Effect of inorganic salts, amino acids and proteins on the degradation of pure magnesium in vitro

Materials Science and Engineering C 29 (2009) 1559–1568

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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m s e c

Effect of inorganic salts, amino acids and proteins on the degradation of pure magnesium in vitro Akiko Yamamoto ⁎, Sachiko Hiromoto Metallic Biomaterials Group, Biomaterials Center, National Institute for Materials Science, 1-1, Namiki, Tsukuba, Ibaraki, 305-0044, Japan

a r t i c l e

i n f o

Article history: Received 12 August 2008 Received in revised form 22 October 2008 Accepted 10 December 2008 Available online 24 December 2008 Keywords: Bioabsorbable metals Biodegradation Magnesium Corrosion Protein adsorption

a b s t r a c t The possibility of magnesium and its alloys in medical applications is actively investigated in these days for the realization of biodegradable metallic devices. However, the degradation behavior and mechanisms of magnesium and its alloys in physiological environment such as inside the human body have not been elucidated. In this study, we performed 14-d long immersion tests of pure magnesium (3N) in 4 kinds of physiological solutions simulating the body fluids to examine the effects of the chemical components of the body fluids on the degradation of magnesium. The degradation rate of pure magnesium was strongly influenced by the kinds of the solution used. The highest degradation rate was obtained in NaCl, followed by E-MEM, Earle's solution, and E-MEM+FBS. The average degradation rate in NaCl for 8–14 d is about 100 times larger than that in E-MEM+FBS, which is the closest solution to human blood plasma. These results show that protein adsorption and insoluble salt formation retarded magnesium degradation, whereas organic compounds such as amino acids encourage the dissolution of magnesium. Buffering the solution also influenced the degradation rate; buffering NaCl with HEPES increased the degradation rate but buffering with NaHCO3 decreased it. Based on these results, the use of appropriate solution such as E-MEM+FBS is important for in vitro evaluation of the magnesium degradation rate under the physiological environment simulating inside the human body. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The development of a new bioabsorbable metal is anticipated for implant devices since bioabsorbable or biodegradable polymers and ceramics have brought us great success in medical fields. Magnesium and its alloys are one of the candidates for bioabsorbable metals because of the existence of magnesium at a relatively high concentraAbbreviations: NaCl, 0.125 M NaCl in water; Earle(+), Earle's solution containing calcium and magnesium salts; E-MEM, Eagle's minimum essential medium; E-MEM+ FBS, E-MEM supplemented with fetal bovine serum to be 10 vol.%; HEPES, N-2hydroxyethylpiperazine-N'-2-ethane sulfonic acid; EGTA, ethylene glycol bis (2-aminoethylether)-N,N'-tetraacetic acid; Ai, total amount of released Mg2+ up to day i (mg); Ci, concentration of Mg2+ in the collected portion at day i (mg/L); Ctotal(B3−), total concentration of the salt H3B, and the ions H2B−, HB2−, and B3−; Ksp(H3B), solubility product of the salt H3B; Ka1(H3B), dissociation constant of the salt H3B at the first dissociation process; Ka2(H2B−), dissociation constant of the salt H3B at the second dissociation process; Ka3(HB2−), dissociation constant of the salt H3B at the second dissociation process; Ri, dissociation ratio of Mg2+ per unit surface area of the specimen per day at day i (mg/mm2 d); R1, initial degradation rate, that is, Mg2+ release from the specimen at day 1 (mg/mm2 d); Rst, steady-state degradation rate, that is, the average of daily Mg2+ release for 8–14 d (mg/mm2 d); Rst', adjusted steady-state degradation rate (mg/mm2 d); S0, surface area of the specimen including its top, bottom, and side (mm2); S8, estimated surface area of the specimen at day 8 (mm2); W0, initial weight of the specimen (mg). ⁎ Corresponding author. Tel.: +81 29 860 4169; fax: +81 29 860 4626. E-mail address: [email protected] (A. Yamamoto). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.12.015

tion in the human body as an essential element [1]. The first study on the application of a magnesium alloy for an orthopedic device was performed in 1907 [2], and then followed up in 1930s [3] and 1940s [4], but they failed because of hydrogen gas generation accompanying to the corrosion of the magnesium alloy. Recent attempts on stent application of conventional magnesium alloys (AE21 [5] and WE43 [6,7]), however, open a new stage for the bioabsorbable magnesium alloys, because a stent is exposed to a blood flow which encourages the diffusion of generated hydrogen gas. Clinical trials for critical limb ischemia [7] and coronary arteries [8] are underway in Europe, suggesting an enormous possibility of bioabsorbable magnesium alloys for stent applications. Another new attempt of magnesium alloys is carried out for orthopedic devices [9–13]. In both cardiovascular and orthopedic applications, implanted devices are expected to support blood vessel wall or fractured bone along their healing process. Therefore, the control of the degradation speed of magnesium alloys is the most important issue for their application as bioabsorbable metals. For the successful control of the degradation speed of magnesium and its alloys, it is important to evaluate their degradability and to understand their degradation behavior and mechanism inside the human body. However, they have not been elucidated since most of the degradation or corrosion studies of magnesium and its alloys were performed in NaCl solution or other simple environment so far [14–19].

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Table 1 The concentration of inorganic salts and some organic components in plasma and 6 kinds of solutions used for immersion tests.

Na+ (mmol/L) K+ (mmol/L) Ca2+ (mmol/L) Mg2+ (mmol/L) Cl− (mmol/L) HCO− 3 (mmol/L) HPO2− 4 (mmol/L) 2− SO4 (mmol/L) Amino acids (mg/L) Dex/Glu (g/L) Proteins (g/L) HEPES (mmol/L) Phenol red (g/L)

Plasma NaCl

NaCl+ HEPES

NaCl+ NaHCO3

Earle (+)

EMEM

E-MEM+ FBS

142 5.0 2.5 1.5 103 27.0 1.0 0.5 nd

125 – – – 125 – – – –

151 – – – 125 26.2 – – –

151 5.37 1.80 0.811 125 26.2 0.897 0.811 –

151 5.37 1.80 0.811 125 26.2 0.897 0.811 0.860

151 5.37 1.80 0.811 125 26.2 0.897 0.811 0.860

nd – 63–80 – – –

– – 10

– – –

1 – –

1 – –

1.13 4.3 –

–

0.1

0.1

0.1

0.1

0.1

125 – – – 125 – – – –

0.1

nd: no data available. Dex: dextran. Glu: glucose.

Blood plasma and intercellular fluid are neutral solutions containing 2− inorganic ions including Mg2+, Ca2+, Cl−, HCO− 3 and HPO4 , as well as organic compounds such as amino acids and proteins [20], so the degradation of magnesium alloys in these fluids is a much complicated phenomenon comparing to that in a simple salt solution. Some corrosion studies were performed in Hank's solution [9,10] or a simulated body fluid [21], but it does not contain organic compounds that influence corrosion of metals [22]. Actually, it is reported that the corrosion rate of magnesium alloys in substitute ocean water (ASTMD1141-98) differed from those in vivo[12]. In addition, in vitro evaluation methods of the degradability of bioabsorbable metals have not been established. In this study, we perform immersion tests using 4 kinds of solution simulating the body fluids to examine the effects of the components in the solution on the degradation of pure magnesium. The solutions used are 0.125 M NaCl, Earle' solution containing calcium and magnesium salts [Earle(+)], Eagle's minimum essential medium (E-MEM), and EMEM supplemented with fetal bovine serum (E-MEM+FBS). The concentrations of major components of these solutions and blood plasma are shown in Table 1. The simplest solution among these is 0.125 M NaCl, which is abbreviated as NaCl in the following. In order to observe the buffering effect of the solution, two additional NaCl solutions are prepared by adding N-2-hydroxyethylpiperazine-N'-2ethane sulfonic acid (HEPES) or sodium bicarbonate (NaHCO3). The former is indicated as NaCl+HEPES, and the latter is indicated as NaCl+NaHCO3, and their components are also shown in Table 1. HEPES is a popular reagent to buffer a cell culture medium in the air. NaHCO3 gives a basic buffer system in the cell culture medium under the atmosphere of 5 vol.% CO2 in the humid air, and is contained in Earle (+), E-MEM, and E-MEM+FBS as well. Using these 6 solutions, the effects of inorganic salts, organic compounds such as amino acids, and protein on the degradation of pure magnesium are investigated. The obtained results will contribute to understand the magnesium's degradation behavior and mechanism as well as to establish in vitro evaluation methods for the degradability of magnesium and its alloys. 2. Materials and methods 2.1. Sample preparation The material used in the present study is pure magnesium (3N, Electronic Space Products International, California, USA). The typical concentrations of major impurities are as follows: 0.02 for Cu, b0.05 for Fe, b0.01 for Mn, b0.001 for Ni, b0.01 for Pb, and b0.01 for Sn in ppm.

An average grain size is 97.5 µm. Disks of 9.5 mm in diameter and 2 mm in thickness were prepared. Every surface of these disks was polished with #600(14 µm) SiC paper in ethanol. The size and the weight of each disk were measured prior to the immersion test, and then, the disk was ultrasonically washed and sterilized with acetone for 5 min. Three disks were prepared for each solution for immersion tests. 2.2. Immersion test Six types of solutions used for the immersion tests and their components are shown in Table 1 as described before. Earle(+), EMEM, and FBS were purchased from Dainippon Sumitomo Pharma. Co. Ltd., Japan, Nissui Pharmaceutical Co. Ltd., Japan, and JRH Biosciences Inc. USA, respectively. Each solution contains 0.1 wt.% phenol red as a pH indicator. All solutions were sterilized by autoclaving or filtration and prepared in an aseptic condition. To perform the immersion test, we set up a simple model for simulating a human body fluid condition. An average adult human has ca. 2.75 L of blood plasma [24] and excretes 1.5 L of urine per day [25], and the scale is reduced as 1/100. A 27.5 mL portion of each solution was poured into a sterile glass container with a lid having a membrane filter (0.22 µm) for ventilation except NaCl and NaCl+HEPES. In the cases of these solutions, the same glass container with a lid having no ventilation filter was used. Then, the glass container was rotated at the speed of 300 rpm under a condition of 37 °C and 5 vol.% CO2 in the humidified air for 4 h prior to the immersion test to adjust the pH of the fluid to be neutral. Then, one disk was immersed into the fluid in the glass container aseptically. Each container was weighed, and kept under the same condition with rotation. At every 24 h period, a 15 mL portion (55 vol.%) of the fluid was aseptically removed and replenished with fresh one in order to simulate the fluid exchange of an adult human by the excretion of urine. Prior to the replacement, each container was weighed again to find the loss of the fluid by evaporation, which is about 50–150 µL per 24 h. Then, the same amount of sterile distilled water was added into the container to compensate the condensation of the fluid and mixed well. In case that the precipitation was observed on the bottom of the glass container, precipitation was re-suspended into the fluid before the 15 mL portion of the fluid was removed. Immersion test was continued up to 14 d. During the immersion, the pH of the fluid was also checked by the color of the pH indicator (phenol red) added into the fluid since it is important to observe the pH change of the solution around the magnesium disk surface as well as to observe the pH change of the whole solution under the atmosphere of 5 vol.% CO2 in the humid air while keeping an aseptic condition. After the immersion, each disk was collected, rinsed with distilled water quickly, and dried under nitrogen flow to observe their surface by an optical microscope. The surface of some of the disks was analyzed by energy dispersive X-ray spectroscopy (EDX) equipped to the scanning electron microscope (Hitachi S-4800). 2.3. Quantification of the Mg2+ ion Quantification of the Mg2+ ion in the collected portion of each fluid during the immersion test was performed by a colorimetric method using xylidyl blue-I [26,27], which is applied for the measurement of magnesium in serum [28]. The kit, “Magnesium B-test Wako” (Wako Chemicals, Tokyo, Japan), presenting a one-reagent mixture containing 0.1 mM of xylidyl blue-1 [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)-N,N'-tetraacetic acid], and non-ionic surfactant Triton X-100, is employed in the present study. The calibration curves were prepared by adding 5–50 mg/L of MgCl2 to each kind of solution before using for immersion test. By mixing the aliquot of the collected fluid to the colorimetric reagent at the ratio of 1:150, the absorbance at 620 nm was measured. Then, the concentration

A. Yamamoto, S. Hiromoto / Materials Science and Engineering C 29 (2009) 1559–1568

Fig. 1. (a, b) Mg2+ release during the immersion into NaCl. (a) Mg2+i dissolution rate per P unit surface area at day i, Ri and (b) accumulated Mg2+ release, Ri :

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amount of the white precipitates on the bottom of the glass container reduced. After 10 d of immersion, the small bubbles of hydrogen gas were again observed around the disk before the exchange of the fluid. After 14 d of immersion, the collected solution was tested by pH paper to show the pH over 9 (lower than 10). The maximum dissolution of Mg2+ was observed at day 1, then, the degradation rate decreased until day 7– 8, and came to be constant during 8–14 d. In Fig. 1(b), the accumulated release of Mg2+ has a flexion point around day 7, and the graph shows almost linear relationship during day 8–14. The microscopic images of the disk after 14 d of immersion into NaCl are shown in Fig. 2. Most of the remaining disk was covered by a white layer, which is considered as Mg(OH)2 because it is thermodynamically stable over pH 8.5 based on the Pourbaix diagram when the concentration of Mg2+ in the solution is about 1 M [29]. In this study, a 55 vol.% portion of the immersion solution was excreted and replenished once per day. Inside the human body, however, excretion of excess Mg2+ as well as diffusion of OH− continuously occurred. Therefore, the pH of the body fluid which is microscopically very close to the magnesium disk surface may increase even in the human body, but the pH of the body fluid far from the disk surface should be kept as ordinary 7.5. In this study, however, the pH increase of the NaCl was observed just after the immersion started. The pH of NaCl decreased gradually during the immersion but still was over 9 after 14 d of immersion even though the 55% portion of the NaCl was exchanged every day. Therefore, in the case of NaCl, our model of immersion solution exchange (a 55% portion once per day) is not good enough to simulate the condition in the human body from the viewpoint of maintaining the pH of the body fluid far from the magnesium disk surface. The exchange of the immersion solution may decrease the pH of the solution once per day, but it should be kept as 7.5 to simulate the condition inside the human body. Therefore, buffering of NaCl solution was attempted by adding HEPES or NaHCO3.

i=1

of the Mg2+ was calculated using each calibration curve. Six replicates were prepared for each collected portion and standard solution for the calibration. The Mg2+ dissolution per each day was given by the equation below; Ai = 0:0275Ci − ð0:0275 − 0:015ÞCi − 1

ð1Þ

where Ci is the concentration of Mg2+ (mg/L) in collected portion at day i, and Ai is the total amount of the released Mg2+ (mg) of day i. The Mg2+ dissolution rate per unit surface area (mg/mm2 d) at day i, Ri, was obtained by the following equation; Ri = Ai = S0

ð2Þ

where S0 is the surface area (mm2) including the top, bottom, and side of the disk before immersion. 3. Results 3.1. Immersion tests in NaCl solutions The Mg2+ release from the disk during the immersion tests into NaCl is shown in Fig. 1. The generation of hydrogen gas bubbles and the red shift of the color of the solution were observed just after the immersion started, indicating the increase of the pH by corrosion reaction. After 5 min of immersion, its color changed into purple, suggesting the pH is over 9. After 2 d of immersion, white precipitates were observed in the bottom of the glass container and the generation of hydrogen gas bubble was not observed before exchanging the fluid. After 7 d of immersion, the color of the solution before exchanging the fluid was purplish red, indicating the decrease of pH comparing to that of the day before. The

Fig. 2. (a, b) Microscopic images of a Mg disk after 14 d of immersion into NaCl. The top (a) and bottom (b).

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increase of the pH (7.5–7.7). Small hydrogen gas bubbles were continuously observed during the immersion. In the case of Earle(+) and E-MEM+FBS, the color of the solution was slightly pinkish after 1 d of immersion, but no color change was observed after 2 d of immersion, indicating the pH of the fluid was maintained as that of the fresh one after 2 d of immersion. Hydrogen gas bubbles disappeared after 1 d of immersion. No precipitate was observed on the bottom of the glass container during the immersion in these three solutions. The tendency of Mg2+ dissolution in these solutions was similar to each other; the maximum dissolutions of Mg2+ were observed at day 1, and then, the dissolution rate decreased until day 5–8. After 7 or 8 d of immersion, the dissolution rate was rather constant. In Fig. 5(b), the accumulated release of Mg2+ in these three solutions shows gradual change of the release rates around day 5–8. The highest Mg2+ dissolution was observed in E-MEM, followed by Earle(+). The microscopic images of the disks after 14 d of immersion into Earle(+), E-MEM, and E-MEM+FBS are shown in Fig. 6. A white and gray layer was observed on the top surface of each disk immersed into these three solutions, but the thickness and the amount of the white layer decreased in the order of E-MEM N Earle(+) N E-MEM+FBS. 3.3. Comparison of the degradation properties in 6 solutions

Fig. 3. (a, b) Mg2+ release during the immersion into NaCl+HEPES and NaCl+NaHCO3. (a) Mg2+ dissolution rate per unit surface area at day i, Ri and (b) accumulated Mg2+ release, i P Ri :

Since the tendency of Mg2+ dissolution in Earle(+), E-MEM and EMEM+FBS is similar to those of the three kinds of NaCl solutions, two parameters were employed to understand the degradation profiles: the Mg2+ release at day 1 and the average of daily Mg2+ release for 8–14 d as “initial degradation rate, R1” and “steady-state degradation rate, Rst”, respectively. Rst was obtained as the slope of the accumulated release curve during day 8–14 by the least square method. The total Mg2+ release, which is the sum of the daily release of Mg2+ during 14 d of immersion, was also used.

i=1

The results of the immersion tests of magnesium disks in these solutions are shown in Fig. 3. The hydrogen gas bubbles were observed just after the immersion started. The color of both solutions shifted to deep red (pH was 7.6–7.8), suggesting their pH increase is much smaller than that of NaCl. In the case of NaCl+HEPES, the hydrogen gas bubbles disappeared and white precipitates were observed in the bottom of the glass containers after 2 d of immersion before the exchange of the fluid. These precipitates disappeared after 4 d of immersion before the exchange of the fluid. In the case of NaCl+NaHCO3, the hydrogen gas bubbles disappeared after 3 d of immersion while no precipitate was observed on the bottom of the glass container during the whole period of immersion. In both cases, further color change of the pH indicator was not observed. For both solutions, the maximum dissolutions of Mg2+ were observed at day 1. Then, the degradation rates decreased until day 2–5, and came to be roughly constant during 5–14 d. In Fig. 3(b), the accumulated amounts of released Mg2+ in NaCl+HEPES and NaCl+NaHCO3 have no clear flexion points; showing a linear relationship during immersion. The difference of the release rates in these solutions is much clear in the accumulated release curves than the daily release curves. The dissolution of Mg2+ is higher in NaCl+HEPES rather than in NaCl+NaHCO3. The microscopic images of the disks after 14 d of immersion into NaCl+ HEPES and NaCl+NaHCO3 are shown in Fig. 4. A white-gray layer covered over the dark-gray surface of the remaining disk, but this white-gray layer seems to be thinner than those on the disks immersed into NaCl. 3.2. Immersion tests in other solutions The results of the immersion tests in the other three solutions are shown in Fig. 5. In all solutions, hydrogen gas bubbles were observed just after immersion started. In the case of E-MEM, the color of the solution changed slightly pinky red after 1 d of immersion, indicating the slight

Fig. 4. (a, b) Microscopic images of Mg disks after 14 d of immersion into NaCl. (a) NaCl+HEPES, and (b) NaCl+NaHCO3 .

A. Yamamoto, S. Hiromoto / Materials Science and Engineering C 29 (2009) 1559–1568

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1/100 of NaCl, 1/5 of E-MEM, and 1/2 of Earle(+). Fig. 8(b) shows the ratio of Rst or R′st to R1 in 6 kinds of solutions, which indicates the decrease of the dissolution rate according to the progress of the immersion. R′st /R1 for NaCl+HEPES is the highest among 6 solutions, followed by NaCl+NaHCO3, NaCl, E-MEM, Earle(+), and E-MEM+FBS. 3.4. Surface analysis of immersed disks by EDX Identification of the surface layer of the disks after the 14 d of immersion is attempted by EDX analysis and its results are shown in Fig. 9 and Table 2. Because of the lack of conductivity and unstableness (detachment of the surface layer by cracking), precise analysis was difficult to perform. However, the quantitative data indicates the incorporation of Ca and P on the surface of the disks immersed into Earle(+), E-MEM, and E-MEM+FBS. The highest concentrations of Ca and P at the surface were observed for E-MEM+FBS, followed by EMEM. On the contrary, the highest concentration of Mg at the surface was observed for NaCl, followed by E-MEM, Earle(+), and E-MEM+FBS

Fig. 5. (a, b) Mg2+ release during the immersion into Earle(+), E-MEM, and E-MEM+FBS. (a) Mg2+ dissolution rate per unit surface area at day i, Ri and (b) accumulated Mg2+ i P release, Ri : i=1

Fig. 7 shows the total Mg2+ release during 14 d of immersion into 6 solutions. The maximum dissolution was observed in NaCl+HEPES, followed by NaCl, NaCl+NaHCO3, E-MEM, Earle(+) and E-MEM+FBS in the order. The Mg2+ dissolutions in the three kinds of NaCl solutions were much higher than those of Earle(+), E-MEM, and EMEM+FBS. As shown in Figs. 2 and 4, the immersed disks in these solutions drastically reduced their volumes, suggesting the decrease of surface area during the immersion from a macroscopic viewpoint. In the present study, Rst was calculated with the initial surface area of the disk, S0, therefore the significant decrease of the surface area gives a smaller value of Rst than it truly is. Consequently, the estimation of the surface area after 8 d of immersion was performed to obtain the true value of Rst, assuming that the disk degrades while keeping its shape in similar to the initial shape. Day 8 is applied because it is the mid point of the immersion period of 14 d. The surface area of the disk at day 8 is estimated by the following equation; #2 = 3 " 8 X S8 = S0 1− Ai =W0 Þ ð3Þ i=1

where W0 indicates the initial weight of the disk, and Ai is given by Eq. (1) as described before. Then, the adjusted steady-state degradation rate, R′st was calculated as follows; 0

Rst = Rst ðS0 = S8 Þ:

ð4Þ

′ for the 6 kinds of solutions. Fig. 8(a) shows R1 and Rst or Rst As expected, Rst′ for the three NaCl solutions were higher than Rst, whereas R′st for other three solutions were almost same to Rst. The highest Rst ′ is obtained in NaCl+HEPES, followed by NaCl+NaHCO3, NaCl, E-MEM, Earle(+), and E-MEM+FBS. R′st for E-MEM+FBS is about

Fig. 6. (a, b, c) Microscopic images of Mg disks after 14 d of immersion into Earle(+) (a), E-MEM (b), and E-MEM+FBS (c).

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Fig. 7. Total Mg2+ release during 14 d of immersion of pure Mg into 6 kinds of solutions.

in the order. Carbon was detected even in the disk immersed into NaCl, which does not contain C, suggesting the possibility of contamination. Therefore, it is not appropriate to judge the incorporation of carbonate into the surface layer in the case of Earle(+), E-MEM, and E-MEM+FBS. 4. Discussion 4.1. Effect of pH and bicarbonate on the degradation of Mg In general, the surface of Mg disk is covered by its oxide in the air at room temperature, but this oxide will be converted into hydroxide in

Fig. 9. (a, b, c) Examples of EDX spectra of the surface of Mg disks after immersion into NaCl (a), Earle(+) (b), and E-MEM+FBS (c) for 14 d.

the existence of water or moisture in the air [14]. Magnesium hydroxide is relatively stable in the basic range of pH values (N8.5–11, depending on the concentration of Mg2+ in the solution), but is soluble in the neutral or acidic ranges of pH [29]. Corrosion of Mg in aqueous solution generally proceeds as two parallel reactions: electrochemical and hydrated-water reaction [30]. Total corrosion reaction of Mg is described below; 2+

Fig. 8. (a, b) Initial degradation rate (R1) and steady-state degradation rate (Rst or Rst′) of Mg immersion into 6 kinds of solution for 14 d (a), and the ratio of Rst or Rst′ to R1 (b). Rst′, which is adjusted steady-state degradation rate, was calculated using estimated surface area at day whereas Rst was calculated using initial surface area.

Mg + 2H2 OYMg

+ 2OH

−

+ H2 :

ð5Þ

Along the progress of corrosion reaction, pH of the solution near the Mg surface will increase with the accumulation of OH−, resulting in

A. Yamamoto, S. Hiromoto / Materials Science and Engineering C 29 (2009) 1559–1568 Table 2 EDX data of the surface of Mg disks after 14 d of immersion into 4 kinds of solution for major elements (atomic %). Solution

Mg

O

Ca

P

C

NaCl Earle(+) E-MEM E-MEM+FBS

17.9 8.8 ± 2.4 12.7 ± 0.9 2.8 ± 0.4

65.8 65.0 ± 4.6 58.0 ± 5.1 59.6 ± 0.2

– 3.9 ± 4.2 10.1 ± 5.8 12.3 ± 0.4

– 5.3 ± 3.8 8.2 ± 1.7 10.0 ± 0.0

15.7 15.3 ± 6.5 8.0 ± 3.9 13.7 ± 0.4

concentration of free Mg2+ in the solution can be estimated by following equations. [32]; h 2 Ksp ðMgCO3 Þ = Mg

2+

Mg

−

+ 2OH YMgðOHÞ2 A:

ð6Þ

The accumulated Mg(OH)2 layer on the Mg surface can act as a barrier against dissolution by preventing mass diffusion between the magnesium substrate and the solution. In the case of NaCl solution, its pH increase was observed just after immersion started, and precipitation was also observed on the disk surface as well as on the bottom of the glass container after 2 d of immersion. The maximum Mg2+ release was obtained at day 1. These facts suggest that the accumulated Mg(OH)2 layer on the disk surface retarded the Mg2+ release after day 2. The EDX analysis found only Mg, O, and C (which may be contaminated) at the surface of the disk after immersion into NaCl, suggesting the accumulation of Mg(OH)2 on the disk surface. The Mg2+ release continuously decreased up to day 8, indicating that not only the decrease of the surface area of Mg disk by corrosion but also the maturing and thickening of accumulated Mg(OH)2 layer contributed to the decrease of Mg2+ release after day 2. Maturing and thickening of the Mg(OH)2 layer is also supported by the value of Rst'/R1 less than 1 as well as the reduction of the release rate around day 8 which appears on the accumulated release curve in Fig. 1(b); the matured and thickened Mg(OH)2 layer physically disturbs dissolution of Mg more effectively than it does in the beginning of the immersion period. To avoid the pH increase by corrosion reaction, buffering reagents were added into NaCl solution. In the case of HEPES addition, pH increase was still observed just after immersion started, but the range of the increase was smaller than that of NaCl. The precipitate on the bottom of the glass container disappeared after 4 d of immersion in the case of NaCl+HEPES, whereas it disappeared after 9 d of immersion in the case of NaCl. Since the solubility product constant, K sp of Mg(OH) 2 in water is 5.61 × 10− 12 [31], the maximum concentration of Mg2+ in water at the pH of 9–10 is 104 times lower than that at the pH of 7–8 based on the Eq. (7) [32]; h i   2 + − 2 ½OH  : ð7Þ Ksp MgðOHÞ2 = Mg These facts indicate that the accumulation of Mg(OH)2 layer on the disk surface in NaCl+HEPES is smaller than that in NaCl. Rst'/R1 in NaCl+HEPES is 0.9, indicating that maturing and thickening of the accumulated Mg(OH)2 layer on the disk does not occur or contribute to the inhibition of Mg dissolution. The linear tendency of the accumulated release curve for NaCl+HEPES as well as the higher value of the total Mg2+ release in NaCl+HEPES than that in NaCl correspond well to the above interpretation. Another buffering reagent is NaHCO3, which gives a major buffering system in a cell culture medium by the combination with H2CO3 derived from dissolved CO2 in a CO2-incubator. However, Ksp of MgCO3 in water is relatively low as 6.82 × 10− 6 [31], therefore MgCO3 can possibly precipitate in NaCl+NaHCO3. MgCO3 and its hydrates were confirmed in the surface film of magnesium and its alloys in the case of atmospheric corrosion, which contains only 0.3 vol.% of CO2 [33,34]. The solubility of MgCO3 in the NaCl+NaHCO3, that is, the

+

ih i 2− CO3

  h i 2− 2− Ctotal CO3 = ½H2 CO3  + ½HCO3 −  + CO3 nh

ð8Þ ð9Þ

i o ½HCO3 −  = ½H2 CO3 

ð10Þ

n h io 2− Ka2 ðHCO3 − Þ = ½H +  CO3 = ½HCO3 − 

ð11Þ

Ka1 ðH2 CO3 Þ =

the precipitation of the Mg(OH)2 on the surface because the solubility of Mg(OH)2 decreases with the increase of OH− concentration [29].

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H

+

where Ka1 and Ka2 are the dissociation constants of H2CO3 and HCO− 3 as 4.47×10− 7 and 4.68×10− 11, respectively [31]. The concentration of CO2− 3 is obtained by the combination of Eqs. (9)–(11); h  h i   i h i 2− 2− + 2 + CO3 + H = Ctotal CO3 Ka1 Ka2 = H Ka1 + Ka1 Ka2 : ð12Þ When the pH of the solution and Ctotal(CO2− 3 ) are assumed as 7.5 and 0.026 M, which is the concentration of NaHCO3 added, [CO2− 3 ] is 3.59 × 10− 5 M, which gives [Mg2+] of 0.19 M. In the CO2-incubator where the concentration of CO2 is kept about 5 vol. % in air, Ctotal will be higher than the assumed value because of the dissolving CO2 into the −5 buffered solution, resulting in the higher [CO2− M, 3 ] than 3.59 × 10 2+ which gives the lower [Mg ] than 0.19 M. In the case of NaCl, the maximum concentration of free Mg2+ in water at the pH of 7.5 is 56.2 M calculated by Eq. (7) and Ksp of Mg(OH)2 as 5.61 × 10− 12 [31]. Therefore, the precipitation of MgCO3 is more likely to occur in NaCl+ NaHCO3 rather than the precipitation of Mg(OH)2 in NaCl even in the neutral range of pH as 7–8. During the immersion, Mg2+ release in NaCl+NaHCO3 steeply decreased from day 1 to day 2, whereas those in NaCl and NaCl+HEPES gradually decreased from day 1 to day 5. This difference in the decrease of Mg2+ release rate indicates that a specific insoluble salt precipitation contributes to the surface layer formation on the magnesium disk in NaCl+NaHCO3, which retards Mg2+ dissolution by inhibiting mass diffusion. The total Mg2+ release and Rst'/R1 in NaCl+ NaHCO3 are lower than those in NaCl+HEPES while the pH of the solutions is buffered at the same level. These results indicate that the precipitation of MgCO3 is effective to improve the corrosion resistance of magnesium in a neutral buffered solution with carbonate– bicarbonate system. It is reported that carbonate ion enhances the de-stabilization of Mg(OH)2/MgO layer when the concentration of carbonate ion is relatively low as 4.76 × 10− 4 M [17], that is about 1/50 of the concentration in the present study. This fact suggests that carbonate ion is effective to retard the Mg dissolution when their concentration is high enough to form insoluble MgCO3 layer on the disk. Comparing to NaCl, the total Mg2+ release in NaCl+NaHCO3 was smaller than those in NaCl, indicating that the surface layer formed in NaCl+NaHCO3 at the beginning of the immersion has a better barrier effect than the surface layer formed in NaCl especially in the early period of immersion. However, Rst′ and Rst′/R1 in NaCl+NaHCO3 were larger than those in NaCl, meaning that the barrier effect of the surface layer of the disk in NaCl+NaHCO3 is lower than that in NaCl during immersion period of 8–14 d. The fact that no clear reduction was observed in the slope of the accumulated release curve for NaCl+ NaHCO3 also supports this suggestion. In other words, the matured and thickened Mg(OH)2 layer formed in NaCl has a better barrier effect rather than that in NaCl+NaHCO3 during the latter half of the immersion period. The Mg(OH)2 layer formed in NaCl slowly matured and thickened, and then, contributed to reduce Mg2+ dissolution whereas the surface layer containing MgCO3 formed in NaCl+NaHCO3 contributed to reduce Mg2+ dissolution from the beginning of the immersion period.

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Fig. 10. Schematic explanation of homeostatic control of magnesium in the human body (modified from [23]).

4.2. Effect of the inorganic and organic components on the degradation of Mg Body fluids such as blood plasma contain various kinds of inorganic salts. Earle's solution contains inorganic salts at similar concentrations to those in blood plasma. It is generally considered that insoluble salt formation and covering specimen surface is effective to retard the corrosion rate of the magnesium and its alloys [33]. Phosphate ion as well as carbonate ion is major inorganic anions in Earle's solution to form insoluble magnesium salts. Phosphate salts may preferentially precipitate in this condition based on the following calculation with solubility products and concentrations of these ions. Since Ctotal(CO2− 3 ) in the Earle's solution is same to that in NaCl+ NaHCO3 as 0.026 M, the equilibrium concentration of CO2− at the pH 3 of 7.5 is 3.59 × 10− 5 M, giving the concentration of free Mg2+ as 0.19 M according to Eqs. (8)–(12). Concerning the phosphate ion, the equilibrium concentration of PO3− 4 is estimated as the followings [32];   h i h i 3− 2− 3− = ½H3 PO4  + ½H2 PO4 −  + HPO4 + PO4 Ctotal PO4 Ka1 ðH3 PO4 Þ =

i nh o + H ½H2 PO4 −  = ½H3 PO4 

Ka2 ðH2 PO4 − Þ =

nh H

+

ih

2−

HPO4

io

ð14Þ

= ½H2 PO4 − 

ð15Þ

  nh ih io h i 2− + 3− 2− = H PO4 = HPO4 Ka3 HPO4 h h i   3− 3− = Ctotal PO4 Ka1 Ka2 Ka3 = H PO4

+

i3

h + H

+

i2

ð13Þ

h Ka1 + H

ð16Þ

+

 i Ka1 Ka2 + Ka1 Ka2 Ka3

ð17Þ where Ka1, Ka2 and Ka3 are the dissociation constants of H3PO4, H2PO4−, −3 and HPO2− , 6.17 ×10− 8 and 4.79×10− 13, respectively [31]. 4 as 6.92 ×10 3− Since Ctotal(PO4 ) in the Earle's solution is 8.97 ×10− 4 M, the equilibrium concentration of PO3− at the pH of 7.5 is 8.98 ×10− 9 M. Ksp of the Mg3 4 (PO4)2 in water is 1.04×10− 24 [31], therefore the maximum concentration of Mg2+ is calculated as 2.34× 10− 3 M with the Eq. (18) [32]. h i h i   2 + 3 3− 2 PO4 : Ksp Mg3 ðPO4 Þ2 = Mg

ð18Þ

The lower limit of free Mg2+ concentration for phosphate than carbonate clearly points out the preferential precipitation of phosphate in Earle(+). Furthermore, calcium and phosphate ions are supersaturated in Earle(+)

based on the solubility constant of Ca3(PO4)2 in pure water [31]. That means, calcium phosphate will precipitate when the pH of the Earle(+) increases or other nucleation opportunities are given. The EDX analysis of the disk after immersion into Earle(+) revealed the incorporation of Ca and P into the surface layer, confirming this hypothesis. These insoluble salt precipitations on the magnesium disk surface will be a major cause of the retardation of magnesium disk degradation in the condition presented in this study. As a result, total Mg2+ release and Rst′/R1 in Earle(+) are much lower than those of all kinds of NaCl solutions, supporting the above discussion about the significant effect of insoluble phosphate formation. E-MEM contains amino acids and some organic components of blood plasma as well as inorganic salts. Amino acids and some organic chelating compounds can form a complex with metal cation, which encourages the dissolution of metal [22,35]. In the case of magnesium dissolution, chelating with an organic compound will inhibit the formation of insoluble salts, suggesting the reduction of the retardation by insoluble salt layer on the magnesium disk surface. The EDX analysis of the disk immersed into E-MEM confirmed the incorporation of Ca and P into the surface layer as well as the disk immersed into Earle(+), however, the concentration of Mg in the surface layer is higher for E-MEM than for Earle(+). This difference may suggest that the difference in the thickness or structure of the surface layer formed in these solutions, but precise analysis is necessary for further discussion of this point. Total Mg2+ release and Rst′/R1 in E-MEM are larger than those in Earle(+), which supports the hypothesis of the reduction of the retardation effect by insoluble salt formation on magnesium dissolution. However, the total Mg2+ release, Rst′, and R1 are still much smaller than those in NaCl, suggesting the encouragement of the dissolution by chelating with organic compounds is relatively small than the retardation effect of insoluble phosphate formation. Blood plasma contains about 6.3–8 wt.% of proteins [36], that is almost twice of E-MEM+FBS, which only contains 4.3 wt.% of proteins. Proteins adsorb on the metal surface, and in some cases, this adsorbed protein layer has a protective effect on metal corrosion such as 316L stainless steel or Ti alloys [22]. In the present paper, total Mg2+ release and Rst′/R1 for E-MEM+FBS are smaller than those for any other solutions. This fact suggests that protein adsorption onto the magnesium disk surface has a significant effect on the retardation of magnesium dissolution. This may be attributed to the contribution of the adsorbed proteins to make insoluble salt layer dense or more effective as a barrier against corrosion. The EDX analysis of the disk immersed into E-MEM+FBS also showed the incorporation of Ca and P into the surface layer at the higher concentrations than those for E-

A. Yamamoto, S. Hiromoto / Materials Science and Engineering C 29 (2009) 1559–1568

MEM and Earle(+), but incorporation of proteins is difficult to discuss with the EDX data. Further investigation is required to clarify this point. Based on the results obtained in the present study, magnesium degradation in the physiological solution simulating a body fluid is strongly influenced by the kind of solution used. The parameter Rst'/R1 is considered to correlate with the effectiveness of the surface layer prepared in the solution for the retardation of Mg corrosion. The difference of Rst'/R1 between NaCl+NaHCO3 and Earle(+) indicates the contribution of insoluble phosphate formation, and that between E-MEM and E-MEM+FBS indicates the contribution of protein adsorption. The concentration of Mg at the disk surface decreased in the following order: NaCl, E-MEM, Earle(+), and E-MEM+FBS, which is the same order of Rst′ and Rst′/R1. This correspondence suggests that the composition and structure of the surface layer formed in the simulating body fluid severely influence the corrosion of Mg. E-MEM+FBS, having the closest composition to the human blood plasma, gave the smallest degradation rate of pure magnesium: Rst′ in E-MEM+FBS is about 1/100 of that in NaCl. This fact suggests the importance to use appropriate solution for in vitro estimation of the degradation rate of magnesium inside the body. 4.3. In vitro immersion test modeling the condition of body fluids Besides the inorganic and organic components of the body fluids, cells are another important factor to control corrosion of metallic materials inside the human body. Macrophages and active oxygen species generated by them are reported to accelerate the corrosion of Ti [37]. Not only the biological factors but also biomechanical environment such as blood flow may influence the corrosion of magnesium inside the body. In this study, the immersion condition was decided in consideration of simulating the condition of the body fluid and homeostasis of Mg2+ in the human body. The homeostasis of Mg2+ in the human body fluid is controlled by the excretion and reabsorption of Mg2+ at kidney and by the uptake of Mg2+ at intestinal tract [23] (see Fig. 10). In a healthy adult human, 2.4–3.5 g of magnesium is filtered from blood plasma through the kidney and ca. 95% of filtered magnesium is reabsorbed during one day [23]. This amount is about 10 and 33 times higher than the amounts of magnesium in extracellular fluid and in blood plasma. When the uptake of Mg2+ at the intestinal tract increases, the reabsorption of Mg2+ at the kidney decreases, that is, the excess amount of Mg2+ is rapidly excreted into urine to maintain the similar concentration of Mg2+ in the body fluid [23]. Supposing the case of a stent, Mg2+ released from the device diffuses into the blood plasma, and then, it is considered to be filtered and excreted into urine rather promptly. Supposing the case of a bone fixing device, Mg2+ released from the device diffuses into extracellular fluid, following by the diffusion into blood plasma due to the capillary blood vessel network, and then, it will be filtered and excreted into urine. Therefore, in this study, we put the priority on simulating blood circulation and excretion by urine rather than the diffusion into intracellular and extracellular fluids for setting up the immersion test condition. The scale of the body fluid was reduced as 1/100, e.g. 27.5 mL of blood plasma and daily replenishment of 55 vol.% of it. Inside the human body, excess Mg2+ is continuously excreted into urine, but we simplify it as to exchange a certain portion of the immersion solution once per day. According to the results of this study, the kind of the simulating body fluid used is very important because it changes the corrosion rate of magnesium 100 times at most via the composition and structure of the surface layer formed in the fluid. The surface layer formed in the fluid is strongly influenced by the concentration of relating ions such 3− as OH−, Mg2+, CO2− 3 , and PO4 , suggesting the importance of the diffusion condition of these ions in the fluid. Blood flow accelerates the mass diffusion around the magnesium device, which may prevent the precipitation of corrosion products, resulting in the acceleration of

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Mg2+ dissolution. Total amount of the body fluid also influences the degradation speed of magnesium; the larger amount accelerates the corrosion comparing to the smaller. That means, the in vivo implantation test into a small animal is not always an appropriate method to estimate the degradation rate of magnesium device in the human body. Based on these discussions, continuous or controlled flow system [38] of a simulating body fluid with replenish system of the 55 vol.% portion of the fluid per day is preferable as the in vitro evaluation method of the degradation rate of magnesium alloys, and of course, it is critical to use the solution such as E-MEM+FBS, having the similar composition to blood plasma, as the simulating body fluid. Further investigation will be necessary to elucidate the effect of the flow rate of the simulating body fluid on the magnesium degradation rate in order to establish the simulating condition of the blood flow in various tissues such as blood vessel, soft tissue, bone marrow, etc., as well as the confirmation of in vitro and in vivo correlation for the degradation rate and behavior of magnesium and its alloys. 5. Conclusions In this study, immersion tests of pure magnesium into NaCl, Earle (+), E-MEM and E-MEM+FBS were performed to examine the effects of inorganic salts and organic compounds on the degradation of magnesium. Immersion condition applied simulates the excretion and replenishment of the body fluid in consideration of the homeostasis of magnesium in the human body. Two kinds of buffering reagents, HEPES and NaHCO3, were added into NaCl to examine the effect of pH control around 7.5. As the results, the kind of the simulated body fluid used drastically changed the degradation rate of pure magnesium. Buffering the NaCl solution with HEPES increased the total Mg2+ release during 14 d of immersion, whereas buffering with NaHCO3 under the atmosphere of 5 vol.% of CO2 decreased the Mg2+ release especially on the beginning of the immersion. The lowest degradation rate was obtained in E-MEM+FBS, followed by Earle(+), E-MEM, NaCl+NaHCO3, NaCl, and NaCl+HEPES in the order. Protein adsorption and precipitation of insoluble salts were effective for the retardation of magnesium degradation in these solutions, whereas organic compounds such as amino acids reduced the barrier effect of insoluble salt layer against dissolution of magnesium. The Rst′ in EMEM+FBS was about 1/100 of that in NaCl. The surface layer formed during the immersion differed with the kind of the fluid used, which controls the degradation of magnesium. Therefore, the use of the solution such as E-MEM+FBS, having the similar composition to blood plasma, is mandatory for in vitro estimation of the magnesium degradation rate inside the human body. Acknowledgement The authors appreciate Dr. H. Somekawa & Dr. T. Mukai for measuring grain size of the material used as well as practical advices. The authors also thank Ms. Y. Kohyama, & Ms. A. Kikuta for their excellent technical assistance. This work was partially supported by Grant-in-Aid for Young Scientists (B) 18700431. References [1] H. Tanaka, Chem. Rev. Q. 27 (1995) 3. [2] G.B. Stroganov, E.M. Savitsky, N.M. Tikhova, V.F. Terekhova, M.V. Volkov, K.M. Sivash, V.S. Borodkin, US Patent 3,687,135 (1972). [3] E.D. McBride, J. Am. Med. Assoc. 111 (1938) 2464. [4] C.P. McCord, Ind. Med. 11 (1942) 71. [5] B. Heublein, R. Rohde, V. Kaese, M. Niemeyer, W. Hartung, A. Haverich, Heart 89 (2003) 651. [6] C. Di Mario, H. Griffith, O. Goktekin, N. Peeters, J. Verbist, M. Bosiers, K. Deloose, B. Heublein, R. Rohde, V. Kasese, C. Ilsley, R. Erbel, J. Interv. Cardiol. 17 (2004) 391. [7] P. Peeters, M. Bosiers, J. Verbist, K. Deloose, B. Heublein, J. Endovasc. Ther. 12 (2005) 1. [8] R. Erbel, C.D. Mario, J. Bartunek, J. Bonnier, B. de Bruyne, F.R. Eberli, P. Erne, M. Haude, B. Heublein, M. Horrigan, C. Ilsley, D. Böse, J. Koolen, T.F. Lüscher, N. Weissman, R. Waksman, Lancet 369 (2007) 1869.

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