Improved blood compatibility and cyto-compatibility of Zn-1Mg via plasma electrolytic oxidation

Materialia 5 (2019) 100244

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Improved blood compatibility and cyto-compatibility of Zn-1Mg via plasma electrolytic oxidation Yinying Sheng a, Hanyu Zhou a, Zhibin Li a, Lianxi Chen b, Xiaojian Wang a,∗, Xueyang Zhao a, Wei Li a,b,∗∗ a b

Institute of Advanced Wear & Corrosion Resistant and Functional Materials, Jinan University, Guangzhou 510632, China National Joint Engineering Center of High-performance Wear-resistant Metallic Materials, Guangzhou 510632, China

a r t i c l e

i n f o

Keywords: Zn-1Mg Plasma electrolytic oxidation (PEO) Biodegradable Blood compatibility Biocompatibility

a b s t r a c t Compared to magnesium (Mg) or iron (Fe) based biodegradable alloys, Zinc (Zn) alloys exhibit moderate degradation rates, thus regarded as promising implant materials for orthopedic and cardiovascular applications. However, Zn2+ released from degradation process may result certain degrees of cytotoxicity. Thus it is necessary to modify the surface of biodegradable Zn alloys for better biocompatibility. In this paper, a porous ceramic coating was successfully prepared on a Zn-1Mg alloy using plasma electrolytic oxidation (PEO). The effect of the PEO coating on the corrosion resistance and in vitro cyto-compatibility of the Zn-1Mg alloy were evaluated. It was noted that the thickness of the PEO coatings could be adjusted from 6.7 ± 0.5 μm to 28.6 ± 2 μm by increasing the oxidation time from 30 s to 120 s. Longer oxidation time led to a more rough and hydrophobic surface, which was consisted of ZnO and Zn2 SiO4 . The stable and protective oxide layer enhanced the corrosion resistance of the Zn-1Mg alloy. The PEO coated Zn-1Mg exhibited better blood compatibility and cyto-compatibility. Decreased hemolytic ratio and lower platelet adhesion was observed on the PEO coated Zn-1Mg sample. The enhanced cyto-biocompatibility is mainly attributed to small amount of Si2+ and Mg2+ released during the early degradation of the PEO coating.

1. Introduction The interest in biodegradable metals and alloys, such as zinc (Zn), iron (Fe) and magnesium (Mg), is mainly related to their potential use as implant materials for orthopedic [1] and cardiovascular applications [2], where a temporary medical device is required. Biodegradable metals and alloys need to provide temporary scaffolding for tissue healing, and then degrade completely after fulfilling their mission [3]. Zinc alloys, compared to other bio-metals such as Mg and Fe alloys, exhibit moderate degradation rate [4–7]. However, pure zinc suffers from low mechanical strength (<30 MPa) and plasticity that are insufficient for most medical device applications. Developing zinc alloys with sufficient mechanical stress and ductility, while retaining its biocompatibility, is still a challenge for biomedical researchers. Alloying pure Zn with biodegradable element Mg has attracted much research interests recently. Caizhen Yao et al. [8] showed that casted Zn– 1 wt%Mg alloys possessed a tensile stress of 180 MPa, which is significantly higher than that of pure Zn (30 MPa). However the strength and elongation of the Zn–Mg alloys decreased when Mg addition increased to 2–3 wt%, due to the formation of brittle eutectic phase Mg2 Zn11 [9].

∗ ∗∗

For biodegradable metals and alloys, metal ions released during degradation will inevitably contact with local cells and tissues. Although Zn and Mg are essential trace elements in the human body and participate in almost all metabolic reactions [10-12], high local ion concentration may possess certain cytotoxicity [13]. Research has revealed that 50% Lethal Dose (LD50) of three kinds of vascular cells to Zn2+ : that is 50 μM for human endothelial cells (hDF), 70 μM for human aortic smooth muscle cells (AoSMC), and 265 μM for human dermal fibroblasts (HAEC) [14–16]. Endothelial cells directly cultured on the polished pure Zn surface resulted in vascular cell attachment, but quickly followed by cell death, which related to the initial quick release of free Zn2+ during degradation [14]. After coating Zn surface with a layer of gelatin, vascular cells attached and proliferated well on the Zn surface. Besides, Zn–Mg alloy is more susceptible to micro-galvanic corrosion, due to larger potential difference between Zn matrix and precipitates, which resulting in faster degradation rate than pure zinc, and higher local ions concentration [17]. Thus it is necessary to modify the surface of biodegradable zinc-based alloys for better biocompatibility [18–21]. As a widely used surface modification technique, PEO has been widely explored in recent years [22], which is suitable for implants with

Corresponding author. Corresponding author at: Institute of Advanced Wear & Corrosion Resistant and Functional Materials, Jinan University, Guangzhou 510632, China. E-mail addresses: [email protected] (X. Wang), [email protected] (W. Li).

https://doi.org/10.1016/j.mtla.2019.100244 Received 15 December 2018; Accepted 2 February 2019 Available online 5 February 2019 2589-1529/© 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Y. Sheng, H. Zhou and Z. Li et al.

irregular shape and complex internal structures. The surface oxide coating provides high corrosion resistance, in the same time, has a strong bonding with the substrate [23]. The research and application of PEO on medical titanium (Ti) alloys and magnesium (Mg) alloys has been explored extensively [24–27]. Studies have reported that porous PEO coating on Ti alloys is beneficial for the growth of osteoblasts [25], improving osteogenic properties subsequently. However, there were only few reports on the surface modification of degradable zinc alloys. Emmanuel et al. [26] and Stojadinovic et al. [27] prepared the PEO of pure zinc in KOH solution and a Na2 SiO3 ·5H2 O and KOH mix solution. Dense ZnO coatings could be obtained. However, a thorough understanding of how PEO coating affect the degradation process of the alloy is still unclear. In addition, the interaction of PEO coating with surrounding cells needs further understanding. Herein, a novel electrolyte containing Na2 SiO3 was developed for the PEO treatment. PEO coatings with different thickness and porous structures were successfully prepared on Zn-1Mg. The influence of PEO coating on the corrosion resistance, hemolytic ratio, platelet adhesion and cyto-compatiblity were investigated.

Materialia 5 (2019) 100244

experiments were conducted at 37 °C and the testing electrolyte was modified simulated body fluids (m-SBF), which has ion concentrations equal to those of blood plasma in total amount except for the HCO− 3 concentration [29]. All the samples were wrapped around the sides with copper wires and then encapsulated in epoxy resin with exposing 1 cm2 of the surface. After 30 min immersion in the m-SBF solution, electrochemical impedance spectroscopy (EIS) was performed in the frequency range from 100 kHz to 100 mHz with a 10 mV altering signal at the open circuit potential (OCP). The polarization curves were acquired at the scanning rate of 0.167 mV·s−1 and range from −300 mV to 500 mV with respect to the OCP. According to ASTM G1202-89 [30], the corrosion rate (Vcorr ) was calculated as following formula: 𝑉𝑐𝑜𝑟𝑟 = 𝐾 × 𝐼𝑐𝑜𝑟𝑟 × 𝐸𝑊 ∕𝜌

(1)

where, K = 3.27 × 10−3 mm g/μA cm yr. Icorr is the corrosion current density in μA/cm2 . EW =32.68 (equivalent weight of Zn). 𝜌=7.14 g/cm3 (the density of Zn-based alloys) [31]. 2.4. Blood compatibility evaluation

2. Materials and experimental methods

Zn-0.1 wt% Mg was prepared by melting of pure Zn (99.995%) and pure Mg (99.90%), and solution treated at 330 °C for 20 h. Zn1Mg alloy after solution treatment was cut into plates with size of 10 mm × 10 mm × 2 mm. Prior to the PEO treatment, the specimens were grounded with a series of 400–2500 SiC abrasive paper respectively. The samples were ultrasonically cleaned in an ethanol bath for 15 min, rinsed in deionized water for 30 s, then dried in air. The PEO treatment was performed with an alternating current-type high power supply (PNⅢ) in a two-electrode cell: the sample was set as the anode and the graphite electrode as the cathode [28]. The electrolytic bath containing 14 g/L Na2 SiO3 , 2 g/L KOH, 15 mL/L glycol and 10 mL/L glycerol was placed in a circulating water cooling tank to keep the electrolyte temperature between 25 °C and 60 °C. The duty cycle and frequency were fixed at 30% and 800 Hz, and the distance between electrodes was fixed at 7 cm. After being treated at 300 V for 30 s and 120 s, the specimens were cleaned with DI water and ethanol, and then air dried.

The hemolytic and platelet adhesiveness test are performed to evaluate the blood compatibility of bare Zn-1Mg and PEO coatings. Healthy rabbit blood collected from a New Zealand White Rabbit was centrifuged first at 1000 rpm for 10 min. Upper platelet-rich plasma (PRP) was removed away and the rest red blood cell solution (RBCs) were diluted with HEPES buffer (10 mM HEPES, 150 mM NaCl, pH=7.0) to 3.3% (v/v) [32]. Samples were cleaned in acetone, ethanol and DI water in turn with an ultrasonic oscillator for 15 min each, and then sterilized by ultraviolet radiation for 30 min. The uncoated and PEO-coated Zn-1Mg were firstly dipped in normal saline, incubated at 37 °C for 24 h, and then placed on a 24-well culture plate. A volume of RBCs was added to each well (solution volume: surface area =1.25 cm−1 ). Normal saline solution and 1% (v/v) Triton X-100were used as a negative control and a positive control, respectively. After incubated at 37 °C for 2 h, the supernatants were transferred to a 96-well plate, and centrifuged for 10 min at 400 rpm. The absorbance (optical density, OD) was read at 545 nm in the automatic microplate reader (THERMO Multiskan MK3, Thermo Scientific., USA). The hemolytic ratio was calculated according to the following formula:

2.2. Characterization of PEO coatings

𝐻% =

Scanning electron microscope (ZEISS SUPRA 40, Oberkochen, Germany) was used to observe the surface and cross-section morphology of PEO coating. The coating composition was determined by energy dispersive X-ray spectroscopy (EDS). The phase compositions and structure of coatings was identified by using X-ray diffraction (XRD, D/Max 2400 V, Rigaku, Japan) with CuK𝛼1 wavelength (𝜆=1.54506 Å) in grazing incidence (𝛼=2°). The XRD spectra were collected in the regular range from 20° to 90° at a scanning rate of 4°/min. An atom force microscope (AFM, MFP-3D-BioAFM, Asylum Research, USA) was performed in the tapping mode to further evaluate the surface morphology. The root-mean-square roughness (RMS) of Zn-1Mg and PEO coatings was examined with the scanning range of 25 μm × 25 μm and calculated by the Asylum Research Software. The contact angle measurements were performed on a contact angle goniometer (JY-82, Shanghai, China) at room temperature using a 10 μL water droplet, and the average value was obtained by nine repeated measurements on three samples.

where H is hemolytic ratio, and ODt , ODpc , and ODnc represent OD values of the sample, the positive control and the negative control, respectively. The blood cells on the surface were fixed by 0.2% glutaraldehyde in PBS for 12 h at 4 °C. The red cells not well adhered on the surface were washed out with sterile phosphate-buffered saline (PBS) and followed by dehydrated with gradient alcohol (30%, 50%, 70%, 90%, 100%), critical point dried and sputter coated with gold for observation using SEM. In parallel to above, the specimens were immersed in the prepared PRP, and incubated at 37 °C for 2 h. After rinsing, fixing and critical point drying, the specimens were coated with gold, and the number and morphology of adherent platelets were observed by SEM.

2.1. The preparation of PEO coating

2.3. Electrochemical studies The degradation behavior was evaluated via electrochemical tests, conducted on an electrochemical workstation (PARSTAT 4000, Princeton) using a three-electrode cell system, with a saturated calomel electrode (SCE) as the reference electrode, a platinum foil as the counter electrode, and the sample as the working electrode, respectively. The

((

) ( )) 𝑂𝐷𝑡 − 𝑂𝐷𝑝𝑐 ∕ 𝑂𝐷𝑛𝑐 − 𝑂𝐷𝑝𝑐 × 100%

(2)

2.5. In vitro cyto-compatibility evaluation The human umbilical vein endothelial cells (HUVECs) were cultured in 1640 Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37 °C in a humidified atmosphere with 5% CO2 . Specimens were sterilized with 70 vol% ethanol for 30 min and rinsed three times with sterile PBS and then placed on a 24-well culture plate. Before seeding, each sample was firstly immersed into DMEM for 24 h in a humidified atmosphere with 5% CO2 at 37 °C (solution volume: surface area =1.25 cm−1 ). The extracts were collected and digested by nitric acid, the concentrations of leached Zn, Si and Mg were monitored by

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Materialia 5 (2019) 100244

Fig. 1. Cross-sections (a, b), surface morphology (d, e) of the PEO coatings and corresponding EDS spectra and quantitative analysis (c, f) after PEO treatment for 30 s (a, d, c) and 120 s (b, e, f).

inductively-coupled plasma optical emission spectroscopy (ICP-OES, PE Optima 2100DV, USA). After that, 5 × 104 HUVECs per well were seeded on the sample surface (2.5 × 104 /cm2 ), as well as the negative control groups with only culture media, while not on the positive control groups. After 24 h incubation, the cell viability were measured with a Cell Counting KIT-8 (CCK-8, Dojindo, Japan), the OD values were monitored at 450 nm on the automatic microplate reader (THERMO Multiskan MK3, Thermo Scientific., USA). The cell viability is expressed following the relationship: Cell viability(%) =

((

) ( )) 𝑂𝐷𝑡 − 𝑂𝐷𝑝𝑐 ∕ 𝑂𝐷𝑛𝑐 − 𝑂𝐷𝑝𝑐 × 100%

(3)

The seeded samples were rinsed with sterile PBS, followed by fixation with 4% paraformaldehyde for 15 min. The phalloidin fluorescein isothiocyanate (R415, Life Technologies, USA) was used to stain cytoskeleton protein F-actin, and 4, 6-diamidino-2- phenylindole (DAPI, Beyotime Institute of Biotechnology, China) was used to stain the nuclei. Finally, the cells were observed under a fluorescence microscope (LSM 700, Carl Zeiss, Germany). 2.6. Statistical methods The experimental data were expressed as mean ± standard deviation (stdev). 3 parallel samples were used for each experiment, and three measurements were performed to do some statistics of the presented values. Statistical analyses were performed using SPSS. ANOVA was performed to determine statistical significance and differences were considered statistically significant at p<0.005. ∗ p<0.05, ∗ ∗ p<0.01,∗ ∗ ∗ p<0.005. 3. Results and discussion 3.1. Characterization of the surface coatings (PEO) The morphology and cross-section of the PEO coatings are shown in Fig. 1. As shown in Fig. 1(a) and (b), the PEO coatings were well adherent to the substrate and consisted of a compact inner barrier layer and a loosely porous outer layer. The overall thickness of the PEO coating was

Table 1 The porosity and pore size analyzed by image pro plus. Sample

PEO-30s PEO-120s

Porosity (%)

16.10 ± 2.56 21.47 ± 3.69

Pore size (μm) min

max

mean

0.52 1.02

3.91 8.58

1.82 3.03

6.7 ± 0.5 μm for the sample treated for 30 s (PEO-30s), and 28.6 ± 2 μm for the sample treated for 120 s (PEO-120s). Five repetitive measurements were conducted to calculate the average of the thickness. The corresponding surface morphologies are shown in Fig. 1(d) and (e) respectively. Typical rough and porous morphology with a small number of micro cracks could be observed, which is similar to the morphology of PEO treated Ti and Mg [9–12]. During the PEO process, the micropores were formed by gas bubbles coming out of discharge channels and the micro-cracks (marked with arrows in Fig. 1) were created due to thermal stress during the solidification of the molten oxide [33]. The porosity and pore size of surface microstructure are analyzed by image pro plus, and the results are listed in Table 1. With the increase of the oxidation time, the porosity of the PEO coating increased and the average pore size increased from 1.82 to 3.03 μm. The EDS spectra of the cross-section and the top surface of the coatings are given in Fig. 1(c) and (f). The main elements in the coating were Zn, O and Si. The AFM topographic images after PEO treatment are shown in Fig. 2. The PEO coatings exhibit volcanic shaped micropores, which in good agreement with the SEM images. The surface roughness increased from 90.2 ± 1.5 μm to 115.5 ± 2.5 μm with the increase of oxidation time. Fig. 3 shows the XRD patterns of the Zn-1Mg and the PEO coatings on Zn-1Mg substrate. Since the solubility of Mg in Zn is only about 0.1%, Zn-1Mg was consisted of Zn and the eutectic phase Mg2 Zn11 , which mainly distributed at grain boundaries. Both the PEO coatings were

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Materialia 5 (2019) 100244

Fig. 2. AFM images of the Zn-1Mg (a), PEO-30 s (b) and PEO-120 s (c) measured at the tapping mode.

Table 2 Ecorr (mV), Icorr (μA/cm2 ) values and Vcorr (mm/year) calculated of different samples in m-SBF calculated from the polarization curves.

E corr (mV) I corr (μA/cm2 ) Vcorr (mm/year)

Fig. 3. XRD spectra of the Zn-1Mg substrate and the PEO coatings with different treating time.

Zn-1Mg

PEO-30s

PEO-120s

−1137 ± 15 28.47 ± 7 0.43 ± 0.10

−1086 ± 9.6 14.70 ± 0.4 0.22 ± 0.04

−1006 ± 6.5 8.071 ± 0.2 0.12 ± 0.02

ther increasing treating time up to 120 s, the water contact angle increased to 124.76°. A larger contact angle is believed having a lower surface energy. The result indicates that the PEO coating surface is more hydrophobic [20]. The higher value of the water contact angle is determined by its surface composition (ZnO) and complex porous morphology. The corrosion medium may be expelled from the material’s surface, as the captured air in capillary system on the hydrophobic surface [34], which will weaken the binding force between liquid and the substrate. 3.2. Degradation behavior of the PEO coatings

Fig. 4. Water contact angles on the Zn-1Mg and the PEO coated samples (∗ p<0.05, ∗ ∗ p<0.01, ∗ ∗ ∗ p<0.005).

composed of zinc oxide (ZnO) and zinc silicate (Zn2 SiO4 ). With the increase of oxidation time from 30 s to 120 s, the characteristic diffraction peaks of ZnO and Zn2 SiO4 became more intense. Fig. 4 reveals the water contact angles on the surface of Zn-1Mg and PEO coatings. The water contact angle for the Zn-1Mg substrate was 101.33°, while that of the PEO-30s increased to 117.87°. Fur-

EIS and polarization tests of the uncoated and coated samples were conducted in m-SBF, after stabilized in the solution for 30 min. The results are shown in Fig. 5(a) and (b) respectively. The corrosion current density (Icorr ) and corrosion potential (Ecorr ) were calculated by Tafel extrapolation, and listed in Table 2. The Ecorr of the Zn-1Mg was −1137 mV, whereas the value after 30 s and 120 s oxidation showed a positive shift to −1086 mV and −1006 mV respectively. The Icorr of the PEO coated samples was an approximately one order magnitude lower than that of bare Zn-1Mg. Lower Icorr and positively shifted Ecorr indicate that PEO coating significantly improved the corrosion resistance of Zn-1Mg, the calculated corrosion rate of PEO coatings was proved to possess the lowest value for about 0.12 (mm/year). The improvement of corrosion resistance could be attributed to the dense compact inner layer in PEO coating that retarded the infiltration of aggressive solution into coating during the corrosion process [24,35]. The EIS results help to further understand the corrosion processes, especially at the electrode/electrolyte interface. According to the Nyquist plots in Fig. 5(b), corresponding electrical equivalent circuits were obtained by fitting the experiment data using ZSimpWin, and the fitted EIS data are listed in Table 3. Before and after the PEO treatment, the EIS spectra and corresponding electrical equivalent circuits were much different for their significant distinct interface conditions. For the uncoated alloy, the spectrum has two semi-circles; the high frequency semicircle is attributed to the charge transfer accompanied by the corrosion products transfer, while the semicircle in low frequency indicates a diffusion process through the porous layer [36]. For the PEO coated samples, the

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Materialia 5 (2019) 100244

Fig. 5. (a) Potentiodynamic polarization curves, (b) Nyquist plots and (c,d) Electrical equivalent circuits used to fit the EIS spectra for bare and PEO-coated Zn-1Mg in m-SBF.

Table 3 Fitted EIS results of samples in m-SBF based on the corresponding equivalent circuit models.

Zn-1Mg PEO-30s PEO-120s

Rs (Ω cm−2 )

CPEcl (μΩ−1 cm−2 s−1 )

n

Rcl (Ω cm−2 )

CPEl1 (μΩ−1 cm−2 s−1 )

n

Rl1 (Ω cm−2 )

CPEl2 (μΩ−1 cm−2 s−1 )

n

Rl2 (Ω cm−2 )

CPEdl (μΩ−1 cm−2 s−1 )

n

Rct (Ω cm−2 )

Chi square

34.93 33.21 32.93

4.915 × 10−6 – –

0.9286 – –

947.3 – –

– 5.297 × 10−4 1.571 × 10−4

– 0.8000 0.7981

– 45.48 82.84

– 4.931 × 10−4 9.033 × 10−4

– 0.6723 0.6747

– 741.5 2842

6.543 × 10−3 1.878 × 10−4 2.926 × 10−4

0.5789 0.5831 0.6862

471.5 6976 5487

1.427 × 10−3 1.858 × 10−4 2.937 × 10−4

semi-circle was incomplete and reached much higher values in the scale. The constant phase element (CPE) is a general diffusion-related element, which is related to an interface feature (special geometry, roughness) of the interface [37]. Rs represents the solution resistance between the reference electrode and sample. For the equivalent circuit of the bare Zn-1Mg, CPEcl and Rcl represents the capacitance of corrosion product layer and resistance, respectively. CPEdl and Rct represent the double layer capacitance and charge transfer resistance, respectively. The corrosion processes are in good agreement with results reported by other investigators [38,39]. The corrosion process could be explained by two partial reactions. The cathodic partial reaction is the reduction of oxygen on the compact layer surface and leads to an increase in pH, as follows: O2 + 2H2 O + 4e → 4OH−

(4)

The anodic reaction involves the dissolution of zinc and leads to a decrease in the sample weight. Zn + 2OH− → Zn(OH)2 + 2e

(5)

Zn(OH)2 + 2H+ → Zn2+ + 2H2 O

(6)

Yang et al. put pure zinc stents in abdominal aortas of rabbits for 1 year, and found ZnO, ZnCO3 and a small amount of amorphous calciumphosphorus complex as the main corrosion products [40]. For the equivalent circuit of the PEO coated samples, Rl1 was the resistance of the outer porous layer coatings in parallel with constant phase element CPEl1 . Rl2 was the resistance of the dense layer coatings in parallel with CPEl2 . Rct and CPEdl represented the corroding interface [41]. Compared to that of the bare Zn-1Mg (1.45 × 103

Y. Sheng, H. Zhou and Z. Li et al.

Fig. 6. Hemolytic ratio of Zn-1Mg and PEO coatings (∗ p<0.05, ∗∗∗ p<0.005).

Materialia 5 (2019) 100244

∗∗

p<0.01,

Ω·cm2 ), the total resistance increased to 7.80 × 103 Ω·cm2 for PEO-30s and 8.44 × 103 Ω·cm2 for PEO-120s respectively. Especially, the Rct increased to 6.98 × 103 Ω·cm2 for PEO-30s and 5.49 × 103 Ω·cm2 for PEO120s respectively, which is one order of magnitude higher than that of the bare Zn-1Mg (471.5 Ω·cm2 ). The EIS results suggested that the PEO coatings greatly enhanced the corrosion resistance of Zn-1Mg alloy in m-SBF. 3.3. Blood compatibility of PEO coatings Fig. 6 shows the hemolytic ratio of the Zn-1Mg and the PEO coatings, the corresponding red blood cells morphology characterization, and platelet adhesion images are shown in Fig. 7. The uncoated Zn-1Mg demonstrated a significantly higher hemolytic ratio (5.01% ± 0.62%), in comparison with the PEO groups (2.05% ± 1.21%), (1.10% ± 0.50%) respectively. In addition, less destroyed blood cells were observed on the surface of the PEO coatings. The increase of coating thickness had little effect on the morphology of blood cells. Wang et al. also found

similar results in AZ31B alloy with Si-containing coatings, and they believed the high activity of the bare alloy surface, as well as the quick release of metal ions were responsible for the damage to red blood cells, which results in the high hemolysis rate [42]. PEO coatings on the surface were shown delayed the release process of Zn2+ and Mg2+ from the substrate [43] and also delayed the increase of pH value of the solution [44]. In addition, the surface with a negative charge will result in improved hemolysis rate. The PEO coating was obtained in an alkaline solution, and the surface tended to be negative charged. The adhesion behavior of platelets is related to the surface features of wettability, hydrophilic/hydrophobic balance and charge interaction. Fig. 7(d)–(f) shows the platelet adhesion and aggregation on the sample surfaces. It was shown that the number of platelets on the Zn-1Mg surface was much higher than that on the PEO coatings. On the surface of Zn-1Mg, the adherent platelets were more activated, where many pseudopodia could be seen. However, the adhered platelets on the PEO coating were generally in rounded shape, which is a typical shape of inactivated platelets. The formation of pseudopodia and deformation of platelets was not observed. According to the results, the surface of the PEO coating can inhibit the adhesion and activation of platelet. Hong et al. concluded that hydrophilic surface tended to have higher binding of platelets [45]. The increase in contact angle after PEO treatment may contribute to the inhabitation of platelet adhesion and activation. The good blood compatibility characteristics of PEO coating may be favorable in the biomedical development of biodegradable Zn-1Mg. 3.4. In vitro cyto-biocompatibility of PEO coatings In order to evaluate the in vitro cyto-compatibility of the PEO coating, HUVECs were directly cultured on the surface of bare and the PEO coated Zn-1Mg for 24 h and the cell viability showed in Fig. 8(a). The cell viability of HUVECs cultured on the control was expressed as 100%. The HUVECs after 24 h incubation with the Zn-1Mg display decreased cell viability (88%), while the PEO coated samples showed significantly higher cell viability than the control. Fig. 8(c) shows the HUVECs morphology on the corresponding samples after 24 h incubation. The HUVECs cells on the PEO coated samples displayed cobblestone appearance with large nuclei. The results showed that the PEO coated Zn-1Mg sam-

Fig. 7. The red blood cells morphology characterization (a–c), and platelet adhesion images (d–e) of Zn-1Mg and PEO coatings.

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Materialia 5 (2019) 100244

Fig. 8. In vitro cell viability of HUVECs cultured directed on the surface of the untreated and PEO samples for 24 h (a), the Zn2+ , Mg2+ and Si the extracts (b) and fluorescent images of cells (c) (∗ p<0.05, ∗ ∗ p<0.01,∗ ∗ ∗ p<0.005).

ples exhibited better cyto-compatibility. It was seen from Fig. 8(b) that the concentration of all the Zn2+ , Mg2+ and Si2+ were increased for the PEO coated samples after 24 h immersion in the DMEM supplemented with 10% FBS, because of chemical dissolution of the coatings. Zn2+ as the main ions released has been proven as a dose-dependent response to different cells [14]. Ma et al. studied the suitable amount of Zn2+ to endothelial cells, and founded that small concentrations of Zn2+ is beneficial for cell adhesion and proliferation and finally improving cytocompatibility [46]. Szuster-Ciesielska et al. confirmed the inhibitory effect of zinc on apoptotic cell death [47]. The concentration of Zn2+ for both the Zn-1Mg and the PEO coated samples were higher than 5 μg/mL after 24 h incubation. The cell viability of Zn-1Mg was lower than that of the control group, while the cell viability of PEO group was significantly higher than that of the control group. The increase in Zn2+ comes from the degradation of ZnO and ZnSiO4 in PEO coating, and not causing damage to the cell health, may be connected to the regulatory effect on cell death confirmed by Szuster-Ciesielska et al. This indicated that the PEO coating promotes cell adhesion and proliferation. Wu et al. also found that Mg and Si ions could stimulate the proliferation of osteoblast cell [48]. Sun et al. showed that the ionic products released from the silicate promoted the proliferation of MG63 cells [49]. Wang et al. concluded that Si ions played an important role in new bone generation [42], as Si could activate osteogenesis-related signaling pathways, and ultimately enhance cell viability and differentiation [50]. In the present work, the increased cell viability for HUVECs on the PEO coatings might be related to the release of low concentrations of Mg and Si. In the extracts of calcium silicate (CS), the expression of vascular endothelial growth factor (VEGF) receptor 2 on HUVEC were enhanced. Si ions (0.7–1.8 μg/ml) [51] are confirmed to regulate the growth factors for osteogenic differentiation and vascularization [52]. Dashnyam

2+

concentration of

et al. also found high cell compatibility in cell proliferation level of HUVECs for Si-contained ionic extracts from the silica-based mesoporous microcarriers. They explained that the Si ions release up-regulates the expression of signaling molecules such as VEGF and FGF2 [53,54]. The PEO coatings containing of ZnO can provide a stable surface. The hydrophobic surface of the PEO coated sample could improve the corrosion resistance. The microstructure of the micro-scale pores formed by plasma electrolytic oxidation may facilitate the cell adhesion. A better cell initial attachment and viability were achieved by the PEO modification of the surface and which will promote endothelialization in the end.

4. Conclusion Porous ceramic coatings containing of ZnO and Zn2 SiO4 were prepared on the Zn-1Mg through PEO treatment. The effect of the PEO coating on the corrosion resistance and in vitro biocompatibility of Zn-1Mg were evaluated. The main conclusions were summarized as follows: 1. The thickness and the surface morphology of the PEO coating could be adjusted by the oxidation time. With the increasing of oxidation time to 120 s, the PEO coating of 28.6 ± 2 μm thick were obtained. 2. The electrochemical results showed that smaller corrosion current density, more positive corrosion potential and higher impedance were detected on the PEO coated Zn-1Mg in m-SBF. Rct increased to 1.83 × 103 Ω·cm2 for PEO-30s and 5.49 × 103 Ω·cm2 for PEO-120 s respectively, which is one order of magnitude higher than that of the bare Zn-1Mg (471.5 Ω·cm2 ). The improved corrosion resistance could be attributed to the stable and protective oxide coating.

Y. Sheng, H. Zhou and Z. Li et al.

3. The PEO coated Zn-1Mg showed a better blood compatibility than the bare Zn-1Mg, including a decreased hemolytic ratio, with a lower lytic activity against red blood cells and lower platelet adhesion. 4. The PEO coated samples also showed higher cell viability and better cell adhesion, indicating a better cyto-compatibility in vitro. The improved cyto-compatibility might have resulted from to the small amount release of Si2+ and Mg2+ during the early degradation of the PEO coating. Our results suggested that PEO treatment is a promising method for Zn based biodegradable materials to improve both the corrosion resistance and in vitro cyto-compatibility. Acknowledgments This work was supported by the Science and Technology Planning Project of Guangdong Province No. 2017B090903005 and the Science and Technology Planning Project of Guangzhou No. 201704030045. XJW acknowledges the financial support from Jinan University (No. 88015139) and the Fundamental Research Funds for the Central Universities (No. 17817024). Declaration of interest All authors have completed the Materialia disclosure form and declare that: (i) no support, financial or otherwise, has been received from any organization that may have an interest in the submitted work; and (ii) there are no other relationships or activities that could appear to have influenced the submitted work. References [1] D. Vojtěch, J. Kubásek, J. Šerák, P. Novák, Mechanical and corrosion properties of newly developed biodegradable Zn-based alloys for bone fixation, Acta Biomater. 7 (2011) 3515–3522. [2] R.J. Guillory, P.K. Bowen, S.P. Hopkins, E.R. Shearier, E.J. Earley, A.A. Gillette, et al., Corrosion characteristics dictate the long-term inflammatory profile of degradable zinc arterial implants, Acs Biomater. Sci. Eng. 2 (2016) 2355–2364. [3] G. Mani, M.D. Feldman, D. Patel, C.M. Agrawal, Coronary stents: a materials perspective, Biomaterials 28 (2007) 1689–1710. [4] E. Mostaed, M. Sikora-Jasinska, J.W. Drelich, M. Vedani, Zinc-based alloys for degradable vascular scent applications, Acta Biomater. 71 (2018) 1–23. [5] P.K. Bowen, J. Drelich, J. Goldman, Zinc exhibits ideal physiological corrosion behavior for bioabsorbable stents, Adv. Mater. 25 (2013) 2577–2582. [6] L. Chen, Y. Sheng, X. Wang, X. Zhao, H. Liu, W. Li, Effect of the microstructure and distribution of the second phase on the stress corrosion cracking of biomedical Mg–Zn–Zr–xSr alloys, Materials 11 (2018) 551–567. [7] L. Chen, Y. Bin, W. Zou, X. Wang, W. Li, The influence of Sr on the microstructure, degradation and stress corrosion cracking of the Mg alloys-ZK40xSr, J. Mech. Behav. Biomed. 66 (2017) 187–200. [8] C. Yao, Z. Wang, S.L. Tay, T. Zhu, W. Gao, Effects of Mg on microstructure and corrosion properties of Zn–Mg alloy, J Alloy Compd. 602 (2014) 101–107. [9] H. Gong, K. Wang, R. Strich, J.G. Zhou, In vitro biodegradation behavior, mechanical properties, and cytotoxicity of biodegradable Zn–Mg alloy, J Biomed. Mater. Res. B 103 (2015) 1632–1640. [10] C.J. Frederickson, K. Jae-Young, A.I. Bush, The neurobiology of zinc in health and disease, Nat. Rev. Neurosci. 6 (2005) 449–462. [11] S.D. Gower‐Winter, C.W. Levenson, Zinc in the central nervous system: from molecules to behavior, Biofactors 38 (2012) 186–193. [12] P.J. Little, R. Bhattacharya, A.E. Moreyra, I.L. Korichneva, Zinc and cardiovascular disease, Nutrition 26 (2010) 1050–1057. [13] X. Tong, D. Zhang, X. Zhang, Y. Su, Z. Shi, K. Wang, et al., Microstructure, mechanical properties, biocompatibility, and in vitro corrosion and degradation behavior of a new Zn–5Ge alloy for biodegradable implant materials, Acta Biomater. 82 (2018) 197–204. [14] E.R. Shearier, P.K. Bowen, W. He, A. Drench, J. Drelich, J. Goldman, et al., In vitro cytotoxicity, adhesion, and proliferation of human vascular cells exposed to zinc, Acs Biomater. Sci. Eng. 2 (2016) 634–642. [15] J. Ma, N. Zhao, D. Zhu, Bioabsorbable zinc ion induced biphasic cellular responses in vascular smooth muscle cells, Sci. Rep.-UK 6 (2016) 26661–26679. [16] D. Zhu, Y. Su, M.L. Young, J. Ma, Y. Zheng, L. Tang, Biological responses and mechanisms of human bone marrow mesenchymal stem cells to Zn and Mg biomaterials, ACS Appl. Mater. Interfaces 9 (2017) 27453–27461. [17] H. Gong, K. Wang, R. Strich, J.G. Zhou, In vitro biodegradation behavior, mechanical properties, and cytotoxicity of biodegradable Zn–Mg alloy, J. Biomed. Mater. Res. B 103 (2015) 1632–1640. [18] J. Cheng, T. Huang, Y.F. Zheng, Relatively uniform and accelerated degradation of pure iron coated with micro-patterned Au disc arrays, Mat. Sci. Eng. C.-Mater. 48 (2015) 679–687.

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