In vitro degradation, hemolysis, and cytocompatibility of PEO/PLLA composite coating on biodegradable AZ31 alloy Zhongling Wei,1 Peng Tian,2 Xuanyong Liu,2 Bangxin Zhou1 1

Institute of Materials, Shanghai University, Shanghai 200072, People’s Republic of China State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China

2

Received 4 November 2013; revised 20 March 2014; accepted 29 April 2014 Published online 29 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33208 Abstract: Magnesium and its alloys have large potential as degradable and absorbable biomaterials because of their mechanical properties and biocompatibility. However, their corrosion resistance is usually inadequate especially in physiological environment, which limits their broad applications in biomedical areas. In this work, plasma electrolytic oxidized/ poly(L-lactide) (PEO/PLLA) composite coating was successfully fabricated on biodegradable AZ31 alloy by combing PEO process and sealing with PLLA. The microstructure, elemental composition, and phase composition of the PEO/PLLA composite coating were investigated. The in vitro degradation of the PEO/PLLA composite coating in simulated body fluid (SBF) was also systematically evaluated. The results revealed that the PEO/PLLA composite coating improved the corrosion resistance of AZ31 alloy significantly. The corrosion potential shifted from 21.663V to more positive position 21.317 V and the corrosion current density was reduced with six-order of magnitude. The Mg21 ions, hydrogen release,

and pH value change of solution caused by degradation were all decreased significantly. Moreover, the PEO process played a critical role in sustaining the integrity of the implant in long-term service. The result of hemolysis test showed that the PEO/PLLA composite coating vested AZ31 alloy a low hemolysis ratio (0.806 6 0.771)%, which is much lower than the safe value of 5% according to ISO 10993-4. For the cytocompatibility test, compared with bare AZ31 alloy and PEO coating, MC3T3-E1 cells showed much better adhesion and proliferation on the PEO/PLLA composite coating with nearly 4-fold increase of cells after 7-day cultivation, indicating that the PEO/PLLA composite coating has good biocompatibility C 2014 Wiley Periodicals, Inc. J for biomedical applications. V Biomed Mater Res Part B: Appl Biomater, 103B: 342–354, 2015.

Key Words: magnesium, plasma electrolytic oxidation, poly(Llactide), biodegradation, hemolysis, cell adhesion

How to cite this article: Wei Z, Tian P, Liu X, Zhou B. 2015. In vitro degradation, hemolysis, and cytocompatibility of PEO/PLLA composite coating on biodegradable AZ31 alloy. J Biomed Mater Res Part B 2015:103B:342–354.

INTRODUCTION

Magnesium and its alloys have large potential being used as degradable and resorbable metallic materials in the medical device applications such as intravascular stents1–4 and orthopedic implants.5–7 Based on the gradually degradation in physiological environment in vivo, long-term adverse effect, or risk of local inflammation caused by the permanent implants which are regarded as foreign body for human tissues will be effectively prevented. With completely degradation after the recovery of the injury tissues, a second surgery will not need to be conducted for implant removal. Repeated surgery not only increases the morbidity rate of the patients, but also results in an increase of health care costs and longer hospitalization.8 The released Mg21 ions are considered as nontoxic in human body. The degradation of magnesium and its alloys will not increase the serum magnesium level.9 Mg21 ions also take part in many

enzymatic reactions in human body and moreover the presence of magnesium benefits the mineralization process of bony tissue.10 Despite of those many advantages of magnesium and its alloys, the rapid degradation in physiological environment is still a main obstacle in promoting their broad applications. Once immersed in aqueous environments, magnesium and its alloys begin to corrode. The overall corrosion reaction of magnesium and its alloys in aqueous environments can be given as below: MgðsÞ 12H2 Oðaq Þ Mg ðOHÞ2ðsÞ 1H2ðgÞ

(1)

This overall reaction may include the following partial reactions: 2 Mg ðsÞ Mg 21 ðaq Þ 12e ðanodic reaction Þ

(2)

Correspondence to: Z. Wei ([email protected]) and X. Liu ([email protected]) Contract grant sponsor: National Basic Research Program of China; contract grant number: 973 Program, 2012CB933600 Contract grant sponsor: National Natural Science Foundation of China; contract grant numbers: 81271704, 31100675, 31200721 Contract grant sponsor: Shanghai Science and Technology R&D Fund; contract grant numbers: 11JC1413700, 11DJ1400302

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2H2 OðaqÞ 12e2 H2ðgÞ 12OH2 ðaqÞ ðcathodic reactionÞ

(3)

2 Mg 21 ðaq Þ 12OH ðaq Þ Mg ðOH Þ2ðSÞ ðproduction formation Þ

(4) Although the magnesium hydroxide accumulated on the underlying magnesium and its alloys can act as a corrosion protective layer, when the chloride concentration in the environment rises above 30 mmol/L,11 the magnesium hydroxide begins to convert into magnesium chloride, which is highly soluble in water. Therefore, severe pit corrosion and rapid degradation can be observed on magnesium and its alloys in vivo where the chloride content of the body fluid is about 150 mmol/L.12 The rapid degradation rate also causes the side effect of accumulation of hydrogen nearby the magnesium-based implant because of the limited hydrogen gas adsorption ability in the tissues.13 To improve the corrosion resistance of magnesium and its alloys in physiological environment so as to extend their service time in human body, many modification methods such as alloying and various surface treatments were conducted. Mg-Zn alloy,14 Mg-Zn-Zr alloy,6 Mg-Ca alloy,15,16 MgMn-Zn alloy,6 and Mg-rare earth alloy17 were especially developed for biomedical applications. The alloying elements should be selected and evaluated carefully considering their potential toxicity for human.18 Apart from alloying, surface modifications such as plasma electrolytic oxidation (PEO),19,20 ion implantation,21,22 Ca-P coatings,23,24 and polymer coatings9,25 have also been widely investigated. Plasma electrolytic oxidation (PEO) can fabricate a porous ceramic coating on magnesium and its alloys with high corrosion resistance.19,26 However, without sealing, the micropores and microcracks on the coating surface will allow the corrosion electrolytes permeating into the coating, reducing its corrosion resistance. Considering the biomedical applications, the biodegradable polymers are the ideal choice to seal PEO coating. Poly(L-lactide) (PLLA) is a biodegradable and biocompatibility material which have been used in many field.27 PEO/PLLA composite coating has also been reported to improve the hemocompatibility and cytocompatibility of WE42 alloy in the field of cardiovascular stents.28,29 Until now, the in vitro degradation with immersion time and the cells cultured directly on the surface of PEO/PLLA coating have not been investigated systematically. In this work, the PEO/PLLA composite coating were fabricated on AZ31 alloy, as shown in Figure 1, aiming at fabricating PEO coating as corrosion inhibitor and PLLA coating as biofunctional layer. Characterization, in vitro degradation, hemocompatibility and cytocompatibility have been systematically investigated for the PEO/PLLA composite coating aiming at obtaining a promising degradation-controlling and biocompatible degradable magnesium implant. MATERIALS AND METHODS

Materials The commercial AZ31 alloy, whose chemical composition is 2.5–3.5 wt % Al, 0.7–1.3 wt % Zn, 0.2–1.0 wt % Mn, 0.05 wt % Si, 0.01 wt % Cu, and Mg balance, was used in this

FIGURE 1. Fabrication process of PEO/PLLA composite coating on AZ31 alloy. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

work. AZ31 alloy plate was cut into samples with a size of 10 3 10 3 2 mm3. They were finely ground with 1000# SiC abrasive paper to remove contaminants and native oxidation layer to obtain relatively smooth surface, ultrasonically cleaned with acetone, ethyl alcohol, and then dried in air prior to plasma electrolytic oxidation (PEO) process. Fabrication of PEO and PEO/PLLA coating Porous PEO coatings on AZ31 alloy were fabricated by PEO equipment (Pulsetech, China) in electrolytes containing 0.05M sodium silicate (Na2SiO39H2O, Sinopharm, China), 0.1M sodium hydroxide (NaOH, Sinopharm, China) and 0.05M sodium fluoride (NaF, Sinopharm, China). PEO process was carried out with a constant current density of 50 mA/cm2, frequency of 800 Hz and duty cycle of 20% for 15 min. AZ31 alloy samples were used as anode, and a spiral steel pipe was used as a cathode while it was also used as a dwelling water pipe to sustain the temperature of the electrolytes less than 30 C in the electrolytic cell during the PEO process. A magnetic stirrer was used to keep the uniform of the components and temperature of the electrolytes. After PEO process, the samples were gently washed with deionized water and dried in air (denoted as AZ31-PEO). The PEO treated samples were then submersed in the 6 wt % PLLA dichloromethane solution. As dichloromethane volatized overnight, PLLA effectively sealed the PEO coatings. In this way, the PEO/PLLA coatings were successfully fabricated on AZ31 alloy (denoted as AZ31-PEO/PLLA). All the samples were sealed by water-proof silica gel, just leaving an area of 1 cm2 for the following tests. Characterization In order to investigate the influence of surface morphologies on corrosion behavior, the surface morphologies of uncoated and coated samples were observed by scanning electron microscopy (SEM, Hitachi-S3800N, Hitachi, Japan). The elemental compositions of the samples were also measured by energy-dispersive X-ray spectrometry (EDS, IXRF-550i, IXRF SYSTEMS, USA) to ensure that no toxic elements were introduced by the coatings fabrication. To determine whether nondegradable phase was formed in the coating or not,

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FIGURE 2. Surface morphologies of AZ31 (A), AZ31-PEO (B), and AZ31-PEO/PLLA (C).

their phase compositions were analyzed by X-ray diffraction (XRD, Diffractometer D8, Bruker, Germany) in the 2h range of 20 90 at a scan rate of 4 /min. Electrochemical tests The corrosion resistance of AZ31-PEO and AZ31-PEO/PLLA as well as bare AZ31 alloy was evaluated by potentiodynamic polarization test through a CHI760C electrochemical analyzer (Shanghai, China) in simulated body fluid (SBF).12 The process was conducted using a conventional threeelectrode electrochemical cell with a saturated calomel electrode (SCE) as the reference electrode, a graphite rod as the counter electrode and the sample with area of 0.255 cm2 as the working electrode. Prior to the potentiodynamic polarization test, the sample was exposed in SBF for 30 min to establish a relatively steady open-circuit potential. All electrochemical tests were conducted at 37 C with a scanning rate of 5 mV/s. The corrosion potential and current density were calculated with the CHI760C software according to Tafel extrapolation. Electrochemical impedance spectroscopy (EIS) analysis was also carried out in SBF. The amplitude of the sinusoidal perturbing signal was 5 mV, and the frequency varied from 100 kHz to 10 mHz for all samples. Prior to the EIS measurement, the sample was also kept in SBF for 30 min to establish a relatively steady open-circuit potential. The DC voltage applied for the EIS tests was open circuit potential. The EIS results were analyzed with ZViewTM software. All the SBF used for the potentiodynamic polarization test and electrochemical impedance spectroscopy tests were under air condition with steady air pressure and temperature. Immersion tests in SBF In order to evaluate the corrosion behavior of the samples, short-term, medium-term, and long-term immersion tests were designed. For short-term evaluation, all samples of AZ31-PEO and AZ31-PEO/PLLA as well as bare AZ31 alloy were immersed in SBF at 37 C for 7 and 14 days. At the

prescribed immersion time, the samples were removed, rinsed with water, and dried in air. The surface morphologies of bare AZ31, AZ31-PEO, and AZ31-PEO/PLLA, both before and after being immersed in SBF, were examined with scanning electron microscopy (SEM, Hitachi-S3800N, Hitachi, Japan). The elemental compositions of the samples were also measured by energy-dispersive X-ray spectrometry (EDS, IXRF-550i, IXRF SYSTEMS, USA). For medium-term evaluation, the pH changes, Mg21 concentration changes of the immersion test solution and the hydrogen evolution were conducted within 28 days. In order to determine the pH changes and the Mg21 concentration changes of the immersion test solution, each sample was placed inside a tube containing 40 mL SBF for 1, 4, 7, 14, 28 days. Three samples were used as parallel ones. After the immersion time, the pH value and the Mg21 concentration of the solutions were measured. The custom designed hydrogen evolution set up was used to collect the hydrogen produced through degradation of samples in SBF. The evolution of hydrogen was tested by placing the samples in SBF at 37 C under the hydrogen collector and measuring the water level intermittently for 28 days. All Samples were packaged by waterproof silica gel with 1 cm2 exposure area. Five samples were placed for each condition, accounting for the low hydrogen evolution anticipated from the coated samples. The ratio of sample exposure area to SBF volume was kept constant at 25 cm2/L. For long-term evaluation, the integrity of the samples was evaluated by volume lost test after the samples immersed in SBF as long as 4 months. After the immersion, they were taken out, washed with distilled water and then dried in air. The surface morphologies of the samples were taken by the digital camera and the volume lost was qualitatively evaluated. Hemolysis test To evaluate the blood compatibility of AZ31, AZ31-PEO, and AZ31-PEO/PLLA, the hemolysis test was conducted according to ISO 10993-4.30 The blood was obtained from healthy

TABLE I. Surface Elemental Compositions of AZ31, AZ31-PEO, and AZ31-PEO/PLLA Samples AZ31 AZ31-PEO AZ31-PEO/PLLA

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C wt %

O wt %

Mg wt %

P wt %

Ca wt %

Si wt %

Al wt %

Zn wt %

4.22 6.06 47.24

1.76 39.06 52.76

91.08 41.78 –

– – –

– – –

– 10.64 –

1.49 0.76 –

1.45 1.70 –

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TABLE II. Electrochemical Parameters of Bare AZ31 Alloy, AZ31-PEO, and AZ31-PEO/PLLA Tested in SBF

Ecorr (V) icorr (lAcm2) Rc (X/cm2) Rct (X/cm2)

FIGURE 3. XRD patterns of AZ31, AZ31-PEO, and AZ31-PEO/PLLA. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

SPF SD rat. 4 mL blood was diluted by 5 mL normal physiological saline (0.9% NaCl). AZ31, AZ31-PEO, and AZ31-PEO/ PLLA samples with 1 3 1 cm2 exposure area were soaked in 1 mL normal physiological saline in 24-well culture plates as test groups and kept at 37 C for 30 min. Untreated normal physiological saline and distilled water served as negative and positive controls. After that 0.02 mL diluted blood was added into each well and kept at 37 C for another 60 min. Subsequently, the solutions were centrifuged at 3000 rpm for 5 min. At last, the optical density (OD) of supernatant fluid was obtained by an enzymelabeling instrument (BIO-TEK, ELX 800) at 570 nm wave length. The hemolysis ratio (HR) was calculated as the following equation: HR5[(ODt2ODn)/(ODp2ODn)]3100%. The ODt means the OD value of tested group. The ODn and ODp were OD values of negative and positive groups, respectively. Three samples were used in each group.

AZ31

AZ31-PEO

AZ31-PEO/PLLA

21.663 290.2 589.3 32,892

21.606 1.1 4,888 58,295

21.317 0.00024 32,892 895,900

Cell adhesion and proliferation Osteoblast-like cell line MC3T3-E1 (Cells Resource Center of Shanghai Institute for Biological Science, Shanghai, China) was used to evaluate the cytocompatibility of AZ31, AZ31PEO, and AZ31-PEO/PLLA. Cells were routinely cultured at 37 C in a humidified atmosphere of 5% CO2 in air. Samples were put into 24-well culture plates (Costar, USA) after sterilized by ultraviolet for 24 h. After rinsed with a phosphate buffered saline solution (pH 5 7.4, PBS) twice, l.0 mL cell suspension with cell density of 5 3 104 cell/mL was added into each well. After the cells were cultured for 1, 4, and 7 days, the samples were taken out and rinsed with a phosphate buffered saline solution (pH 5 7.4, PBS) twice to remove the unattached cells. All samples were fixed with a 2.5% glutaraldehyde solution in water for 12 h at 4 C. The samples were then dehydrated in a grade ethanol series (30, 50, 75, 90, 95, and 100% v/v) for 10 min, respectively, with final dehydration conducted in absolute ethanol twice followed by drying in the hexamethyldisilizane ethanol solution series (1 : 2, 2 : 1, pure hexamethyldisilizane). After gold sputtering, the cell morphology was observed by scanning electron microscopy (SEM, HitachiS3800N, Hitachi, Japan). The proliferation and vitality of cells cultured on the samples were determined using alamarBlueTM assay. The assays were performed after the cells were cultured for 1, 4, and 7 days, respectively. At the end of each culture time, the culture medium was removed and 0.5 mL fresh medium with 10% alamarBlueTM was added to each well. After incubation for another 4 h, culture medium (100 lL) was transferred to 96-well culture plate for measurement. Accumulation of reduced alamarBlueTM in culture medium was determined by an enzyme-labeling instrument (BIOTEK, ELX 800) at extinction wavelengths of 570 and 600 nm. Cell proliferation and viability were calculated according to the formula.31 Three samples were used in each group for each culture time for statistical analysis. RESULTS

FIGURE 4. Potentiodynamic polarization curves of AZ31-PEO and AZ31-PEO/PLLA as well as bare AZ31 in SBF at 37 C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

Characterization Figure 2 shows the surface views of AZ31, AZ31-PEO, and AZ31-PEO/PLLA. It can be observed that there were many micro pores on the PEO coating [Figure 2(B)]. These pores may give the chance for the corrosive fluid to penetrate in the coating and contact with the substrate. After sealed with PLLA, the micro pores were filled successfully, showing a flat outmost surface [Figure 2(C)].The compact PEO/PLLA composite coating may prevent the corrosive fluid from contacting with the substrate.

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FIGURE 5. EIS results of AZ31-PEO and AZ31-PEO/PLLA as well as bare AZ31 in SBF at 37 C (a), the magnification of the red block in (a) shown in (b), the magnification of the red block in (b) shown in (c), (d) equivalent circuit for EIS fitting for AZ31, AZ31-PEO, and AZ31-PEO/PLLA. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

The elemental compositions of AZ31, AZ31-PEO, and AZ31-PEO/PLLA detected by EDS are summarized in Table I. The AZ31 mainly consisted of Mg, Al, and Zn. The C and O elements maybe contribute to the oxidation and adsorption in the air. After the PEO process, the surface elemental composition came to change. More O element was introduced because of the oxidation of AZ31. Furthermore, Si element was observed attributing to the addition of Na2SO3 in the electrolytes. When sealed with PLLA, the C and O elements became the main compositions of the surface, which are the two main compositions of PLLA. No Mg element was detected, implying the effective sealing of PLLA for PEO coating and protection for the substrate. The XRD patterns of AZ31, AZ31-PEO, and AZ31-PEO/ PLLA are presented in Figure 3. All patterns in Figure 3 showed the feature peaks of Mg (PDF#35-0821), attributing to the AZ31 substrate. The feature peaks of magnesium oxide (MgO, PDF#77-2364) were observed in the XRD patterns of AZ31-PEO and AZ31-PEO/PLLA, proving the formation of MgO coating by PEO process, which was consist with the result of superficial O element increase in Table I. Moreover, the intensity of the feature peaks of either Mg or MgO was relatively weaker for AZ31-PEO/PLLA, showing the sealing effect of outmost PLLA. No feature peaks of PLLA were observed in the XRD pattern of AZ31-PEO/PLLA, implying that no crystallization of PLLA was achieved with this sealing process.

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Electrochemical tests Figure 4 shows potentiodynamic polarization curves of AZ31, AZ31-PEO, and AZ31-PEO/PLLA in SBF at 37 C. Generally, the cathodic polarization curve represents the cathodic hydrogen evolution while the anodic one represents the dissolution of Mg.25 Table II summarizes the corrosion potential (Ecorr) and corrosion current density (icorr) obtained by Tafel extrapolation. It is clear that the corrosion resistance of AZ31-PEO and AZ31-PEO/PLLA were both significantly improved compared to the bare AZ31, which can be observed by a shift of polarization curve towards the region of lower current density and higher potential. AZ31PEO/PLLA showed the best corrosion resistance for protection of the AZ31 alloy substrate. The typical Nyquist diagrams of EIS for AZ31, AZ31PEO, and AZ31-PEO/PLLA are shown in Figure 5. Compared with bare AZ31, the capacitive loops of AZ31-PEO and AZ31-PEO/PLLA were both enlarged, indicating that they have higher corrosion resistance, which implies the effective protection for the substrate of the two kinds of coatings.32 An equivalent circuit was used to fit the EIS spectrums of AZ31, AZ31-PEO, and AZ31-PEO/PLLA, which was shown in Figure 5(D). The Rs is the resistance of the solution. The CPEc and Rc are the capacitance and resistance of the corrosion production or coatings on AZ31, respectively. The CPEdl represents the double electric layer in the interface between the surface and electrolyte, while the Rct is the charge

PEO/PLLA COMPOSITE COATING ON BIODEGRADABLE AZ31 ALLOY

ORIGINAL RESEARCH REPORT

FIGURE 6. Surface morphologies of bare AZ31 (A), AZ31-PEO (B), and AZ31-PEO/PLLA (C) after immersed in SBF for 7 (D7) and 14 (D14) days.

transfer resistance. The electrochemical parameters obtained from the equivalent circuit are also listed in Table II. The results of EIS showed the same trend with the results of polarization curves, i.e., the Rc and Rct of the AZ31-PEO and AZ31-PEO/PLLA were both higher than those of bare AZ31 alloy, indicating the protection of the two kinds of coatings for substrate.

Immersion tests in SBF In order to directly observe the degradation behavior of the magnesium and its alloys in SBF, immersion test was used. Figure 6 shows the surface morphologies of AZ31PEO and AZ31-PEO/PLLA as well as bare AZ31 after degradation for a period of time in SBF. For bare AZ31 [Figure 2(A)], after immersed in SBF, severe corrosion happened.

TABLE III. Surface Elemental Compositions of Bare AZ31, AZ31-PEO, and AZ31-PEO/PLLA After Immersed in SBF for 7 and 14 Days Immersion Time in SBF 7 days

14 days

Samples

C wt %

O wt %

Mg wt %

P wt %

Ca wt%

Si wt %

AZ31 AZ31-PEO AZ31-PEO/PLLA AZ31 AZ31-PEO AZ31-PEO/PLLA

14.66 7.27 48.03 12.60 11.57 48.30

46.01 47.18 51.97 45.86 44.50 51.70

17.34 23.82 – 17.15 24.01 –

13.44 4.89 – 14.45 5.20 –

8.55 3.32 – 9.94 3.24 –

– 13.52 – – 11.48 –

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FIGURE 7. pH values of the SBF solution containing bare AZ31, AZ31PEO, and AZ31-PEO/PLLA over a period of 28 days (Data presented as mean 6 SD, n 5 3). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Many corrosion pits and cracks can be observed on their surface after degradation in SBF for 7 and 14 days [Figure 6(A–D7, (A–D14)]. For PEO coating, after degradation in SBF, there were also a lot of cracks forming on its surface [Figure 6(B–D7), (B–D14)]. These cracks maybe unavoidably weaken the protection ability of the coating for substrate. After sealed with PLLA, AZ31-PEO/PLLA showed rather flat surface structure [Figure 2(C)], successfully inhibiting the contact of the corrosive fluid and the substrate. After immersed in SBF for 7 and 14 days, the surface morphologies of AZ31-PEO/PLLA had nearly no change [Figure 6(C–D7), (C–D14)]. Over the degradation in SBF, the surface elemental concentrations of the samples changed. Table III summarizes the elemental compositions of AZ31, AZ31-PEO, and AZ31PEO/PLLA over the immersion period. The changes of Al and Zn elements were not considered in this condition. Over a period of immersion in SBF, less Mg element and more O, P, and Ca elements can be observed for bare AZ31

FIGURE 8. Mg21 ion concentration of the SBF solution containing bare AZ31, AZ31-PEO, and AZ31-PEO/PLLA over a period of 28 days (Data presented as mean 6 SD, n 5 3). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 9. Cumulative hydrogen volume released per cm2 of AZ31, AZ31-PEO, and AZ31-PEO/PLLA in SBF as a function of immersion time. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

and AZ31-PEO while hardly little Mg element can be found on the surface during 14 days and P and Ca elements nearly didn’t deposit on its surface for AZ31-PEO/PLLA. Figure 7 shows the pH change in the SBF solution containing AZ31, AZ31-PEO, and AZ31-PEO/PLLA. It can be seen that the pH values of the SBF solution containing AZ31 and AZ31-PEO increased significantly with the immersion time. The pH value of the SBF solution containing AZ31PEO/PLLA nearly kept constant with the immersion time, showing little influence of AZ31-PEO/PLLA on the surrounding environment. Figure 8 presents the Mg21 ion concentration changes in the SBF solution containing AZ31, AZ31-PEO, and AZ31PEO/PLLA. Just like the change of pH values (Figure 7), the solutions containing AZ31 and AZ31-PEO underwent rapid increase of Mg21 ion concentration while the Mg21 ion concentration of solution containing AZ31-PEO/PLLA showed a relatively gentle increase. Figure 9 shows the cumulative volume of hydrogen released from the SBF solution containing AZ31, AZ31-PEO, and AZ31-PEO/PLLA for up to 28 days. The untreated AZ31 as expected showed a quick and copious amount of cumulative hydrogen evolution with immersion time. However, the release rate decreased slightly with the immersion time. The PEO coating showed protective ability and slowed the hydrogen release rate of the substrate for the early 14 days. But after 14 days, the hydrogen volume released from AZ31-PEO samples exceeded that from the untreated AZ31 alloy. This result agreed with the pH change (Figure 7) and Mg21 ions concentration change (Figure 8). There were nearly no hydrogen released from the PEO/PLLA coated samples until immersed in SBF for 18 days and then hydrogen released slowly after that time, indicating that the PLLA begins to lose its protection ability. To evaluate the long service performance of the implants, a long period of 4 months immersion in SBF was conducted. AZ31, AZ31-PEO, and AZ31-PEO/PLLA samples showed different performance in maintaining their integrity,

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FIGURE 10. Surface appearances of AZ31, AZ31-PEO, and AZ31-PEO/PLLA before and after immersion in SBF for 4 months.

as shown in Figure 10. Severe corrosion can be seen on bare AZ31 after the long period of immersion, with nearly half volume lost compared with that of as-prepared AZ31 sample. With PEO coating protection, AZ31-PEO showed much better corrosion resistance and almost maintained the integrity of the sample. AZ31-PEO/PLLA showed the best performance in corrosion resistance in SBF, with only the degradation and peeling of the outmost PLLA, which is usually observed in the condition that degradable polymer is used as corrosion rate controller on Mg alloys.25 The inner layer of PEO coating and the substrate were nearly not affected. Hemolysis test Table IV shows the result of the hemolysis test of AZ31, AZ31-PEO, and AZ31-PEO/PLLA. Compared with AZ31, the hemolysis ratio of AZ31-PEO was lowered to 23.419 6 1.565, which is still up 5% and means that it will still lead to severe hemolysis when contacted with the blood. When sealed with PLLA, AZ31-PEO/PLLA exhibited the hemolysis ratio of 0.806 6 0.771, which is much lower than 5%. Cell adhesion and proliferation The morphologies of MC3T3-E1 cells cultured on AZ31, AZ31-PEO, and AZ31-PEO/PLLA for 1, 4, and 7 days are presented in Figures 11–13, respectively. After 1 day of culture, it is clear that the cells cultured on AZ31-PEO/PLLA exhibited good adhesion and displayed numerous filopodia extensions [Figure 13(D1)]. Although the cells cultured on AZ31 and AZ31-PEO also adhered to the surface and filopoTABLE IV. Hemolysis Ratio of the Bare AZ31, AZ31-PEO, and AZ31-PEO/PLLA (the Size of Samples: 1 3 1 cm2, data presented as mean 6 SD, n 5 3) Sample AZ31 AZ31-PEO AZ31-PEO/PLLA

Hemolysis Ratio (%) 93.290 6 0.782 23.419 6 1.565 0.806 6 0.771

dia protrusions were also observed [Figures 11(D1) and 12(D1)], their growth seemed to be inhibited. After 3 and 7 days of culture, MC3T3-E1 cells proliferated fast on the PEO/PLLA composite coating, nearly covering the surface for 7 days [Figure 13(D7)]. However, nearly no more cells can be observed on the bare AZ31 and PEO coating compared with 1 day culture, showing their poor cytocopatibility for MC3T3-E1 cells. The alamarBlueTM assay result implied the proliferation and vitality of MC3T3-E1 cells cultured on AZ31, AZ31-PEO, and AZ31-PEO/PLLA, as shown in Figure 14. The results revealed the same trend as that observed by SEM. MC3T3E1 cells on the PEO/PLLLA coating showed an obviously higher proliferation rate and vitality than that on both bare AZ31 and PEO coating. So AZ31 and AZ31-PEO couldn’t provide a proper environment for the cell growth because of the cell culture condition change caused by their fast degradation. DISCUSSION

Recently, magnesium and its alloys attract much attention as biodegradable materials. As biodegradable materials, magnesium and its alloys have some advantage over commonly used biodegradable materials such as polymer, ceramics or glasses especially on mechanical properties. However, the rapid degradation of magnesium and its alloys in physiological environment usually causes adverse effects such as large amount of Mg21 release, hydrogen accumulation and local alkalization, which are the most critical obstacles for their utilization as biodegradable implant in vivo.1,7 So controlling the degradation rate is critical in the utilization of magnesium and its alloys for biomedical applications. For this end, a surface coating is an effective method to be applied on surface modification of magnesium and its alloys. Considering the eventually degradation of the implant in human body, the protective coating on magnesium alloys should be designed to be also biodegradable in the physiological environment. The plasma electrolytic oxidation (PEO)

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FIGURE 11. Morphologies of MC3T3-E1 cells cultured on the bare AZ31 samples for 1, 4, and 7 days.

coating mainly consists of MgO (Figure 3). The MgO is likely to change into soluble Mg21 by the aid of Cl2.33 So the PEO coating will not influence the eventually degradation of the implant when inserted in human body. However, a constant protection for magnesium substrate will not achieved just by the protection of PEO coating because of its porous structure and gradually degradation in vivo. The effective seal of the microcracks and micro pores on the surface of PEO coating can further improve the protection ability for the substrate. Poly(L-lactide) (PLLA) is common used as biodegradable materials. Its degradation bases on the hydrolysis of ester bonds. The degradation product of PLLA is Llactide which will take part in metabolism in human body. As biodegradable and biocompatible materials, PLLA is a proper choice for sealing PEO coating. This kind of composite coating has been investigated in WE42 alloy as stent application.28,29 In our present work, the PEO/PLLA composite coating were fabricated on AZ31 alloy, corrosion behavior with immersion time and cells adhesion and prolif-

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eration directly on the coating surface were systematically investigated. In electrochemical measurements, the potentiodynamic polarization and electrochemical impedance spectroscopy test are both used to characterize the corrosion behavior of samples. In potentiodynamic polarization curve, the corrosion potential (Ecorr) is representative for the trend of corrosion occurrence. The more positive of the Ecorr the material shows, the more stable the material is in corrosive fluid. The corrosion current icoor can be related to the corrosion rate of the substrate or coatings. As summarized in Table II, the AZ31-PEO/PLLA showed more positive Ecoor and much smaller icoor, which indicating that the composite coating showed good protection for the substrate. Representative EIS of AZ31 alloy substrate, AZ31-PEO and AZ31-PEO/PLLA samples immersed in SBF are shown in Figure 5. The capacitive loop at high and intermediate frequencies can be attributed to the charge transfer reaction of the magnesium corrosion process. Its evolution could be

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ORIGINAL RESEARCH REPORT

FIGURE 12. Morphologies of MC3T3-E1 cells cultured on AZ31-PEO samples for 1, 4, and 7 days.

interpreted by the variations of dielectric property and thickness of anodic film. The loop at low frequencies associates with a diffusion process across the corrosion layer and the adsorption of the corrosion products. It is evident that the diameter of the loop is proportional to the value of the transfer resistance Rct. The larger the value of Rct, the better is the corrosion protective property of the coating.34 The transfer resistance Rct whose unit is X/cm2 could be calculated from EIS, as shown in Table II. As compared with the Rct value of bare AZ31 alloy, the Rct values of AZ31-PEO and AZ31-PEO/PLLA both increased. The AZ31-PEO/PLLA showed the biggest Rct, which implies that the coating could reduce the biodegradation rate of magnesium. As we know, if a magnesium implant is inserted in the human body, it will degrade by the attack of body fluid. Many cracks formed on the surface of AZ31 alloy after immersed in SBF for 7 and 14 days [Figure 6(A)]. Although the results of electrochemical measurements for AZ31-PEO showed better corrosion resistance, severe corrosion also

happened after immersed in SBF for a certain time [Figure 6(B)]. The anticorrosion behavior of the PEO coating is considered to be due to the compact inner layer which could interface the corrosion electrolyte ions. The porosity and microcracks on the surface of PEO coating could increase the surface area contacted with corrosion ions. So although the inner compact layer of the PEO coating could improve the corrosion resistance, the outer layer would permit more corrosive electrolyte adsorbed into the PEO coating and decrease the corrosion resistance of the PEO coating. For Mg and MgO, the reaction of them with water could transferred them to Mg(OH)2 film. The formed Mg(OH)2 connected with some H2O molecule to form hydrate of Mg(OH)2nH2O. When the samples were dried in air, the film shrank due to dehydration thus forming many cracks.35 These cracks could further reduce the corrosion resistance of the coating. The rapid degradation of magnesium will give rise to a local increase in pH value and Mg21 ion concentration of

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FIGURE 13. Morphologies of MC3T3-E1 cells cultured on AZ31-PEO/PLLA samples for 1, 4, and 7 days.

body fluid nearby. The MgO could also be dissolved in SBF easily and accordingly resulting in a high pH value and Mg21 ion concentration. This has been confirmed by this study (Figures 7 and 8). The following reactions may occur. MgO1H2 O ! Mg ðOH Þ2

(5)

Mg ðOH Þ2 ! Mg 21 12OH 2

(6)

Local alkalization will make it possible for phosphate ions and Ca21 in SBF to deposit with OH2.36 Ca-P deposition can be observed on both AZ31 and AZ31-PEO, also indicating that the PEO coating couldn’t prevent the substrate from corrosion effectively (Table III). The Ca-P deposit and the corrosion products mainly consisting of Mg(OH)2 will in turn influence on the consecutive degradation of the substrate as a protection layer. The increase rate of pH value and Mg21 ion concentration was lowered which can be observed in Figures 7 and 8. After sealed with PLLA,

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the local alkalization and Mg21 ion enrichment were prevented effectively, showing the little influence of AZ31-PEO/ PLLA on the immersion solution. No Ca-P deposition also indicates the deficient bioactivity of PLLA.37 The excess gas evolution than the ability of tissue absorption and dissolution caused by the rapid degradation of magnesium will lead gas cavity around the implant in vivo. So controlling the gas evolution in a proper level is also important in utilization of magnesium implant in vivo. Figure 9 shows the hydrogen released from the samples immersed in SBF. AZ31 alloy released big volume of hydrogen and the hydrogen release rate was reduced with the immersion time as the corrosion products formed on the surface. The PEO coating reduced the released hydrogen for the early immersion time but more hydrogen was released from the PEO coating protected substrate, indicating its insufficient control of hydrogen release in the long period. After sealed with PLLA, the hydrogen release was controlled in a low level for a quite long period.

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TM

FIGURE 14. Results of alamarBlue assay showing the MC3T3-E1 cell proliferation on bare AZ31, AZ31-PEO, and AZ31-PEO/PLLA. (Data presented as mean 6 SD, n 5 3 and analyzed using a two-way ANOVA, *p < 0.05, ***p < 0.001). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Interestingly, up to 4 months immersion in SBF, the PEO coating showed its advantage in protection for the AZ31 alloy substrate (Figure 10). The integrity of the sample was mostly maintained because of the PEO coating protection. After sealed with PLLA, AZ31-PEO/PLLA showed better corrosion resistance for either short or long period of immersion in SBF. Local alkalization and Mg21 ions enrichment will make a significant influence on the physiological balance. It is believed that the magnesium based materials degraded too fast and the high concentration of magnesium in the solution should be responsible for the high hemolysis rate.38 Although the PEO coating reduced the hemolysis ratio to (23.419 6 1.565) %, which is much lower than that of AZ31 ((93.290 6 0.782) %), it is not suitable for utilization as blood contacting materials for the potential danger of erythrocytes. After sealed with PLLA, AZ31-PEO/PLLA with a hemolsis ratio of (0.806 6 0.771) % will have little influence on the blood. It is well known that cells are very sensitive to the change of the surrounding environment such as the sharp change of pH value and Mg21 ion concentration. So the sharp increase of pH value and enrichment of Mg21 ion concentration by the degradation of AZ31 and AZ31-PEO may be unfavourable for the cell growth (Figures 11 and 12). As biodegradable materials, PLLA not only works as anticorrosion layer but also provides a suitable condition for cells adhesion and proliferation on AZ31 alloy (Figure 13). Actually, combing plasma electrolytic oxidation with poly(L-lactide) coating can take advantage of the superiority of the two coating on biodegradable magnesium alloy. For PEO coating only, just as discussed above, the porous surface structure was not proper as short-term and mediumterm anticorrosion layer as corrosion and side effects were not effectively inhibited. However, the PEO coating showed its advantage in long-term corrosion resistance. Polymer coatings have been widely applied as protective coating on

magnesium alloys. However, without anticorrosion coating as substrate, although providing initial protection for the substrate, the polymer coatings did not maintain a reduction in corrosion resistance rate for long term and inhomogeneous coating durability with gas pocket formation in the polymer coating will result in eventual detachment from the alloy surface.39 So the PEO coating played a critical role as interface layer between the magnesium alloy substrate and polymer coating. Furthermore, as a kind of polymer, the PLLA coating is easily further modified for various biological applications.40 Moreover, compared with nondegradable coatings,41,42 the PEO/PLLA composite coating will eventually degrade after implantation with PLLA hydrolysis and the following PEO coating dissolving. So although reducing the corrosion rate of the magnesium implant to maintain its performance for quite a long time, the PEO/PLLA composite coating will not influence the eventually degradation of the implant. CONCLUSIONS

To obtain a proper biodegradable implant, the PEO/PLLA composite coating was fabricated on biodegradable AZ31 alloy by plasma electrolytic oxidation and sealing with PLLA. The results of in vitro degradation tests showed that the PEO/PLLA composite coating protected AZ31 from fast degradation in physiological environment effectively. The PEO process played a critical role in sustaining the integrity of implant in long service. The hemolysis ratio of the PEO/ PLLA composite coating was (0.806 6 0.771)%, which is much lower than the safe value of 5%, implying that the PEO/PLLA composite coating can be used as blood contacting protective layer on biodegradable AZ31 alloy. The MC3T3-E1 cells could adhere and proliferate well on the PEO/PLLA composite coating, showing that the composite coating can provide a proper environment for cells growth. Therefore, the PEO/PLLA composite coating has large potential being used as protective layer on biodegradable AZ31 alloy for biomedical applications. REFERENCES 1. Waksman R. Promise and challenges of bioabsorbable stents. Catheterization Cardiovascular Interv 2007;70:407–414. 2. Erbel R, Di Mario C, Bartunek J, Bonnier J, de Bruyne B, Eberli € se D, FR, Erne P, Haude M, Heublein B, Horrigan M, Ilsley C, Bo Koolen J, L€ uscher TF, Weissman N, Waksman R. Temporary scaffolding of coronary arteries with bioabsorbable magnesium stents: A prospective, non-randomised multicentre trial. Lancet 2007;369:1869–1875. € rk H. Bioabsorbable 3. Schranz D, Zartner P, Michel-Behnke I, Akintu metal stents for percutaneous treatment of critical recoarctation of the aorta in a newborn. Catheterization Cardiovascular Interv 2006;67:671–673. 4. Mario CD, Grifftths H, Goktekin O, Peeters N, Verbist J, Bosiers M, Deloose K, Heublein B, Rohde R, Kasese V, Ilsley C, Erbel R. Drugeluting bioabsorbable magnesium stent. J Interv Cardiol 2004;17: 391–395. 5. Zhang E, Xu L, Yu G, Pan F, Yang K. In vivoevaluation of biodegradable magnesium alloy bone implant in the first 6 months implantation. J Biomed Mater Res Part A 2009;90:882–893. 6. Xu L, Yu G, Zhang E, Pan F, Yang K. In vivo corrosion behavior of Mg-Mn-Zn alloy for bone implant application. J Biomed Mater Res Part A 2007;83:703–711.

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