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Influence of Mg2+ concentration, pH value and specimen parameter on the hemolytic property of biodegradable magnesium
Materials Science and Engineering B 176 (2011) 1823–1826
Contents lists available at ScienceDirect
Materials Science and Engineering B journal homepage: www.elsevier.com/locate/mseb
Influence of Mg2+ concentration, pH value and specimen parameter on the hemolytic property of biodegradable magnesium Ying Chen, Shaoxiang Zhang, Jianan Li, Yang Song, Changli Zhao, Hongju Wang, Xiaonong Zhang ∗ State Key Laboratory of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China
a r t i c l e
i n f o
Article history: Received 8 October 2010 Received in revised form 12 February 2011 Accepted 8 March 2011 Available online 13 April 2011 Key words: Biodegradable Magnesium MgZn Hemolytic rate
a b s t r a c t In this study, we first evaluated the hemolytic rate of pure magnesium and MgZn alloy, and the experimental results indicated that both of these two materials showed severe hemolysis. Furthermore, a revised hemolysis test was designed to assess the impact of the Mg2+ concentration and the pH value on the hemolytic rate of magnesium. It was found that a Mg2+ concentration of 11.4 ppm in the normal saline extract with a pH value of 7.35 or 4.93 did not demonstrate any hemolysis. However, the extract with the same Mg2+ concentration but a pH value of 12.01 or 2.48 could result in heavy hemolysis. In addition, compared to the pervious study, it was suggested that there should be a preferred choice between the mass and the surface area of the specimen that used in the hemolysis test according to its different medical applications. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Cardiovascular stents fabricated with inert metals such as stainless steel or Nitinol alloy have been broadly used to treat coronary diseases due to their excellent mechanical properties over decades [1]. Nevertheless, it is regarded that the role of strutting is provisional and the persistent existence of these corrosion resistant implants seems needless [2–4]. In fact, the permanent implanted stents are prone to delay the arterial remodeling and induce chronic inflammatory reaction as well as physical irritation. Furthermore, some metallic ions (e.g. Ni) that are toxic to the human body can release from the normally incorrodible stents [5]. Therefore, magnesium and magnesium alloys have attracted great attention as metallic biomaterials to manufacture biodegradable stents due to their biodegradability, biocompatibility as well as fairly good mechanical properties [4–8]. The ideal magnesium stents are expected to thoroughly degrade or be absorbed after the healing of the pathological tissue and avoid the disadvantages brought by traditional permanent stents. Previous studies have also shown promising results [4–8]. The reported hemolytic rates of magnesium alloys, however, generally exceed 5% very much [9,10], which means they have a fatal impact on red blood cells according to ISO10993-4:2002 [11]. This is regarded to be one of the major obstacles for the applications of magnesium alloys as biodegradable blood contact
∗ Corresponding author. Tel.: +86 21 3420 2759; fax: +86 21 3420 2759. E-mail address: [email protected] (X. Zhang). 0921-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mseb.2011.03.002
materials. Magnesium alloys are highly susceptible to the corrosion in the physiological conditions according to the following equation [12]: Mg + 2H2 O → Mg(OH)2 + H2 ↑
(1)
The existing Cl− is able to transfer Mg(OH)2 into soluble MgCl2 , resulting in the release of OH− to the nearby corrosion media and thus a rise of the pH value in the surrounding environment [13]. Moreover, the degradation mechanism of magnesium stents in vascular environment can be more complicated. On one hand, blood is constantly flowing and applies shear stress on the surface of the stent. On the other hand, blood contains considerable proteins and cells and is buffered by CO2 as well as bicarbonates. In addition, the implanted material will be integrated to the surrounding tissue after some time, so hemolysis may only be a short term problem.In general, Mg2+ concentration and pH value are two typical factors considered to be potentially responsible for the high hemolytic rate of the biodegradable magnesium alloys, but the actually reason was divergent. On the other hand, due to the variation of the selected experimental parameters among different researchers, it is difficult to judge whose results are more reasonable and convincing. In this study, we investigated the hemolytic property of MgZn alloy and pure magnesium at first. Then, a modified method was designed to study the influence of Mg2+ concentration and pH value on the hemolytic rate. At last, we discussed the impact of specimen parameters on hemolysis of magnesium and provided suggestions about the selection of specimens for hemolysis tests.
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Fig. 2. Hemolytic rate of pure magnesium, MgZn alloy and extracts with different pH values.
Fig. 1. Optical image of the MgZn alloy after electropolishing.
2. Materials and methods 2.1. Preparation of material The solid solution treated Mg–6 wt.% Zn alloy was used in this study and its microstructure, compositions and fabrication process were reported in Ref. [14]. Pure magnesium (as-cast) with a purity of 99.99% was used as the counterpart. Each specimen was cut into a circular tube and its outer diameter, wall thickness and height was 11.3 mm, 0.5 mm and 41.2 mm, respectively, in order to reach a total surface area of 30 cm2 . The specimen was firstly drilled and then linearly cut from their bulks. Three replicates were prepared for each group and the experimental data were averaged. Specimens were then ground with SiC paper progressively up to 1200 grit and then electropolished and dried at room temperature. 2.2. Hemolysis test Anti-coagulated rabbit blood containing sodium citrate (3.8 wt.%) in the ratio of 9:1 was obtained and diluted by normal saline (4:5 ratio by volume). MgZn alloy and pure magnesium specimens were individually soaked in 10 ml normal saline (3 cm2 /1 ml) in a standard tube at 37 ◦ C in a water bath. After 0.5 h incubation, 0.2 ml diluted blood was added into each tube was then maintained at 37 ◦ C for 1 h. Normal saline was used as the negative control and distilled water as the positive control, respectively. After that, the tubes were all centrifuged at 2500 rpm for 5 min and the supernatant was carefully transferred to a chromometer to analyze the optical density (OD) by an ELISA Reader. The pH value and the Mg2+ concentration in the normal saline solution were also measured by PHS-3C pH meter (Lei-ci, Shanghai) and inductively coupled plasma-atomic emission spectrometry (ICP-AES) (Iris Advantage 1000), respectively. The hemolytic rate (HR) of each specimen was calculated by the following equation: HR =
ODt − ODn × 100% ODp − ODn
was adjusted to different values. Specifically, four pure magnesium specimens were separately incubated for 0.5 h in four different tubes that previously contained 10 ml normal saline at 37 ◦ C, and then all the specimens were removed. After that, the Mg2+ concentration and the pH value of each extract were measured. In fact, it could be believed that the saline in every tube had the identical Mg2+ concentration and the pH value because of the same experimental parameters. Then three tubes were randomly picked up and the pH value of the selected extracts was adjusted to be 7.35, 4.93 and 2.48 by diluted hydrochloric acid, respectively. After that, 0.2 ml diluted anti-coagulated blood was added into each extract followed by 1 h incubation at 37 ◦ C in the water bath. At last, the ODs of all extracts were measured and the HRs were calculated. 3. Results and discussion Fig. 1 is an optical image of the MgZn alloy after electropolishing. It was shown that the specimen had a bright and clean surface. Generally, HR is used to evaluate the destructive degree of the implant material to red blood cells. Fig. 2 shows the HRs of specimens and normal saline extracts. It was seen that the HR of pure magnesium, MgZn alloy, the untreated extract and the extract with a pH of 2.48 was 52.263%, 68.337%, 45.168% and 38.4%, respectively, which exceeded the recommended value of 5% considerably and meant that all of them would lead severe hemolysis according to ISO10993-4:2002. However, the extract with a pH of 7.35 or 4.93 did not demonstrate any hemolysis.
(2)
where ODt means the OD value of the test group, and ODn and ODp were the OD value of negative and positive groups, respectively. Specimens after the hemolysis test were observed under a JSM-7401F scanning electron microscope (SEM) at an accelerating voltage of 5 kV. Furthermore, a modified hemolysis test was also carried out in order to assess the impact of the Mg2+ concentration and the pH value on the hemolytic properties of pure magnesium. During the test, the Mg2+ concentration was fixed and the pH of normal saline
Fig. 3. SEM image of pure magnesium specimen after hemolysis test.
Y. Chen et al. / Materials Science and Engineering B 176 (2011) 1823–1826
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Fig. 4. Mg2+ concentration (a) and pH value (b) of the normal saline after 0.5 h and 1.5 h immersion.
Fig. 3 shows the surface morphology of pure magnesium after the hemolysis test. The cracked degradation layer indicated the presence of Mg(OH)2 on the surface. The minor deposits on the corrosion layer were probably remains of blood. Fig. 4 exhibits the Mg2+ concentration (Fig. 4a) and the pH value (Fig. 4b) of the normal saline incubating with pure magnesium for 1.5 h and 0.5 h, respectively. It is apparent that when the immersion time increased from 0.5 h to 1.5 h, there was only a minor increase of the Mg2+ concentration (11.4–12.02 ppm) and the pH value (12.01–12.43) of the normal saline. This phenomenon was mainly because of the formation of degradation layer on the surface of the specimen reduced its degradation rate as the immersion time extended. On the other hand, the soared pH value was also beneficial to the decline of the corrosion speed of magnesium. Combining the data above, it was found that there was no hemolysis when the Mg2+ concentration reached 11.4 ppm in the normal saline with a moderate pH value (7.35 or 4.93), while the extracts with the same Mg2+ concentration but a lower or higher pH (2.48 or 12.01) could cause a HR that surpassed 5% very much. Considering the released Mg2+ concentration within 1.5 h was 12.02 ppm (Fig. 4a) that was only a bit more than that within 0.5 h, it was implied that the high pH value (12.43) was probably the main factor that resulted in the excessive hemolysis of pure magnesium. This result contradicted Gao’s result that the high Mg2+ concentration released into the environment should be responsible for the high HR of magnesium [15]. It should be noted that in one of our previous investigations, the MgZn alloy was reported to possess a HR of 3.4% and had no destructive influence on erythrocyte [16]. This seemed to be conflicted with the current experimental result that the HR of MgZn alloy was 68.377%. In order to clarify this sharp contrast, it is necessary to carefully examine the details of these two experiments. Table 1 lists the specific parameters of the specimens used in the two hemolysis tests. In fact, the blood donors in these two hemolysis tests were both healthy New Zealand albino rabbits, and the procedure of the two tests was identical. Obviously, the distinction merely consisted in surface areas and masses of the specimens. According to ISO 10993-4:2002, the specimen used in the hemolysis test is recommended to be either a weight of 5 g or a total surface area of 30 cm2 . But our former tests manifested that the MgZn alloy with a weight of 5 g (with a surface area of 11.76 cm2 ) had a much lower HR compared to the one with a surface area of Table 1 Parameters of the specimens used in hemolysis test. Specimen
Surface area (cm2 )
Mass (g)
Hemolytic rate
MgZn MgZn
30 11.76
2.675 5
68.377% 3.4%
30 cm2 (with a mass of 2.675 g) used in this test. This was probably because a larger surface meant more contact between the specimen and the solution, thus leading to a more sufficient reaction and higher pH values and HRs. Therefore, it can be suggested that the specimen preparation for the hemolysis test should be dependent on its medical applications. The orthopedic implants are generally bulks, so they have smaller specific surface areas and larger masses than the cardiovascular stents which are thin tubes of metallic mesh. As a result, it is purposed that for the implant material potentially used as orthopedic implants, a whole weight of 5 g is a preference for the hemolysis test. In contrast, for the hemolysis test of stent materials, the entire surface area of 30 cm2 may be more reasonable. 4. Conclusion In this study, we investigated the HR of pure magnesium, MgZn alloy and the normal saline extracts of pure magnesium with various pH. It was found that pure magnesium, MgZn alloy and the extract with a pH of 12.01 or 2.48 possessed HRs much higher than 5%, but the extract with a pH of 7.35 or 4.93 did not showed any hemolysis. This indicated that the high HR of biodegradable magnesium was probably due to the high pH instead of dissolved Mg2+ . On the other hand, it was suggested that there should be a preference when considering the parameters of the specimen for the hemolysis test according to its potential medical applications. Acknowledgements The authors are grateful for the supports from the Natural Science Foundation of China (No. 30772182 and No. 30901422), Shanghai Jiao Tong University Interdisciplinary Research Grants (Grant No. YG2009MS53) and the “863” High-Tech Plan of China (No. 2009AA03Z424). References [1] R. Balcon, R. Beyar, S. Chierchia, I. De Scheerder, P.G. Hugenholtz, F. Kiemeneij, et al., European Heart Journal 18 (1997) 1536–1547. [2] P.H. Grewe, D. Thomas, A. Machraoui, J. Barmeyer, K.M. Muller, American Journal of Cardiology 85 (2000) 554–558. [3] M. Bosiers, P. Peeters, O. D’Archambeau, J. Hendriks, E. Pilger, C. Düber, et al., Cardiovascular and Interventional Radiology 32 (2009) 424–435. [4] R. Erbel, C. Di Mario, J. Bartunek, J. Bonnier, B. de Bruyne, F.R. Eberli, et al., Lancet 369 (2007) 1869–1875. [5] G. Mani, M.D. Feldman, D. Patel, C.M. Agrawal, Biomaterials 28 (2007) 1689–1710. [6] B. Heublein, R. Rohde, V. Kaese, M. Niemeyer, W. Hartung, A. Haverich, Heart 9 (2003) 651–656. [7] C.J. McMahon, P. Oslizlok, K.P. Walsh, Catheterization and Cardiovascular Interventions 69 (2007) 735–738. [8] P. Peeters, M. Bosier, J. Verbist, K. Deloose, B. Heublein, Journal of Endovascular Therapy 12 (2005) 1–5.
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[9] X.N. Gu, Y.F. Zheng, C. Yan, S.P. Zhong, T.F. Xi, Biomaterials 30 (2009) 484–498. [10] E.L. Zhang, D.S. Yin, L.P. Xu, L. Yang, K. Yang, Materials Science and Engineering: C 29 (2009) 987–993. [11] ISO 10993-4:2002, The International Organization for Standardization, 2002. [12] G. Song, A. Atrens, D. StJohn, J. Nairn, Y. Li, Corrosion Science 39 (1997) 855–875. [13] G. Song, A. Atrens, D. StJohn, X. Wu, J. Nairn, Corrosion Science 39 (1997) 1981–2004.
[14] S.X. Zhang, X.N. Zhang, C.L. Zhao, J.N. Li, Y. Song, C.Y. Xie, et al., Acta Biomaterialia 6 (2010) 626–640. [15] J.C. Gao, L.Y. Qiao, C.Y. Li, Y. Wang, Transactions of Nonferrous Metals Society of China 16 (2006) 588–592. [16] S.X. Zhang, J.N. Li, Y. Song, C.L. Zhao, X.N. Zhang, C.Y. Xie, et al., Materials Science and Engineering: C 29 (2009) 1907–1912.
Contents lists available at ScienceDirect
Materials Science and Engineering B journal homepage: www.elsevier.com/locate/mseb
Influence of Mg2+ concentration, pH value and specimen parameter on the hemolytic property of biodegradable magnesium Ying Chen, Shaoxiang Zhang, Jianan Li, Yang Song, Changli Zhao, Hongju Wang, Xiaonong Zhang ∗ State Key Laboratory of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China
a r t i c l e
i n f o
Article history: Received 8 October 2010 Received in revised form 12 February 2011 Accepted 8 March 2011 Available online 13 April 2011 Key words: Biodegradable Magnesium MgZn Hemolytic rate
a b s t r a c t In this study, we first evaluated the hemolytic rate of pure magnesium and MgZn alloy, and the experimental results indicated that both of these two materials showed severe hemolysis. Furthermore, a revised hemolysis test was designed to assess the impact of the Mg2+ concentration and the pH value on the hemolytic rate of magnesium. It was found that a Mg2+ concentration of 11.4 ppm in the normal saline extract with a pH value of 7.35 or 4.93 did not demonstrate any hemolysis. However, the extract with the same Mg2+ concentration but a pH value of 12.01 or 2.48 could result in heavy hemolysis. In addition, compared to the pervious study, it was suggested that there should be a preferred choice between the mass and the surface area of the specimen that used in the hemolysis test according to its different medical applications. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Cardiovascular stents fabricated with inert metals such as stainless steel or Nitinol alloy have been broadly used to treat coronary diseases due to their excellent mechanical properties over decades [1]. Nevertheless, it is regarded that the role of strutting is provisional and the persistent existence of these corrosion resistant implants seems needless [2–4]. In fact, the permanent implanted stents are prone to delay the arterial remodeling and induce chronic inflammatory reaction as well as physical irritation. Furthermore, some metallic ions (e.g. Ni) that are toxic to the human body can release from the normally incorrodible stents [5]. Therefore, magnesium and magnesium alloys have attracted great attention as metallic biomaterials to manufacture biodegradable stents due to their biodegradability, biocompatibility as well as fairly good mechanical properties [4–8]. The ideal magnesium stents are expected to thoroughly degrade or be absorbed after the healing of the pathological tissue and avoid the disadvantages brought by traditional permanent stents. Previous studies have also shown promising results [4–8]. The reported hemolytic rates of magnesium alloys, however, generally exceed 5% very much [9,10], which means they have a fatal impact on red blood cells according to ISO10993-4:2002 [11]. This is regarded to be one of the major obstacles for the applications of magnesium alloys as biodegradable blood contact
∗ Corresponding author. Tel.: +86 21 3420 2759; fax: +86 21 3420 2759. E-mail address: [email protected] (X. Zhang). 0921-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mseb.2011.03.002
materials. Magnesium alloys are highly susceptible to the corrosion in the physiological conditions according to the following equation [12]: Mg + 2H2 O → Mg(OH)2 + H2 ↑
(1)
The existing Cl− is able to transfer Mg(OH)2 into soluble MgCl2 , resulting in the release of OH− to the nearby corrosion media and thus a rise of the pH value in the surrounding environment [13]. Moreover, the degradation mechanism of magnesium stents in vascular environment can be more complicated. On one hand, blood is constantly flowing and applies shear stress on the surface of the stent. On the other hand, blood contains considerable proteins and cells and is buffered by CO2 as well as bicarbonates. In addition, the implanted material will be integrated to the surrounding tissue after some time, so hemolysis may only be a short term problem.In general, Mg2+ concentration and pH value are two typical factors considered to be potentially responsible for the high hemolytic rate of the biodegradable magnesium alloys, but the actually reason was divergent. On the other hand, due to the variation of the selected experimental parameters among different researchers, it is difficult to judge whose results are more reasonable and convincing. In this study, we investigated the hemolytic property of MgZn alloy and pure magnesium at first. Then, a modified method was designed to study the influence of Mg2+ concentration and pH value on the hemolytic rate. At last, we discussed the impact of specimen parameters on hemolysis of magnesium and provided suggestions about the selection of specimens for hemolysis tests.
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Fig. 2. Hemolytic rate of pure magnesium, MgZn alloy and extracts with different pH values.
Fig. 1. Optical image of the MgZn alloy after electropolishing.
2. Materials and methods 2.1. Preparation of material The solid solution treated Mg–6 wt.% Zn alloy was used in this study and its microstructure, compositions and fabrication process were reported in Ref. [14]. Pure magnesium (as-cast) with a purity of 99.99% was used as the counterpart. Each specimen was cut into a circular tube and its outer diameter, wall thickness and height was 11.3 mm, 0.5 mm and 41.2 mm, respectively, in order to reach a total surface area of 30 cm2 . The specimen was firstly drilled and then linearly cut from their bulks. Three replicates were prepared for each group and the experimental data were averaged. Specimens were then ground with SiC paper progressively up to 1200 grit and then electropolished and dried at room temperature. 2.2. Hemolysis test Anti-coagulated rabbit blood containing sodium citrate (3.8 wt.%) in the ratio of 9:1 was obtained and diluted by normal saline (4:5 ratio by volume). MgZn alloy and pure magnesium specimens were individually soaked in 10 ml normal saline (3 cm2 /1 ml) in a standard tube at 37 ◦ C in a water bath. After 0.5 h incubation, 0.2 ml diluted blood was added into each tube was then maintained at 37 ◦ C for 1 h. Normal saline was used as the negative control and distilled water as the positive control, respectively. After that, the tubes were all centrifuged at 2500 rpm for 5 min and the supernatant was carefully transferred to a chromometer to analyze the optical density (OD) by an ELISA Reader. The pH value and the Mg2+ concentration in the normal saline solution were also measured by PHS-3C pH meter (Lei-ci, Shanghai) and inductively coupled plasma-atomic emission spectrometry (ICP-AES) (Iris Advantage 1000), respectively. The hemolytic rate (HR) of each specimen was calculated by the following equation: HR =
ODt − ODn × 100% ODp − ODn
was adjusted to different values. Specifically, four pure magnesium specimens were separately incubated for 0.5 h in four different tubes that previously contained 10 ml normal saline at 37 ◦ C, and then all the specimens were removed. After that, the Mg2+ concentration and the pH value of each extract were measured. In fact, it could be believed that the saline in every tube had the identical Mg2+ concentration and the pH value because of the same experimental parameters. Then three tubes were randomly picked up and the pH value of the selected extracts was adjusted to be 7.35, 4.93 and 2.48 by diluted hydrochloric acid, respectively. After that, 0.2 ml diluted anti-coagulated blood was added into each extract followed by 1 h incubation at 37 ◦ C in the water bath. At last, the ODs of all extracts were measured and the HRs were calculated. 3. Results and discussion Fig. 1 is an optical image of the MgZn alloy after electropolishing. It was shown that the specimen had a bright and clean surface. Generally, HR is used to evaluate the destructive degree of the implant material to red blood cells. Fig. 2 shows the HRs of specimens and normal saline extracts. It was seen that the HR of pure magnesium, MgZn alloy, the untreated extract and the extract with a pH of 2.48 was 52.263%, 68.337%, 45.168% and 38.4%, respectively, which exceeded the recommended value of 5% considerably and meant that all of them would lead severe hemolysis according to ISO10993-4:2002. However, the extract with a pH of 7.35 or 4.93 did not demonstrate any hemolysis.
(2)
where ODt means the OD value of the test group, and ODn and ODp were the OD value of negative and positive groups, respectively. Specimens after the hemolysis test were observed under a JSM-7401F scanning electron microscope (SEM) at an accelerating voltage of 5 kV. Furthermore, a modified hemolysis test was also carried out in order to assess the impact of the Mg2+ concentration and the pH value on the hemolytic properties of pure magnesium. During the test, the Mg2+ concentration was fixed and the pH of normal saline
Fig. 3. SEM image of pure magnesium specimen after hemolysis test.
Y. Chen et al. / Materials Science and Engineering B 176 (2011) 1823–1826
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Fig. 4. Mg2+ concentration (a) and pH value (b) of the normal saline after 0.5 h and 1.5 h immersion.
Fig. 3 shows the surface morphology of pure magnesium after the hemolysis test. The cracked degradation layer indicated the presence of Mg(OH)2 on the surface. The minor deposits on the corrosion layer were probably remains of blood. Fig. 4 exhibits the Mg2+ concentration (Fig. 4a) and the pH value (Fig. 4b) of the normal saline incubating with pure magnesium for 1.5 h and 0.5 h, respectively. It is apparent that when the immersion time increased from 0.5 h to 1.5 h, there was only a minor increase of the Mg2+ concentration (11.4–12.02 ppm) and the pH value (12.01–12.43) of the normal saline. This phenomenon was mainly because of the formation of degradation layer on the surface of the specimen reduced its degradation rate as the immersion time extended. On the other hand, the soared pH value was also beneficial to the decline of the corrosion speed of magnesium. Combining the data above, it was found that there was no hemolysis when the Mg2+ concentration reached 11.4 ppm in the normal saline with a moderate pH value (7.35 or 4.93), while the extracts with the same Mg2+ concentration but a lower or higher pH (2.48 or 12.01) could cause a HR that surpassed 5% very much. Considering the released Mg2+ concentration within 1.5 h was 12.02 ppm (Fig. 4a) that was only a bit more than that within 0.5 h, it was implied that the high pH value (12.43) was probably the main factor that resulted in the excessive hemolysis of pure magnesium. This result contradicted Gao’s result that the high Mg2+ concentration released into the environment should be responsible for the high HR of magnesium [15]. It should be noted that in one of our previous investigations, the MgZn alloy was reported to possess a HR of 3.4% and had no destructive influence on erythrocyte [16]. This seemed to be conflicted with the current experimental result that the HR of MgZn alloy was 68.377%. In order to clarify this sharp contrast, it is necessary to carefully examine the details of these two experiments. Table 1 lists the specific parameters of the specimens used in the two hemolysis tests. In fact, the blood donors in these two hemolysis tests were both healthy New Zealand albino rabbits, and the procedure of the two tests was identical. Obviously, the distinction merely consisted in surface areas and masses of the specimens. According to ISO 10993-4:2002, the specimen used in the hemolysis test is recommended to be either a weight of 5 g or a total surface area of 30 cm2 . But our former tests manifested that the MgZn alloy with a weight of 5 g (with a surface area of 11.76 cm2 ) had a much lower HR compared to the one with a surface area of Table 1 Parameters of the specimens used in hemolysis test. Specimen
Surface area (cm2 )
Mass (g)
Hemolytic rate
MgZn MgZn
30 11.76
2.675 5
68.377% 3.4%
30 cm2 (with a mass of 2.675 g) used in this test. This was probably because a larger surface meant more contact between the specimen and the solution, thus leading to a more sufficient reaction and higher pH values and HRs. Therefore, it can be suggested that the specimen preparation for the hemolysis test should be dependent on its medical applications. The orthopedic implants are generally bulks, so they have smaller specific surface areas and larger masses than the cardiovascular stents which are thin tubes of metallic mesh. As a result, it is purposed that for the implant material potentially used as orthopedic implants, a whole weight of 5 g is a preference for the hemolysis test. In contrast, for the hemolysis test of stent materials, the entire surface area of 30 cm2 may be more reasonable. 4. Conclusion In this study, we investigated the HR of pure magnesium, MgZn alloy and the normal saline extracts of pure magnesium with various pH. It was found that pure magnesium, MgZn alloy and the extract with a pH of 12.01 or 2.48 possessed HRs much higher than 5%, but the extract with a pH of 7.35 or 4.93 did not showed any hemolysis. This indicated that the high HR of biodegradable magnesium was probably due to the high pH instead of dissolved Mg2+ . On the other hand, it was suggested that there should be a preference when considering the parameters of the specimen for the hemolysis test according to its potential medical applications. Acknowledgements The authors are grateful for the supports from the Natural Science Foundation of China (No. 30772182 and No. 30901422), Shanghai Jiao Tong University Interdisciplinary Research Grants (Grant No. YG2009MS53) and the “863” High-Tech Plan of China (No. 2009AA03Z424). References [1] R. Balcon, R. Beyar, S. Chierchia, I. De Scheerder, P.G. Hugenholtz, F. Kiemeneij, et al., European Heart Journal 18 (1997) 1536–1547. [2] P.H. Grewe, D. Thomas, A. Machraoui, J. Barmeyer, K.M. Muller, American Journal of Cardiology 85 (2000) 554–558. [3] M. Bosiers, P. Peeters, O. D’Archambeau, J. Hendriks, E. Pilger, C. Düber, et al., Cardiovascular and Interventional Radiology 32 (2009) 424–435. [4] R. Erbel, C. Di Mario, J. Bartunek, J. Bonnier, B. de Bruyne, F.R. Eberli, et al., Lancet 369 (2007) 1869–1875. [5] G. Mani, M.D. Feldman, D. Patel, C.M. Agrawal, Biomaterials 28 (2007) 1689–1710. [6] B. Heublein, R. Rohde, V. Kaese, M. Niemeyer, W. Hartung, A. Haverich, Heart 9 (2003) 651–656. [7] C.J. McMahon, P. Oslizlok, K.P. Walsh, Catheterization and Cardiovascular Interventions 69 (2007) 735–738. [8] P. Peeters, M. Bosier, J. Verbist, K. Deloose, B. Heublein, Journal of Endovascular Therapy 12 (2005) 1–5.
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[9] X.N. Gu, Y.F. Zheng, C. Yan, S.P. Zhong, T.F. Xi, Biomaterials 30 (2009) 484–498. [10] E.L. Zhang, D.S. Yin, L.P. Xu, L. Yang, K. Yang, Materials Science and Engineering: C 29 (2009) 987–993. [11] ISO 10993-4:2002, The International Organization for Standardization, 2002. [12] G. Song, A. Atrens, D. StJohn, J. Nairn, Y. Li, Corrosion Science 39 (1997) 855–875. [13] G. Song, A. Atrens, D. StJohn, X. Wu, J. Nairn, Corrosion Science 39 (1997) 1981–2004.
[14] S.X. Zhang, X.N. Zhang, C.L. Zhao, J.N. Li, Y. Song, C.Y. Xie, et al., Acta Biomaterialia 6 (2010) 626–640. [15] J.C. Gao, L.Y. Qiao, C.Y. Li, Y. Wang, Transactions of Nonferrous Metals Society of China 16 (2006) 588–592. [16] S.X. Zhang, J.N. Li, Y. Song, C.L. Zhao, X.N. Zhang, C.Y. Xie, et al., Materials Science and Engineering: C 29 (2009) 1907–1912.