In Vitro Corrosion and Cytocompatibility of a Microarc Oxidation

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In Vitro Corrosion and Cytocompatibility of a Microarc Oxidation Coating and Poly(L-lactic acid) Composite Coating on Mg-1Li-1Ca Alloy for Orthopaedic Implants Rongchang Zeng, Lanyue Cui, Ke Jiang, Rui Liu, Baodong Zhao, and Yufeng Zheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00527 • Publication Date (Web): 29 Mar 2016 Downloaded from http://pubs.acs.org on April 4, 2016

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ACS Applied Materials & Interfaces

In Vitro Corrosion and Cytocompatibility of a Microarc Oxidation Coating and Poly(Llactic acid) Composite Coating on Mg-1Li-1Ca Alloy for Orthopaedic Implants

Rong-Chang Zeng a, b, *, Lan-yue Cui a, b, Ke Jiang a, b, Rui Liu c, Bao-Dong Zhao c, *, Yu-Feng Zheng d a

College of Materials Science and Engineering, Shandong University of Science and Technology, Qingdao,

266590, China; b

State Key Laboratory of Mining Disaster Prevention and Control Co-founded by Shandong Province and the

Ministry of Science and Technology, Shandong University of Science and Technology, Qingdao, 266590, China; c

d

Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, 266590, China; State Key Laboratory for Turbulence and Complex Systems and Department of Materials Science and

Engineering, College of Engineering, Peking University, Beijing, 100871, China. KEYWORDS: Magnesium alloy; Degradation; Micro-arc oxidation; Poly (L-lactic acid); Biomaterial

ABSTRACT: Manipulating the degradation rate of biomedical magnesium alloys poses a challenge. The characteristics of a micro-arc oxidation (MAO), prepared in phytic acid, and poly(L-lactic acid) (PLLA) composite coating, fabricated on a novel Mg-1Li-1Ca alloy, were investigated through field emission scanning electron microscopy (FE-SEM), electron probe X-ray microanalysis (EPMA), energy dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD). The corrosion behaviours of the samples were evaluated via hydrogen evolution, potentiodynamic polarization and electrochemical impedance spectroscopy in Hanks’ solution. The results indicated that the MAO/PLLA composite coatings significantly enhanced the corrosion resistance of the Mg-1Li-1Ca alloy. MTT and ALP assays using MC3T3 osteoblasts indicated that the MAO/PLLA coatings greatly improved the cytocompatibility, and the morphology of the cells cultured on

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different samples exhibited good adhesion. Haemolysis tests showed that the composite coatings endowed the Mg-1Li-1Ca alloys with a low haemolysis ratio. The increased solution pH resulting from the corrosion of magnesium could be tailored by the degradation of PLLA. The degradation mechanism of the composite coatings was discussed. The MAO/PLLA composite coating may be appropriate for applications on degradable Mg-based orthopaedic implants.

1 Introduction The regeneration of bone defects using biomedical materials such as bioceramics, stainless steel, titanium, polymers and composites has been extensively studied in recent years 1-4. In comparison with these materials, magnesium (Mg) alloys show great potential in clinical applications because their good biocompatibility, degradability and mechanical properties are similar to those of human bone 5-7. However, the poor corrosion resistance of Mg alloys leads to a rapid increase in the pH value and hydrogen collection in the microenvironment surrounding the implant 8-10. Therefore, it is of importance to control the degradation of Mg alloys to an appropriate rate. Usually, the corrosion resistance of Mg alloys can be improved by means of purifying and alloying 2, 11-13, refinement of microstructure by post-processing 14, 15 and surface modification, such as phosphate conversion coatings 16, 17, micro-arc oxidation (MAO) coatings 18, 19, polymeric coatings 19, silane-modified coatings 20, layer-by-layer assembly 21 and layered double hydrotalcite 26-27. Numerous investigations have been made on biomedical Mg alloys, such as Mg-Al (AZ31), Mg-Zn (Mg6Zn, Mg-Zn-Nd), Mg-Mn, Mg-RE (WE43), Mg-Ca (Mg-0.8Ca) and Mg-Li (LAE442) alloys 11-15. Ca-bearing Mg alloys (Mg-Ca) have attracted particular attention because Ca is a major element of human bone and can refine the microstructure of Mg alloys 11. However, Mg-Ca alloys exhibit poor corrosion resistance 11, 13. Additionally, Mg-Li alloys are remarkably malleable and ultralight due to the alloying element Li. Li is the only element that can change the microstructure of Mg alloys from HCP crystalline into α (-Mg), α+β (-Li) and β phases with increasing Li concentration

10, 11, 14

. Usually, β (-Li) phase has a body-centered cubic


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structure and better ductility than α (-Mg) phase. Higher Li content leads to an enhancement in deformability of binary Mg-Li alloys. The introduction of Ca into Mg-Li alloys gives rise to an increase in tensile strength. In addition, Li possesses higher activity than Mg, and exerts a pronounced influence on the corrosion resistance. Li concentration below 9 wt. % in Mg is beneficial for the corrosion resistance; however, increasing Li concentration significantly accelerates the corrosion of Mg alloys 28. Trace of Li element has a necessary and beneficial function for human body. Mg-Li-Al-RE alloy (LAE442) demonstrates superior in vivo corrosion resistance and good biocompatibility 10. The Li concentration in bone slightly increased after implantation. The contents of Li in liver and bone of rabit are 1.4 μg kg-1 and 0.08 mg kg-1, respectively; far below any physiological risk level 29. However, the alloy has higher contents of Al and RE elements. The Al element is reported to be predominantly accumulated in the nervous system and has been implicated in the pathogenesis of Alzheimer’s disease, and the RE elements including Ce, Pr and Y exhibit hepatotoxicity 15. Thus, Al- and RE- free Mg-Li-Ca alloys may be more suitable for degradable biomaterials. The earlier researches of our group show that the Mg-Li-Ca alloy exhibits a higher in vitro corrosion resistance than Mg-Ca alloys due to the formation of a compact corrosion product layer

11, 14

. The

microstructure of the dual phase Mg-9Li-1Ca alloy is characterized by α (-Mg) and β (-Li) phases and intermetallic compound Mg2Ca particles, and the oxide films, composed of Mg- and Li-bearing compounds acting as barrier layers, greatly improve the corrosion performance of the alloy 14. Among the various surface modifications, i.e., the MAO coating, also known as plasma electrolytic oxidation (PEO) coatings, have been widely employed on Mg alloys. MAO/PEO refers to a high-voltage plasma-assisted anodic oxidation process in which plasma discharges cause the partial short-term melting of the oxide layer and promote the formation of a highly adherent ceramic oxide coating. This coating possesses advantages such as high hardness, good wear resistance and moderate corrosion resistance 30. MAO coatings prepared in different solutions, e.g., silicate, phosphate and fluoride electrolytes, exhibit different morphological characteristics, porosities, thicknesses and corrosion rates

31, 32

. MAO coatings

prepared in phytic acid (C6H18O24P6) solution exhibit a better pore uniformity and corrosion resistance than those produced in silicate solution 33, 34. Despite the remarkably advantageous properties of the MAO coating,


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the presence of micro-pores and cracks generated during the micro-arc discharges provides paths for aggressive ions to penetrate into and react with the substrate, thereby accelerating corrosion. Fischerauer et al. 35

investigated the degradation rates of uncoated and MAO-coated ZX50 Mg alloy implants in rat femurs and

found that in the first 4 weeks, the degradation rate for the uncoated Mg alloys is higher than for the MAOcoated ones. However, the reverse trend happens in the subsequent 4 weeks. The result reveals that porous MAO coatings only improve corrosion performance for a limited period 36-38. In other words, it is necessary to seal the pores of the MAO coatings to enhance the corrosion resistance and provide a longer protection. Poly(lactic acid) (PLA), as a biodegradable and biocompatible polymer, has been approved for human clinical uses including small load-bearing bone implants and cardiovascular scaffolds

39-41

. It undergoes a

degradation process by the simple hydrolysis of an ester bond, and the final products are water and carbon dioxide. A significant drawback of PLA is that its degradation generates acidic products that lower the local solution pH, accelerating further degradation and triggering inflammatory and foreign body reactions in vivo 42-44

. Substantial investigations, involving those on the corrosion performance of PLA coatings on Mg and its

alloys, have been carried out. Taking the degradation features of Mg alloys and PLA into consideration, PLA polymeric coatings on Mg alloys have become a potential option to maintain the pH values at a normal level as well as to control the corrosion rate. Abdal-hay et al. 21, 45 found that polymeric membranes of pristine and hydroxyapatite-doped PLA strongly enhance the corrosion resistance and bending strength of the Mg substrate and ameliorates cell growth. Xu et al. 46, 47 prepared poly(L-lactic acid) (PLLA) and poly(ε-caprolactone) (PCL) coatings of both high molecular weight and low molecular weight on pure Mg, revealing that both films significantly improve cytocompatibility and suppress the pH increase of the medium during Mg degradation. Chen et al. 48 reported that PLA and PCL coatings on high-purity Mg (HP-Mg) improve the corrosion resistance. In addition, some attempts have been made to apply organic coatings for sealing the micro-pores and cracks of MAO coatings on Mg alloys. MAO/PLLA composite coatings on WE42, AZ31 and Mg-Li-Ca-Y alloys present excellent cytocompatibility and corrosion resistance 49-52. Nevertheless, the degradation mechanism of the MAO/PLLA film on Mg alloys remains unclear.


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The purpose of this investigation is to fabricate a porous MAO/PLLA composite coating on the novel Mg alloy Mg-1Li-1Ca and to obtain insight into the degradation mechanism and cytocompatibility of the Mg-1Li1Ca alloy with and without the MAO/PLLA coating. 2. Materials and methods 2.1 Samples and coating preparation procedures An as-extruded Mg-1Li-1Ca (1.26 wt. % Li, 0.95 wt. % Ca, and balanced Mg) alloy was chosen as the substrate. The material was cut into square specimens with dimensions of 20 mm × 20 mm × 4 mm for in vitro degradation in Hanks’ balanced salt solution (HBSS, 8 g L-1 NaCl, 0.4 g L-1 KCl, 0.14 g L-1 CaCl2, 0.35 g L-1 NaHCO3, 1 g L-1 glucose, 0.1 g L-1 MgCl2•6H2O, 0.1 g L-1 MgSO4•7H2O, 0.06 g L-1 KH2PO4, and 0.126 g L-1 Na2HPO4•12H2O) with pH=7.4 and 10 mm × 10 mm × 4 mm for cytocompatibility tests. Prior to the coating preparation, the samples were mechanically ground with 400-2500 grit SiC paper and then ultrasonically cleaned in acetone for 2 min, washed with deionized (DI) water and dried in a dry chamber. The pretreated specimens were anodized by self-made MAO equipment, consisting of a power supply, a stainless steel barrel, and a stirring and cooling system. The MAO treatment was carried out for 600 s using the stainless steel barrel as the cathode at a frequency of 100Hz alternating current with a bridge-rectifier waveform, a breakdown voltage of 170-180 V, and a duty cycle of 50 %. The electrolyte, comprising 10g L-1 NaOH and 8g L-1 phytic acid, was continuously stirred during the anodization process. After the preparation of the coating, the samples were rinsed with DI water and dried by warm air. PLLA (MW=200,000, Shandong Institute of Medical Instruments, China) was dissolved in dichloromethane with continuously stirring for a 5 wt. % solution. The MAO-treated substrates were then immersed into the polymeric solution for 60 s, withdrawn and dried in a self-made freeze-drying device. The prepared samples were hung in a pre-cooled airtight container using dryice. The air was then evacuated, and the container was put in a dry chamber at 55 °C for 12 h to allow solvent evaporation as well as to achieve a


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porous film. Prior to the dipping process, all of the specimens were placed in a dry chamber at 120 °C for 10 min to remove the moisture from the porous MAO coating. 2.2 Characterization of coatings and in vitro degradation 2.2.1 Surface analysis The surface and cross-sectional morphologies of the MAO/PLLA coating were investigated using a fieldemission scanning electron microscope (FE-SEM, Nova NanoSEM 450, USA). All samples for the SEM observation were sputtered with gold. Both the coated and bare samples before and after the in vitro degradation tests were examined by a X-ray diffraction meter (XRD, Rigaku D/Max 2500PC, Japan) with a Cu target (=0.154 nm) at a scanning rate of 0.02 s–1 in the 2θ range of 10° ~ 90°. The chemical composition of the coating was inspected through energy-dispersive X-ray spectroscopy (EDS, Oxford Isis) affiliated with electron probe X-ray microanalysis (EPMA, JXA-8230, Japan). 2.2.2 Electrochemical corrosion test The potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) were performed on a potentiostat (PARSTAT 2273) in HBSS. All of the electrochemical tests were conducted in a classical three-electrode system that consists of the sample as the working electrode (1 cm2), a platinum plate as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. The PDP curves were recorded with a sweep rate of 2 mV s-1, and EIS measurements were acquired from 105 to 10–2 Hz using a 5 mV amplitude perturbation. 2.2.3 In vitro immersion tests The substrate, MAO and MAO/PLLA-coated substrates were prepared in triplicate and immersed in 300 ml HBSS. The ratio of the sample surface area to solution volume was 1: 25 cm2 ml-1, and the temperature was controlled at 37  0.2 °C by a water bath. The samples were taken out after immersion for 140 h, with the pH values of the solution and the hydrogen evolution volume were recorded every hour during the immersion. The schematic illustration of the hydrogen evolution test was shown in Figure 1. The hydrogen evolution rate (HER)


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as a function of immersion time can be related through

HER 

VH

st

(1) 2

where VH is the hydrogen evolution volume (mL), s and t are the exposed area (cm ) and immersion time (h), respectively.

The pH value of the solution was measured by a pH meter (PH-400). Immersion period of 140 h was predetermined upon the possible observation on the failure of the MAO/PLA coating, and for the insight into the corrosion mechanism of the MAO/PLA coating.

Figure 1 Schematic illustration of the hydrogen evolution test. 2.3 Cytocompatibility and haemolysis tests 2.3.1 Cell culture and preparation of extracts Murine MC3T3-E1 osteoblast-like cells (Cells Resource Centre of Shanghai Institute for Biological Science, Shanghai, China) were utilized for cell culture studies. Cells were cultured in minimum essential medium alpha (α-MEM, Hyclone) containing 1 % penicillin/streptomycin antibiotics and 10 % foetal bovine serum (Gibco, Australia) at 37 °C in a humidified atmosphere of 5 % CO2. At 78-80 % confluence, the cells


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were enzymatically detached with a minimum amount of trypsin-EDTA (Sigma-Aldrich, USA) and resuspended in culture medium. The cells were then re-cultured until the third passage, which was used for the experiments. The specimens were sterilized by ethylene oxide and then placed in clean centrifuge tubes with added α-MEM at a ratio of 3ml cm-2. After 24 h of incubation at 37 °C, the samples were removed, and the media containing the extracts of different samples were collected for the following tests. 2.3.2 MTT assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the proliferation and viability based on the reductive cleavage of MTT (a yellow salt) to formazan (a dark blue/purple compound) by the mitochondrial dehydrogenase of living cells. The cell suspension was adjusted to a cell density of 1 × 105 mL-1, and then 200 μL was added into each well of 96-well plates, which were sealed by phosphate buffered solution (pH 7.4, PBS, Solibo) in the outer 40 wells. The wells with the cell suspension were divided into 4 groups: the Mg-1Li-1Ca substrate group, MAO group, MAO/PLLA group, control group (culture media containing cell suspension) and blank group (culture media alone), each with 5 wells. After 24 h of incubation, the culture media were removed, together with the leaching liquor of the substrates. The bare substrate, MAO and MAO/PLLA-coated samples were added into the wells of the corresponding groups in volumes of 100 μL; while equivalent volumes of culture media were supplied to the control group and blank group. The culture medium of each well was changed every other day during the testing period, and 20 μL MTT solution was injected on each of days 1, 3 and 5. The 96-well plates were kept in the incubator for 4 h. After incubation, the medium was removed, and the formazan crystals were solubilized in DMSO. The absorbance (A) was measured by a universal microplate spectrophotometer (722, Shanghai Precision Science Instrument, China) at a wavelength of 490 nm, and the relative growth rate (RGB %) of the cells was calculated by the relation below

RGB(%) 

At  100% Ac

(2)


8

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where At is the absorbance of the testing specimen and Ac is the absorbance of the control groups. The results are expressed as the mean ± standard deviation of five samples per group and were compared using Student’s t-test, with a significance level of P < 0.05. 2.3.3 Alkaline phosphatase (ALP) assay An alkaline phosphatase colorimetric assay kit (Nanjing Jiancheng, China) was used to evaluate the osteoblast differentiation. The cell suspension was adjusted to a cell density of 1 × 105 mL-1, and 2 mL of the suspension was added into the first four wells of a 6-well plate. After being seeded for 24 h, the culture media were removed and rinsed twice by PBS. Then, α-MEM and the extracts of the bare substrates, the MAO-coated samples, and the MAO/PLLA-coated samples were added to each well in sequence, and the plate was incubated under standard cell culture conditions. The culture media were discarded, and the wells were rinsed twice by PBS after 5 and 7 days of incubation. Then, 500 μL of 1.5 % Triton X-100 solution was added into each well, and the plate was placed into a refrigerator for 10 h at 4 °C. The four groups of the Mg-1Li-1Ca substrates, the MAO and MAO/PLLA coated samples, and the control were settled with 5 wells. The corresponding supernatants were supplemented by the volume of 50 μL per well in a 96-well plate. The addition of reagents was following the instructions of the ALP kit, and the absorbance was measured using a universal microplate spectrophotometer at a wavelength of 520 nm and converted into the amount of ALP (U L-1). 2.3.4 Cellular adhesion and morphology imaging A 1.5 mL suspension was seeded onto the substrates, the MAO and MAO/PLLA-coated samples after sterilization by ethylene oxide at a cell density of 5 × 104 cells per well in 24-well plates. After 24 h of culturing in a cell incubator, the specimens were taken out and immobilized by 4 % glutaraldehyde for 12 h and then dehydrated with ethanol step by step. Lyophilization was applied in the dehydration to maintain the structure and morphology of the cells. Following the drying process, the samples were coated with platinum, and the cell morphology was observed using FE-SEM. 2.3.5 Haemolysis test


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The haemolysis test in vitro was conducted as follows: 10 mL fresh rabbit arterial blood from a healthy New Zealand white rabbit, containing 0.5 mL potassium oxalate (20g L-1) anticoagulant was added into the culture media with different extracts prepared in the previous step, kept at 37 °C for 60 min, and then centrifuged at 3000 rpm for 5 min. Three sets of parallel tests were settled, and the A of the supernatant solution was measured using the spectrophotometer at a wavelength of 545 nm. The haemolytic ratio (HR) was obtained by the following equation

HR(%) 

At  Anc  100% Apc  Anc

(3)

where At is the A of the testing specimen; Anc is the A of the negative control group (10 mL culture medium with 0.2 mL diluted blood), and Apc is the A of the positive control group (10 mL distilled water with 0.2 mL diluted blood). 3 Results and discussion 3.1 Microstructure of the alloy and morphologies of the coatings The microstructure of the Mg-1Li-1Ca alloy after etching was characterized by the α-Mg phase and the intermetallic compounds Mg2Ca dispersed in the grain interiors and at the grain boundaries (Figure 2), in which the Li atoms are solid-soluted. The Mg2Ca phase, with a higher corrosion potential than the α-Mg matrix, may lead to micro-galvanic corrosion between the Mg2Ca phase and its neighbouring α-Mg matrix 14. The typical morphology of the MAO coating in Figures 3a and c demonstrates the presence of micro-pores and micro-cracks. Interestingly, a porous PLA film was also prepared on the MAO coating, as depicted in Figures 3b and d. The EDS result of the MAO coating, as shown in the inset of Figure 3a, demonstrates that the MAO coating is mainly composed of Mg, O, and traces of Ca and P, indicating the presence of MgO and calcium phosphates 51. It is likely that the Ca and P of the MAO coating are from the substrate and electrolytes used in the formation of the coating, respectively, while the presence of C and O designates the MAO/PLLA coating, as shown in the inset of Figure 3b.


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Figure 2 Optical morphology of the microstructure of the Mg-1Li-1Ca alloy.

Figure 3 SEM morphologies of (a) the MAO coatings; (b) the MAO/PLLA composite coatings formed on the Mg-1Li-1Ca alloys, (c) and (d) the high magnification of (a) and (b), respectively. The insets in (a) and (b) correspond to the EDS spectra of the MAO and MAO/PLLA coatings, respectively.


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Figure 4 shows the cross-sectional SEM micrographs and elemental mapping of the substrates with MAO/PLLA films. The MAO and the PLLA coatings have thicknesses of approximately 3 µm and 10 µm, respectively. As illustrated in Figure 4b, the intensity of C decreased when the EDS scan line approached the PLLA/MAO interface, comparing with the reverse trend for Mg and O. This is due to the different chemical compositions of each layer, which are consistent with the EDS spectrum in the insets of Figure 3. Note that in Figure 3a the MAO coating contains traces of Ca and P elements, which originate from the Mg-1Li-1Ca alloy and the solution with phytic acid, respectively. Thus, the line scan of both Ca and P can be ignored; and the concentration profiles of the C, O and Mg elements from the substrate to resin can be evident. Nevertheless, it can be recognized that C diffused into the MAO layer, indicating that the PLLA sealed the porous film and the PLLA coating and MAO coating interlocked.

Figure 4 (a) SEM cross-sectional morphology, and (b) the EDS line scan mapping across MAO/PLLA coating. 3.2 Hydrogen evolution rates and pH measurements


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The hydrogen evolution rate (HER) vs. immersion time curves of the coated and uncoated samples are shown in Figure 5. In the initial stage of immersion, the HER of the substrates is much higher than that of the coated samples, and some bubbles can be seen on the surfaces of the substrates, while no bubbles were observed on the coated ones. However, the HER of the MAO-coated specimens began to rapidly increase at an immersion of 50 h and finally became slightly higher than that of the substrates after 85 h of immersion, compared with the constant HER of the MAO/PLLA-coated samples. It is noted that simialr degradation behaviour occurs on the MAO coating on Mg-1.21Li-1.12Ca-1.0Y alloy based on our previous study 51. Namely, the HERs of the MAO coating on Mg-1.21Li-1.12Ca-1.0Y alloy (0.142 mL·cm-2·h–1) surpasses that of the substrate after an immersion time of 9 h. Interestingly, the HERs of the MAO coating on the Mg-1Li-1Ca alloy (0.004 mL·cm-2·h–1) is much lower than that on the Mg-1.21Li1.12Ca-1.0Y alloy, and it is until 85 h that the HERs of the MAO coating surpasses that of the substrate, although the MAO thickness (about 3 μm) on the Mg-1Li-1Ca alloy is much lower than that (approximately 10 μm) on the former alloy 51. The result designates that the MAO coating on the Mg-1Li-1Ca alloy possesses superior corrosion resistance to that on the Mg-1.21Li-1.12Ca-1.0Y alloy, that is, the thickness of MAO is not a critical factor influcencing the corrosion resistance. This abnormal scenario may be attributable to the different chemicals used in the prepared solutions. The MAO coatings were prepared in a silicate electrolyte containing 20 g/L NaOH, 20 g/L Na2SiO3, 15g/L NaB4O7 and 10 g/L C6H5Na3O7 together with 7.5 g/L phytic acid for the Mg-1.21Li-1.12Ca-1.0Y alloy, and in an electrolyte comprising 10g L-1 NaOH and 8g L-1 phytic acid for the Mg-1Li-1Ca alloy. As a result, a thinner and more compact MAO coating formed on the Mg-1Li1Ca alloy (Fig. 4a and c). After 140 h of immersion, the average HER of the substrate, the MAO and MAO/PLLA-coated samples were 2.12 mL·cm-2·h–1, 1.58 mL·cm–2·h–1, and 0.56 mL·cm–2·h–1, respectively. The HER indicate that the MAO/PLLA coating greatly enhanced the corrosion resistance of the Mg-1Li-1Ca alloy and also reveal that the disadvantage of the MAO coatings in the corrosion process that could be improved by PLLA modification are consistent with Fischerauer’s research 35.


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Figure 5 HER of the substrate, MAO coating and MAO/PLLA coating immersed in HBSS for 140 h. Figure 6 designates the changes in the pH value of the solutions with the immersion time. The process can be divided into four stages. In stage one, there was a significant increase in the solution pH for each sample, resulting from the corrosion of the Mg alloys. For the bare substrate and the MAO-coated samples, this was followed by a decrease that led to a period of stable pH values, which can be defined as stage two. During this stage, stable oxide films have been formed, so the reaction slowed down after the initial 10 h of immersion. However, it is noteworthy that H2O and Cl– can diffuse into the PLLA coating and react with the MAO coating and substrate before the hydrolysis degradation of the polymer films in the first 10 h of immersion, which can explain the slight increased state of the pH value of the MAO/PLLA samples. Interestingly, for the PLLA coating, the solution pH decreased to the lowest value of 7.3 after 82 h of immersion, followed by a slow continuous increase, which can be defined as stage three. This can be the result of the chemical corrosion of the MAO coating and the electrochemical corrosion of the substrate, which released alkaline corrosion products into the solution after the delamination and collapse of the PLLA coating. After 110 h of immersion, the pH value of the MAO samples surpassed that of the bare substrates, which exhibited a similar trend to the HER. However, the average pH value of the MAO/PLLA-coated sample is lower than that of the bare substrates and the MAO-coated samples during the immersion. This result is probably attributable to the degradation of PLLA, which neutralizes the hydroxyls released during the corrosion of Mg with the lactic acid produced after the hydrolysis of the ester bond. Fortunately, our previous study, regarding the corrosion of MAO/PLLA on Mg-Li-Ca-Y alloy, has confirmed the acidicity of PLLA coating; a linear decrease in solution


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pH over immersion time for the PLLA coating on the PTFE plate discloses the formation of acidic products during its degradation 51.

Figure 6 Changes in the pH value of HBSS with immersion for the coated and uncoated samples. 3.3 Electrochemical behaviour PDP curves obtained for the coated and uncoated samples in HBSS with and without immersion are shown in Figure 7, showing that the structures and properties of the surfaces changed during the immersion time. The free corrosion potential (Ecorr) and corrosion current density (Icorr) were derived from these data by the Tafel extrapolation method and are summarized in Table 1. It can be seen that the Ecorr of the samples without the immersion follows this consequence: MAO/PLLA > MAO > bare substrate. This indicates that the coated samples exhibited a lower thermodynamic tendency towards electrochemical corrosion, the MAO/PLLA coating showed the lowest tendency to participate in the anodic reaction, and the Icorr of the MAO/PLLA-coated sample is about two orders of magnitude lower than that of the bare substrate, which indicates that the corrosion resistance is effectively improved. After immersion in HBSS for 140 h, the Ecorr decreases in the order of MAO/PLLA > substrate > MAO, so that the MAO-coated sample exhibits a stabilized thermodynamic stability during the immersion time. Compared with the substrate, an increase in Icorr for the MAO coating is in pronounced agreement with the results from HER (Figure 5) and the reverse change in solution pH (Figure 6), implying that the MAO coating suffered from severe attack from the solution and the delamination of the MAO coating. Similar scenario is also discerned on the degradation of the MAO/PLLA


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coating on the Mg-Li-Ca-Y alloy 51. The Ecorr and Icorr of the bare substrate were, however, enhanced and lowered, respectively, which is probably related to the protection of the compact corrosion product layer. The MAO/PLLA-coated sample retains significant corrosion resistance, which is consistent with the HERs. The PDP curve of the MAO/PLLA coating, neverthelss, show no significant difference among the three samples after immersion in HBSS for 140 h, which are ascribed to the peeling-off of the MAO coating and the swelling of the PLLA coating after a long period of immersion 53.

Figure 7 PDP curves obtained for the substrate, MAO coating and MAO/PLLA coating with and without immersion of 140 h. Table 1 Parameters of the PDP curves of the samples with and without immersion of 140 h. Sample

Immersion time (h)

Ecorr (V vs. SCE)

Icorr (A cm-2)

Substrate–0

0

–1.66

2.48×10–5

MAO–0

0

–1.50

1.60×10–6

MAO/PLLA–0

0

–1.46

2.96×10–7

Substrate–140

140

–1.61

7.21×10–6

MAO–140

140

–1.62

1.03×10–5

MAO/PLLA–140

140

–1.60

4.19×10–6


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Figure 8a shows Nyquist plots of the uncoated and coated samples with and without immersion in HBSS for 140 h. All of the Nyquist plots were fitted with the corresponding equivalent circuits, which are also designated in Figure 8 (b-g). As shown in the Nyquist plots, the MAO/PLLA-coated samples exhibited the best corrosion resistance, and the impedance of the MAO-coated and immersion treated MAO/PLLA-coated samples decreased, while that of the bare substrate increased after being immersed for 140 h. The experimental data were processed using ZSimpWin 3.4, and the chi-squares of the fitting results are within a permissible range (Table 2).


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Figure 8 (a) Nyquist curves for the coated and uncoated samples; (b-g) fitted curves and equivalent circuits for the (b) substrate, (d) MAO coating and (f) MAO/PLLA coating without immersion; (c) substrate, (e) MAO coating and (g) MAO/PLLA coating with immersion of 140 h.


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Table 2 Electrochemical data obtained by equivalent circuit fitting of EIS curves of uncoated and coated samples with and without immersion for 140 h

Rs

Rt

Rp

Rf

Cdl

Cf

Samples 2

2

2

2

-2

-2

Ω·cm

Ω·cm

Ω·cm

Ω·cm

F·cm

F·cm

70.2

384.5

-

908.9

-

127.3

2.3×104

1174

6248

95.4

1.2×104

1.9×104

179.9

2722

142.3

184.7

CPE1

CPE2

CPE3

Ω-

Ω-

Ω-

W RL

L 2

Chi Ω-

2

Ω·cm

H·cm

2.6×10-5

122

586.4

-

2.0×10-4

-

-

-

-

-

2.6×10-4

3.7×10-7

-

-

-

-

-

4.3×10-4

-

3.5×10-7

-

-

-

-

3.7×10-4

3.8×10-4

4.4×10-6

-

6.1×10-5

-

-

3139

2.6×104

-

5.5×10-4

-

2.2×10-9

9.8×10-6

1.6×10-5

-

3.2×104

1.0×105

-

2.0×10-4

1

·Sn·cm-2

1

·Sn·cm-2

1

-

1.5×10-8

1.7×10-3

5.4×10-7

2.9×10-8

1.5×10-6

7.5×104

1.3×10-5

3.6×10-10

4810

-

1.1×10-4

670.6

1556

-

2.3×104

334

2895

Substrate-0

·Sn·cm-2

1

·S0.5·cm-2

-squared

h MAO-0 h MAO/PLLA -0 h Substrate140 h MAO-140 h MAO/PLLA -140 h


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Figures 8b, d and f disclose the Nyquist plots and corresponding equivalent circuits before immersion. It can be seen that the Nyquist plots consist of two capacitive loops at high and middle frequencies, followed by an inductive loop in the low-frequency range. The capacitive loop in the high-frequency range corresponds to the charge transfer resistance (Rt) and interface diffusion constant phrase element (CPE2) in Figure 8b and the interface double-layer capacity (Cdl) in Figures 8d and f. The latter represents constant phase elements, defined as ZCPE=[Y0(jω)n]−1

(4)

Y0 is the constant of the CPE, jω is the complex variable for sinusoidal perturbations with ω=2πf, and n is the exponent of CPE with values between 0 and 1. The CPE acts as a pure resistor when n equals 0 and as an ideal capacitor when n equals 1. The value of n is associated with the non-uniform distribution of current as a result of both the degree of roughness of the surface as well as the degree of the oxide's heterogeneity. The middle capacitive loop is attributed to the film resistance (Rf) and the film capacitance (CPE1) of the electrolyte penetrating through the corrosion product layer on the surface of the substrate. The increased Rf from 908.9 Ω·cm2 to 6248 Ω·cm2 and 75490 Ω·cm2 confirms that the MAO/PLLA-coated sample retains good corrosion resistance. The inductive loop, corresponding to an inductor L and a resistor RL at the low frequency, is ascribed to the absorption and peeling of the corrosion products such as Mg(OH)2 54. Figures 8d and f show similar Nyquist plots, which can be fitted with one equivalent circuit, exhibiting the multiple-layer structure of both coatings. The circuit contains the Rt, Cdl, Rf, and the polarization resistance (Rp), the outer porous layer capacitance (Cf), and the inner barrier layer capacitance (CPE1). Nyquist plots and equivalent circuits of the bare substrate and coated samples with 140 h of immersion in HBSS are shown in Figures 8c, e and g, respectively. The Nyquist plot of the substrates demonstrates a capacitive loop at a high frequency in Figure 8c, representing the charge transfer process, and an oblique line in a middle low frequency range, corresponding to the corrosion reaction being controlled by the diffusion process 55. Therefore, as depicted in Figure 8c, the equivalent circuit of the substrate after immersion is similar to that of the substrate without immersion, with replacement of the inductor with a Warburg resistance. The reason for this change is that as the immersion time is prolonged, the produced corrosion products cover the


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surface of the substrate, leading to the formation of a compact corrosion product layer that isolates the solution from the substrates. Therefore, the diffusion process plays an important role in parallel with the absorption and delamination of the corrosion products without immersion, which can be equivalent to an inductor. As for the Nyquist plots in Figures 8e and g, an inductor L and a resistor RL simultaneously appear at the low frequencies due to the formation of corrosion pits on the coatings. However, the MAO/PLLA coating maintained its integrity, whereas serious corrosion occurred on the MAO coatings due to a hydration reaction and the dissolution of Mg, resulting in localized destruction and the loss of the protective layer. 3.4 Cytocompatibility and haemolysis tests Table 3 shows the result of the haemolysis test of the bare substrate, MAO and MAO/PLLA coated samples. Compared with the bare substrate, the HR of the MAO coating was lowered by up to 12.48 %, indicating that it may lead to severe haemolysis when contacted with the blood. The MAO/PLLA coating exhibited a HR of 0.17 %, which is much lower than 5 % (a judging criterion for excellent blood compatibility), indicating the excellent blood compatibility of the composite coatings. Table 3 HR (%) of the Mg-Li-Ca substrate, MAO, and MAO/PLLA (n=5). Samples

HR (%)

Substrate

61.35 ±0.36

MAO

12.48 ±0.18

MAO/PLLA

0.17 ±0.04

The cytotoxicity of the MAO and MAO/PLLA-coated samples was determined using MC3T3-E1 cells and the MTT assay (Figure 9). The relative growth ratio (RGR) of the three groups is less than 100 % after 24 h of culture, while the RGR of the MAO/PLLA coating slightly exceeds 100 % after a culture of 72 h. MC3T3-E1 cells in the extract media of the MAO/PLLA coating showed an obviously higher proliferation rate and vitality than those on the Mg-1Li-1Ca substrates and the MAO coating. It is noteworthy that the lowest


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cytotoxicity of the MAO-coated samples may be ascribed to the influence of the electrolytes, which persisted in the micropores after MAO process. It is also interesting to note that the RGR of the Mg-1Li-1Ca substrates is close to that of the composite coatings due to the protective film generated during the extraction process, which is consistent with the results of the ions released during immersion, such as Mg2+, Ca2+ and Li+, which are of small volume less than the tolerated contents of osteoblasts 28.

Figure 9 RGR of MC3T3-E1 cells cultured for different times in extract media from the substrates, MAO and MAO/PLLA coated samples compared to in α-MEM culture medium. Error bars represent ± S for n=5 and P < 0.05, as indicated by the asterisk (*). ALP activity is one of the most widely used markers for early osteoblastic differentiation 56, 57. ALP is a ubiquitous enzyme that catalyses the hydrolysis of phosphate esters at alkaline pH. The differentiation of MC3T3 cells on differently treated samples was evaluated in up to 7 days of culture using ALP as an early phase marker. Figure 10 shows the ALP activity of the osteoblast culture in the extract media of the Mg-1Li1Ca substrates, MAO and MAO/PLLA-coated samples, as well as pure culture media as a control. The osteoblasts cultured in the extracts of the MAO/PLLA coatings displayed a highest ALP expression among the samples, which indicates a stronger ability for cell differentiation 56, 57. The ALP expression of the substrates and the MAO coatings shows a parallel trend to the MTT assay.


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Figure 10 Proliferation and differentiation of MC3T3-E1 cells cultured for different times in extracts from the substrates, MAO and MAO/PLLA-coated samples as well as α-MEM culture medium. Error bars represent ± S for n=3 and P < 0.05, as indicated by the asterisk (*). The morphologies of MC3T3-E1 cells cultured on the coated and uncoated samples for 24 h are presented in Figure 11. After 24 h of the culture, it is clear that the cells cultured on the MAO/PLLA coatings exhibited good adhesion, which can be identified by the number of spreading cells as well as the spreading shape and areas. Additionally, the cells displayed numerous filopodia extensions (Figures 11 e-f). It is especially noteworthy that the edges of the pores on the MAO/PLLA coatings can be easily attached to by the filopodia, which strongly enhances the cell adhesion. Although the cells cultured on the substrates and the MAO coatings also adhered to the surface and filopodia protrusions were also observed in Figures 11 a-d, their growth seemed to be inhibited.


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Figure 11 SEM morphologies of MC3T3-E1 cells cultured on (a-b) the Mg-Li-Ca alloys, (c-d) the MAO coating and (e-f) the MAO/PLLA coating for 24 h. 4. Discussion The surface appearances of the coated and uncoated samples after immersion in HBSS for 140 h are shown in Figure 12. A compact corrosion layer with micro cracks is formed on the surface of the substrate (Figure 12a). Corrosion pits on the MAO coating destroyed its integrity (Figure 12b). However, the surface of the MAO/PLLA coating showed no obvious variation on most of its surface (Figure 12c) with few corrosion


24

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pits covered by white corrosion products. With the integrity and mechanical property of the duplex coating being undermined during the immersion by the diffusion of H2O, solution ions and the release of H2, the corrosion products centralized under the polymeric PLLA film. Then, the delamination and collapse of the PLLA coating occurred, which was demonstrated by the distinct shearing boundary on the MAO/PLLA surface (Figure 12d). The EDS spectra of each sample after immersion in HBSS for 140 h is also demonstrated by the insets in Figure 12, revealing that the MAO/PLLA coating had similar elemental compositions on the substrate and the MAO coating, mainly containing O, Mg, P and Ca (Figures 12a, b).

Figure 12 SEM morphologies and EDS element analysis of surface of the samples after 140 h of immersion: (a) the substrate; (b) the MAO coatings; (c, d) the MAO/PLLA coatings. Interestingly, the centralization of corrosion products around the corrosion pit in Figure 12d was destroyed by the stream of electrons released during the immersion, exposing the corrosion pit to the microscope. An opening pore can be clearly observed in Figure 13, and C, O, Mg, Ca and P appear to be


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uniformly distributed in the corrosion products. It is noteworthy that around the corrosion pit, the concentrations of Mg and O are much higher than those of Ca and P, and the distribution of Ca corresponds to that of P. The elemental distribution resulted from the formation of different corrosion products, including Mg(OH)2, which centralized around the corrosion pit, and hydroxyapatite (HA) 16.

Figure 13 SEM morphology of a corrosion pit on the MAO/PLLA surface after 140 h of immersion: (a) SEM micrographs; the elemental mappings of EDS: (b) C, (c) O, (d) Mg, (e) Ca and (f) P. Figure 14 shows the cross-sectional SEM morphology and EDS elemental mapping of the MAO/PLLA coating after immersion. The composite film remained integrated and compact after immersed in HBSS for 140 h, with a small quantity of corrosion pits beneath the polymeric coating, that were produced by the diffusion of H2O and Cl- through the PLLA and MAO coating into the substrate and the dissolution of Mg, thus resulting in the blistering of the PLLA coating due to the hydrogen evolution. As shown in the EDS elemental mapping, C, O and Mg can be detected on the cross-section of the corrosion pit, suggesting the formation of the hydrolysates of the PLLA coating and the Mg(OH)2 corrosion products of the alloy.


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Figure 14 Cross-sectional morphology of MAO/PLLA coating after 140 h of immersion in HBSS: (a, b) crosssectional morphologies; EMPA image of cross-section of blister: (c) elemental mappings of EDS: (d) C, (e) O, and (f) Mg. Figure 15 shows the XRD patterns of the coated and uncoated samples with and without immersion for 140 h. The presence of MgO indicates that the ceramic coatings are formed during MAO treatment in


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electrolytes containing phytic and NaOH, which is different from the coatings formed in silicate electrolytes 27. Interestingly, the presence of calcium lactate ((C3H5O3)2Ca) can be detected on the MAO/PLLA coating samples after immersion in HBSS, demonstrating the reactions between chemical species such as Ca2+ from the corrosion medium and the dissolution of the Mg-1Li-1Ca alloy, and oligomers or monomers such as lactide and lactic acid released by the degradation of the PLLA film.

Figure 15 XRD patterns of the substrate, MAO coatings and MAO/PLLA coatings before and after immersion for 140 h. The cytotoxicity and biocompatibility of clinical materials are also greatly influenced by the corrosion. During the corrosion process, local alkalization and enrichment in Mg2+ exert a significant impact on the physiological balance. It is believed that Mg-based materials degrade too fast, and the high concentration of Mg2+ in the solution is responsible for the high haemolysis rate 58. Although the MAO coatings reduced the HR to 12.85 %, which is much lower than that of the Mg-1Li-1Ca substrates (61.35 %), it is not suitable for utilization as blood-contacting materials for the potential danger to erythrocytes. After being sealed with PLLA, MAO/PLLA coatings, with a HR of 0.17 %, would have little influence on the blood. It is well known that cells are very sensitive to changes in the micro-surrounding environment, such as the sharp changes in the pH value and Mg2+ concentration. The sharp increase in the pH value and concentration of Mg2+ released by the degradation of the substrates and the MAO coatings may hinder cell growth (Figures 9-11). As a biodegradable material, PLLA not only works as a physical barrier layer but also provides a


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suitable condition for cell adhesion and proliferation on the Mg-1Li-1Ca alloys (Figures 11 e-f). Combining MAO with PLLA can take advantage of the superiority of the two coatings on biodegradable Mg alloys. As is well known, PLA-based materials belong to the family of aliphatic polyesters, so their ester groups are hydrolytically degraded in the presence of water according to the following reaction 59: –COO– + H2O → –COOH+ OH–

(5)

Thus, PLA-based materials degrade via a hydrolytic autocatalytic degradation process at the chain end and in the middle of the chain, which are called exo-chain cleavage and endo-cleavage, respectively. However, the cleavage mechanism of PLA dissolved in solution depends on the media pH such that the hydrolytic degradation proceeds with a chain-end scission mechanism in lactyl monomer unit, forming lactic acid in acidic solution, whereas in alkaline solution, hydrolytic degradation takes place via backbiting to form a lactide unit, which is further hydrolysed to give lactic acid 60. Degradation of the PLA coating in HBSS at 37 °C and pH 7.4 is similar to in vivo degradation. The degradation in neutral solution seems to be selective and often occurs in amorphous regions, producing hydrophilic groups (-OH and -COOH). The catalytic group (-COOH) is subsequently condensed in the amorphous region between crystalline regions, resulting in the acceleration of the hydrolytic degradation in a restricted area 61. The degradation mechanism of PLA-based materials include three models

62

: a surface corrosion

mechanism, bulk corrosion mechanism and core-accelerated bulk corrosion mechanism, with a critical thickness (Lcritical) of polymeric films that can be used as a criterion index to evaluate which corrosion takes place. With the decrease of the thickness of the polymer films, the hydrolytic degradation rate of the material surface slows down, compared with the diffusion rate of water molecules or catalytic substances within the material, and the mechanism model turns from surface corrosion to bulk corrosion. The PLLA coating in this experiment has a thickness (approximately 10 μm) that is lower than Lcritical of 0.5-2 mm. Therefore, the bulk corrosion occurred during the immersion test, which can be divided into three stages 63: (1) initial water diffusion, absorption and hydration of the PLLA coatings, (2) gradual decrease in molecular weight, and (3) formation and dissolution of water-soluble oligomers and monomers. Accompanied with a decrease in the


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molecular weight and crystallinity, the thickness of the films decreased, causing a release of formed oligomers and monomers to the solution, resulting in a reduced autocatalytic effect and a lower degradation rate. Furthermore, the MAO film can significantly influence the degradation behaviour of the polymer film. As is well known, Mg is highly susceptible to galvanic corrosion 64, leading to the formation of Mg(OH)2 during the chemical reactions, and the presence of Cl− can dissolve Mg(OH)2, leading to the release of OH− to the corrosion media. These reactions follow as 65 Mg → Mg2+ + 2e–

(6)

2H2O + 2e– → 2OH– + H2↑

(7)

Mg + 2H2O → Mg(OH)2 + H2↑

(8)

Mg(OH)2 + 2Cl− → MgCl2 + 2OH−

(9)

Therefore, the degradation of Mg eventually results in a locally alkaline environment, which can result in the consumption of the acidic products released by the degradation of the PLLA coatings, accelerating and promoting the hydrolytic degradation of the polymers as well as the loss of the Mg(OH)2 layers. Our earlier study 14 demonstrates that the microstructure of Mg-1Li-1Ca is composed of an α-Mg phase and intermetallic compound Mg2Ca particles, which result in an initial corrosion around the Mg2Ca particles, generating hydrogen bubbles and Mg(OH)2. The corrosion products gradually concentrate on the interface of the MAO coatings and substrate during the reaction and destroy the MgO layer as MgO + H2O → Mg(OH)2

(10)

Simultaneously, the H2 liberated also increases the internal stress under the MAO coatings and PLLA coatings and undermines the mechanical properties of the coatings, leading to the generation of peeled off pores and cracks on the PLLA films and finally disintegration into fragments (Figure 12) 65, 66. The detachment of the PLLA coating may be detrimental to application as stent materials, but the composite coating still shows great potential in bone regeneration applications. Above all, the MAO/PLLA composite coating degraded predominately at the inner layer and exhibited features of localized corrosion and pitting corrosion, resulting in the concentration of corrosion products inside and on the surface of coatings, finally leading to localized


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destruction and collapse. Thus, a degradation mechanism is schematically illustrated in Figure 16a and Figure 16b, indicating that the degradation of PLLA on the Mg-1Li-1Ca alloy experienced four steps: (1) water diffusing through the PLLA film onto the MAO coating and the substrate due to the diffusion rate of water being faster than the degradation of the PLLA film, (2) chemical corrosion of the MAO coating (Equation (9)), (3) electrochemical corrosion of the substrate (Equation (5)), and (4) the swelling, hydrolysis (Equation (4)) and rupture of the PLLA film.

Figure 16 Schematic illustrations of degradation mechanism of the porous MAO/PLLA composite coatings on the Mg-1Li-1Ca alloys in HBSS: (a) the swelling of PLLA and corrosion of the substrate at the initial stage, (b) the blistering and final peeling-off of PLLA under the pressure of hydrogen gas and corrosion products.


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5 Conclusions Porous MAO/PLLA composite coatings were fabricated by dip-coating and freeze-drying to improve the corrosion resistance of Mg-1Li-1Ca alloys, and the corrosion behaviours of the coatings in HBSS were investigated by SEM, EPMA, XRD and corrosion measurements. The conclusions are as follows: (1) The MAO/PLLA coatings significantly enhanced the corrosion resistance, increasing the Ecorr from -1.66 V to -1.44 V and decreasing the Icorr by approximately two orders of magnitude. (2) The corrosion predominantly occurred at the interface of the MAO coating and the substrate, and the accumulation of corrosion products as well as hydrogen bubbles caused swelling and blistering of the PLLA coating due to the faster penetration rate of water into the PLLA coating than its degradation rate. (3) The acidic and alkaline products, respectively resulting from the degradation of PLLA and the corrosion of Mg, neutralized each other to maintain a stable and appropriate pH value in HBSS. (4) The MAO/PLLA coating on Mg alloys greatly enhances its cytocompatibility and blood compatibility, and provides an appropriate solution pH and porous micro-structure, which is helpful for the attachment of cells. The MAO/PLLA coating may be a promising approach for bone tissue regeneration engineering. AUTHOR INFORMATION Corresponding Author * E-mail address: [email protected] (R. C. Zeng); [email protected] (B. D. Zhao). Tel. +86-532-80681226; fax: +86-532-86057920; ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (51571134), SDUST (Shandong University of Science and Technology) Research Fund (2014TDJH104), Joint Innovative Centre for Safe and Effective Mining Technology and Equipment of Coal Resources, Shandong Province. REFERENCES 1. Zheng, Y. F.; Gu, X. N.; Witte, F. Biodegradable Metals. Mater. Sci. Eng. R 2014, 77, 1-34.


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2. Witte, F. The History of Biodegradable Magnesium Implants: A Review. Acta Biomater. 2010, 6, 16801692. 3. Zeng, R. C.; Dietzel, W.; Witte, F.; Hort, N.; Blawert, C. Progress and Challenge for Magnesium Alloys as Biomaterials. Adv. Eng. Mater. 2008, 10, B3-B14. 4. Staiger, M. P.; Pietak, A. M.; Huadmai, J.; Dias, G. Magnesium and Its Alloys as Orthopedic Biomaterials: A Review. Biomaterials 2006, 27, 1728-1734. 5. Baino, F. Biomaterials and Implants for Orbital Floor Repair. Acta Biomater. 2011, 7, 3248-3266. 6. Witte, F.; Feyerabend, F.; Maier, P.; Fischer, J.; Störmer, M.; Blawert, C. Biodegradable Magnesiumhydroxyapatite Metal Matrix Composites. Biomaterials 2007, 28, 2163-2174. 7. Witte, F.; Hort, N.; Vogt, C.; Cohen, S.; Kainer, K. U.; Willumeit, R. Degradable Biomaterials Based on Magnesium Corrosion. Curr. Opin. Solid State Mater. Sci. 2008, 12, 63-72. 8. Witte, F.; Ulrich, H.; Rudert, M.; Willbold, E. Biodegradable Magnesium Scaffolds: Part 1: Appropriate Inflammatory Response. J. Biomed. Mater. Res. Part A 2007, 81A, 748-756. 9. Gu, X. N.; Zheng, Y. F.; Zhong, S. P.; Xi, T. F.; Wang, J. Q.; Wang, W. H. Biocorrosion and Cytotoxicity Evolution of Mg-Zn-Ca Bulk Metallic Glass. Biomaterials 2010, 39, 1093-1103. 10. Witte, F.; Kaese, V.; Haferkamp, H.; Switzer, E.; Meyer-Lindenberg, A.; Wirth, C. J. In Vivo Corrosion of Four Magnesium Alloys and the Associated Bone Response. Biomaterials 2005, 26, 3557-3363. 11. Zeng, R. C.; Guo, X.; Liu, C.; Cui, H. Z.; Tao, W.; Liu, Y.; Li, B. Study on Corrosion of Medical Mg-Ca and Mg-Li-Ca Alloys. Acta Metall. Sin. 2011, 47, 1477-1482. 12. Zeng, R. C.; Wang, L. D.; Zhang, F.; Cui, H. Z.; Han, E. H. In Vitro Corrosion of Mg-6Zn-1Mn-4Sn1.5Nd/0.5Y Alloys. Front. Mater. Sci. 2014, 8, 230-243.


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13. Gu, X.; Zheng, Y.; Cheng, Y.; Zhong, S.; Xi, T. In Vitro Corrosion and Biocompatibility of Binary Magnesium Alloys. Biomaterials 2009, 30, 484-498. 14. Zeng, R. C.; Sun, L.; Zheng, Y. F.; Cui, H. Z.; Han, E. H. Corrosion and Characterisation of Dual Phase Mg-Li-Ca alloy in Hank’s Solution: The influence of Microstructural Features. Corros. Sci. 2014, 79, 6982. 15. Gu, X. N.; Li, S. S.; Li, X. M.; Fan, Y. B. Magnesium Based Degradable Biomaterials: A Review. Front. Mater. Sci. 2014, 8, 200-218. 16. Zeng, R. C.; Lan, Z. D.; Kong, L.; Huang, Y.; Cui, H. Z. Characterization of Calcium-modified Zinc Phosphate Conversion Coatings and Their Influences on Corrosion Resistance of AZ31 Alloy. Surf. Coat. Technol. 2011, 205, 3347-3355. 17 Zeng, R. C.; Zhang, F.; Lan, Z. D.; Cui, H. Z.; Han, E. H. Corrosion Resistance of Calcium-modified Zinc Phosphate Conversion Coatings on Magnesium-aluminium Alloys. Corros. Sci. 2014, 88, 452-459. 18. Gu, X. N.; Li, N.; Zhou, W. R.; Zheng, Y. F.; Zhao, X.; Cai, Q. Z. Corrosion Resistance and Surface Biocompatibility of a Microarc Oxidation Coating on a Mg-Ca Alloy. Acta Biomater. 2011, 7, 1880-1889. 19. Liang, J.; Zhang, R. H.; Peng, Z. J.; Liu, B. X. One-step Electrochemical Fabrication of Bilayered MgO/Polymer Coating on Magnesium Alloy. Front. Mater. Sci. 2014, 8, 307-312. 20. Badar, M.; Lünsdorf, H.; Evertz, F.; Rahim, M. I.; Glasmacher, B.; Hauser, H. The Formation of An Organic Coat and the Release of Corrosion Microparticles from Metallic Magnesium Implants. Acta Biomater. 2013, 9, 7580-7589 21. Abdal-hay, A.; Barakat, N. A. M.; Lim, J. K. Hydroxyapatite-doped Poly(lactic acid) Porous Film Coating for Enhanced Bioactivity and Corrosion Behavior of AZ31 Mg Alloy for Orthopedic Applications. Ceram. Int. 2013, 39, 183-195.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


22. Wong, H. M.; Yeung, K. W. K.; Lam, K. O.; Tam, V.; Chu, P. K.; Luk, K. D. K. A Biodegradable Polymer-based Coating to Control the Performance of Magnesium Alloy Orthopaedic Implants, Biomaterials 2010, 31, 2084-2096. 23. Zeng, R. C.; Liu, L. J.; Pang, T. T.; Zhang, F.; Zhang, W. W. Corrosion Resistance of Silane-modified Hydroxide Zinc Carbonate Film on AZ31 Magnesium Alloy. Acta Metall. Sin. (Engl. lett.) 2015, 28, 373380. 24. Zeng, R. C.; Liu, L. J.; Li, S. Q.; Zou, Y. H.; Zhang, F. Self-assembled Silane Film and Silver Nanoparticles Coating on Magnesium Alloys for Corrosion Resistance and Antibacterial Applications. Acta Metall. Sin. (Engl. Lett.) 2013, 26, 681-686. 25. Liu, X.; Yue, Z.; Romeo, T.; Weber, J.; Scheuermann, T.; Moulton, S. Biofunctionalized Anti-corrosive Silane Coatings for Magnesium Alloys. Acta Biomater. 2013, 9, 8671-8677. 26. Zeng, R. C.; Liu, Z. G.; Zhang, F.; Li, S. Q.; Cui, H. Z. Corrosion of Molybdate Intercalated Hydrotalcite Coating on AZ31 Mg Alloy. J. Mater. Chem. A 2014, 2, 13049-13057. 27. Zhang, F.; Liu, Z. G.; Zeng, R. C.; Li, S. Q.; Cui, H. Z. Corrosion Resistance of Mg-Al-LDH Coating on Magnesium Alloy AZ31. Surf. Coat. Technol. 2014, 258, 1152-1158. 28. Ding, Y. F.; Wen, C.; Hodgson, P.; Li, Y. C. Effects of Alloying Elements on the Corrosion Behavior and Biocompatibility of Biodegradable Magnesium Alloys: A Review. J. Mater. Chem. B 2014, 2, 1912-1933. 29. Vogt, C.; Bechstein, K.; Gruhl, S.; Lange, M.; Druecker, H.; Witte, F.; Vogt, J. Investigation of the Degradation of Biodegradable Mg Implant Alloys in Vitro and in Vivo by Analytical Methods. Proceeding of the 8th International Conference on Magnesium Alloys and Their Applications 2010, 11621174. 30. Sankara Narayanan, T. S. N.; Park, I. S.; Lee, M. H. Strategies to Improve the Corrosion Resistance of Microarc Oxidation (MAO) Coated Magnesium Alloys for Degradable Implants: Prospects and Challenges. Prog. Mater. Sci. 2014, 60, 1-71.


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31. Gao, Y.; Yerokhin, A.; Matthews, A.; DC Plasma Electrolytic Oxidation of Biodegradable Cp-Mg: In-vitro Corrosion Studies. Surf. Coat. Technol. 2013, 234, 132-142. 32. Němcová, A.; Skeldon, P.; Thompson, G. E.; Pacal, B. Effect of Fluoride on Plasma Electrolytic Oxidation of AZ61 Magnesium Alloy. Surf. Coat. Technol. 2013, 232, 827-838. 33. Zhang, R. F.; Xiong, G. Y.; Hu, C. Y. Comparison of Coating Properties Obtained by MAO on Magnesium Alloys in Silicate and Phytic Acid Electrolytes. Curr. Appl. Phys. 2010, 10, 255-259. 34. Zhang, R. F.; Zhang, S. F.; Duo, S. W. Influence of Phytic Acid Concentration on Coating Properties Obtained by MAO Treatment on Magnesium Alloys. Appl. Surf. Sci. 2009, 255, 7893-7897. 35. Fischerauer, S. F.; Kraus, T.; Wu, X.; Tangl, S.; Sorantin, E.; Hänzi, A. C. In Vivo Degradation Performance of Micro-arc Oxidized Magnesium Implants: A Micro-CT Study in Rats. Acta Biomater. 2012, 9, 5411-5420. 36. Han, X. G.; Zhu, F.; Zhu, X. P.; Lei, M. K.; Xu, J. J. Electrochemical Corrosion Behavior of Modified MAO Film on Magnesium Alloy AZ31 Irradiated by High-intensity Pulsed Ion Beam. Surf. Coat. Technol. 2013, 228, S164-S170. 37. Liu, G. Y.; Hu, J.; Ding, Z. K.; Wang, C. Bioactive Calcium Phosphate Coating Formed on Micro-arc Oxidized Magnesium by Chemical Deposition. Appl. Surf. Sci. 2011, 257, 2051-2057. 38. Ivanou, D. K.; Starykevich, M.; Lisenkov, A. D.; Zheludkevich, M. L.; Xue, H. B.; Lamaka, S. V.; Plasma Anodized ZE41 Magnesium Alloy Sealed with Hybrid Epoxy-silane Coating. Corros. Sci. 2013, 73, 300308. 39. Garlotta, D. A.; Literature Review of Poly (lactic acid). J. Polym Environ. 2001, 9, 63-84. 40. Anderson, J. M.; Shive, M. S.; Biodegradation and Biocompatibility of PLA and PLGA Microspheres. Adv. Drug Delivery Rev. 1997, 28, 5-24.


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41. Zhou, H.; Lawrence, J. G.; Bhaduri, S. B. Fabrication Aspects of PLA-CaP/PLGA-CaP Composites for Orthopedic Applications: A Review. Acta Biomater. 2012, 8, 1999-2016. 42. Bergsma, J. E.; De Bruijn, W. C.; Rozema, F. R.; Bos, R. R.; Boering, G. Late Degradation Tissue Response to Poly (L-lactide) Bone Plates and Screws. Biomaterials 1995, 16, 25-31. 43. Lu, L.; Garcia, C. A.; Mikos, A. G. In Vitro Degradation of Thin Poly (DL-lactic-co-glycolic acid) Films. J. Biomed. Mater. Res. 1999, 46, 236-244. 44. Van der Elst, M.; Klein, C. P.; De Blieck-Hogervorst, J. M.; Patka, P.; Haarman, H. J. Bone Tissue Response to Biodegradable Polymers Used for Intramedullary Fracture Fixation: A Long-term in Vivo Study in Sheep Femora. Biomaterials 1999, 20, 121-128. 45. Abdal-hay, A.; Barakat, N. A. M.; Lim, J. K. Influence of Electrospinning and Dip-coating Techniques on the Degradation and Cytocompatibility of Mg-based Alloy. Colloid Surf. A-Physicochem. Eng. Asp. 2013, 420, 37-45. 46. Xu, L.; Yamamoto, A. In Vitro Degradation of Biodegradable Polymer-coated Magnesium under Cell Culture Condition. Appl. Surf. Sci. 2012, 258, 6353-6358. 47. Xu, L.; Yamamoto, A. Characteristics and Cytocompatibility of Biodegradable Polymer Film on Magnesium by Spin Coating. Colloid Surf. B-Biointerfaces 2012, 93, 67-74. 48. Chen, Y.; Song, Y.; Zhang, S.; Li, J.; Zhao, C.; Zhang, X. Interaction Between A High Purity Magnesium Surface and PCL and PLA Coatings During Dynamic Degradation. Biomed. Mater. 2011, 6, 025005. 49. Guo, M.; Cao, L.; Lu, P.; Liu, Y.; Xu, X. Anticorrosion and Cytocompatibility Behavior of MAO/PLLA Modified Magnesium Alloy WE42. J. Mater. Sci. -Mater. Med. 2011, 22, 1735-1740. 50. Lu, P.; Cao, L.; Liu, Y.; Xu, X.; Evaluation of Magnesium Ions Release, Biocorrosion, and Hemocompatibility of MAO/PLLA-modified Magnesium Alloy WE42. J. Biomed. Mater. Res. Part BAppl. Biomater. 2011, 96B, 101-109.


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51. Zeng, R. C., Qi, W. C., Song, Y. W., He, Q. K.; Cui, H. Z. In Vitro Degradation of MAO/PLA Coating on Mg-1.21Li-1.12Ca-1.0Y Alloy. Front. Mater. Sci. 2014, 8, 343-353. 52. Wei, Z. L.; Tian, P.; Liu, X. Y.; Zhou, B. X. In Vitro Degradation, Hemolysis, and Cytocompatibility of PEO/PLLA Composite Coating on Biodegradable AZ31 Alloy. J. Biomed. Mater. Res. Part B -Appl. Biomater. 2015, 103, 342-354. 53. Song, G. L.; Atrens, A. Corrosion Mechanisms of Magnesium Alloys. Adv. Eng. Mater. 1999, 1, 11-33. 54. Song, Y.; Han, E. H.; Shan, D.; Yim, C. D.; You, B. S. The Effect of Zn Concentration on the Corrosion Behavior of Mg-xZn Alloys. Corros. Sci. 2012, 65, 322-330. 55. Zhang, J.; Dai, C.; Wei, J.; Wen, Z.; Zhang, S.; Chen, C. Degradable Behavior and Bioactivity of Microarc Oxidized AZ91D Mg Alloy with Calcium Phosphate/Chitosan Composite Coating in M-SBF. Colloid Surf. B: -Biointerfaces 2013, 111, 179-187. 56. Shu, Y.; Ou, G. M.; Wang, L.; Zou, J. C.; Li, Q. L. Surface Modification of Titanium with Heparinchitosan Multilayers via Layer-by-layer Self-assembly Technique. J. Nanomater. 2011, 2011, 1-8. 57. Takagishi, Y.; Kawakami, T.; Hara, Y.; Shinkai, M.; Takezawa, T.; Nagamune, T. Bonelike Tissue Formation by Three-dimensional Culture of MG63 Osteosarcoma Cells in Gelatin Hydrogels Using Calcium-enriched Medium. Tissue Eng. 2006, 12, 927-937. 58. Gao, J. C.; Qiao, L. Y.; Li, L. C.; Wang, Y. Hemolysis Effect and Calcium-phosphate Precipitation of Heat-organic-film Treated Magnesium. Trans. Nonferrous Met. Soc. China 2006, 16, 539-544. 59. Tokiwa, Y.; Calabia, B. P. Biodegradability and Biodegradation of Poly (lactide). Appl. Microbiol. Biotechnol. 2006, 72, 244-251. 60. Jong, S. J. de; Arias, E. R.; Rijkers, D. T. S.; Van Nostrum, C. F.; Kettenes-van den Bosch, J. J.; Hennink, W. E. New Insights into the Hydrolytic Degradation of Poly (lactic acid): Participation of the Alcohol Terminus. Polymer 2001, 42, 2795-2802.


38

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


61. Van Nostrum, C. F.; Veldhuis, T. F. J.; Bos, G. W.; Hennink, W. E. Hydrolytic Degradation of Oligo(lactic acid): A Kinetic and Mechanistic Study. Polymer 2004, 45, 6779-6787. 62. Burkersroda, F.; Schedl, L.; Göpferich, A. Why Degradable Polymers Undergo Surface Erosion or Bulk Erosion. Biomaterials 2002, 23, 4221-4231. 63. Piemonte, V.; Gironi, F. Kinetics of Hydrolytic Degradation of PLA. J. Polym. Environ. 2012, 21, 313-318. 64. Zeng, R. C.; Zhang, J.; Huang, W. J.; Dietzel, W.; Kainer, K. U.; Blawert, C. Review of Studies on Corrosion of Magnesium Alloys. Trans. Nonferrous Met. Soc. China 2006, 16, s763-s771. 65. Yuan, X.; Mak, A. F. T.; Kwok, K. W.; Yung, B. K. O.; Yao, K. Characterization of Poly (L-lactic acid) Fibers Produced by Melt Spinning. J. Appl. Polym. Sci. 2001, 81, 251-260. 66. Tomihata, K.; Suzuki, M.; Ikada, Y. The pH Dependence of Monofilament Sutures on Hydrolytic Degradation. J. Biomed. Mater. Res. 2001, 58, 511-518.


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Graphic Abstract 57x21mm (300 x 300 DPI)


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Figure 2 Optical morphology of the microstructure of the Mg-1Li-1Ca alloy. 63x47mm (300 x 300 DPI)



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Figure 3 SEM morphologies of (a) the MAO coatings; (b) the MAO/PLLA composite coatings formed on the Mg-1Li-1Ca alloys, (c) and (d) the high magnification of (a) and (b), respectively. The insets in (a) and (b) correspond to the EDS spectra of the MAO and MAO/PLLA coatings, respectively. 108x74mm (600 x 600 DPI)


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Figure 4 (a) SEM cross-sectional morphology, and (b) the EDS line scan mapping across MAO/PLLA coating. 110x144mm (300 x 300 DPI)



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Figure 5 HER of the substrate, MAO coating and MAO/PLLA coating immersed in HBSS for 140 h. 59x41mm (300 x 300 DPI)


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Figure 6 Changes in the pH value of HBSS with immersion for the coated and uncoated samples. 59x41mm (300 x 300 DPI)



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Figure 7 PDP curves obtained for the substrate, MAO coating and MAO/PLLA coating with and without immersion of 140 h. 59x41mm (600 x 600 DPI)


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Figure 8 (a) Nyquist curves for the coated and uncoated samples; 59x41mm (600 x 600 DPI)



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Figure 8 (b-g) fitted curves and equivalent circuits for the (b) substrate, (d) MAO coating and (f) MAO/PLLA coating without immersion; (c) substrate, (e) MAO coating and (g) MAO/PLLA coating with immersion of 140 h. 101x65mm (600 x 600 DPI)


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Figure 9 RGR of MC3T3-E1 cells cultured for different times in extract media from the substrates, MAO and MAO/PLLA coated samples compared to in α-MEM culture medium. Error bars represent ± S for n=5 and P < 0.05, as indicated by the asterisk (*). 59x41mm (600 x 600 DPI)



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Figure 10 Proliferation and differentiation of MC3T3-E1 cells cultured for different times in extracts from the substrates, MAO and MAO/PLLA-coated samples as well as α-MEM culture medium. Error bars represent ± S for n=3 and P < 0.05, as indicated by the asterisk (*). 59x41mm (600 x 600 DPI)


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Figure 11 SEM morphologies of MC3T3-E1 cells cultured on (a-b) the Mg-Li-Ca alloys, (c-d) the MAO coating and (e-f) the MAO/PLLA coating for 24 h. 157x157mm (300 x 300 DPI)



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Figure 12 SEM morphologies and EDS element analysis of surface of the samples after 140 h of immersion: (a) the substrate; (b) the MAO coatings; (c, d) the MAO/PLLA coatings. 118x88mm (300 x 300 DPI)


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Figure 13 SEM morphology of a corrosion pit on the MAO/PLLA surface after 140 h of immersion: (a) SEM micrographs; the elemental mappings of EDS: (b) C, (c) O, (d) Mg, (e) Ca and (f) P. 78x39mm (300 x 300 DPI)



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Figure 14 Cross-sectional morphology of MAO/PLLA coating after 140 h of immersion in HBSS: (a, b) crosssectional morphologies; EMPA image of cross-section of blister: (c) elemental mappings of EDS: (d) C, (e) O, and (f) Mg. 175x195mm (300 x 300 DPI)


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Figure 15 XRD patterns of the substrate, MAO coatings and MAO/PLLA coatings before and after immersion for 140 h. 885x618mm (96 x 96 DPI)



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Figure 16 Schematic illustrations of degradation mechanism of the porous MAO/PLLA composite coatings on the Mg-1Li-1Ca alloys in HBSS: (a) the swelling of PLLA and corrosion of the substrate at the initial stage 118x89mm (300 x 300 DPI)


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Figure 16 Schematic illustrations of degradation mechanism of the porous MAO/PLLA composite coatings on the Mg-1Li-1Ca alloys in HBSS: (b) the blistering and final peeling-off of PLLA under the pressure of hydrogen gas and corrosion products. 118x88mm (300 x 300 DPI)


In Vitro Corrosion and Cytocompatibility of a Microarc Oxidation

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