded with cells for 1, 4, 24 hours, respectively. At the scheduled time, the culture mediums were removed and the samples were washed with PBS. Then cells were fixed with 4% paraformaldehyde (PFA) diluent, permeabilized with

0.1%

(v/v) Triton X-100 (Amresco, USA) and blocked with 1 wt % bovine serum protein (BSA, Sigma, USA). Subsequently, Rhodamine-Phalloidin was used to stain F-actin and DAPI to stain nucleus. Finally, cells adhesion processes on different samples were observed by confocal laser scanning microscopy (CLSM, Leica SP8, Germany). 2.5 rBMSCs proliferation and morphology observation


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Cells were cultured on the samples for 1, 3 and 5 days (four parallel samples were used). Cell proliferation rate was detected via alamarBule assay. At each detected time, one specimen of each group was taken out and immersed in 2.5 v% glutaraldehyde to fix the cells. The fixed cells were dehydrated with graded ethanol series, dried with hexamethyldisilizane ethanol solution, and then observed by SEM. 2.6 Live-dead cell staining Cells were cultured on the specimens for 5 days. Afterwards, samples were rinsed with PBS for twice and added with 100 μL fluorescence dying solution (5 μM propidium iodide and 2 μM calcium-AM). After cultured for another 15 mins, samples were washed with PBS and kept in PBS before observed with CLSM. 2.7 Immunocytochemical staining Immunocytochemiacry was performed for studying osteopontin (OPN). Cells were cultured on the specimens for 14 days. Afterwards, the samples were rinsed with PBS, and then fixed in 4% PFA, followed by permeabilized in 0.1 v/v % Triton X-100, blocked with 10% bovine serum albumin. Then, cells were cultured with the primary antibodies of anti-OPN and kept at 4 °C for 16 h. At the scheduled time, goat anti-rabbit IgG A488, conjugated to fluorescein isothiocyanate, were added and incubated for 1 h. Afterwards, F-actin was stained with Rhodamine-Phalloidin and nucleus were stained with DAPI. CLSM was used to observe the stained samples. To obtain a quantitative data, the ratio of fluorescence intensity per unit of OPN staining was measured using Image J software. 2.8 Alkaline phosphatase (ALP) activity assay



Cells were cultured on the specimens for 7 days and14 days, respectively (four parallel samples were used). After cultured for scheduled times, the cells on the specimen were dissociated with LBWPI (mix-solution of 1% lgepal, 10 mM Tris-HCl and 1 mM MgCl2). The obtained dissociated solutions were certificated (4 °C, 8000 rpm) and the supernatant was collected for the ALP measurement. The supernatant were added with p-nitrophenyl phosphate, and 30 mins later, its optical density was detected at 405 nm. ALP activity was calculated according to the standard curve. Meanwhile, BCA protein assay was applied to measure the protein content of the supernatant. The relative activity of ALP was acquired by normalizing the ALP activity against the total protein content. 2.9 Antibacterial activity evaluation Gram-positive S. aureus was used to evaluate the antibacterial activity of various samples. Each sample was added with 800 μL bacterial suspension (107 CFU/mL) and incubated at 37 °C. After 1 day, the samples were transferred to tubes with 4 mL 0.9 wt% NaCl and then dissociated adhered bacteria. Afterwards, the solutions were diluted 10 times and then 100 μL diluted solution was dropped on standard agar culture plate. The culture plate was incubated for 16 h, and then the active microbes were counted and the antibacterial ratio was figured according to the formula: Antibacterial ratio = (A-B)/A × 100% where A is the average number of bacteria on the PEO/LDH sample, B is the average number of bacteria on the Zn-1#, Zn-2# and Zn-3# samples. Moreover, bacteria on the samples were fixed in 2.5 v% glutaraldehyde, followed by


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dehydrated in ethanol and dried in hexamethyldisilizane ethanol solution. The bacterial morphology was observed by SEM. 2.10 Statistical analysis Statistical analysis was performed using one-way ANOVA or two-way ANOVA. All of the data are expressed as the mean ± standard deviation (SD). Values of *p < 0.05, **p < 0.01 or ##p < 0.01 were considered statistically significant.

3. Results and discussion Figure 1a shows the surface morphology of PEO/LDH, Zn-1#, Zn-2# and Zn-3# samples. All the composite coatings exhibit similar morphology. At a low magnification, the pore structures formed during PEO process can be observed. However, at a high magnification, it is obvious that the pores were all sealed with nanosheet structures. The EDS spectra of corresponding SEM images are showed in Figure 1b. O, F, Mg, Si, Al element signals were detected at the spectra of PEO/LDH sample, among which F, Si was ascribed to the process of PEO and Al was ascribed to hydrothermal treatment. For Zn-1#, Zn-2# and Zn-3# samples, a strong Zn element signal was detected, meaning the successful introduction of Zn. The quantitative element contents were detected by EDS maps and are showed in Figure 1c. As the increase of Zn ions in the hydrothermal solution, the Zn content in the coating increased (1.17 at%, 1.56 at% and 2.13 at%, respectively). The mapping results, showed in Figure R1, further confirmed the conclusion. Moreover, the element compositions of different samples were detected by



XPS (Table S1), which also suggested the same trend. The coatings with two layer structure were confirmed by the cross-sectional images ( Figure S2).

Figure 1. Morphological evaluation of various samples by SEM (a), EDS spectra of corresponding SEM images (b), and elemental compositions of various samples by EDS maps (c). Figure 2 shows the XRD patterns of all the coated samples. All the patterns showed similar peaks, indicating the similar structure compositions. For all patterns, the peak around 42° ascribed to MgO phase, and the peaks around 32.3°, 34.4° and 36.7° were ascribed to Mg phase. The peak detected around 11.3° in PEO/LDH sample’s pattern represented Mg-Al LDH structure (PDF 50-1684). When Zn ions were added into the hydrothermal solution, the formed LDH structure was Mg-Zn-Al LDH. Many literatures reported that Mg-Zn-Al LDH can be prepared by coprecipitation method in the solution containing Zn2+, Mg2+ and Al3+.44-45 In the present work, Zn2+ and Al3+ were added into


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the hydrothermal solution, and Mg2+ would release from the PEO substrate. The existence of Zn was confirmed by the result of EDS, but we did not detected the peak of ZnO and Zn(OH)2 in the XRD pattern, whose strongest peak appeared at 36o (PDF 36-1451) and 30.6o (PDF 38-0356), respectively. Thus, it is believed that Zn element existed in the LDH structure, namely the formed nanosheet was Mg-Zn-Al LDH.

Figure 2. XRD patterns of different samples.

The clinical applications of Mg-based implants are mainly blocked by its fast degradation rate. Thus, the first principle of surface modification of biomedical Mg is to enhance its corrosion resistance. In our previous work, PEO/Mg-Al LDH composite coating could protect the Mg substrate from fast corrosion.43 In this work, PEO/Mg-Zn-Al LDH composite coatings were constructed on AZ31 alloy and their



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corrosion resistance were evaluated via potentiodynamic polarization curve (Figure 3a) and hydrogen evolution (Figure 3b). The corresponding corrosion parameters, which were obtained from the polarization curve, are shown in Table 1. Comparing to PEO/LDH sample, all the Zn-incorporated samples showed higher corrosion potential (Ecorr) and lower corrosion current density (jcorr), suggesting better corrosion resistance. Among three Zn-incorporated samples, Zn-3# sample exhibited best corrosion resistance, because it possessed the lowest jcorr value and highest polarization resistance (Rp). Table 1 Corrosion potential (Ecorr), corrosion current density (jcorr) and polarization resistance (Rp) calculated according to the potentiodynamic polarization curves. AZ31 PEO/LDH Zn-1# Zn-2# Zn-3#

Ecorr (V/SCE) -1.61 -1.33 -0.60 -0.70 -0.68

jcorr (A/cm2) 2.76×10-5 2.37×10-5 1.56×10-5 1.28×10-5 8.59×10-6

Rp (kΩ/cm2) 40.92 45.76 64.06 83.38 140.35

Figure 3. Polarization curves of various samples detected in PBS (a) and hydrogen evolution of various samples detected in 0.35 wt% NaCl (b).


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When Mg corrodes in liquid, hydrogen would release from Mg substrate. Thus, hydrogen evolution can represent the corrosion rate of the sample. Cl- ions play a vital role in the corrosion process of Mg. When Cl- concentration is higher than 30 mM, Mg(OH)2 would convert to soluble MgCl2 and then accelerate pit corrosion.46 The hydrogen evolution test was applied in 0.35 wt% NaCl solution, because the Clconcentration in body fluid is about 100 mM. At the attack of Cl-, a quick release of hydrogen from the AZ31 alloy was observed. After modified with composite coatings, the amount of released hydrogen was sharply decreased. Less hydrogen was released from Zn-incorporated samples than PEO/LDH composite film. It is worth noting that Zn-3# samples showed the lowest hydrogen evolution throughout the test period, indicating its best corrosion resistance. The above results were consistence with the results of electrochemical analysis. Cytocompatibility is an essential factor for biomedical implants. The surface of implants should be beneficial for cell adherence, spreading and proliferation. Figure 4 shows the adhere process of rBMSCs on various surfaces. As we observed in our previous work,43 even after cultured for 24 h, cells still showed round shapes and hardly spreading on AZ31 alloy, indicating no cells can survive on the surface of bare AZ31 alloy. At the initial 1 h, cells on all the composite coating showed spreading and round shapes. After adhering for 4 h, cells presented larger round morphology. For Zn-incorporated coated samples, there were a few cells showed polygon morphology



(indicated by yellow arrow in Figure 4), suggesting their better spreading status. After 24 h, cells on coated samples all showed polygon morphology with numerous filopodia and lamellipodia. The spreading area of Zn-2# and Zn-3# samples were larger than PEO/LDH and Zn-1# samples. The results mentioned above indicate that introducing Zn element to the composite coating would not do disadvantage to cells adhere and spreading process, but promote the process.

Figure 4. CLSM images of cells cultured on various surface for 1, 4 and 24 h with F-actin stained with Rhodamine-Phalloidin (red) and nucleus stained with DAPI (blue).

Figure 5a shows the proliferation rate of rBMSCs cultured on PEO/LDH, Zn-1#, Zn-2# and Zn-3# samples. After incubated for 3 days, compared with other samples, cells on Zn-2# sample showed the highest proliferation rate. When the culture time prolonged to 5 days, the number of cells on Zn-2# sample still was the most, while the number of


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cells on Zn-3# sample was significantly lower than that on PEO/LDH and Zn-1# samples. A qualitative analysis was observed by SEM and the result is presented in Figure 5d. The qualitative observation result was consistent with the quantitative result. Figure 6 displays the result of live/dead staining after cultured for 5 days. For PEO/LDH, Zn-1# and Zn-2# samples, cells almost cover the sample surface. However, cells cultured on Zn-3# samples showed a lower density, indicating a worse cytocompatibility of Zn-3# sample than the other three samples.

Figure 5. The proliferation rate (a) and SEM images (d) of cells cultured on various surfaces, the amount of releasing Mg2+ (b) and Zn2+(c) after immersing in culture



medium for 1 day.

Figure 6. CLSM images of live/dead staining of cells after cultured on various surfaces for 5 days (green represent live cells and red represent dead cells).

For biomedical implants, it is well recognized that surface phase compositions and morphology are the main factors that influencing cells behavior. The corrosion resistance of Mg-based implants, which is closely correlated with the surface phase, plays a vital role in cells behavior. Our previous study already proved that the corrosion resistance of PEO/LDH samples is well enough for cell adhesion and proliferation.43 Furthermore, the corrosion analysis (Figure 3a and b) suggested that Zn-incorporated samples showed better anticorrosion than PEO/LDH. Thus, in the view of corrosion resistance, the surfaces of all four samples are suitable for cells proliferation. The morphologies of various samples were all similar (Figure 1a). The surface phase compositions of four samples were all LDH structures, and the difference is that the surface phase composition of PEO/LDH was Mg-Al LDH while that of Zn-incorporated samples were Mg-Zn-Al LDH (Figure 2). The surface phase compositions determine species and amount of releasing ions, and further cause different cell behaviors. Figure 5b and c show the


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amount of Mg2+ and Zn2+ releasing from the samples after immersing in culture medium for 1 day. The amount of releasing Mg2+ from various samples was at the same level, while the amount of releasing Zn2+ increased with the increase of Zn content in the composite coatings (Zn-3# ＞ Zn-2# ＞ Zn-1# ＞ PEO/LDH). Therefore, the different amount of releasing Zn2+ in the culture medium of various samples was the main reason for different cell behaviors. Literatures reported that the inhibition of cell proliferation was observed when the Zn2+ concentration in the culture medium was over 100 μM (6.4 ppm).47-48 In the present study, the releasing amount of Zn2+ on the first day was all below the limitation, thus there was no difference of proliferation of cells cultured on various sample on the first day. After cultured for 3 days, the proper accumulative amount of Zn2+ from the Zn-2# sample was beneficial for cells proliferation, while Zn2+ ions from Zn-1# samples was too low and Zn2+ ions from Zn-3# sample was too high. The trend continued until the fifth day except that the suppressed cell proliferation was observed on Zn-3# samples, which might be attributed to the excess releasing amount of Zn2+. Thus, it can be concluded that, compared with PEO/LDH sample, Zn-1# sample showed comparable cytocompatibility and Zn-2# sample showed better short-term cytocompatibility.

Meanwhile, Zn-3# showed worse cytocompatibility, which means it

might not be suitable to use as implants. Many researchers also incorporated Zn element into the surface of biomedical metals, and their cytocompatibility results are consistent with our results mentioned above. Qiao et al. prepared Zn-incorporated TiO2 coating via different methods.35 Their results suggested that the coating with more Zn2+ release



showed lower cell proliferation. Liu et al. constructed Zn-incorporated chitosan/gelatin surfaces on Ti substrate via layer-by-layer self-assembly technique.49 They found significantly reduced cell viability when the Zn content of the coating was more than 6.14 at%. For bone-related implants, well osteogenic differentiation ability is required to obtain desirable osseointegration. The osteogenic activities of various samples were evaluated by measuring the expression of OPN protein and the relative activity of ALP. After cultured for 14 days, the OPN protein of the cells was stained and the results are showed in Figure 7a, and the relative fluorescent intensity is given in Figure 7b. Owing to that F-actin and nucleus were also stained, the data can also reflect the long-term cytocompatibility of rBMSCs cultured on various samples. Nearly no cells were detected in the surface of Zn-3# sample, indicating its serve cytotoxicity. As a consequence, it is meaningless to evaluate its osteogenic activity. In contrast to the proliferation rate of the cell after cultured for 5 days (Figure 5a), cells were not cover the surface of Zn-2# sample after cultured for 14 days, suggesting a poor long-term cytocompatibility. Cells on PEO/LDH and Zn-1# samples showed high cell viability with cells spreading over the entire surfaces. The fluorescent of OPN on Zn-1# and Zn-2# samples were significantly stronger than that on PEO/LDH samples, suggesting their better osteogenic differentiation ability. Figure 8 shows the ALP level of cells cultured on various samples. At day 7, cells on various samples showed the same level of ALP. At day 14, cells on Zn-1# sample exhibited the highest ALP level, while on Zn-2# sample showed the lowest


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ALP level. The ALP levels were acquired by normalizing ALP activity against the total protein content. For Zn-2# sample, the dead cells would raise the total protein content, resulting in a relative lower ALP level. It can be concluded from the above data that Zn-1# sample shows a long-term cytocompatibility and better osteogenic activity against PEO/LDH sample.

Figure 7. OPN protein expression of rBMSCs cultured on various samples for 14 days (a), and the corresponding fluorescent intensity (b).

Figure 8. Relative ALP activity of rBMSCs after cultured on various samples for 7 and



14 days.

Just as discussed in the previous section, Zn2+ takes the responsibility for cell behavior. Furthermore, Zn element is closely related with bone growth, as it is an important component of bone. When human body is in a Zn-deficient condition, bone would grow slowly.50-51 Many studies revealed that a proper Zn2+ would stimulate bone formation and mineralization.19, 39 Huo et al. suggested that the excellent osteogenesis of Zn-incorporated biomaterials is related to ERK1/2 signaling.33 The releasing of Mg2+ is beneficial for the osteogenic activity of cells cultured on PEO/LDH sample, but cells cultured on Zn-1# sample, with the appropriate amount of releasing Zn2+ and Mg2+, showed better osteogenic activity. In addition, during a 14-day incubation period, Zn-2# and Zn-3# samples would release too much Zn2+, which is disadvantage for cell growth. It worth noting that cells on Zn-2# sample showed the best OPN expression among all the samples, but considering its cytocompatibility, it is not suitable to work as bone implants. The antibacterial effect of various samples was evaluated via culturing S. aureus on sample surfaces. After cultured for 24 h, S. aureus was dissociated off from the samples and re-cultured on agar culture medium. The typical photographs of bacteria colonies are displayed in Figure 9a, and the results of bacteria counting are shown in Figure 9b. Dense bacteria colonies were observed on agar culture medium of Zn-3# sample. For Zn-1# and Zn-2# samples, significantly less dense bacteria colonies grown on the agar culture medium, while no bacterial colonies were observed on agar culture medium of Zn-3#


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sample. It can be seen that all Zn-incorporated samples can potently inhibit the proliferation of S. aureus. The counting results suggest that, compared with PEO/LDH sample, the bacterial colonies of Zn-1# and Zn-2# samples reduced about 80%, and Zn-3# sample showed a 100% antibacterial ability. The statistical bacteriostatic results were further demonstrated by SEM observation. Figure 10 shows the morphology of S. aureus after adhering to samples for 24 h. Numerous S. aureus, with plum morphology, were seen on PEO/LDH sample. A large number of S. aureus were connected with each other, indicating it is easy to form biofilms. However, much fewer S. aureus was observed on Zn-1#, Zn-2# and Zn-3# samples. Though the bacteria on these samples showed integrity membranes, no significant biofilms were observed, suggesting their effective antibacterial ability. The antibacterial behavior of various samples against E.coli is showed in Figure S3, which also indicated that Zn-incorporated coating show enhanced antibacterial ability.



Figure 9. Photographs of re-cultivated S.aureus colonies on agar culture plates (a) and analysis of reduction percentages of bacterial colonies (b).

Figure 10. SEM images of S. aureus after cultured on PEO/LDH (a), Zn-1# (b), Zn-2# (c), and Zn-3# for 24 h. Compared with antibiotic-based implants,27,

52

which are concerned by explosive

releasing and antibiotic resistance, inorganic antibacterial agents show higher application value in the clinic. Zn2+ is a universally known inorganic antibacterial agent, which can kill both oxybiontic bacterial and anaerobes.39, 49 The exactly mechanism is not clearly so far, but serval hypotheses are put forward to explain the phenomenon. One of the hypotheses ascribes the antibacterial activity of Zn2+ to its positive charge. Because the membrane of bacteria is negative, Zn2+ would adhere to the membrane, whose charge distribution would be changed immediately. Finally, the substance in and out of the membrane would be infected and lead to bacterial apoptosis. Another hypothesis believes that Zn would complex with O and N elements, which are the main component of proteins, or substitute the metal ions that excite enzyme activity. Both the processes would influence bacterial normal physiological activities. There is also hypothesis believes that Zn2+ inhibit bacterial proliferation via bonding with nucleic acid. In the present study, Zn-incorporated coatings can release Zn2+ in the physiological medium.


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The releasing Zn ions would inhibit bacterial adhesion and proliferation.

4. Conclusions PEO/Mg-Zn-Al LDH composite coating with different Zn contents was prepared on AZ31 alloy by plasma electrolytic oxidation and hydrothermal treatment. The Zn-incorporated coatings showed enhanced corrosion resistance, comparing to PEO/LDH coating. The composite coating with low Zn content showed no cytotoxicity, but significantly improved osteogenic activity. However, high Zn content in the composite coating would severely inhibit the cell proliferation. All the Zn-incorporated composite coatings showed effective antibacterial ability. In conclusion, when introducing proper Zn element to the composite coating, it is promising to obtain Mg-based implant with enhanced corrosion resistance, osteogenic activity and antibacterial ability. ASSOCIATED CONTENT Supporting information Figure S1, Elemental distributions of different samples. Table S1, Surface elemental compositions of different coatings detected by XPS. Figure S2, Cross-sectional images of coated samples. Figure S3, SEM images of E. coli after cultured on PEO/LDH, Zn-1#, Zn-2#, and Zn-3#for 24 h. Figure S4. Inhibition zones around different samples against S.aureu.

ACKNOWLEDGMENTS This work is financially supported by National Natural Science Foundation of China (31771044), the National Key Research and Development Program of China No.



2016YFC1100604, National Natural Science Foundation for Distinguished Young Scholars of China (51525207), Shanghai Committee of Science and Technology, China (18YF1426900) and International Partnership Program of Chinese Academy of Sciences Grant No.GJHZ1850.

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Enhanced antibacterial and osteogenic activitiies by PEO/Mg-Zn-Al LDH composite coating. 35x15mm (600 x 600 DPI)



Figure 1. Morphological evaluation of various samples by SEM (a), EDS spectra of corresponding SEM images (b), and elemental compositions of various samples by EDS maps (c). 94x56mm (300 x 300 DPI)


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Figure 2. XRD patterns of different samples. 114x82mm (600 x 600 DPI)



Figure 3. Polarization curves of various samples detected in PBS (a) and hydrogen evolution of various samples detected in 0.35 wt% NaCl (b). 65x26mm (600 x 600 DPI)


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Figure 4. CLSM images of cells cultured on various surface for 1, 4 and 24 h with F-actin stained with Rhodamine-Phalloidin (red) and nucleus stained with DAPI (blue). 93x54mm (300 x 300 DPI)



Figure 5. The proliferation rate (a) and SEM images (d) of cells cultured on various surfaces, the amount of releasing Mg2+ (b) and Zn2+(c) after immersing in culture medium for 1 day. 142x127mm (300 x 300 DPI)


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Figure 6. CLSM images of live/dead staining of cells after cultured on various surfaces for 5 days (green represent live cells and red represent dead cells). 49x15mm (300 x 300 DPI)



Figure 7. OPN protein expression of rBMSCs cultured on various samples for 14 days (a), and the corresponding fluorescent intensity (b). 86x47mm (300 x 300 DPI)


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Figure 8. Relative ALP activity of rBMSCs after cultured on various samples for 7 and 14 days. 55x31mm (600 x 600 DPI)



Figure 9. Photographs of re-cultivated S.aureus colonies on agar culture plates (a) and analysis of reduction percentages of bacterial colonies (b). 95x56mm (300 x 300 DPI)


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Figure 10. SEM images of S. aureus after cultured on PEO/LDH (a), Zn-1# (b), Zn-2# (c), and Zn-3# for 24 h. 31x6mm (300 x 300 DPI)


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