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Surface modification of anodized Mg in ammonium hydrogen fluoride by various voltages
SCT-19287; No of Pages 8 Surface & Coatings Technology xxx (2014) xxx–xxx
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Surface modification of anodized Mg in ammonium hydrogen fluoride by various voltages Heng Bo Jiang a, Yu Kyoung Kim a, Jeong Hui Ji a, Il Song Park b, Tae Sung Bae a, Min Ho Lee a,⁎ a Department of Dental Biomaterials and Institute of Biodegradable material, Institute of Oral Bioscience and Brain Korea 21 plus Project, School of Dentistry, Chonbuk National University, Jeonju 561-756, South Korea b Division of Advanced Materials Engineering, Research Center for Advanced Materials Development and Institute of Biodegradable Materials, Chonbuk National University, Jeonju, 561-756, South Korea
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Article history: Received 16 October 2013 Accepted in revised form 18 March 2014 Available online xxxx Keywords: Magnesium MgF2 coating Micro arc fluorination Biodegradable Corrosion resistance
a b s t r a c t In recent years, extensive research has been carried out on the potential of using magnesium for biodegradable implants. However, the fast rate of corrosion of magnesium remains one of the major limitations of using Mg and Mg alloys as implants. The present work aimed to deposit a corrosion-resistant coating onto pure Mg (≥ 99.93%) in a saturated ammonium hydrogen fluoride solution at 120–200 V by micro arc fluorination (MAF) to improve resistance to corrosion. The treated samples were then characterized by SEM, EDS and XRD to assess morphological, chemical and structural characteristics. Electrochemical corrosion and immersion tests were used to evaluate the level of corrosion resistance. To test the coatings in vitro, MC3T3-E1 cells were cultured on the surface and cell viability was analyzed. A layer of dense ceramic coating with micro pores was observed on the surface. The thickness of the coatings ranged from approximately 2.5 to 5.5 μm. XRD and EDS analyses indicated that the coatings formed on the magnesium surface were composed of MgF2. Electrochemical and immersion tests performed in the SBF proved that the anodic fluorination coatings significantly improved the corrosion resistance of pure Mg. In the in vitro assay, both the proliferation and adherence rates of cells grown on the MgF2 coatings improved compared with the untreated group. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The development of biodegradable implants is an important goal within the field of biomaterials. Magnesium is one biodegradable material that could gradually be dissolved and remain harmless after implantation [1–4]. Compared with traditional implant materials, magnesium is superior due to its mechanical properties, which are similar to those of human bone [5–7]. However, magnesium corrodes very rapidly in the human body and loses its mechanical properties. So, pure magnesium is hard to use as a medical implant material [8,9]. Fortunately, some methods of forming biodegradable films can control corrosion rates and allow for the maintenance of mechanical properties over a period of time. These include alkaline treatment [10–12], metal plating [13–15], fluoride conversion treatment [16–20] and some biodegradable coating techniques [21–25]. Recently, the process of electrochemical application has been widely used for coating the ⁎ Corresponding author. E-mail address: [email protected] (M.H. Lee).
surface of magnesium [10–12], and this method has been proven to produce material that forms a high quality protective film [15,26,27]. Micro-arc oxidation (MAO) is a significant new surface-treatment technology that utilizes a high-voltage [28–33]. Significantly, MAO allows for adjustment of the film thickness, favors cellular adhesion to post-treatment coatings, creates a uniform film and involves short processing times and the formation of porosity, all of which are similar to the qualities of bone tissue [31–35]. To create a strong structure with good biocompatibility, the MAO-treated magnesium should be uniform. However, following oxidation via MAO treatment, the films often crack [30,31], thereby decreasing the corrosion resistance. A classical method in the field of fluoride treatment involves soaking magnesium in hydrogen fluoride acid, creating a coating of MgF2 [16]. Some researchers [16–18] confirmed that fluoride-treated magnesium is resistant to corrosion in vitro, and the fluoride-coated implants have been shown to possess good clinical tolerance in vivo [36]. Though this seems to be a simple process, the surface created by this method is too smooth and the treatment takes more than 24 h [16,36]. This smooth surface does not meet the demands of modern biomaterials.
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In this work, we used the micro-arc oxidation technique [28] to improve the properties of magnesium metals combined with fluoride treatment to form full magnesium fluoride ceramic coatings. We named this method micro arc fluorination (MAF). The MAF method was conducted within the range of 120–200 V in a fluoride electrolyte. MAF-treated Mg was analyzed and evaluated for corrosion resistance in simulated body fluid (SBF). The biocompatibility of the MAF-treated Mg was evaluated by testing the proliferation and attachment of MC3T3-E1 cells to the coating. 2. Material and methods 2.1. Sample Preparation Pure magnesium (≥99.93%) was cut into pieces with dimensions of 10 × 10 × 3 mm, polished with 1200-grit SiC paper and cleaned in ethanol for 1 min. SBF was made by dissolving 0.185 g/L calcium chloride dehydrate, 0.098 g/L magnesium sulfate, 0.35 g/L sodium hydrogen carbonate and 9.5 g/L Hanks' Balanced Salt Solution (HBSS) in distilled water. The pH of the SBF was adjusted to 7.4 using HCl solution. 2.2. Micro arc fluorination (MAF) The Mg sample, used as the anode, and a carbon rod, used as the cathode, were soaked in saturated NH4HF2-solution, and voltages of 120, 160 and 200 V were applied for 3 min using constant-voltage mode. During this process, the solution was stirred with a magnetic bar. After treatment, the samples were rinsed with distilled water and dried. The conditions of MAF treatment are shown in Table 1. 2.3. Surface characterization The surface morphologies and elementary compositions were observed by scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS). The phases of MAF-treated Mg were determined by X-ray diffraction (XRD), using a scan rate of 1°/min by the Cu Kα line, generated at 40 kV and 30 mA.
the surfaces of the samples were observed by optical microscopy (Leica Microsystem ZE4D) and SEM. 2.6. In vitro test MC3T3-E1 cells were seeded onto pure Mg and MAF-treated Mg at a cell density of 2 × 104 cells/ml. Culture wells were used for the control group. Cell proliferation was evaluated by measuring mitochondrial dehydrogenase activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) after culturing the cells for 1, 2 and 3 days. For each group, samples were evaluated in triplicate. To prepare the MTT solution, MTT was first dissolved in PBS and then diluted by adding 100 ml of the solution to 900 ml of serum free a-MEM. Using the diluted solution, 1 ml was added to each sample in 24-well plates to form formazan. After 4 h at 37 °C in a CO2 incubator, the solution was aspirated and blue formazan crystals were dissolved in 1 ml DMSO for 20 min. Subsequently, 200 μl of the resulting solution was transferred to 96-well plates, and five data points were obtained from each sample. The optical density of the solution in each well was measured at a wavelength of 570 nm using a Microplate Reader (Molecular Devices, Sunnyvale). To observe the cell morphology after 3 days in culture, the samples were rinsed with PBS, fixed first in 2.5% glutaraldehyde solution for 2 h and then in 1% osmium tetroxide for 2 h, dehydrated in a gradient of ethanol/distilled water mixtures (30%, 50%, 60%, 70%, 80%, 90%, absolute ethanol) for 10 min each and then dried. Cell morphology was observed by SEM. 2.7. Statistical analysis Three replicate experiments were performed, all of which yielded comparable results. Unless otherwise specified, all data are expressed as the mean ± standard deviation (SD) of three independent experiments. Statistical analysis was conducted by one-way analysis of variance (ANOVA). Values of p b 0.05 were considered significant. 3. Results
2.4. Corrosion resistance test
3.1. Characterization of pure and MAF-treated Mg
The corrosion resistances of pure magnesium and MAF-treated Mg were measured by the potentiodynamic polarization test (PARSTAT2273). The Mg samples served as the working electrode, an Ag/AgCl/sat KCl electrode served as the reference electrode, and a platinum sample served as the counter electrode. The samples were immersed in SBF at room temperature, and the scanning rate was 3 mV/s.
Fig. 1 shows the surface morphologies of the samples treated by MAF at 120, 160 and 200 V. The samples showed a typical ceramic surface morphology formed by MAF, consisting of a porous and uniform fluoride layer formed on the magnesium surface. The MAF coating process started at 120 V, according to the observation of a micro arc on the Mg surface. The MAF120 surface had a long-slot shape structure, while the MAF160 and MAF200 surfaces were uniform and porous. As shown in Fig. 1, MAF200 was rougher and had bigger pores than MAF120 and MAF160. Fig. 2 shows the chemical compositions of the MAF-treated Mg by EDS. Magnesium treated at different voltages in the fluoride electrolyte solution showed the presence of both F and Mg peaks. This suggested that magnesium fluoride was formed in the coating layer by MAF. Fig. 3 shows the cross-section morphologies and EDS-mapping scanning results of the pure magnesium after MAF treatments under different conditions. Based on the morphology of MAF-treated Mg and the element distributions of F and Mg, it was possible to distinguish between the MAF-treated Mg coating layer and the substrate. The thickness of the MAF-treated Mg coating increased as the applied voltage increased. The thicknesses of the MAF120, MAF160 and MAF200 coatings were approximately 2.5 μm, 3.5 μm and 5.5 μm, respectively. Fig. 4 shows the XRD patterns of the pure and MAF-treated Mg samples. Compared with pure Mg, the patterns observed in the MAF-treated Mg samples clearly indicated the presence of tetragonal MgF2. The MgF2 phase was increased as the applied voltage increased (Fig. 3).
2.5. SBF immersion test In the SBF immersion test, pure Mg and MAF-treated Mg were vertically immersed in SBF and incubated at 37 °C. The ratio of SBF volume to sample area was 40 ml/cm2 [37]. pH values were measured using a pH meter (Thermo Electron Corporation Orion-525Aplus). To determine the rate of weight loss, the SBF was changed every week. Every week, one group of samples (n = 4) was taken out and cleaned in chromium trioxide (CrO3) solution, as recommended in ASTM G1-03 [38]. Then,
Table 1 Sample code and conditions of the pure magnesium and MAF-treated Mg. Sample code
Treatment process
Time (min)
Pure MAF120 MAF160 MAF200 MAF210
– Anodized by constant-voltage at 120 V Anodized by constant-voltage at 160 V Anodized by constant-voltage at 200 V Anodized by constant-voltage at 210 V
– 3 3 3 3
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Fig. 1. Surface morphology of the MAF-treated Mg of (a) MAF120, (b) MAF160, (c) MAF200 and (d) MAF210.
3.2. Corrosion behavior of pure and MAF-treated Mg Fig. 5 shows the results of the potentiodynamic polarization test. E0, icorr and Tafel analysis were used to evaluate the corrosion resistance of
the MAF-treated Mg. Higher E0 values indicate increasing corrosion, while icorr is inversely proportional to the amount of corrosion. The corrosion rate of the samples was calculated by Tafel analysis expression [39]. At a potential of − 1.38 V, the current of MAF120 rapidly
Fig. 2. EDS spectra of (a) pure magnesium, (b) MAF120, (c) MAF160, and (d) MAF200.
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Fig. 3. Cross-sectional image and EDS mapping assay of the MAF-treated Mg samples, (a) MAF120, (b) MAF160, and (c) MAF200.
increased up to −1.34 V. The sudden increase in the current means that the oxidation reaction is occurring too quickly. For MAF160 and MAF200, there was no sudden increase in the current, meaning that
Fig. 4. XRD patterns of (a) pure magnesium, (b) MAF120, (c) MAF160, and (d) MAF200.
corrosion occurred uniformly in these samples. The calculated corrosion rates are shown in Table 2. MAF200 showed the lowest current density and the highest potential, and the corrosion rate calculated by Tafel analysis was 0.019 mm/year. The corrosion rates of MAF160 and MAF120 were 0.024 mm/year and 0.030 mm/year, respectively.
Fig .5. Potentiodynamic polarization plot recorded for (a) pure magnesium, (b) MAF120, (c) MAF160, and (d) MAF200.
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Table 2 Results of the potentiodynamic corrosion test on pure magnesium and MAF-treated Mg of MAF120, MAF160, and MAF200. Code
E0 (V; I = 0)
icorr (μA/cm2)
CR (mm/year)
Pure MAF120 MAF160 MAF200
–1.842 –1.573 –1.558 –1.547
5.064 0.301 0.238 0.187
0.505 0.030 0.024 0.019
Fig. 6 shows the pH variation of pure and MAF-treated Mg immersed in SBF solution over the various immersion times. When magnesium is dissolved in SBF solution, the pH value of the solution rises. The pH value was highest in the solution containing pure magnesium, increasing from 7.4 to approximately 8.9 in the initial 2 days following immersion. After the initial 2 days, the pH value of the SBF increased slowly with increasing immersion time in all groups. In contrast, the pH values of the SBF solution with MAF-treated samples immersed were lower than that with pure Mg. Among the MAF-treated samples, the solution with MAF200 exhibited the lowest pH value, whereas that was MAF120 had the highest pH value, followed by that with MAF160. The increasing pH value indicates that Mg was dissolved in the SBF solution; the less Mg dissolved, the slower the pH increased. In other words, a slower increase in pH indicates better corrosion resistance. Fig. 7 shows the weight loss percentage of pure and MAF-treated Mg after immersion in the SBF solution. The weight loss value of pure Mg was clearly increased as immersion time increased. In contrast, the MAF-treated Mg prepared at a higher voltage had a slower rate of degradation. After immersion for 8 weeks, the weight loss values of pure Mg, MAF120, MAF160 and MAF200 were 55.6%, 5.3%, 4.9% and 1.5%, respectively. Fig. 8 shows the morphologies of pure and MAF-treated Mg after weight loss tests following immersion for 8 weeks. For pure magnesium, very serious corrosion was observed. For the MAF-treated Mg, large holes of corrosion were observed around the fluoride film on the MAF120 and MAF160 surface, and these holes were similar to those observed in pure Mg. However, for MAF200 surface, the fluoride film was uniform, without any obvious corrosive holes or cracks. 3.3. Cell proliferation on pure Mg and MAF-treated Mg Fig. 9 and Table 3 show the cell proliferation data of MC3T3-E1 cells cultured on pure magnesium and MAF200 for 1, 2 and 3 days. Cells grown in culture wells were used for the control group. Cell
Fig. 7. Weight loss variation over time of (a) pure magnesium, (b) MAF120, (c) MAF160, and (d) MAF200.
proliferation on the pure Mg decreased with as culture time increased. However, for MAF200, both cell adhesion and proliferation were significantly improved (p b 0.05). Cell viability on the MAF200 surface was greater than in the control and pure Mg groups, which is attributable to the anti-corrosive, micro-porous surface promoting cell growth. Fig. 10 shows the morphology of MC3T3-E1 cells cultured on pure magnesium and MAF200 for 3 days. There is an obvious difference in the response of the cells to pure Mg and MAF200: pure Mg generated grossly observable corrosion products and blocked adherence of MC3T3-E1 cells to the surface, while the cells on the MAF200 surface exhibited stretched shapes and several pseudopodia contacting the surface, indicating both increased adherence and survival. 4. Discussion Magnesium has strong chemical activity and low corrosion resistance. Therefore, surface treatment is necessary to improve the corrosion resistance. Most anodizing treatments for magnesium, such as MAO, generate an oxide coating layer, The MAO-treated Mg has porosity and high mechanical properties, but the layer created by this treatment has some drawbacks: the layer formed often has cracks [30,31], and magnesium oxides can react with bodily fluids. The main contents of bodily fluids are water and chlorine ions, which cause the severe corrosion [32,40]. On the other hand, the protective function of the fluxes can be explained by the Pilling and Bedworth factor [41,42]. F¼
Wo dm Wm do
where: Wo Wm dm do
Fig. 6. pH variation of (a) pure magnesium, (b) MAF120, (c) MAF160, and (d) MAF200. Samples were immersed SBF for 14 days. n = 3 samples per group. Values are expressed as the mean ± SD.
molecular weight of the surface oxide atomic weight of the metal density of the metal density of the oxide
According to this equation, a surface oxide is protective against a circumstance if the factor is above 1. Table 4 shows that the protection coefficient of an MgO film is less than 1. This might explain the formation of cracks on MAO-treated Mg, and cannot be used for bioimplants. In Table 4, the protection coefficient of the MgF2 film is greater than 1, meaning that MgF2 has a strong protective effect.
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Fig. 8. (a–d) SEM and (e–h) optical images of (a, e) pure magnesium, (b, f) MAF120, (c, g) MAF160 and MAF200 (d, h) after the weight loss test conducted for 8 weeks.
In the preliminary experiments, magnesium in the electrolyte began to spark at 120 V. The sparks indicated that micro arc oxidation of the magnesium surface had begun. Brighter, more intense sparks were observed when the voltage increased to 200 V. When 210 V was applied, the electrolyte began to boil and the sample was destroyed during the process (Fig. 1(d)), indicating that magnesium cannot be treated by MAF in saturated NH4HF2-solution at 210 V. Therefore, for this paper we chose the voltage range of 120–200 V. Micro arc discharge was maintained for 20–30 s with applied voltages of 120–200 V. After this period of time, the discharge phenomenon disappeared, indicating that in constant voltage mode, a fluoride coating of regular thickness had formed on the magnesium surface. This coating created a double electrochemical layer, thereby decreasing the electrochemical reactivity of the magnesium surface. In the MAF process, fluoride ions move to the anode and react with Mg to form the MgF2 coating layer. At high-voltage conditions, the anode surface had a higher current and sparked more intensely, resulting in the formation of a ceramic coating. In this study, pure and porous MgF2 coatings were successfully fabricated (Figs. 3–5) by MAF processing in saturated NH4HF2-electrolyte
solution. The porous surface has greater biological adaptability than the smooth surface because surface area affects the ability of cells to attach. The larger surface area could cause faster adherence of surrounding tissue to the surface [43,44]. Further, compared with a smooth surface, a rough surface can allow cells to stick more easily and begin to grow [45]. In addition, compared with the processing time of soaking Mg in HF solution [16–18], MAF treatment greatly reduced the processing time, which is highly convenient for research and development. In vitro corrosion test data showed that the fluoride-coated Mg and its alloy had great corrosion resistance. The results of the corrosion test allow us to conclude that that the corrosion resistance increased with the coating thickness. This phenomenon has also been reported in other literature about fluoride coatings [17,31]. Tingting Yan et al. [17] and X.N. Gu et al. [31] found that increasing the thickness of fluoride coating had an impact on the Mg surface, improving the corrosion resistance. According to the literature [8], hydrogen gas bubbles were observed just after Mg was immersed in SBF solution. However, in this study, gas bubbles were not observed when MAF-treated Mg was immersed into SBF, indicating that the releasing rate of gas can be controlled by MAF treatment. The human body endogenously produces gas that is slowly discharged from the body via sweat glands and bodily fluids. Therefore, if the amount of gas released from the implants is small, it will be easily discharged from the body. There have been some reports in the literature [46,47] concerning Mg implants given to rats by μ-CT. A large amount of gas was formed near the implants, and the gas slowly dissipated over time. In other words, as MAF-treated Mg improved corrosion resistance, the negative influence of gas will no longer be a problem for the surrounding tissue. For the MAFtreated Mg, pitting corrosion was found after the 8 week immersion test for MAF120 (Fig. 8(f)) and MAF160 (Fig. 8(g)). However, MAF200 showed more mild and uniform corrosion (Fig. 8(h)). Therefore, corrosion resistance increased with increasing MAF thickness. In this work, MAF200 was better than the others in all aspects of performance. The corrosion rate and weight loss values for MAF200 were obviously the smallest, and only some bubble attached on the surface of MAF200
Table 3 MTT results of the mean OD value on the pure magnesium and MAF200 with control for 1, 2 and 3 days.
Fig. 9. Cell proliferation of MC3T3-E1 cells cultured for 1 day on pure magnesium, MAF200, and the control surface. *p b 0.05.
Group
1 day
2 days
3 days
Control Pure MAF200
0.691 0.173 0.755
1.228 0.148 1.416
1.378 0.131 1.428
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Fig. 10. Cell morphology of cultured MC3T3-E1 cells after 3 days on (a) pure magnesium, and (b) MAF200; high magnification (a-1) pure magnesium and (b-1) MAF200.
when put into the simulated body fluid for 8 weeks. Thus, we show that MAF200 is suitable for use as an implant material. Toxicology issues related to corrosion products from highly biodegradable metals require special attention. In the results shown here, the cell survival rate, as measured by an MTT assay, decreased on pure Mg. It is possible that Mg has some toxic effects on cells. In highly alkaline solutions, increasing the pH value results in cell death. X.N. Gu et al. [31] also tested the cytotoxicity of Mg and its alloy in an indirect assay and found that cell proliferation was not decreased. This implies that the decrease in cell viability is due to the increased pH and not to Mg toxicity. If a physical reaction occurs on the wall surface, cell growth on the surface will be affected. In the results of this paper, cells were not found growing on pure Mg, likely due to the large amount of bubbles generated from the Mg surface upon immersion into the medium [8]. Additionally, the Mg surface was highly corrosive in the medium and constantly dissolved. So, the cells may fall off of the Mg surface due to the bubbles and corrosion products, explaining the decreased growth in the pure Mg group seen in the MTT results. X.N. Gu et al. [31] observed no adherent cells and a decreased MTT value on untreated Mg-Ca alloy. However, in our experiment, the cell adhesion and proliferation behaviors are significantly improved for MAF200 compared with pure Mg and the control
group (p b 0.05), indicating that the MAF-treated Mg provides a biologically favorable environment. Because the MAF-treated Mg is mainly composed of MgF2 formed on the Mg substrate, it has high corrosion resistance. In addition, fluorine has anti-bacterial properties and can be beneficial for cell growth [48,49]. Martina Thomann et al. [36] found in clinical trials that new bone tissue was generated around MgF2-coated Mg implants. Therefore, the MgF2 coating raised the corrosion resistance of the magnesium metal, thereby improving biocompatibility. 5. Conclusions In this work regarding MAF treatment of Mg, we conclude the following: The MgF2 layer coating the pure Mg was successfully fabricated by MAF treatment. The MAF-treated Mg coating is porous with a thickness ranging from 2.5 to 5.5 μm. The MAF-treated Mg, in comparison with other samples, showed significantly improved corrosion resistance. After the immersion test in SBF, the MAF120 and MAF160 samples showed pitting corrosion, while the MAF200 sample had a more mild and uniform surface in comparison. Cell proliferation on the MAF-treated Mg was improved over that on the pure Mg. Acknowledgments
Table 4 Protection coefficient of surface films on magnesium melts [42]. Surface film formed
Protection coefficient
Mg + O = MgO Mg + H2O = MgO + H2 3Mg + N2 = Mg3N2 Mg + S = MgS 3Mg + 2BF3 = 3MgF2 + 2B Mg + 2HF = MgF2 + H2 3MgO + 2BF3 = B2O3 + 3MgF2 Mg + CO2 = 2MgO + C 2Mg + CO = MgO + C
0.71 0.71 0.79 1.26 1.32 1.32 3.1 0.9 1.08
This work was financially supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2011-0028709) and by the Basic Science Research Program through the NRF, which was funded by the Ministry of Education, Science and Technology (2010-0023901) and Regional Strategic Industry project (2013-R0002274). References [1] J. Vormann, Mol. Asp. Med. 24 (2003) 27–37. [2] S. Zhang, et al., Acta Biomater. 6 (2010) 626–640.
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Surface modification of anodized Mg in ammonium hydrogen fluoride by various voltages Heng Bo Jiang a, Yu Kyoung Kim a, Jeong Hui Ji a, Il Song Park b, Tae Sung Bae a, Min Ho Lee a,⁎ a Department of Dental Biomaterials and Institute of Biodegradable material, Institute of Oral Bioscience and Brain Korea 21 plus Project, School of Dentistry, Chonbuk National University, Jeonju 561-756, South Korea b Division of Advanced Materials Engineering, Research Center for Advanced Materials Development and Institute of Biodegradable Materials, Chonbuk National University, Jeonju, 561-756, South Korea
a r t i c l e
i n f o
Article history: Received 16 October 2013 Accepted in revised form 18 March 2014 Available online xxxx Keywords: Magnesium MgF2 coating Micro arc fluorination Biodegradable Corrosion resistance
a b s t r a c t In recent years, extensive research has been carried out on the potential of using magnesium for biodegradable implants. However, the fast rate of corrosion of magnesium remains one of the major limitations of using Mg and Mg alloys as implants. The present work aimed to deposit a corrosion-resistant coating onto pure Mg (≥ 99.93%) in a saturated ammonium hydrogen fluoride solution at 120–200 V by micro arc fluorination (MAF) to improve resistance to corrosion. The treated samples were then characterized by SEM, EDS and XRD to assess morphological, chemical and structural characteristics. Electrochemical corrosion and immersion tests were used to evaluate the level of corrosion resistance. To test the coatings in vitro, MC3T3-E1 cells were cultured on the surface and cell viability was analyzed. A layer of dense ceramic coating with micro pores was observed on the surface. The thickness of the coatings ranged from approximately 2.5 to 5.5 μm. XRD and EDS analyses indicated that the coatings formed on the magnesium surface were composed of MgF2. Electrochemical and immersion tests performed in the SBF proved that the anodic fluorination coatings significantly improved the corrosion resistance of pure Mg. In the in vitro assay, both the proliferation and adherence rates of cells grown on the MgF2 coatings improved compared with the untreated group. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The development of biodegradable implants is an important goal within the field of biomaterials. Magnesium is one biodegradable material that could gradually be dissolved and remain harmless after implantation [1–4]. Compared with traditional implant materials, magnesium is superior due to its mechanical properties, which are similar to those of human bone [5–7]. However, magnesium corrodes very rapidly in the human body and loses its mechanical properties. So, pure magnesium is hard to use as a medical implant material [8,9]. Fortunately, some methods of forming biodegradable films can control corrosion rates and allow for the maintenance of mechanical properties over a period of time. These include alkaline treatment [10–12], metal plating [13–15], fluoride conversion treatment [16–20] and some biodegradable coating techniques [21–25]. Recently, the process of electrochemical application has been widely used for coating the ⁎ Corresponding author. E-mail address: [email protected] (M.H. Lee).
surface of magnesium [10–12], and this method has been proven to produce material that forms a high quality protective film [15,26,27]. Micro-arc oxidation (MAO) is a significant new surface-treatment technology that utilizes a high-voltage [28–33]. Significantly, MAO allows for adjustment of the film thickness, favors cellular adhesion to post-treatment coatings, creates a uniform film and involves short processing times and the formation of porosity, all of which are similar to the qualities of bone tissue [31–35]. To create a strong structure with good biocompatibility, the MAO-treated magnesium should be uniform. However, following oxidation via MAO treatment, the films often crack [30,31], thereby decreasing the corrosion resistance. A classical method in the field of fluoride treatment involves soaking magnesium in hydrogen fluoride acid, creating a coating of MgF2 [16]. Some researchers [16–18] confirmed that fluoride-treated magnesium is resistant to corrosion in vitro, and the fluoride-coated implants have been shown to possess good clinical tolerance in vivo [36]. Though this seems to be a simple process, the surface created by this method is too smooth and the treatment takes more than 24 h [16,36]. This smooth surface does not meet the demands of modern biomaterials.
http://dx.doi.org/10.1016/j.surfcoat.2014.03.032 0257-8972/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: H.B. Jiang, et al., Surf. Coat. Technol. (2014), http://dx.doi.org/10.1016/j.surfcoat.2014.03.032
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In this work, we used the micro-arc oxidation technique [28] to improve the properties of magnesium metals combined with fluoride treatment to form full magnesium fluoride ceramic coatings. We named this method micro arc fluorination (MAF). The MAF method was conducted within the range of 120–200 V in a fluoride electrolyte. MAF-treated Mg was analyzed and evaluated for corrosion resistance in simulated body fluid (SBF). The biocompatibility of the MAF-treated Mg was evaluated by testing the proliferation and attachment of MC3T3-E1 cells to the coating. 2. Material and methods 2.1. Sample Preparation Pure magnesium (≥99.93%) was cut into pieces with dimensions of 10 × 10 × 3 mm, polished with 1200-grit SiC paper and cleaned in ethanol for 1 min. SBF was made by dissolving 0.185 g/L calcium chloride dehydrate, 0.098 g/L magnesium sulfate, 0.35 g/L sodium hydrogen carbonate and 9.5 g/L Hanks' Balanced Salt Solution (HBSS) in distilled water. The pH of the SBF was adjusted to 7.4 using HCl solution. 2.2. Micro arc fluorination (MAF) The Mg sample, used as the anode, and a carbon rod, used as the cathode, were soaked in saturated NH4HF2-solution, and voltages of 120, 160 and 200 V were applied for 3 min using constant-voltage mode. During this process, the solution was stirred with a magnetic bar. After treatment, the samples were rinsed with distilled water and dried. The conditions of MAF treatment are shown in Table 1. 2.3. Surface characterization The surface morphologies and elementary compositions were observed by scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS). The phases of MAF-treated Mg were determined by X-ray diffraction (XRD), using a scan rate of 1°/min by the Cu Kα line, generated at 40 kV and 30 mA.
the surfaces of the samples were observed by optical microscopy (Leica Microsystem ZE4D) and SEM. 2.6. In vitro test MC3T3-E1 cells were seeded onto pure Mg and MAF-treated Mg at a cell density of 2 × 104 cells/ml. Culture wells were used for the control group. Cell proliferation was evaluated by measuring mitochondrial dehydrogenase activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) after culturing the cells for 1, 2 and 3 days. For each group, samples were evaluated in triplicate. To prepare the MTT solution, MTT was first dissolved in PBS and then diluted by adding 100 ml of the solution to 900 ml of serum free a-MEM. Using the diluted solution, 1 ml was added to each sample in 24-well plates to form formazan. After 4 h at 37 °C in a CO2 incubator, the solution was aspirated and blue formazan crystals were dissolved in 1 ml DMSO for 20 min. Subsequently, 200 μl of the resulting solution was transferred to 96-well plates, and five data points were obtained from each sample. The optical density of the solution in each well was measured at a wavelength of 570 nm using a Microplate Reader (Molecular Devices, Sunnyvale). To observe the cell morphology after 3 days in culture, the samples were rinsed with PBS, fixed first in 2.5% glutaraldehyde solution for 2 h and then in 1% osmium tetroxide for 2 h, dehydrated in a gradient of ethanol/distilled water mixtures (30%, 50%, 60%, 70%, 80%, 90%, absolute ethanol) for 10 min each and then dried. Cell morphology was observed by SEM. 2.7. Statistical analysis Three replicate experiments were performed, all of which yielded comparable results. Unless otherwise specified, all data are expressed as the mean ± standard deviation (SD) of three independent experiments. Statistical analysis was conducted by one-way analysis of variance (ANOVA). Values of p b 0.05 were considered significant. 3. Results
2.4. Corrosion resistance test
3.1. Characterization of pure and MAF-treated Mg
The corrosion resistances of pure magnesium and MAF-treated Mg were measured by the potentiodynamic polarization test (PARSTAT2273). The Mg samples served as the working electrode, an Ag/AgCl/sat KCl electrode served as the reference electrode, and a platinum sample served as the counter electrode. The samples were immersed in SBF at room temperature, and the scanning rate was 3 mV/s.
Fig. 1 shows the surface morphologies of the samples treated by MAF at 120, 160 and 200 V. The samples showed a typical ceramic surface morphology formed by MAF, consisting of a porous and uniform fluoride layer formed on the magnesium surface. The MAF coating process started at 120 V, according to the observation of a micro arc on the Mg surface. The MAF120 surface had a long-slot shape structure, while the MAF160 and MAF200 surfaces were uniform and porous. As shown in Fig. 1, MAF200 was rougher and had bigger pores than MAF120 and MAF160. Fig. 2 shows the chemical compositions of the MAF-treated Mg by EDS. Magnesium treated at different voltages in the fluoride electrolyte solution showed the presence of both F and Mg peaks. This suggested that magnesium fluoride was formed in the coating layer by MAF. Fig. 3 shows the cross-section morphologies and EDS-mapping scanning results of the pure magnesium after MAF treatments under different conditions. Based on the morphology of MAF-treated Mg and the element distributions of F and Mg, it was possible to distinguish between the MAF-treated Mg coating layer and the substrate. The thickness of the MAF-treated Mg coating increased as the applied voltage increased. The thicknesses of the MAF120, MAF160 and MAF200 coatings were approximately 2.5 μm, 3.5 μm and 5.5 μm, respectively. Fig. 4 shows the XRD patterns of the pure and MAF-treated Mg samples. Compared with pure Mg, the patterns observed in the MAF-treated Mg samples clearly indicated the presence of tetragonal MgF2. The MgF2 phase was increased as the applied voltage increased (Fig. 3).
2.5. SBF immersion test In the SBF immersion test, pure Mg and MAF-treated Mg were vertically immersed in SBF and incubated at 37 °C. The ratio of SBF volume to sample area was 40 ml/cm2 [37]. pH values were measured using a pH meter (Thermo Electron Corporation Orion-525Aplus). To determine the rate of weight loss, the SBF was changed every week. Every week, one group of samples (n = 4) was taken out and cleaned in chromium trioxide (CrO3) solution, as recommended in ASTM G1-03 [38]. Then,
Table 1 Sample code and conditions of the pure magnesium and MAF-treated Mg. Sample code
Treatment process
Time (min)
Pure MAF120 MAF160 MAF200 MAF210
– Anodized by constant-voltage at 120 V Anodized by constant-voltage at 160 V Anodized by constant-voltage at 200 V Anodized by constant-voltage at 210 V
– 3 3 3 3
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Fig. 1. Surface morphology of the MAF-treated Mg of (a) MAF120, (b) MAF160, (c) MAF200 and (d) MAF210.
3.2. Corrosion behavior of pure and MAF-treated Mg Fig. 5 shows the results of the potentiodynamic polarization test. E0, icorr and Tafel analysis were used to evaluate the corrosion resistance of
the MAF-treated Mg. Higher E0 values indicate increasing corrosion, while icorr is inversely proportional to the amount of corrosion. The corrosion rate of the samples was calculated by Tafel analysis expression [39]. At a potential of − 1.38 V, the current of MAF120 rapidly
Fig. 2. EDS spectra of (a) pure magnesium, (b) MAF120, (c) MAF160, and (d) MAF200.
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Fig. 3. Cross-sectional image and EDS mapping assay of the MAF-treated Mg samples, (a) MAF120, (b) MAF160, and (c) MAF200.
increased up to −1.34 V. The sudden increase in the current means that the oxidation reaction is occurring too quickly. For MAF160 and MAF200, there was no sudden increase in the current, meaning that
Fig. 4. XRD patterns of (a) pure magnesium, (b) MAF120, (c) MAF160, and (d) MAF200.
corrosion occurred uniformly in these samples. The calculated corrosion rates are shown in Table 2. MAF200 showed the lowest current density and the highest potential, and the corrosion rate calculated by Tafel analysis was 0.019 mm/year. The corrosion rates of MAF160 and MAF120 were 0.024 mm/year and 0.030 mm/year, respectively.
Fig .5. Potentiodynamic polarization plot recorded for (a) pure magnesium, (b) MAF120, (c) MAF160, and (d) MAF200.
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Table 2 Results of the potentiodynamic corrosion test on pure magnesium and MAF-treated Mg of MAF120, MAF160, and MAF200. Code
E0 (V; I = 0)
icorr (μA/cm2)
CR (mm/year)
Pure MAF120 MAF160 MAF200
–1.842 –1.573 –1.558 –1.547
5.064 0.301 0.238 0.187
0.505 0.030 0.024 0.019
Fig. 6 shows the pH variation of pure and MAF-treated Mg immersed in SBF solution over the various immersion times. When magnesium is dissolved in SBF solution, the pH value of the solution rises. The pH value was highest in the solution containing pure magnesium, increasing from 7.4 to approximately 8.9 in the initial 2 days following immersion. After the initial 2 days, the pH value of the SBF increased slowly with increasing immersion time in all groups. In contrast, the pH values of the SBF solution with MAF-treated samples immersed were lower than that with pure Mg. Among the MAF-treated samples, the solution with MAF200 exhibited the lowest pH value, whereas that was MAF120 had the highest pH value, followed by that with MAF160. The increasing pH value indicates that Mg was dissolved in the SBF solution; the less Mg dissolved, the slower the pH increased. In other words, a slower increase in pH indicates better corrosion resistance. Fig. 7 shows the weight loss percentage of pure and MAF-treated Mg after immersion in the SBF solution. The weight loss value of pure Mg was clearly increased as immersion time increased. In contrast, the MAF-treated Mg prepared at a higher voltage had a slower rate of degradation. After immersion for 8 weeks, the weight loss values of pure Mg, MAF120, MAF160 and MAF200 were 55.6%, 5.3%, 4.9% and 1.5%, respectively. Fig. 8 shows the morphologies of pure and MAF-treated Mg after weight loss tests following immersion for 8 weeks. For pure magnesium, very serious corrosion was observed. For the MAF-treated Mg, large holes of corrosion were observed around the fluoride film on the MAF120 and MAF160 surface, and these holes were similar to those observed in pure Mg. However, for MAF200 surface, the fluoride film was uniform, without any obvious corrosive holes or cracks. 3.3. Cell proliferation on pure Mg and MAF-treated Mg Fig. 9 and Table 3 show the cell proliferation data of MC3T3-E1 cells cultured on pure magnesium and MAF200 for 1, 2 and 3 days. Cells grown in culture wells were used for the control group. Cell
Fig. 7. Weight loss variation over time of (a) pure magnesium, (b) MAF120, (c) MAF160, and (d) MAF200.
proliferation on the pure Mg decreased with as culture time increased. However, for MAF200, both cell adhesion and proliferation were significantly improved (p b 0.05). Cell viability on the MAF200 surface was greater than in the control and pure Mg groups, which is attributable to the anti-corrosive, micro-porous surface promoting cell growth. Fig. 10 shows the morphology of MC3T3-E1 cells cultured on pure magnesium and MAF200 for 3 days. There is an obvious difference in the response of the cells to pure Mg and MAF200: pure Mg generated grossly observable corrosion products and blocked adherence of MC3T3-E1 cells to the surface, while the cells on the MAF200 surface exhibited stretched shapes and several pseudopodia contacting the surface, indicating both increased adherence and survival. 4. Discussion Magnesium has strong chemical activity and low corrosion resistance. Therefore, surface treatment is necessary to improve the corrosion resistance. Most anodizing treatments for magnesium, such as MAO, generate an oxide coating layer, The MAO-treated Mg has porosity and high mechanical properties, but the layer created by this treatment has some drawbacks: the layer formed often has cracks [30,31], and magnesium oxides can react with bodily fluids. The main contents of bodily fluids are water and chlorine ions, which cause the severe corrosion [32,40]. On the other hand, the protective function of the fluxes can be explained by the Pilling and Bedworth factor [41,42]. F¼
Wo dm Wm do
where: Wo Wm dm do
Fig. 6. pH variation of (a) pure magnesium, (b) MAF120, (c) MAF160, and (d) MAF200. Samples were immersed SBF for 14 days. n = 3 samples per group. Values are expressed as the mean ± SD.
molecular weight of the surface oxide atomic weight of the metal density of the metal density of the oxide
According to this equation, a surface oxide is protective against a circumstance if the factor is above 1. Table 4 shows that the protection coefficient of an MgO film is less than 1. This might explain the formation of cracks on MAO-treated Mg, and cannot be used for bioimplants. In Table 4, the protection coefficient of the MgF2 film is greater than 1, meaning that MgF2 has a strong protective effect.
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Fig. 8. (a–d) SEM and (e–h) optical images of (a, e) pure magnesium, (b, f) MAF120, (c, g) MAF160 and MAF200 (d, h) after the weight loss test conducted for 8 weeks.
In the preliminary experiments, magnesium in the electrolyte began to spark at 120 V. The sparks indicated that micro arc oxidation of the magnesium surface had begun. Brighter, more intense sparks were observed when the voltage increased to 200 V. When 210 V was applied, the electrolyte began to boil and the sample was destroyed during the process (Fig. 1(d)), indicating that magnesium cannot be treated by MAF in saturated NH4HF2-solution at 210 V. Therefore, for this paper we chose the voltage range of 120–200 V. Micro arc discharge was maintained for 20–30 s with applied voltages of 120–200 V. After this period of time, the discharge phenomenon disappeared, indicating that in constant voltage mode, a fluoride coating of regular thickness had formed on the magnesium surface. This coating created a double electrochemical layer, thereby decreasing the electrochemical reactivity of the magnesium surface. In the MAF process, fluoride ions move to the anode and react with Mg to form the MgF2 coating layer. At high-voltage conditions, the anode surface had a higher current and sparked more intensely, resulting in the formation of a ceramic coating. In this study, pure and porous MgF2 coatings were successfully fabricated (Figs. 3–5) by MAF processing in saturated NH4HF2-electrolyte
solution. The porous surface has greater biological adaptability than the smooth surface because surface area affects the ability of cells to attach. The larger surface area could cause faster adherence of surrounding tissue to the surface [43,44]. Further, compared with a smooth surface, a rough surface can allow cells to stick more easily and begin to grow [45]. In addition, compared with the processing time of soaking Mg in HF solution [16–18], MAF treatment greatly reduced the processing time, which is highly convenient for research and development. In vitro corrosion test data showed that the fluoride-coated Mg and its alloy had great corrosion resistance. The results of the corrosion test allow us to conclude that that the corrosion resistance increased with the coating thickness. This phenomenon has also been reported in other literature about fluoride coatings [17,31]. Tingting Yan et al. [17] and X.N. Gu et al. [31] found that increasing the thickness of fluoride coating had an impact on the Mg surface, improving the corrosion resistance. According to the literature [8], hydrogen gas bubbles were observed just after Mg was immersed in SBF solution. However, in this study, gas bubbles were not observed when MAF-treated Mg was immersed into SBF, indicating that the releasing rate of gas can be controlled by MAF treatment. The human body endogenously produces gas that is slowly discharged from the body via sweat glands and bodily fluids. Therefore, if the amount of gas released from the implants is small, it will be easily discharged from the body. There have been some reports in the literature [46,47] concerning Mg implants given to rats by μ-CT. A large amount of gas was formed near the implants, and the gas slowly dissipated over time. In other words, as MAF-treated Mg improved corrosion resistance, the negative influence of gas will no longer be a problem for the surrounding tissue. For the MAFtreated Mg, pitting corrosion was found after the 8 week immersion test for MAF120 (Fig. 8(f)) and MAF160 (Fig. 8(g)). However, MAF200 showed more mild and uniform corrosion (Fig. 8(h)). Therefore, corrosion resistance increased with increasing MAF thickness. In this work, MAF200 was better than the others in all aspects of performance. The corrosion rate and weight loss values for MAF200 were obviously the smallest, and only some bubble attached on the surface of MAF200
Table 3 MTT results of the mean OD value on the pure magnesium and MAF200 with control for 1, 2 and 3 days.
Fig. 9. Cell proliferation of MC3T3-E1 cells cultured for 1 day on pure magnesium, MAF200, and the control surface. *p b 0.05.
Group
1 day
2 days
3 days
Control Pure MAF200
0.691 0.173 0.755
1.228 0.148 1.416
1.378 0.131 1.428
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Fig. 10. Cell morphology of cultured MC3T3-E1 cells after 3 days on (a) pure magnesium, and (b) MAF200; high magnification (a-1) pure magnesium and (b-1) MAF200.
when put into the simulated body fluid for 8 weeks. Thus, we show that MAF200 is suitable for use as an implant material. Toxicology issues related to corrosion products from highly biodegradable metals require special attention. In the results shown here, the cell survival rate, as measured by an MTT assay, decreased on pure Mg. It is possible that Mg has some toxic effects on cells. In highly alkaline solutions, increasing the pH value results in cell death. X.N. Gu et al. [31] also tested the cytotoxicity of Mg and its alloy in an indirect assay and found that cell proliferation was not decreased. This implies that the decrease in cell viability is due to the increased pH and not to Mg toxicity. If a physical reaction occurs on the wall surface, cell growth on the surface will be affected. In the results of this paper, cells were not found growing on pure Mg, likely due to the large amount of bubbles generated from the Mg surface upon immersion into the medium [8]. Additionally, the Mg surface was highly corrosive in the medium and constantly dissolved. So, the cells may fall off of the Mg surface due to the bubbles and corrosion products, explaining the decreased growth in the pure Mg group seen in the MTT results. X.N. Gu et al. [31] observed no adherent cells and a decreased MTT value on untreated Mg-Ca alloy. However, in our experiment, the cell adhesion and proliferation behaviors are significantly improved for MAF200 compared with pure Mg and the control
group (p b 0.05), indicating that the MAF-treated Mg provides a biologically favorable environment. Because the MAF-treated Mg is mainly composed of MgF2 formed on the Mg substrate, it has high corrosion resistance. In addition, fluorine has anti-bacterial properties and can be beneficial for cell growth [48,49]. Martina Thomann et al. [36] found in clinical trials that new bone tissue was generated around MgF2-coated Mg implants. Therefore, the MgF2 coating raised the corrosion resistance of the magnesium metal, thereby improving biocompatibility. 5. Conclusions In this work regarding MAF treatment of Mg, we conclude the following: The MgF2 layer coating the pure Mg was successfully fabricated by MAF treatment. The MAF-treated Mg coating is porous with a thickness ranging from 2.5 to 5.5 μm. The MAF-treated Mg, in comparison with other samples, showed significantly improved corrosion resistance. After the immersion test in SBF, the MAF120 and MAF160 samples showed pitting corrosion, while the MAF200 sample had a more mild and uniform surface in comparison. Cell proliferation on the MAF-treated Mg was improved over that on the pure Mg. Acknowledgments
Table 4 Protection coefficient of surface films on magnesium melts [42]. Surface film formed
Protection coefficient
Mg + O = MgO Mg + H2O = MgO + H2 3Mg + N2 = Mg3N2 Mg + S = MgS 3Mg + 2BF3 = 3MgF2 + 2B Mg + 2HF = MgF2 + H2 3MgO + 2BF3 = B2O3 + 3MgF2 Mg + CO2 = 2MgO + C 2Mg + CO = MgO + C
0.71 0.71 0.79 1.26 1.32 1.32 3.1 0.9 1.08
This work was financially supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2011-0028709) and by the Basic Science Research Program through the NRF, which was funded by the Ministry of Education, Science and Technology (2010-0023901) and Regional Strategic Industry project (2013-R0002274). References [1] J. Vormann, Mol. Asp. Med. 24 (2003) 27–37. [2] S. Zhang, et al., Acta Biomater. 6 (2010) 626–640.
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