Biocompatibility and in Vitro Degradation Behavior of Magnesium

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Biocompatibility and in vitro degradation behaviour of magnesium-calcium alloy coated with calcium phosphate using an unconventional electrolyte Bobby Kannan Mathan, Rhys Walter, and Akiko Yamamoto ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00343 • Publication Date (Web): 24 Nov 2015 Downloaded from http://pubs.acs.org on December 3, 2015

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ACS Biomaterials Science & Engineering

Biocompatibility and in vitro degradation behaviour of magnesium-calcium alloy coated with calcium phosphate using an unconventional electrolyte M. Bobby Kannan1, 2, R. Walter1, A. Yamamoto2 1

Biomaterials and Engineering Materials (BEM) Laboratory,

College of Science, Technology and Engineering, James Cook University, Townsville, Queensland 4811, Australia 2

Biometals Group, International Centre for Materials Nanoarchitectonics,

National Institute for Materials Science (NIMS), Tsukuba, Ibaraki 305-0044, Japan

Abstract: Calcium phosphate (CaP) was electrochemically coated on a magnesium-calcium (Mg-Ca) alloy using an unconventional electrolyte and a pulse-potential method. The CaP particles of the coating were relatively large, flat and irregularly oriented, however covered the entire alloy surface with a coating thickness of 5 µm. Cytocompatibility tests using L929 cells inoculated in Eagle minimum essential medium supplement with 10% (v/v) fetal bovine serum (E-MEM+FBS) revealed that CaP coating improvement the cytocompatibility of the alloy. It also showed effective suppression of Mg2+ ion release from the substrate of the coated alloy and consequently reduced the pH increase of the medium. In-vitro degradation experiments using electrochemical techniques in simulated body fluid (SBF) also suggested significant enhancement of the alloy degradation resistance by CaP coating. Potentiodynamic polarisation results showed that the corrosion current density (icorr) of the coated alloy was 1

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~95 % lower than that of the bare metal. Electrochemical impedance spectroscopy (EIS) results revealed that the polarisation resistance (RP) of the coated alloy was more than an order of magnitude higher than that of the bare metal after 2 h immersion in SBF. Interestingly, after 72 h immersion, the measured RP had decreased by ~82 % and the coating appeared cracked and damaged. The results suggest that SBF is more aggressive than EMEM+FBS cell culture medium. Key Words: Magnesium alloy, Biomaterials, Cytocompatibility, Calcium phosphate, Degradation

INTRODUCTION Biodegradable metallic materials have been gaining a significant amount of interest over the past few years for mini-implant applications1. In fact, metallic magnesium is in the forefront due to its attractive mechanical and biocompatible properties for such applications. A significant amount of work has been done on alloying magnesium to reduce its rapid degradation under physiological conditions. While alloying with elements such as aluminium, zinc and rare-earth elements improves the degradation resistance2-4, the localised degradation susceptibility of these alloys is a major concern. Corrosion pits can potentially act as stress risers and lead to the propagation of cracks and implant failure. The phenomenon is commonly known as environment-assisted cracking (EAC), and unfortunately magnesium and its alloys are prone to EAC in chloride-containing environments5, 6. In vitro degradation studies have also shown that localized degradation affects the mechanical integrity of magnesium-based materials2, 7. In order to delay the general and localized degradation of magnesium and its alloys, there has been growing interest in biocompatible and biodegradable coatings. A wide range of coatings has been studied to this end, including plasma electrolytic oxidation8-10, biodegradeable

2


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polymers11-13, and calcium phosphate deposition14-16. Calcium phosphates (CaPs) are wellknown for their high bioactivity and biocompatibility, and have already been used as coating materials on metallic implant materials such as titanium to improve osteointegration and osteoconductivity17, 18. There are various methods available for the coating of CaP on metallic substrates, such as high-temperature sputtering and plasma spraying19, 20. However, these are often high temperature techniques, potentially resulting in a decomposition and nonuniformity of the coating when used on a magnesium substrate. Electrochemical coating of CaP is attractive for magnesium-based materials since it can be done rapidly and at room temperature, and possible to coat complex geometries such as plates and screws implants. There has been some preliminary work done on galvanostatic21,

22

and potentiostatic7,

23

deposition of CaP on magnesium and its alloys. However, a limitation of these electrochemical techniques is that the rapid deposition of CaP results in the evolution of large amounts of hydrogen gas (equation 2), as can be understood from the following reactions24: → +

(1)

2 + 2 → + 2

(2)

+ → +

(3)

+ + 2 → · 2

(4)

The hydrogen gas bubbles build-up on the surface potentially detaches and damages the CaP layer, resulting in a loosely packed and inhomogeneous structure. Further, in the case of potentiostatic deposition, a negatively charged layer forms across the surface, which impedes the adherence of ions and results in poor deposition25. These issues can be overcome by utilising a pulsed-potential waveform, since the TimeOFF results in dissipation of the

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negatively charged layer around the cathode, and allows the ions to migrate to depleted areas in the bath25. Kannan and Wallipa26 compared constant and pulsed-potential CaP coatings on AZ91 magnesium alloy. It was reported that the pulsed-potential technique produced more densely packed coating, which was able to provide significant improvement in the degradation resistance when compared to potentiostatic deposition. Another study by Kannan14 showed that CaP coating produced on AZ91 magnesium alloy in an electrolyte containing ethanol in addition to Ca(NO3)2 and NH4H2PO4 improved the degradation resistance when compared to the conventional aqueous solution coating. Further, the same author15 reported the synergistic effect of pulsed-potential technique and the electrolyte containing ethanol on CaP formation and its performance on AZ91 magnesium alloy. There has been a recent shift toward aluminium-free magnesium alloys for biodegradable implant applications due to potential toxicity of aluminium27. Magnesium-calcium (Mg-Ca) alloys have become one of the popular base materials studied for biodegradable implant applications due to the attractive properties of Ca as an alloying element, such as good biocompatibility28,

29

. Chun-Yan et al.30 compared potentiostatically coated brushite

(CaHPO4·2H2O) coatings on AZ31 and Mg-1.0Ca alloys in Hank’s solution. Although they reported that the coatings improved the degradation resistance of both the alloys, the CaPcoated Mg-1.0Ca alloy exhibited inferior resistance to degradation (based on hydrogen evolution measurements) as compared to the uncoated AZ31 sample. This suggests that the degradation resistance of the base metal is critical for forming CaP coating with better performance. AZ series alloys are relatively more passive than Mg-Ca alloys due to the presence of aluminium, and hence will undergo less dissolution during the coating process and produce a better coating. It is hypothesized that a less aggressive coating electrolyte, 4


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instead of the conventional aqueous solution, could produce a better coating on Mg-Ca alloy. Hence, the aim of this study was to produce a high-performance CaP coating on Mg-Ca substrate using an unconventional electrolyte and a pulsed-potential method. The cytocompatibility of the coating was evaluated using murine fibroblast L929 by WST-1 assay. The in-vitro degradation behaviour was investigated using electrochemical techniques. MATERIALS AND METHODS Material and CaP coating In this study, an Mg-Ca alloy (1 wt. % Ca) was used as the substrate material (Source: Helmholtz-Zentrum Geesthacht, Germany). CaP coating was done using a typical threeelectrode system, with an Ag/AgCl (in saturated KCl) reference electrode and graphite counter electrode. The coating solution was 0.1 M Ca(NO3)2, 0.06 M NH4H2PO4 and 30 % v/v ethanol15. Coating was carried out under a pulsed potential of -3/0 V with a 35 % duty cycle (10/18.5 ms ON/OFF) for 60 minutes. Prior to the coating, the samples were incrementally ground from 120 to 1200 grit SiC paper. After grinding, the samples were ultrasonically cleaned in ethanol and dried. The chemical nature of the coating was determined using X-ray diffraction (XRD) analysis (Diffractometer Model: Rint-Ultima III, Rigaku Co., Tokyo, Japan; operated at 40 kV-40 mA with Cu-Kα for the diffraction angle in the range from 10◦ to 60◦) and Fourier transform infrared (FTIR) spectroscopy analysis (Perkin Elmer spectrum 100 FTIR spectrometer). The coating thickness was measured using a Dual Scope® coating thickness gauge (measurement accuracy ±1 µm), and the coating morphology prior to and following immersion tests was studied using a Jeol JSM5410L Scanning Electron Microscope (SEM). Cytocompatibility testing

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The cytocompatibility of the coated samples was tested using L929 murine fibroblasts, which is one of the recommended cell lines by ISO10993-5, “Biological Evaluation of Medical Devices-Part 5: Tests for in vitro cytotoxicity”. For comparison, the cytocompatibility of the base materials was also tested. Following sterilization by ethylene oxide gas, the samples were placed in a deep glass dish, and L929 cells were inoculated at a density of 1,000 cells/ml in 27.5 ml of Eagle minimum essential medium supplemented with 10% (v/v) fetal bovine serum (E-MEM+FBS). The formulation of E-MEM used in this study (Eagle’s MEM “Nissui”1, Nissui Pharmaceutical Co. Ltd., Japan) is shown in Table 1. As a control, uncoated and coated samples were prepared in the same manner and immersed in the same amount of E-MEM+FBS. The samples were incubated under cell culture condition (37 °C, 5 % CO2) for 1, 4 and 7 days without medium exchange. Table 1. The formulation of Eagle’s minimum essential medium (Eagles’ MEM “Nissui”1). Components CaCl2 KCl MgSO4 NaCl NaHCO3 NaH2PO4 D-Glucose Phenol Red

Conc. (mg/L) Components Conc. (mg/L) 200 L-Threonine 48 400 L-Tryptophan 10 93.5 L-Tyrosine 36 6800 L-Valine 46 2200 Succinic acid 75 115 Sodium Succinate·6H2O 100 1000 D-Ca Pantothenate 1 6 Choline Bitartrate 1.8 126 Folic Acid 1

L-Arginine·HCl L-Cystine·HCl·H2O L-Glutamin L-Histidine·HCl·H2O L-Isoleucine L-Leucine L-Lysine·HCl L-Methionine L-Phenylalanine

31.4 292 42 52 52 73 15 32

i-Inositol Niacinamide Pyridoxal HCl Riboflavin Thiamine HCl Biotin kanamycin

2 1 1 0.1 1 0.02 60(titer)

6


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After a certain incubation period, 5.4 mL of the supernatant liquid was poured into a new culture vessel, and then 0.6 mL of a mixed solution of 1 mM WST-1 [2-(4-iodophenyl)-3-(4nitrphenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]42 and 0.2 mM 1methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS) in PBS(-) was added. Each sample was then aseptically transferred into the vessel containing corresponding supernatant liquid and placed into a CO2 incubator for 4 h. After the second incubation, the absorbance of the supernatant liquid was measured at 450 nm by a microplate reader (Multiskan FC, Thermo Scientific, USA) to estimate the relative viability of cells (RVC) on the sample by following equation: RVC = As/Ss – Ac/Sc, where As and Ac are the absorbance of the supernatant liquid in the sample and control wells, respectively. Ss and Sc are the top surface areas of the samples with and without cells, respectively. Experiments were performed in triplicate for each incubation period with and without cells. To examine the morphology of cells, the specimens after cell culture were fixed with 25% glutaraldehyde solution for 10 min and stained by 10% (v/v) Giemsa’s staining solution (Giemsa’s azur eosin methylene blue solution for microscopy, MERCK Art.1.09204, Germany) for 15 min43. An optical microscope (Axiotech 100, Carl Zeiss AG, Germany) was used to observe the air-dried stained samples, and digital images were recorded by a CCD camera (DS-5M, Nikon Co. Ltd, Japan). Quantification of the Mg2+ ions in the collected portions of E-MEM+FBS during the cell culture/immersion was performed by a colorimetric method using xylidyl blue-I31, 32, which is applied for the measurement of magnesium in serum33. The kit, “Magnesium B-test Wako” (Wako Chemicals, Tokyo, Japan), comprised of a one-reagent mixture containing 0.1 mM of xylidyl

blue-I

[1-azo-2-hydroxy-3-(2,4-dimethylcarboxyanilido)

naphthalene-1-(2-

hydroxybenzene-5-sulfonate], 0.045 mM of EGTA [ethylene glycol bis (2-amino-ethylether)7



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N,N'-tetraacetic acid], and non-ionic surfactant Triton X-100, was used. The calibration curve was prepared by adding MgSO4·7H2O to be 5–50 µg Mg/mL in E-MEM+FBS. After mixing the aliquot of the collected E-MEM+FBS to the colorimetric reagent at the ratio of 1:150, the absorbance at 620 nm was measured. The concentration of the Mg2+ ion was then calculated using the calibration curve. Control samples following the immersion test under cell culture condition were observed by SEM (Mini-scope TM3000, Hitachi High Technologies Co. Ltd., Hitachi, Japan) and analyzed using XRD. The pH of E-MEM+FBS (27.5ml) was monitored by a pH meter (TPX-999i, Toko Kagaku Co. Ltd. Tokyo) under cell culture condition (37 °C, 5 % CO2) for the first 3 days of immersion. The pH electrode was set 1 mm above of the immersed sample. Experiments were performed in duplicate to confirm reproducibility. In-vitro degradation testing Electrochemical degradation experiments were carried out in triplicate using a model ACM Gill AC potentiostat. The electrolyte was simulated body fluid (SBF) buffered with tris(hydroxymethyl)aminomethane (TRIS) buffer at 37 °C and pH 7.40. The composition of SBF is shown in Table 234. Samples were immersed in SBF for 2 h prior to testing for establishing a relatively stable open circuit potential. Electrochemical impedance spectroscopy (EIS) was performed over the frequency range of 105 Hz to 10-2 Hz at 5 mV AC amplitude. Equivalent circuit modelling of the EIS results was done using ZSimpWin V. 3.21 software. Potentiodynamic polarisation experiments were carried out at a scan rate of 0.5 mV/s. Post-degradation analysis was carried out using XRD, FTIR and SEM. Table 2. Chemical composition of the simulated body fluid (SBF)34. Reagent

Amount (/L)

8


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NaCl

8.036 (g)

NaHCO3

0.352 (g)

KCl

0.225 (g)

K2HPO4·3H2O

0.230 (g)

MgCl2·6H2O

0.311 (g)

1 M HCl

40 (mL)

CaCl2

0.293 (g)

Na2SO4

0.072 (g)

TRIS buffera

6.063 (g)

a

TRIS buffer = tris(hydroxylmethylaminomethane)

RESULTS Coating characterisation

The average coating thickness of the deposited CaP layer was measured to be 5.68 ± 0.38 µm. The FTIR spectra (Fig. 1a) of the coating showed strong bands at 1122, 1052 and 984 cm−1 corresponding to phosphate35, 36. Also, bands at 1631 cm-1 and 874 cm-1 corresponding to hydroxide and carbonate groups, respectively, were also observed36. XRD analysis (Fig. 1b) confirmed that the compound is CaP, i.e., CaHPO4.2H2O (ICDD PDF#00-009-0077). However, the hydroxide and carbonate groups observed in the FTIR spectra, which could be Mg(OH)2 and MgCO3, were not evident in the XRD probably due to the amorphous nature of the products. The morphology of the CaP coating obtained from SEM imaging is shown in Fig. 2, which reveals large, flat and irregularly oriented CaP particles. However, the base metal was completely covered by the CaP particles. Interestingly, CaP layer coated under similar conditions on AZ91 alloy showed much denser packing and the particles were larger 9



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in size15. This suggests that the morphology of the CaP coating using electrochemical method depends on the electrochemical behaviour of the base material.

Figure 1. (a) FTIR and (b) XRD spectra of the CaP coating on Mg-Ca alloy.

Figure 2. SEM micrographs of the CaP coating on Mg-Ca alloy: (a) low magnification shows complete coverage of the base material, and (b) high magnification shows large, flat and irregularly oriented particles.

Cytocompatibility testing

Fig. 3 shows the optical micrographs of the coated and bare samples after the cytocompatibility test. As expected, bare alloy showed poor cytocompatibility; only a few 10


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L929 cells could be observed on the bare alloy surface and the number of cells did not increased along the incubation period. However, the CaP coating significantly improved the cytocompatibility of the base material. Quantification of cell growth by WST-1 assay (Fig. 4) also confirms the higher cytocompatibility of CaP-coated alloys as compared to that of the bare alloy. This is mainly attributed to the degradation behavior of the coated alloy. Fig. 5 shows that in the case of bare alloy, the pH of the culture medium increased, whereas for the CaP-coated alloy the pH was around 7.4 remaining relatively stable. The results of Mg2+ release for the bare and CaP-coated alloy with cells in this condition are shown in Fig. 6. The Mg2+ release was much higher for the bare alloy than the CaP-coated alloy, which is in agreement with the higher pH of the culture medium immersing bare alloy (Fig. 5).

Figure 3. Optical micrographs of: (a-c) CaP-coated Mg-Ca alloy and (d-f) bare Mg-Ca alloy, after staining cells in blue with a dye.

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Figure 4. Results of the WST-1 assay for bare alloy and CaP-coated Mg-Ca alloy samples (mean±SD, n=3).

Figure 5. Typical results of the pH measurement of cell culture medium during initial 3 d of immersion under cell culture condition (without cells).

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Figure 6. Mg2+ release into the culture medium from bare alloy and CaP-coated Mg-Ca alloy samples from cell culture tests (mean±SD, n=3).

The SEM observation of the bare alloy after 7 d immersion in the E-MEM+FBS under cell culture condition revealed the occurrence of pitting attack and formation of insoluble salt layers on the surface (Fig. 7a, b). However, the CaP-coated alloy after 7 d immersion revealed the partial dissolution of CaP coating layer (Fig. 7c, d). The formation of insoluble salt layers with needle or hair-like shapes was also observed. XRD analysis of the samples immersed into E-MEM+FBS for 7 d showed reduction of the CaP coating peak after immersion (Fig. 8). Interestingly, the insoluble salts considered being phosphate and/or carbonate37 were not detected by XRD analysis, possibly due to their low crystallinity.

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Figure 7. SEM micrographs of: (a, b) CaP-coated alloy, and (c, d) bare Mg-Ca alloy after 7 d of immersion in E-MEM+FBS under cell culture condition.

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Figure 8. XRD spectra of CaP-coated and bare Mg-Ca alloy before and after 7 d of immersion in E-MEM+FBS under cell culture condition (without cells).

In-vitro degradation

The potentiodynamic polarisation curves of the bare alloy and CaP-coated alloy are shown in Fig. 9 and the corresponding electrochemical data are given in Table 3. CaP coating to the alloy significantly improved the degradation performance, i.e., by decreasing the corrosion current density (icorr) from 90 µA/cm2 to 4.1 µA/cm2, which is ~95 % reduction. The coating also shifted the corrosion potential (Ecorr) towards the noble direction, i.e., -1.64 VAg/AgCl to 1.46 VAg/AgCl. A paired two-tailed t-test showed a not quite significant shift in the Ecorr (p=0.0529), however there was a highly significant reduction in the icorr (p=0.0005).

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Figure 9. Potentiodynamic polarisation curves for the bare alloy and CaP-coated Mg-Ca alloy in SBF (mean±SD, n=3).

Table 3. Electrochemical corrosion parameters of bare alloy and CaP-coated alloy obtained from potentiodyanamic polarisation curves in SBF (mean±SD, n=3). Sample

Ecorr (VAg/AgCl)

icorr (µA/cm2)

Mg-Ca alloy

-1.63±0.06

90±14.1

CaP-coated alloy

-1.46±0.09

4.1±1.7

Fig. 10 shows the EIS plots from 2-72 h immersion of bare metal and CaP-coated alloy in SBF. The polarisation resistance (RP) calculated based on equivalent circuit modelling is 16


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shown in Fig. 11, and the open-circuit potential (OCP) measurements are shown in Fig. 12. The complete data of the modelling is presented in Table 4. The Nyquist plots for both the bare and CaP-coated Mg-Ca alloy were modelled using the equivalent circuit Rs(Q1(R1(Q1R1))) as shown in Fig. 11, where R represents resistors and Q represents constant phase elements. For the bare metal, Rs is the solution resistance, Q1 represents double layer capacitance, R1 represents charge transfer resistance, and R2 and Q2 represent film effects. For the CaP-coated Mg alloy, Rs is the solution resistance, Q1 and R1 represent the porous outer layer, and R2 and Q2 represent the compact inner layer. It can be seen that for the bare alloy, the RP initially starts at ~413 Ω.cm2, and increases to ~1305 Ω.cm2 after 24 h immersion. The RP then decreases across the remaining immersion time to ~695 Ω.cm2 after 72 h. All the plots show a high frequency capacitive loop, which correspond to charge transfer resistance and double layer capacitance4. There is a mid-frequency capacitive loop for the 2 h immersion curve, which is indicative of the relaxation of mass transport through the corrosion product layer4. The mid-frequency loop is no longer visible from 8 h onwards, suggesting that the partially protective layer is no longer present. The RP for the CaP-coated alloy was initially much higher than the bare alloy, at an RP of ~6452 Ω.cm2 (~15 times higher). This value rapidly decreased by 82% over the immersion period, exhibiting an RP of 1185 Ω.cm2 after 72 h immersion, which was only ~70 % higher than the bare alloy. A paired two-tailed t-test between the bare and coated samples showed a significant result of p=0.0052 for the initial 2 h measurements. While the RP values began to converge over the immersion period, the measured difference was still significant after 72 h immersion (p=0.0434). All EIS plots for the CaP-coated alloy showed a similar shape; a single high and mid-frequency capacitive loop corresponding to an outer layer and compact inner layer characteristic of ceramic coatings22, 38.

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Figure 10. EIS plots for: (a) bare alloy, and (b) CaP-coated Mg-Ca alloy over the 72 h immersion period in SBF.

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Figure 11. Polarisation resistance (RP) vs. time from EIS data of bare alloy and CaP-coated Mg-Ca alloy in SBF (mean±SD, n=3).

Figure 12. Open-circuit potential (OCP) measurements of the bare alloy and CaP-coated MgCa alloy during immersion in SBF. 19



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Table 4. Modelling data from the EIS plots of bare alloy and CaP-coated alloy (mean±SD, n=3).

Time

Mg-Ca alloy

R1

CPE1

CPE2

n

Sample (h)

(Ω-1.cm-2.s-n x10-6)

2

815.5 ± 110.3

8

R2

RP

(kΩ.cm2)

(kΩ.cm2)

n (kΩ.cm2)

(Ω-1.cm-2.s-n x10-6)

0.52

0.021 ± 0.015

17.8 ± 4.4

0.88

0.392 ± 0.003

0.413 ± 0.011

95.2 ± 20.0

0.53

0.113 ± 0.050

7.0 ± 2.4

0.98

1.077 ± 0.229

1.190 ± 0.140

24

45.7 ± 32.0

0.77

0.062 ± 0.020

25.0 ± 11.0

0.88

1.243 ± 0.020

1.305 ± 0.003

48

180.9 ± 127.2

0.59

0.041 ± 0.016

64.0 ± 8.7

0.80

0.791 ± 0.067

0.832 ± 0.051

72

0.8 ± 0.1

1.00

0.016 ± 0.001

88.5 ± 12.2

0.74

0.679 ± 0.020

0.695 ± 0.021

2

6.6 ± 4.6

0.81

0.338 ± 0.111

22.6 ± 7.5

0.66

6.114 ± 0.405

6.452 ± 0.294

8

22.5 ± 34.2

0.58

0.331 ± 0.148

12.5 ± 1.5

0.80

3.785 ± 0.017

4.116 ± 0.131

24

12.5 ± 7.4

0.71

0.074 ± 0.006

58.8 ± 1.5

0.75

2.063 ± 0.079

2.137 ± 0.074

48

0.4 ± 0.1

1.00

0.071 ± 0.002

58.2 ± 2.1

0.72

2.680 ± 0.203

2.751 ± 0.201

72

28.7 ± 4.5

0.63

0.053 ± 0.007

88.1 ± 15.0

0.74

1.132 ± 0.012

1.185 ± 0.058

CaP-coated Mg-Ca alloy

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Post-degradation characterisation The FTIR spectra and XRD analysis of the CaP-coated alloy after in-vitro degradation are shown in Fig. 13 The hydroxide and carbonate bands at 1631 cm-1 and 874 cm-1 observed in Fig. 1 are no longer visible. The strong phosphate bands have also merged, showing only a broad phosphate band at approximately 1000 cm-1. This reduction in bands suggests that there has been a change in the coating structure, due to either incongruent dissolution and/or reprecipitation of new CaP phases onto the surface22, 39. This was likely caused by localised pH increase due to hydrogen evolution reaction (equation 6) as a consequence of alloy dissolution. This reduces the solubility of calcium and phosphate ions in the solution, resulting in a subsequent precipitation onto the surface39. XRD analysis (Fig. 13) did not shown any strong peak of CaHPO4.2H2O, which suggests that the precipitates/layer on the surface are/is possibly amorphous in nature.

Figure 13. (a) FTIR and (b) XRD spectra of the CaP-coated Mg-Ca alloy following 72 h immersion in SBF.

The morphology of the CaP coating following 72 h immersion is shown in Fig. 14 a, b. It can be seen that there was noticeable damage to the coating across the entire surface, resulting in 21



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a high amount of cracking and skeletal, frond-like areas. This change in the morphology of the coating occurs once substrate dissolution initiates, forming Mg(OH)2, which provides favourable sites for the reprecipitation of hydroxyapatite nuclei28, 40. These areas are points of rapid and dense crystal growth resulting in the tightly packed frond areas, while the surrounding areas become cracked as Mg(OH)2 converts to highly soluble MgCl2. A comparison with a corroded bare alloy (Fig. 14 c, d) suggests that that there is no substrate surface directly exposed. The bare alloy underwent significant damage across the entire surface, with some areas of heavy pitting attack visible. It follows that since the CaP layer is still covering the entire substrate, the drop in RP observed in Fig. 10 is due to penetration of electrolyte through the coating, rather than direct dissolution of the coating itself.

Figure 14. SEM micrographs of: (a, b) CaP-coated alloy, and (c, d) bare Mg-Ca alloy following 72 h immersion in SBF.

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DISCUSSION

The degradation behaviour of the bare metal follows the progression typically seen in magnesium alloys, i.e., an increase in RP early on in the immersion due to film formation which is partially protective, followed by a slight decrease over time as this layer is deteriorated. The CaP layer, however, displays a much more rapid loss in its protection than expected. Calcium phosphates have been shown to have a very low rate of dissolution under physiological conditions40, 41. Conversely, the EIS results from this study show rapid drops in the measured resistance. This suggests penetration of the electrolyte through the CaP layer. Once the electrolyte comes into contact with the substrate, Mg dissolution would result in H2 evolution, as shown in the half reactions below:

Mg → Mg + + 2e

(5)

2H O + 2e → H + 2OH

(6)

As the H2 gas detaches, it breaks through the coating layer, compromising the protective nature of the CaP coating. There was possible penetration of the electrolyte through the CaP layer, which is supported by the rest potential measurements (Fig. 12) prior to each EIS tests. Across the entire immersion period, there is a relatively stable separation in potentials (~100 mV) between the bare and the coated alloy. If there was a significant breakdown of the CaP layer to explain the rapid drop in RP seen in Fig. 12, the rest potentials would begin to converge to that of the bare alloy. However, the near constant 100 mV potential difference between the two samples, which suggests that the CaP layer is still intact, albeit penetrated by the electrolyte.

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Regardless, this layer is effective enough to improve the initial cytocompatibility of the MgCa alloy. It is reported that the suppression of initial pH increase due to Mg dissolution is a major source of improvement in the cytocompatibility of pure Mg and Mg alloys44,

45

. The

same mechanism is also suggested in this study since suppression of Mg2+ release and pH increase was observed for CaP-coated samples in Figs. 5 and 6.

Interestingly, it was noted that the CaP coating on the alloy was more stable in E-MEM+FBS solution as compared to that in SBF. E-MEM was basically developed based on human blood plasma, and maintains a neutral pH of 7.4 via a carbonate buffer system. The bicarbonate concentration in E-MEM is much higher (2200 mg/L) when compared to SBF (352 mg/L), and also 5% CO2 atmosphere is mandatory in the former. Supplementation with FBS adds serum proteins to E-MEM, which makes it closer to human blood plasma37. It is reported that addition of FBS suppressed the dissociation of pure Mg and encouraged the precipitation of insoluble salts containing Ca and P37. However, SBF is a simple solution with inorganic salts and buffered with TRIS to maintain the pH of the solution. It is clear that absence of serum proteins and 5% CO2 atmosphere has led to slightly higher degradation of the CaP-coated alloy in SBF. This confirms that SBF is more aggressive than E-MEM+FBS cell culture medium. However, evaluation of magnesium-based materials in SBF is quick and less expensive, and further the trend in the degradation behavior of the alloy in the two solutions was not contradicting, hence SBF can be used in the initial screening of magnesium-based materials. In the present study, electrochemical measurement of uncoated and CaP-coated samples was performed in a SBF up to 3 days. Cytocompatibility evaluation of the samples was carried out up to 7 days of incubation. Both evaluation techniques successfully proved the effectiveness of CaP coating for the suppression of initial degradation of the substrate and subsequent 24


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improvement in the cytocompatibility. However, in case of biodegradable device applications such as bone fixture or cardiovascular stents, the implantation period is much longer than 7 days. Microvascular circulation, surrounding tissue reaction and other effects may influence the degradation behavior of the implants. The electrochemical measurement is useful to detect the electrochemical reaction at the sample surface, but may not accurately reflect the conditions in the human body. In this study, these measurements were performed in a SBF only containing inorganic salts. In the cytocompatibility evaluation, the experiment was performed in cell culture medium similar to the human body fluid under 5% CO2 atmosphere (to enforcing carbonate buffer system) and with living cells. This environment is very close to that of the implanting situation, and applying this cell culture technique to electrochemical measurement can be another new standard to evaluating the corrosion behavior of Mg alloy in a biological environment. Preliminary results of electrochemical impedance measurement of Mg alloys with living cells have been reported elsewhere, showing an initial protective effect of cells up to 6 hrs46 and an accelerating effect afterward47. However, the majority of cell culture testing is performed with a limited period of incubation since culture medium exchange is required to maintain the cells in that culture condition. In human tissue, however microvascular circulation plays this role. Solving this issue will enable a more precise simulation of the human tissue environment and thus contribute to the better understanding of biodegradation of Mg and surface engineered Mg alloys.

CONCLUSIONS

In this study, CaP was successfully coated on a magnesium-calcium alloy using a pulsepotential method in an ethanol-containing solution. Cytocompatibility tests in L929 cells 25



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inoculated E-MEM+FBS solution showed that CaP coating remarkably improved the cytocompatibility of the alloy. The Mg2+ ion release from the substrate also decreased in the coated alloy. In-vitro degradation study in SBF showed that CaP coating significantly increased the degradation resistance of the alloy, and especially reduced the localized degradation susceptibility of the alloy. In comparison with E-MEM+FBS cell culture medium, the SBF was more aggressive environment for magnesium-calcium alloy.

ACKNOWLEDGEMENTS The authors would like to thank the National Institute for Materials Science (NIMS) for the MANA-NIMS Fellowship (MBK) and JSPS KAKENHI (Grant number 26282151) for partial financial support, and Helmholtz-Zentrum Geesthacht (HZG), Germany for providing the magnesium alloy. The authors would also like to thank Ms. Hadis Khakbaz for helping with schematic drawing.

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Table of Contents Graphic

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Biocompatibility and in Vitro Degradation Behavior of Magnesium

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