Enhanced Corrosion Resistance and Biocompatibility of Magnesium

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Enhanced Corrosion Resistance and Biocompatibility of Magnesium Alloy by Mg-Al Layered Double Hydroxide Feng Peng, Hua Li, Donghui Wang, Peng Tian, Yaxin Tian, Guangyin Yuan, Demin Xu, and Xuanyong Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12974 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 10, 2016

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ACS Applied Materials & Interfaces

Enhanced Corrosion Resistance and Biocompatibility of Magnesium Alloy by Mg-Al Layered Double Hydroxide

⊥

Feng Peng†,£, Hua Li‡, Donghui Wang†,£, Peng Tian†, Yaxin Tian†,£, Guangyin Yuan , Demin Xu‡,*, Xuanyong Liu†,* †

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

Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China £

University of Chinese Academy of Sciences, Beijing 100049, China

‡

Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032，

China ⊥

National Engineering Research Center of Light Alloys Net Forming, Shanghai Jiao Tong

University, Shanghai 200240, China

ACS Paragon Plus Environment


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ABSTRACT: Magnesium (Mg) and its alloys have been suggested as revolutionary biodegradable materials. However, fast degradation hinders its clinic application. To improve the corrosion resistance and biocompatibility of Mg-Nd-Zn-Zr alloy (JDBM), magnesium-aluminum layered double hydroxide (Mg-Al LDH) was successfully introduced into Mg(OH)2 coating by hydrothermal treatment. The anions in the interlayer of Mg-Al LDH can be replaced by chloride ions, resulting in a relatively low chloride ion concentration near the surface of the coating. The favorable corrosion resistance of the coating was proved by polarization curves and hydrogen collection test. The Mg-Al LDH significantly promoted cell adhesion, migration and proliferation in vitro. In addition, the coating almost fulfilled the request of the clinical application in the hemolysis ratio test. Finally, in vivo results indicated that the coating offered the greatest long-lasting protection from corrosion and triggered the mildest inflammation comparing to the pure Mg(OH)2 coatings and untreated magnesium alloy. Mg(OH)2 coating containing Mg-Al LDH in the present study shows a promising application in improving anticorrosion and biocompatibility of Mg alloys, and might act as a platform for a further modification of Mg alloys ascribed to its special layer structure. Keywords: magnesium alloys, layer double hydroxide, corrosion, biocompatibility, ion exchange


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1. INTRODUCTION As biodegradable materials, magnesium (Mg) and its alloys have attracted attention of many researchers.1 With the complete degradation of Mg alloys after the tissue recovery, the long-term foreign-body reaction or the subsequent operation to remove the implantation can be avoided. Furthermore, the suitable strength and toughness of Mg alloys make them admirable candidates as load bearing orthopedic implants and coronary stents.2-4 However, the fast degradation of Mg alloys in vivo is the main obstacle of its clinical application. It will result in local accumulation of alkaline ions and hydrogen bubbles, which will induce local inflammation and destruction of surrounding tissue.5 It also means that Mg alloys will lose the mechanical tension before the expected end of service. One of the strategies to improve the corrosion resistance of Mg alloys is to regulate its element composition.6-8 Yuan et al. developed a novel Mg alloy, Mg-Nd-Zn-Zr (named JDBM), by adding neodymium (Nd), zinc (Zn), zirconium (Zr) element into Mg.9 Compared with pure Mg, it shows enhanced antibacterial properties, biocompatibility and corrosion resistance. For this perspective, JDBM alloy was chosen as the research object.10-11 Surface modification is an alternative strategy to improve the performance of Mg alloys,4 including hydrothermal treatment, plasma electrolytic oxidation (PEO),12-15 electron beam treatments,

16

ion implantation,17 apatite coating,14 and

organic polymer coating,18-19 etc. Hydrothermal treatment is one of the commonly studied surface modifications and its aim is to prepare a compacted Mg(OH)2 coating



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on the surface of Mg alloys.20-22 It is well recognized that the corrosion of Mg alloys starts with the emerging of pits which will rapidly propagate and develop pitting corrosion afterward. When SO42- and HCO3-, especially Cl- contact with the Mg(OH)2 coating , the Mg(OH)2 layer will be broken down and convert to soluble MgCl2, which speeds up the corrosion.1 For this reason, Mg(OH)2 coating is not sufficient to endow Mg alloys a long-lasting anticorrosion property, although it can successfully separate substrate from aqueous solution or physiological environment. Layered double hydroxides (LDHs) have a interlayer structure which can stockpile anions, and may be used to block chloride ions.23 LDHs are a family of compound whose structure corresponds to hydrotalcite. It makes up of positively charged brucite-like layers with an interlayer region containing various anions and solvation molecules. Its chemical formula is [M2+1−xM3+x(OH)2][An−]x/n·zH2O, where M2+ represent bivalent cations, such as Mg2+, Zn2+ or Ni2+, and M3+ represent trivalent cations such as Al3+,Fe3+ or Mn3+.24 The applications of LDHs mostly focus on catalysts, catalyst precursors,

anion

exchangers,

CO2

absorbents,

bioactive

nanocomposites,

electroactive and photoactive materials.25-27 There were also many studies focusing on the biological application of this structure. In our previous work, we fabricated a Ni-Ti LDH on the surface of nitinol and found that the LDH can control the release of nickel ion, and the released nickel ions could be utilized to kill tumor cell.28 Yao et al. grew Mg-Al LDH micropatterned arrays on gold substrate which can promote cell adhesion and spreading.29 Li et al. employed LDH nanoparticles to simultaneously deliver an anticancer drug 5-fluorouracil (5-FU) and Allstars Cell Death siRNA


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(CD-siRNA) to overcome the drug resistance and enhance cancer treatment.30 However, there is lack of detailed research about the potential anticorrosion and biocompatibility effects of LDH coating. In this study, pure Mg(OH)2 films with different morphologies and film composing of Mg(OH)2 and Mg-Al LDH were synthesized on the surface of JDBM alloy via a simple hydrothermal treatment. The corrosion resistance of the films was detected in vitro and in vivo, cytocompatibility and inflammation response of the films were also evaluated.

2. MATERIALS AND METHODS 2.1. Substrate Material and Coatings Fabrication JDBM alloy, a gift from Shanghai Jiao Tong University, was cut into 10 mm × 10 mm × 1 mm. Before coated, they were ground with 1000# SiC abrasive paper, and then ultrasonically cleaned with ethyl alcohol, dried in the air. The pure Mg(OH)2 coatings were prepared on JDBM alloys by hydrothermal method in 50 mL deionized water (pH = 10 or 12, adjusted by NaOH), and the samples were designed as HT1# and HT2#, respectively. The Mg-Al LDH coating was synthesized by treating the samples in 0.02 M aluminum nitrate solutions (pH = 12.8, adjusted by NaOH), and designed as HT3#. The reaction was kept in a teflon-lined stainless at 120 oC for 12 h. Then samples were taken out and rinsed with deionized water. 2.2. Coatings Characterization The surface morphologies and cross-sectional morphologies of the samples were



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investigated by field emission scanning electron microscopy (FE-SEM; S-4800, HITACHI, Japan). X-ray diffraction (XRD; D/Max, RIGAKU, Tokyo, Japan) was used to identify the crystalline phases of the samples. The surface chemical composition of the specimens was measured by X-ray photoelectron spectroscopy (XPS, PHI-5000C ESCA system PerkinElmer, USA). 2.3. In Vitro Corrosion Behavior Evaluation 2.3.1. Electrochemical Test Electrochemical corrosion of the samples was tested using a CHI760C electrochemical analyzer (Shanghai, China) in phosphate buffer saline (PBS). The process was conducted in a three-electrode electrochemical cell with a saturated calomel electrode (SCE) as the reference electrode, a graphite rod as the counter electrode and the sample with exposing area of 0.255 cm2 as working electrode. Ahead of the test, the sample was stabilized in PBS for 400 seconds. The test was conducted at a scanning rate of 10 mV/s with a temperature of 37 oC. The corrosion potential (Ecorr), current density (icorr) and polarization resistance (Rp) were calculated according to Tafel extrapolation. 2.3.2. Hydrogen Evolution Test To evaluate the long-term corrosion resistance of the samples, hydrogen evolution test was conducted. Three parallel samples for each group were placed in a beaker with 300 mL PBS at 37 oC. The volume of released hydrogen was recorded up to 28 d. According to the volume of hydrogen, the corrosion rate of tested samples can be calculated via the following formula:


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r =

PV M × RT AT

(1)

where r is the corrosion rate (mg·cm-2·d-1), P is standard atmospheric pressure (Pa), V is volume of H2 (mL), R is 8.314 J/(mol·K), T is the temperature (K), M is the molar mass (g/mol), A is the original surface area (cm2), t is the exposure time (day). 2.4. In Vitro Cytocompatibility and Hemocompatibility Evaluation 2.4.1.

Cytotoxicity Evaluation

Human umbilical vein endothelial cells (HUVECs, ScienceCell, USA) were used in the test. After sterilized by ultraviolet irradiation, the samples were incubated in Endothelial Cell Medium (ECM) for 24 h and the sample-area-extraction-medium ratio was 0.25 cm2/mL. The extracted solution was designated as 100%, and was diluted to 90%, 60% and 30% with ECM medium. Meanwhile, 100 µL cell suspensions with a cell density of 5×104 cell/mL were added to each well of a 96-well culture plate. After 24 h, 100 µL extracted solution with different concentrations replaced the culture medium and incubated for another 1 and 4 days. ECM medium without extract served as the control group. Cells number was tested by the alamarBlue assay (AbD Serotec Ltd, UK) according to the manufacturer’s instruction. The viability of cells was calculated using the following equation:

Viability =

FS × 100% FC

(2)

Where Fs is the fluorescence intensity of the sample groups and Fc is the fluorescence intensity of the control groups. 2.4.2.

Cell Migration



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Extracted solution was obtained as mentioned above. 1 mL HUVECs suspension with a cell density of 5× 104 cell/mL was added to 24-well culture plates. After 4 d cultivation, lines crossing the cell well were drawn by a 200 µL pipet tip, and the cell culture medium was replaced with 100% extracted solution. After cultivation for 4, 8, and 12 h, the cells were stained with FITC-Phalloidin (Sigma, USA) and DAPI (Chemical International) at room temperature. Subsequently the cytoskeletal actin and cell nuclei were observed by fluorescence microscopy (Olympus, Japan). Numbers of cells migrating into the blank area were also counted. 2.4.3.

Cell Adhesion

Osteoblast-like cell line MC3T3-E1 was obtained from Cells Resource Center of Shanghai Institute for Biological Science. The MC3T3-E1 cells were seeded on the samples at a density of 1 ×105 cells/well. After 1, 4 and 24 h, the samples were rinsed three times with PBS. Then cells were fixed, permeabilized and blocked successively by 4% paraformaldehyde (PFA) diluent, 0.1% (v/v) Triton X-100 (Amresco, USA) and 1 wt % bovine serum protein(BSA, Sigma, USA) respectively. Then FITC phalloidin was added to stain F-actin, and DAPI stain nucleus. Samples were rinsed with PBS after each step. Finally, specimens were observed by confocal laser scanning microscopy (CLSM, Leica SP8, Germany). 2.4.4.

Cell Morphology and Proliferation

The MC3T3-E1 cells were seeded on the specimens with a density of 5×104 cell/mL. After 4 and 7 d, the samples were taken out and rinsed with PBS twice to remove the unattached cells. All samples were fixed with a 2.5% glutaraldehyde


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solution for 12 h at 4 oC. 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). Morphology of the cells was detected by SEM. The proliferation rate of the cells cultured on the samples was determined by alamarBlue assay. 2.4.5.

Hemolysis Ratio Test

The human blood was obtained from healthy adult donors. The samples were placed in a 24-well plate with 1.5 mL 0.9 wt% NaCl solution and keep at 37 oC for 30 min. Untreated 0.9 wt% NaCl and distilled water served as negative and positive controls respectively. After that, the solution was replaced with 30 µL diluted blood (0.8mL whole blood was diluted with 1 mL 0.9 wt% NaCl solution) and incubated for 1 h at 37 oC. Subsequently, the solution was centrifuged at 3000 rmp for 5 min. The optical density of the supernatant was measure at 545 nm by an enzyme-labeling instrument. The HR was calculated using the following equation:

HR =

AS 545 − AN 545 × 100% AP545 − AN 545

(3)

where AS545 is the absorption value of the samples, AN545 is the absorption value of the negative control and AP545 is the absorption value of the positive control. 2.5 .Animal Experiments The animal model of subcutaneous implantation was utilized to evaluate corrosion behavior of the JDBM, HT1#, HT2# and HT3# in vivo. Six adult SD rats (purchased from the Experimental Animal Centre of Zhongshan hospital), each



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weighing ~200 g, were anesthetized with pentobarbital sodium (40 mg/Kg) by intraperitoneal injection. Eight parallel incisions in two rows were made on the back of each rat. One sample was implanted in subcutaneous pocket through one incision. Each rat carried two parallel samples of all the four types of samples. The samples on each two rats were harvested at 10, 20, and 40 d after implantation, which provide four samples for each type of sample at each time point. After the capsule tissues containing the samples were resected, they were fixed in 10% neutral formaldehyde solution for 2 h. Then the tissues of capsule wall were stripped from the samples and sequentially fixed. The samples were cleaned by chromic acid solution (200 g of CrO3 and 10 g of AgNO3 per liter of water). The mass losses were measured by electronic balance (AL204-IC Mettler Toledo). The changes of surface topography of samples were characterized by an optical camera. The tissues were dehydrated and embedded in paraffin. Histological cross-sections (~5 µm) were stained with hematoxylin-eosin. Images were obtained by a bright-field microscope (DM6M, Leica). The experiment was approved by the Animal Care and Use Committee of Zhongshan Hospital, Fudan University (Shanghai, China) and performed in strict accordance with the recommendations from the Guide for Animal Management Rules from the Ministry of Health of the People’s Republic of China.

3. RERSULTS AND DISCUSSION 3.1. Coating Characterization The surface morphologies of HT1#, HT2# and HT3# are depicted in Figure 1a, b


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and c. Nanoflake-like structures were observed on all of the treated samples. Compared with HT1#, the nanoflake-like structures on the surface of HT2# and HT3# were smaller and more compact, especially for HT2#. The cross-sectional morphologies of the three treated samples shown in Figure 1d, e and f indicated that the adhesion between the coating and substrate of HT2# was worse than that of HT1# and HT3#, the thickness of HT1#, HT2# and HT3# were about 1.8 µm, 2.8 µm and 2.6 µm, respectively. Figure 1g shows the XRD pattern of the samples. Only feature peaks of Mg were observed in the pattern of JDBM alloy. The crystalline phase of Mg(OH)2 appeared in the samples of HT1# and HT2#. Obvious peaks centered at 12° and 24° were detected in HT3#, which means Mg-Al LDH obtained. According to the strength and breadth of Mg(OH)2 peaks, it can be deduced that introducing Mg-Al LDH into the coating will not destroy the crystallization of Mg(OH)2.31, 33 As for HT1# and HT2#, Mg2+ releasing from magnesium substrate tends to react with OH-, forming the crystal nucleus of Mg(OH)2. The higher pH value means more OH- in the solution, resulting in more Mg(OH)2 nucleus. Therefore, the nanoflake-like structures formed in the solution with higher pH value will be smaller and more compact. With regard to HT3#, Al3+ first forms AlO2- in alkali aqueous via following reactions: Al3++OH-→Al(OH)3↓

(4)

Al(OH)3↓+ OH-→AlO2-

(5)

Then complexation between OH- and AlO2- occurs, producing [Al13(OH)32(H2O)] 7+.



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When Mg(OH)2 form on the surface of the substrate, Al3+ complex in [Al13(OH)32(H2O)]

7+

will immediately transport into the lattice of Mg(OH)2 and

replace Mg2+, forming Mg-Al LDH. The extra positive charges will be balanced by OH-, NO3-, and CO32-. After all of the Al3+ are introduced into the Mg-Al LDH, superfluous Mg2+ will only form Mg(OH)2, resulting in the mixed phases of Mg(OH)2 and Mg-Al LDH. The specimens were further investigated by XPS and the results are presented in Figure 2. According to the XPS full spectra (Figure 2a and b) and detailed elemental compositions of the coatings (Table S1 in the Supporting Information), the element Al was only detected in the sample of HT3#. Figure 2c and d show the high-resolution spectra of O1s and Al 2p of HT3#. O 1s peak can be divided into two peaks centered at 530.9 eV and 531.9eV corresponding to oxygen peaks in hydroxyl bonding with Mg and Al, respectively. Al 2p peak centered at 74.42eV can be ascribed to aluminum peak bonding with hydroxyl. The XPS results further confirm the existence of Mg-Al LDH on the surface of HT3#, while pure Mg(OH)2 on the surface of HT1# and HT2#. 3.2. Corrosion Resistance The polarization curves are presented in Figure 3a and resulting data are exhibited in Table 1. It is clear that the corrosion resistance of HT1#, HT2# and HT3# were significantly superior to JDBM alloy. Compared with untreated JDBM alloy, the Ecorr values of treated samples were more positive, while the jcorr values were more negative, and the Rp values were increased with about one magnitude. HT3# showed the best anticorrosion property. Figure 3b and c exhibit the hydrogen evolution of the


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samples and the corrosion rate calculated according to the collected hydrogen. As expected, JDBM alloy showed the fastest releasing of hydrogen followed by HT2#, HT1# and HT3# respectively. It can be concluded that at the beginning of the corrosion, the coating of HT1# and HT3# showed best protection ability. When the time came to 21 d, there was little difference between the corrosion rate of JDBM alloy, HT1# and HT2#, while HT3# still kept a relatively low corrosion rate. The aforementioned results were consistent with the consequence of the electrochemical test. The corrosion rate of Mg alloys is closely related to the surface phase compositions and morphology. However, although the surface morphology of HT2# was much more compact than that of HT1#, the corrosion behaviors of them were nearly the same. It implies that the surface morphology of Mg(OH)2 coating may not be the critical factor of corrosion resistance. Due to the high reactivity of magnesium, untreated magnesium will rapidly dissolve in aqueous solutions, especially in one containing chloride ions.1 The results of corrosion tests suggested that Mg(OH)2 created by de novo corrosion of Mg alloy substrate was loose and unable to provide a sufficient protection from corrosion. Although Mg(OH)2 coatings of HT1# and HT2# prepared by hydrothermal treatment was compacted, they still couldn’t resist the corrosion of soluble chloride ions. Comparing to HT1#, the slightly worse anticorrosion of HT2# might result from the poorer combination of Mg(OH)2 to the substrate, despite it was more compact. However, the introduction of Mg-Al LDH into Mg(OH)2 significantly improved the



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corrosion resistance. Based on the stability of anions (CO32-> OH- > Cl- > NO3-) in the interlayer of Mg-Al LDH, an ion exchange mechanism depicted in Scheme 1 might explain the differences in corrosive behaviors of the coated samples.31 The high concentration of soluble chloride ions in physiological conditions will destroy Mg(OH)2 coating , exposing the JDBM substrate to corrosive solution.2 But with regard to HT3#, the surrounding Cl- could be easily exchanged by NO3- in the interlayer of Mg-Al LDH. Furthermore, some OH- and CO32- in the interlayer might also be substituted for Cl- according to the theory of thermal motion. Both these would result in a lower concentration of Cl- near the surface of the coating and ease the damage of Mg(OH)2 caused by high concentration of Cl-. 3.3. Cell Viability In Vitro Figure 4 exhibits the viability of cells cultured in different extracts. After incubated for 1 d (Figure 4a) with the extracts, there was no significant difference among all four samples. When incubation time extended to 4 d (Figure 4b), the differences among JDBM alloy, HT1# and HT2# were still not obvious, while the viability of cells in HT3# extracts significantly exceeded the others. In addition, 100% extracts of HT3# showed no obvious cytotoxicity while the viability of cells in other samples’ extracts was less than 80%. It can be concluded that Mg-Al LDH reduces the cytotoxicity of Mg(OH)2 coatings. The low cytotoxicity of HT3# extract might result from the favorable changes (pH value, element compositions) of the microenvironment. Plasson et al. reported that cell growth rate was optimal around pH 7.2.35 According to the study of Zhao et


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al.,36 Mg ions at a low concentration had a positive effect on cells proliferation. The best anticorrosion property of HT3# means that the change of pH value and the increasing of released Mg ions in the HT3# extract are the lowest, which results in the minimal toxicity. Migration of endothelial cell is closely related to wound healing process after injury or biomaterials implanted. In this study, a scarification method was used to evaluate the motility of endothelial cell. The qualitative and quantitative results of cell migration are expressed in Figure 5. Figure 5a shows the images of HUVECs cells migrating into the blank area at 4, 8 and 12 h. At the first 4 h, only a handful of cells in all of the extracts migrated to the blank area. However, when time extended to 8 h, the blank areas in HT1# and HT2# extracts were significantly narrower than JDBM alloy, which means cells migrate faster. In the case of HT3# extract, the cells had almost migrated across the blank area. After 12 h, there was still a small gap in JDBM, HT1# and HT2# extracts, while the cells in HT3# extract covered almost all the blank area. The numbers of cells migrating into the blank area are displayed in Figure 5b. It is clear that extracts extracted from coated samples are more beneficial for cell migration than JDBM alloy, and cells in HT3# extract show the fastest migration rate. According to the work reported by Zhao et al,36 low concentration of Mg ions (10mM to 20mM) increased the migration of endothelial cells. Besides, as a signaling molecule, nitric oxide plays a pivotal role in many physiological processes.32, 37-38 Nitrate released from the interlayer of Mg-Al LDH could act as a nitrogen source for cell to produce nitric oxide. The fast migration rate of endothelial cell in HT3# extract



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may attribute to the synergistic effect of the low concentration of Mg ions and the production of the nitric oxide. To explore the initial cell adherence process, the cytoskeletons of MC3T3-E1 cells seeded on the samples were observed by CLSM. The results are showed in Figure 6. After 1 h of incubation, the expression of filamentous F-actin for cells on HT2# and HT3#, especially on HT3#, were better than that on JDBM and HT1#. The trend was remained after culturing for 4 h. After 24 h of incubation, cells on JDBM, HT1# and HT2# presented a round and less spread morphology. However, cells on HT3# showed a more spread morphology with a larger number of filopodia and lamellipodia. The results of this test are believed to relate to the degradation process of the coatings. Within the initial 4 h, the Mg(OH)2 coating of HT1# and HT2# could protected the substrate from a serious degradation, there were lesser changes of pH value and chemical components in the culture medium comparing to JDBM, which leaded to a better level of spreading and surviving of the adhesive cells. Such protection failed at 24 h due to the damage of the coating. However, with the introduction of Mg-Al LDH, the coating of HT3# was still intact after 24 h in the culture medium (Figure S1 in the Supporting Information), on which the adhesive cells maintained a well spread morphology. Figure 7a shows the morphologies of MC3T3-E1 cells cultured on the samples. After 4 d of culture, cells on the surface of JDBM alloy exhibited poor adhesion and few filopodia extensions. In addition, there was little precipitation on the surface of


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JDBM alloy. The precipitation was easily washed away during the process of cell dehydration ascribed to its poor adhesion to the surface of JDBM alloy. No obvious cells could be observed on the surfaces of HT1# and HT2# because they were covered by Mg(OH)2 and MgCO3 precipitation. However, cells exhibited well adhesion on the surface of HT3# and display numerous filopodia protrusions, which means a well cells growth. Furthermore, nanosheet morphology could be found in the area which had not been covered by cells. It implies that the coating of HT3# was stable in psychological environment. After 7 d of culture, Mg(OH)2 and MgCO3 precipitation were also observed on the surface of JDBM alloy, and as expected, no cells could be observed on JDBM alloy, HT1# and HT2# while cells grew all over the surface of HT3#. The results suggest that HT3# is more suitable for cell adhesion and growth comparing with the other samples, which might attribute to the more stable structure of HT3# observed in the test. The proliferation rate of MC3T3-E1 cells on the samples is exhibited in Figure 7b. The results showed the same trend as that observed by SEM. After 4 d of culture, the cells grew better on the surface of HT2# and HT3# than JDBM alloy and HT1#. However, cells number of HT3# significantly exceeded the others at 7 d. The growth of mammalian cells undergo a process of substrate adhesion, spreading, cytoskeleton development, survival and finally proliferation.39 The above mentioned experiments certified that the adhesion, spreading, and survival of cells on the surface of HT3# were better than the others. Furthermore, the fast degradation of JDBM alloy and Mg(OH)2 coatings would lead to the accumulation of the corrosion



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products, which would precipitate on the membrane of the cells (as shown in Figure 7a) and damage the cellular metabolism. Such harmful effect was decreased to the lowest level by the introduction of Mg-Al LDH. All these factors have contributed to the significantly higher proliferation rate of cells grown on the HT3#. 3.4. Hemolysis Ratio Hemolysis ratio (HR) is a vital evaluation for blood-contacting materials since high hemolysis ratio would cause great damage to the erythrocyte, and rapid release of magnesium ions with fast corrosion would cause severe hemolysis reaction.40 As shown in Figure 8, HR values of treated samples were significantly lower than JDBM alloy. Though the HR values of JDBM alloys were improved by Mg(OH)2 coating , they were still much higher than 5% (an acceptable value for clinical application). After introducing Mg-Al LDH structure into Mg(OH)2 coating, the HR value was further improved and almost reaches the acceptable value. 3.5. In Vivo Evaluation The in vivo corrosive environment is different from in vitro.41-43 The in vivo corrosion tests were carried out by subcutaneous implantation. Figure 9a shows the morphology of the JDBM, HT1#, HT2# and HT3# after subcutaneous implantation. It can be found that the degradation increase over time in the sample of JDBM, HT1# and HT2#. Comparing to JDBM, the corrosions of HT1# and HT2# were slightly milder at 20 d, but there were no differences at 40 d. However, the corrosions are barely seen in HT3# at any time point. Figure 9b shows the mass losses of the samples after subcutaneous implantation. The mass losses of HT1# and HT2# were


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smaller than the bare alloy at 20 d, but became similar to JDBM alloy at 40 d. However, the mass loss of HT3# increased insignificantly with the time prolonging of implantation and obviously lower than that of JDBM alloy even after 40 d. It implied that HT1# and HT2# provide relatively short-time anticorrosion function in vivo, which was lost at 40 d due to the degradation of pure Mg(OH)2 coating. However, the introduction of Mg-Al LDH into Mg(OH)2 provides the persistent protection from corrosion in vivo. The subcutaneous capsules surrounding the implanted samples are shown in Figure 10. All implants degraded and produced hydrogen gas in vivo. Gas accumulated within the capsules and formed bubbles. Judging by the sizes of bubbles, HT3# produced the least amount of hydrogen at all the time points, which implied that the gaseous corrosive product of HT3# was the smallest. Since the capsule was filled with gas, part of the capsule wall did not attach to the surface of samples. Only the capsule wall attaching to the sample (Figure S2 in the Supporting Information) was collected and analyzed. The histological cross-sections are shown in Figure 11. Local tissue inflammation was triggered by all samples at the early stage and gradually eased overtime. The capsule tissue gradually matured over time, indicated by the increasing differentiation of fibroblasts into fibrocytes and the accumulation of extracellular matrix. The capsule wall of HT3# presented the mildest inflammation reactions among four groups, indicated by fewer macrophages (indicated by blue arrow) and fewer leukocytes (indicated by green arrow) at 10 and 20 d. The mildest inflammation observed in the capsule of HT3# suggested that the HT3# possesses the



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best histocompatibility in vivo. At 20 d and 40 d, there were accumulations of excessive products of corrosion within some capsules (Figure S3 in the Supporting Information). Acute inflammation (indicated by black arrow in Figure 12) triggered by corrosive products are found sporadically in the cross-sections of four groups. HT3# showed a milder acute inflammatory reaction comparing to ones of the others. It might result from the least amount of corrosive products with HT3#. The study of Bowen at el. revealed that magnesium degraded faster in vitro than in vivo.41 In this study, the in vitro differences of the corrosion behavior and biocompatibility among samples could still be observed in vivo. It implies that the ion exchange mechanism works under the in vivo environment.

CONCLUSIONS Mg-Al LDH was successfully introduced into Mg(OH)2 coating on biodegradable magnesium alloy (JDBM) by a simple hydrothermal treatment method. After incorporated with Mg-Al LDH, in vitro and in vivo corrosion resistance of the Mg(OH)2 coating was significantly improved. Cell adhesion, migration and proliferation were also greatly enhanced and hemolysis ratio was decreased to a level for clinical application. Finally, the Mg(OH)2 coating with Mg-Al LDH exhibited better histocompatibility than pure Mg(OH)2 coatings and untreated magnesium alloy in vivo. Favorable corrosion resistance of coating containing Mg-Al LDH was supposed to be a result of ion exchange process, and enhanced corrosion resistance were supposed to be responsible for its admirable biocompatibility.


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ASSOCIATED CONTENT Supporting Information Surface elemental compositions of the coating samples, surface morphology of all the samples immersed in α-MEM medium for 1 d, the cross section of a typical capsule with bubble, excessive products of corrosion within the capsule, electrochemical impedance spectroscopy (EIS) test and results. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Prof. Xuanyong Liu. E-mail: [email protected]. Tel.: +86 21 52412409. Fax: +86 21 52412409. *Prof. Demin Xu. E-mail: [email protected]. Tel.: +86 21 64041990. Fax: +86 21 64223006. Author Contributions Feng Peng and Hua Li contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS Financial support from the National Science Foundation for Distinguished Young



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Scholars of China (51525207), National Natural Science Foundation of China (31570973), Shanghai Committee of Science and Technology, China (15441904900, 14XD1403900) are acknowledged. Reference (1) Gu, X.-N.; Li, S.-S.; Li, X.-M.; Fan, Y.-B. Magnesium Based Degradable Biomaterials: A Review. Frontiers of Materials Science 2014, 8 200-218. (2) Staiger, M. P.; Pietak, A. M.; Huadmai, J.; Dias, G. Magnesium and Its Alloys as Orthopedic Biomaterials: A Review. Biomaterials 2006, 27 1728-34. (3) Geetha, M.; Singh, A. K.; Asokamani, R.; Gogia, A. K. Ti Based Biomaterials, the Ultimate Choice for Orthopaedic Implants – a Review. Prog. Mater. Sci. 2009, 54 397-425. (4) Wang, J.; Tang, J.; Zhang, P.; Li, Y.; Wang, J.; Lai, Y.; Qin, L. Surface Modification of Magnesium Alloys Developed for Bioabsorbable Orthopedic Implants: A General Review. J Biomed Mater Res B Appl Biomater 2012, 100 1691-701. (5) Zhen, Z.; Xi, T.-f.; Zheng, Y.-f. A Review on in Vitro Corrosion Performance Test of Biodegradable Metallic Materials. Trans. Nonferrous Met. Soc. China 2013, 23 2283-2293. (6) Homayun, B.; Afshar, A. Microstructure, Mechanical Properties, Corrosion Behavior and Cytotoxicity of Mg–Zn–Al–Ca Alloys as Biodegradable Materials. J. Alloys Compd. 2014, 607 1-10. (7) Leng, Z.; Zhang, J.; Yin, T.; Zhang, L.; Guo, X.; Peng, Q.; Zhang, M.; Wu, R. Influence of Biocorrosion on Microstructure and Mechanical Properties of Deformed Mg-Y-Er-Zn Biomaterial Containing 18r-Lpso Phase. J Mech Behav Biomed Mater 2013, 28 332-9. (8) Zhao, N.; Watson, N.; Xu, Z.; Chen, Y.; Waterman, J.; Sankar, J.; Zhu, D. In Vitro Biocompatibility and Endothelialization of Novel Magnesium-Rare Earth Alloys for Improved Stent Applications. PLoS One 2014, 9 e98674. (9) Qin, H.; Zhao, Y.; An, Z.; Cheng, M.; Wang, Q.; Cheng, T.; Wang, Q.; Wang, J.; Jiang, Y.; Zhang, X.; Yuan, G. Enhanced Antibacterial Properties, Biocompatibility, and Corrosion Resistance of Degradable Mg-Nd-Zn-Zr Alloy. Biomaterials 2015, 53 211-20. (10) Zhang, X.; Yuan, G.; Mao, L.; Niu, J.; Fu, P.; Ding, W. Effects of Extrusion and Heat Treatment on the Mechanical Properties and Biocorrosion Behaviors of a Mg-Nd-Zn-Zr Alloy. J Mech Behav Biomed Mater 2012, 7 77-86. (11) Zong, Y.; Yuan, G.; Zhang, X.; Mao, L.; Niu, J.; Ding, W. Comparison of Biodegradable Behaviors of AZ31 and Mg–Nd–Zn–Zr Alloys in Hank's Physiological Solution. Materials Science and Engineering: B 2012, 177 395-401. (12) Cui, X.-J.; Liu, C.-H.; Yang, R.-S.; Li, M.-T.; Lin, X.-Z. Self-Sealing Micro-Arc Oxidation Coating on Az91d Mg Alloy and Its Formation Mechanism. Surf. Coat. Technol. 2015, 269 228-237. (13) Dong, K.; Song, Y.; Shan, D.; Han, E.-H. Corrosion Behavior of a Self-Sealing Pore Micro-Arc Oxidation Film on AM60 Magnesium Alloy. Corros. Sci. 2015, 100 275-283. (14) Tian, P.; Liu, X.; Ding, C. In Vitro Degradation Behavior and Cytocompatibility of Biodegradable AZ31 Alloy with PEO/HT Composite Coating. Colloids Surf B Biointerfaces 2015, 128 44-54. (15) Wei, Z.; Tian, P.; Liu, X.; Zhou, B. In Vitro Degradation, Hemolysis, and Cytocompatibility of PEO/PLLA Composite Coating on Biodegradable AZ31 Alloy. J Biomed Mater Res B Appl Biomater 2015,


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Table 1 Corrosion potential (Ecorr), corrosion current density (icorr) and polarization resistance (Rp) calculated according to the polarization curves. Samples

JDBM

HT1#

HT2#

HT3#

jcorr(A/cm2)

1.562 10-5

7.365 10-7

1.247 10-6

3.626 10-7

Ecorr(V)

-1.7749

-1.5861

-1.6056

-1.5277

Rp(Ω)

1.190 106

2.516 107

1.343 107

4.775 107



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Figure 1. Surface morphologies of HT1# (a), HT2# (b), HT3# (c), and cross-sectional morphologies of HT1# (d), HT2# (e), HT3# (f); XRD patterns of all the coating samples (g). 160x160mm (300 x 300 DPI)


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Figure 2.XPS full spectra of HT1#, HT2# and HT3#(a); amplification of red rectangle in (a) shown in (b); high resolution of O1s (c) and Al2p (d) of HT3#. 102x80mm (600 x 600 DPI)



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Figure 3.Polarization curves (a), hydrogen evolution (b) and corrosion rate (c) of JDBM, HT1#, HT2# and HT3#. 41x10mm (600 x 600 DPI)


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Figure 4. Viability of HUVECs incubated for 1 d (a) and 4 d (b) with different concentration extracts of JDBM, HT1#, HT2# and HT3#. 64x26mm (600 x 600 DPI)



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Figure 5. Migration of HUVECs after incubation different time in extracts of JDBM, HT1#, HT2# and HT3# (The scratch area is between the two white dotted lines.) (a), and numbers of cells migrating into the blank area (b). 160x158mm (300 x 300 DPI)


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Figure 6. CLSM images of MC3T3-E1 cells cultured on JDBM alloy (a), HT1# (b), HT2# (c) and HT3# (d) for 1 h (i-1), 4 h (i-4) and 24 h (i-24) (i stands for a, b, c and d) with actin stained with FITC (green) and the nucleus stained with DAPI (blue). 122x93mm (300 x 300 DPI)



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Figure 7. Surface morphologies of JDBM, HT1#, HT2# and HT3# after culturing MC3T3-E1 cells for 4 and 7 days (a), and the proliferation rate of MC3T3-E1 cells (b). 130x105mm (300 x 300 DPI)


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Figure 8. Hemolysis ratio of JDBM, HT1#, HT2# and HT3#. 57x40mm (300 x 300 DPI)



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Figure 9. Corrosion morphology (a) and mass loss (b) of JDBM, HT1#, HT2# and HT3# after subcutaneous implantation for 10, 20 and 40 d. 206x265mm (300 x 300 DPI)


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Figure 10. The optical photograph of subcutaneous capsules of JDBM, HT1#, HT2# and HT3# after subcutaneous implantation for 10, 20 and 40 d. 160x124mm (300 x 300 DPI)



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Figure 11. Photomicrographs of histological sections of JDBM, HT1#, HT2# and HT3# after subcutaneous implantation for 10, 20, and 40 d. The green arrow indicated leukocytes; blue arrow indicated macrophages. 160x182mm (200 x 200 DPI)


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Figure 12. Acute inflammation triggered by corrosive products at 40 d and black arrow indicated purulent. 160x29mm (300 x 300 DPI)



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Scheme 1.Enhanced corrosion resistance of Mg(OH)2 coating by introducing Mg-Al LDH via an ion exchange process. 124x97mm (300 x 300 DPI)


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Table of Contents Graphic 35x15mm (600 x 600 DPI)


Enhanced Corrosion Resistance and Biocompatibility of Magnesium

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