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In vitro corrosion and biocompatibility study of phytic acid modified WE43 magnesium alloy
Applied Surface Science 258 (2012) 3420–3427
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In vitro corrosion and biocompatibility study of phytic acid modified WE43 magnesium alloy C.H. Ye a , Y.F. Zheng a,b , S.Q. Wang c , T.F. Xi a,c,∗ , Y.D. Li d a
Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China School of Information & Engineering, Wenzhou Medical College, Zhejiang 325035, China d DongGuan EONTEC Co., Ltd., Guangdong 523000, China b c
a r t i c l e
i n f o
Article history: Received 2 October 2011 Received in revised form 15 November 2011 Accepted 17 November 2011 Available online 26 November 2011 Keywords: Magnesium alloy Phytic acid Corrosion Biocompatibility
a b s t r a c t Phytic acid (PA) conversion coating on WE43 magnesium alloy was prepared by the method of immersion. The influences of phytic acid solution with different pH on the microstructure, properties of the conversion coating and the corrosion resistance were investigated by SEM, FTIR and potentiodynamic polarization method. Furthermore, the biocompatibility of different pH phytic acid solution modified WE43 magnesium alloys was evaluated by MTT and hemolysis test. The results show that PA can enhance the corrosion resistance of WE43 magnesium especially when the pH value of modified solution is 5 and the cytotoxicity of the PA coated WE43 magnesium alloy is much better than that of the bare WE43 magnesium alloy. Moreover, all the hemolysis rates of the PA coated WE43 Mg alloy were lower than 5%, indicating that the modified Mg alloy met the hemolysis standard of biomaterials. Therefore, PA coating is a good candidate to improve the biocompatibility of WE43 magnesium alloy. © 2012 Published by Elsevier B.V.
1. Introduction As we all know coronary stent placement has replaced balloon angioplasty as the standard of care for percutaneous coronary intervention. But at the same time in-stent restenosis (ISR) has become a widespread problem because of the permanent nature of the metallic stents, they posed the risk of a continuous interaction between the stent and the surrounding tissue, leading to long-term endothelial dysfunction or chronic inflammatory reaction. Although the usage of rapamycin or paclitaxel can reduce late in-stent restenosis rates [1], thrombosis or the inflammatory reaction caused by the long-time implanted stent or the polymer restricted the usage of drug eluting stent still exist. Stack et al. [2] have taken the lead in developing biodegradable stent (BDS) in 1988, and the animal experiments revealed that it can significantly reduce the in-stent restenosis rates. The BDSs under investigation mainly include (1): biodegradable magnesium alloy [3–6]; (2): biodegradable polymer, for example, PLA/PGA [7,8], PHBV [9], PCL [10] etc.; (3): biodegradable iron alloy [11,12].
∗ Corresponding author at: Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. Tel.: +86 10 6275 3404; fax: +86 10 6275 3404. E-mail address: [email protected] (T.F. Xi). 0169-4332/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.apsusc.2011.11.087
For biodegradable magnesium alloy, the current research mainly focused on AE21 [13], WE43 [14,15], AZ91 [16], etc. Di Mario et al. [17] studied the in vivo corrosion of magnesium stents made of WE43 alloy implanted in minipigs. Their animal and clinical experiment results showed that the WE43 alloy stents possessed sufficient anti-proliferative properties and drastically reduced the restenosis rate in comparison to conventional stainless steel stents. Loos et al. [18] also found that reduced WE43 magnesium alloy will reduce the viability and proliferation of smooth muscle cells compared with clinically applied steel stent (316L). However, the magnesium alloys corrodes too quickly in the physiological environment. To improve the corrosion resistance of the Mg alloys, various surface modification techniques, including alkaline treatment [19], heat treatment [20], microarcoxidation [21] and electrodeposition [22] have been developed recently. One surface treatment technique, phytic acid (PA) treatment has been investigated as a simple and effective methods for Mg alloy [23–25]. Firstly, phytic acid (C6 H18 O24 P6 ) is a natural and nontoxic, organic large molecular compound which present in most legumes, including corn, soy beans and nuts [26,27]. On the other hand, recent studies have shown its ability of alteration in signal transduction [28], stimulation of genes toward greater cell differentiation [29], antioxidant as food additive [30,27], and anticancer [31,32]. Even so, how is about the corrosion
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Fig. 1. The FTIR characteristic bands of phytic acid and phytic acid modified WE43 Mg alloy with different pH.
resistance of phytic acid modified magnesium alloy in simulated body fluid and the biocompatibility of phytic acid are still unknown. In this paper, in vitro corrosion and biocompatibility of phytic acid modified WE43 magnesium alloy was studied. The chemical nature of this conversion coating was investigated by Fourier transform infrared spectroscopy (FTIR) and ESEM equipped with the energy dispersive spectrum (EDS). The corrosion resistance was examined by potentiodynamic polarization method and the immersion test was performed in simulated body fluid (SBF). Furthermore, the biocompatibility of different pH phytic acid solution modified WE43 magnesium alloys was evaluated by MTT and hemolysis test.
2. Material and methods 2.1. Preparation of specimens The as-extruded WE43 alloy (91.35 wt.% Mg, 4.16 wt.% Y, 3.80 wt.% RE, 0.36 wt.% Zr, 0.20 wt.% Zn, 0.13 wt.% Mn) provided by Changchun Zhong-Ke-Xi-Mei Magnesium Alloy Co. Ltd., China, was cut into specimens with diameter of 8 mm and the height of 1.5 mm, then polished successively with 800#, 1200#, and 2000# grit SiC paper, pretreated with acetone and deionized water and dried with hot air, and treated in phytic acid solution. Phytic acid is chemical reagent, purity > 75%, and the other materials are all analytical reagent, purity ≥ 99%. The conversion coatings were prepared by immerging the substrate in the phytic acid solutions with different pH (pH 3, 5, 8 and 10, adjusted by ammonia water) at 40 ◦ C. After immersion, the samples were thoroughly washed with deionized water, dried overnight at 50 ◦ C and the untreated WE43 Mg alloy sample is taken as control. 2.2. Microstructural analysis of the specimens The microstructure was characterized using environmental scanning electron microscope equipping with the energy dispersive spectrum and Fourier transform infrared spectroscopy spectrophotometer (Nicolet 750, Nicolet Co.) in the spectral range 650–4000 cm−1 using attenuated total reflectance mode.
2.3. Immersion test The immersion test was identical to the hydrogen evolution method according to Lin [33]. The samples were put into the center of the beaker filled with SBF solution, and the hydrogen formed in the process of magnesium alloy corrosion was collected in the above volume of the funnel which was vertically laid above the sample surface. The corrosion resistance of samples could be evaluated based on the change of the rate of hydrogen evolved at interval time. 2.4. Electrochemical test The electrochemical measurement was carried out in the SBF solution and the potentiodynamic polarization curves of the modified WE43 magnesium alloy samples were obtained on a three-electrode system and the immersion tests were carried out in SBF buffer. The working electrode is WE43 Mg alloy samples with the exposed area of 1 cm2 . The temperature was kept to be 37 ± 0.5 ◦ C using a water bath. 2.5. Biocompatibility evaluation The biocompatibility of the WE43 magnesium alloy with or without phytic acid modified was evaluated by cytotoxicity experiment and blood hemolysis experiment. L929 cells were used to evaluate the cytotoxicity of the magnesium alloy with or without phytic acid modified samples by the indirect assay. The extracts were prepared using Dulbecco’s Modified Eagle’s Medium (DMEM) serum free medium with the surface area of extraction medium ratio 1.5 ml/cm2 in a humidified atmosphere with 5% CO2 at 37 ◦ C for 72 h. The supernatant fluid was withdrawn after centrifugation and then refrigerated at 4 ◦ C. L929 cells were incubated in 96-well culture plates at 1 × 104 /ml for 24 h to allow attachment and the medium was then replaced with 100 l extract. After 1, 2 and 4 days culture, 10 l MTT was added to each well cultured for 4 h and then 100 ml of the supernatant was measured spectrophotometrically at 570 nm by Elx800 (BioTek instruments). The background with MTT results of extracts was subtracted. In order to evaluate the blood compatibility of the samples, the samples were soaked in 10 ml normal physiological saline in a tube
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Fig. 2. SEM micrographs and the EDS spectrum of the phytic acid modified WE43 magnesium alloy with the solution of (a) pH 3, (b) pH 5, (c) pH 8, (d) pH 10.
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and kept at 37 ◦ C for 30 min. Eight milliliter arterial blood of the volunteer was diluted by 10 ml normal physiological saline that contained 0.5 ml potassium oxalate (20 g/l) decoagulant. After that 0.2 ml diluted blood was added into the tube and kept at 37 ◦ C for 60 min. Then the tube was centrifuged at 2500 rpm for 5 min. At last, the optical density (OD) was obtained by a spectrophotometer (722, Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China) at 545 nm wave length. The negative and positive groups were 10 ml normal physiological saline with 0.2 ml diluted blood and 10 ml distilled water with 0.2 ml diluted blood, respectively. The hemolysis ratio (HR) was calculated according to the equation: HR = [(ODt − ODn)/(ODp − ODn)] × 100%. The ODt means the OD value of tested sample. The ODn and ODp were OD value of negative and positive groups, respectively. 3. Results 3.1. Microstructure of the specimens The characteristic bonds of pure phytic acid and phytic acid with different pH modified WE43 Mg alloy are shown in Fig. 1. The characteristic bands of phytic acid centers at 995 cm−1 , 1462 cm−1 and 3302 cm−1 , corresponding to PO4 3− , HPO4 2− and OH− respectively. PO4 3− can be identified from the FTIR spectra of phytic acid conversion coating, which was shown at about 1200–940 cm−1 . Though some bands have shifted a little, the functional groups of conversion coatings formed in different pH of phytic acid solution are much close to the constituent of phytic acid. But as the conversion coating was dried at 50 ◦ C overnight, OH– (at about 3400 cm−1 ) was not obvious on the phytic acid treated WE43 magnesium alloy samples by FTIR method. Fig. 2 shows the morphology and the element compositions of the coatings formed in different pH of phytic acid solutions. It can be seen that the coatings presents bulk feature, and plenty of microcracks exist on the surface of the film. But the cracks are much bigger in Fig. 2(a) and (b) which corresponding the pH 3 and pH 5 modification solutions than that in Fig. 2(c) and (d), while the film of the latter groups are smoother. The chemical composition of the film was analyzed by EDS as shown on the right of the morphology picture. From the element compositions we can see that the coatings was mainly composed of Mg, O, P, and C elements, and the presence of P showed that phytic acid was successfully coated on the Mg alloy surface, in the formation of the coatings. But its content changed greatly, with the pH of the coating solution changed from 3, 5, 8 to 10, P element content (wt.%) changed from 11.47%, 11.67%, 7.22%, to 5.87% correspondingly, which illustrated that more phytic acid were coated on the pH 3 and pH 5 solution, while the solution of pH 8 and pH 10 formed less phytic acid coatings. 3.2. Immersion test Fig. 3 shows the hydrogen evolution rate of the bare WE43 magnesium alloy and the modified WE43 magnesium alloy with phytic acid solution in SBF solution for about 120 h. The overall corrosion reaction of magnesium at its free corrosion potential can be expressed as follows: Mg + 2H2 O = Mg2+ + 2OH− + H2 ↑
(1)
One mole hydrogen evolution corresponding to 1 mole WE43 magnesium alloy has been corroded, which can be taken as a good indicator to evaluate the corrosiveness of Mg alloy. The results showed that (1) the sample modified in pH 10 group exhibit faster corrosion rate than the other groups; (2) the samples modified in pH 8 and pH 3 groups have the similar corrosion rate with the control; (3) pH 5 group has the lowest corrosion rate.
Fig. 3. The hydrogen evolution volumes of the phytic acid modified WE43 Mg with different pH and the control samples as a function of the immersion time in SBF.
Fig. 4. Potentiodynamic polarization curves of the phytic acid modified WE43 Mg samples and the control in SBF solutions at 37 ◦ C.
Table 1 The average electrochemical parameters for the electrochemical polarization curves of different samples immersed in SBF solutions at 37 ◦ C.
pH 3 pH 5 pH 8 pH 10 Control
Potential (V)
Logi (A cm−2 )
−1.84 −1.79 −1.85 −2.08 −1.85
5.26 × 10−4 8.08 × 10−4 5.08 × 10−4 3.07 × 10−3 5.09 × 10−4
3.3. Electrochemical test Fig. 4 presents the electrochemical polarization curves of different samples immersed in SBF solutions at 37 ◦ C. The average electrochemical parameters are also show in Table 1. The results showed that (1) the order of the corrosion rate is similar to the hydrogen results; (2) the pH 5 group has the best corrosion resistance, while the pH 10 group has the fast corrosion rate.
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Fig. 5. L-929 cell viability expressed as a percentage of the viability of cells in the control and phytic acid modified WE43 Mg extraction solution after 1, 2 and 4 days of culture.
solution pH has a great effect on the modification effect. Especially when the reaction solution’s pH 5, the modified Mg alloy has the best corrosion resistance and biocompatibility. The FTIR analysis (Fig. 1) indicated that phytic acid could modify WE43 Mg alloy whether the solution is in acidic or basic conditions. Cui [34] has given the simple mechanism of the chelate process of the phytic acid on magnesium alloy. In short, a phytic has 12 hydroxyl groups and 6 phosphate carboxyl groups (Fig. 7), which makes it have a powerful capability of chelating with many metal ions in a wide pH scope, for example, copper [35], magnesium [34], zinc [36]. In aqueous solution, the phosphate group of the phytic acid will ionize to form hydronium ion, phytic acid ion with different number of phosphate ion (PO4 3− ) and hydrogen phosphate ion (HPO4 2− ) according to the different pH condition. At the same time, a large number of magnesium ions are ionized at the incipient stage when the WE43 magnesium alloy is immersed in phytic acid solution. These magnesium ions will combine with phytic acid ion to form phytic–Mg phytate complex, which then deposited on the surface of the WE43 magnesium alloy substrate (Fig. 8(a)). As the phytic acid have 12 hydroxyl groups, one phytic acid can chelate with different number of magnesium ions at different solution conditions which will result in different modification effect. The reaction can be referred as the following equation [38]: −iMgn+ + Hj Phy(12−j)− = Mgi Hj (12−2i−j)− Phy
Fig. 6. Hemolysis percentage of the WE43 Mg alloy and the phytic acid modified WE43 Mg alloy.
3.4. Biocompatibility evaluation Fig. 5 illustrates the viability of L-929 expressed as a percentage of the viability of cells cultured in negative control after cultured in DMEM and the phytic acid modified Mg alloys extraction medium solutions for different periods (1, 2 and 4 days). It means that all the modified groups have a better cell viability than the unmodified control. Among the modified groups, the pH 5 group has the highest viability in the three time point ranging from 60% to 75% while the control group’s cell viability is only from 28% to 34%. For the hemolysis test, compared with the control group, the rate of hemolysis decreased (the hemolysis of modified WE43 magnesium alloy changed from 9.27% to about 2% as shown in Fig. 6) after the WE43 Mg alloy immersed in the PA solution with different pH values. All the hemolysis rates of the PA coated WE43 magnesium alloys were lower than 5%, indicating that the modified WE43 magnesium alloy met the hemolysis standard of biomedical materials. 4. Discussion Our study has demonstrated that phytic acid modified WE43 Mg alloy has a good corrosion resistance and biocompatibility compared with the bare WE43 Mg alloy. In addition, the aqueous
(2)
in which Phy referred as phytic acid ions, i and j referred as the different reaction content, and this reaction was influenced by the following factors [37–40]: (1) the pH of the solutions; (2) the concentration of magnesium ions; (3) the conformation of phytic acid. In brief, the more Mg2+ and more phytic acid ions will accelerate this reaction to form the chelate complex’s, but the complex was not stable in a too low pH or a too high pH solution [38], though this condition will produce more Mg2+ or phytic acid radical ion compare with the moderate solution. So for a given pH solution and the magnesium alloy, the conformation of the phytic acid and the concentration of Mg2+ were the only two factors that strongly influence the reaction. But on the other hand, the magnesium ions can obtained only by the WE43 Mg alloy corrosion, from the Eq. (1) we know that the magnesium alloy corrosion rate was controlled by the pH and is increased with the decrease of solution pH, Ambat et al. [41] have also proved this conclusion. In Fig. 2 we can see in pH 3 and pH 5 solution, the coating has a higher content of P which suggests the higher level of Phy–Mg complex on the surface of the Mg alloy, in contrast the pH 8 and pH 10 solution have the less content of this complex. This can be explained that in pH 3 solution the strong acid environment can produce more Mg2+ , but Davidsson et al. [40] have proved that the Phy–Mg complex was partly soluble in pH 3 solution, so under this condition, there will be a little chelate complex on the surface of the WE43 Mg alloy. But for the pH 5 the acid of the solution condition was not so strong, this condition can give rise to enough Mg2+ to react with the deionizated hydroxyl groups in phytic acid, and the complex–Mg complex is stable in this pH condition, so we can see the more P content in pH 5 group. But in pH 8 solution, the basic condition will slow down Mg alloy corrosion rate which will inhibit the Mg2+ release though the phytic acid can produce many hydroxyl ions groups, which is similar with the solution in pH 10, so in pH 8 group, there were less Phy–Mg complex on the coating, the corrosion resistance only slightly higher (Fig. 2) compared with the two acid groups. The difference between the pH 10 solution and the other experimental groups is that the conformation of the phytic acid will change to 5 ax/1 eq from 1 ax/5 eq when the pH changed from below 9.0 to above 9.0 [39] (‘ax’ is the axial bond which is the bond between a carbon and a substituent that is projected vertically up or down on a ring conformation. ‘eq’ is the equatorial band which is the bond between a carbon and a
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Fig. 7. The structure of the phytic acid.
Fig. 8. (a) The mechanism of the Phy–Mg complex formation; (b) the conformation of the phytic acid in different solution condition.
substituent that extends out of a ring of carbons in a ring conformation). As the eq band is unstable, this conformation will change back to 1 ax/5 eq when the solution pH below 9.0, so in the reaction solution, the deprotonated Phy will react with the trace amounts of Mg2+ to form Phy–Mg complex, but when the modified alloy was placed into the SBF testing solution, the conformation of the Phy which was deposited onto the surface of the alloy will quickly reverts to 1 ax/5 eq conformation. During this change, the chelate bond between Mg2+ with phosphate groups, especially the phosphate group in the meta position will break, which will result in the migration of phytic acid to the SBF solution, and because the SBF testing solution has more Ca2+ than Mg2+ (2.5 mM vs 1.5 Mm) and the stability of the calcium–Phy is higher than the magnesium–Phy [37], the Phy after the bond cleavage will react with the calcium in the SBF testing solution to form Ca–Phy complex. But as the phytic acid resolved from the coating is very little, so little phytic acid chelate complex will precipitate on the surface, during this balance reaction, the Phy–Mg chelate complex resolved to phytic acid and Mg2+ would be the main form, which will accelerate Mg alloy corrosion. From Figs. 2 and 3 we can see the samples modified in the pH 5 has the best corrosion resistance, but samples modified in the pH 10 has the fastest corrosion rate.
As the phytic acid is widely present in nature (plants, animals and soils), mainly as calcium, magnesium and potassium mixed salts (also called phytines) and its important biological activity and the high number of applications, since the last century it has been the subject of investigation for many scientists in different fields, for example, enhanced immunity [42], anticancer including breast [43], colon [44], liver [45,46], prostate [47], rhabdomyosarcoma [48], antioxidant properties [49], etc. Without a doubt, the biocompatibility of the phytic acid must be very good, which is also proved by the present results of the MTT test and hymolysis. 5. Conclusions The present study investigated the corrosion resistance and the biocompatibility of the phytic acid modified WE43 magnesium alloy. The hydrogen evolution and the potentiodynamic polarization results show an improved corrosion resistance for phytic acid treated WE43 magnesium alloy in SBF solution compared with the untreated sample. For the SEM and EDS results, we found that the different solution coating will form a film with different P contents, as the P element was only formed though the phytic acid,
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so in the four modified groups, samples treated with phytic acid solution with pH 3 and pH 5 have high P contents which corresponding less phytic acid coating, though the surfaces have big crack compared with the pH 8 and pH 10 groups. But from the hydrogen evolution and the potentiodynamic polarization results we know that the corrosion rate of the phytic acid treated samples followed the ranking order pH 5 < pH 3 < pH 8 < pH 10. These results also indicate the more phytic–Mg complex on the surface of the magnesium alloy, the more better corrosion resistance will be obtained in SBF solution. The cytotoxicity evaluation results show that the biocompatibility of the phytic acid treated WE43 Mg alloys is much better than the blank WE43 magnesium alloy. Among the modified groups, the pH 5 solution modified has the best cell viability which suggests that the modified samples with more Phy–Mg complex will have a better biocompatibility. The hemolysis results also show that the pH 5 group has the smallest hemolysis rate, which proved once more that the phytic acid have a good biocompatibility. Therefore, phytic acid treatment might be a promising technique to improve the corrosion resistance and biocompatibility of biomedical WE43 alloy. Acknowledgements This work was supported by the National Basic Research Program of China (973 Program) (no. 2012CB619102), National High Technology Research and Development Program of China (863 Program) under grant number 2011AA030103 and Guangdong innovation R&D team project (no. 201001C0104669453). References [1] L.M. Khachigian, H.C. Lowe, S.N. Oesterle, Coronary in-stent restenosis: current status and future strategies, J. Am. Coll. Cardiol. 39 (2002) 183–193. [2] R.S. Stack, R.M. Califf, H.R. Phillips, D.B. Pryor, P.J. Quigley, R.P. Bauman, J.E. Tcheng, J.C. Greenfield, Interventional cardiac-catheterization at DukeMedical-Center, Am. J. Cardiol. 62 (1988) F3–F24. [3] H. Eggebrecht, J. Rodermann, P. Hunold, A. Schmermund, D. Bose, M. Haude, R. Erbel, Novel magnetic resonance-compatible coronary stent-The absorbable magnesium-alloy stent, Circulation. 112 (2005) E303–E304. [4] G. Ghimire, J.R. Spiro, R.K. Kharbanda, M. Roughton, et al., Evidence for the return of coronary vasoreactivity following absorption of a bioabsorbable magnesium alloy coronary stent, Am J Cardiol. 100 (2007) 146l. [5] M. Maeng, L.O. Jensen, L. Thuesen, Constrictive vascular remodeling after implantation of a bioabsorbable magnesium alloy stent in porcine coronary arteries, J Am Coll Cardiol. 51 (2008) B91. [6] R. Waksman, R. Pakala, D. Hellinga, R. Baffour, P. Kuchulakanti, R. Seabron, F. Tio, Effect of bioabsorbable magnesium alloy stent on neointimal formation in a porcine coronary model, Eur Heart J. 26 (2005) 417. [7] T.C. Robey, T. Valimaa, H.S. Murphy, P. Tormala, D.J. Mooney, R.A. Weatherly, Use of internal bioabsorbable PLGA finger-type stents in a rabbit tracheal reconstruction model, Arch. Otolaryngol. 126 (2000) 985–991. [8] S.S. Venkatraman, L.P. Tan, J.F.D. Joso, Y.C.F. Boey, X.T. Wang, Biodegradable stents with elastic memory, Biomaterials 27 (2006) 1573–1578. [9] Z. Li, L.A. Xue, S.Y. Dai, Biodegradable shape-memory block co-polymers for fast self-expandable stents, Biomaterials 31 (2010) 8132–8140. [10] J.W. Jukema, N.M.M. Pires, B.L. van der Hoeven, M.R. de Vries, L.M. Havekes, B.J. van Vlijmen, W.E. Hennink, P.H.A. Quax, Local perivascular delivery of antirestenotic agents from a drug-eluting poly(epsilon-caprolactone) stent cuff, Biomaterials 26 (2005) 5386–5394. [11] R. Waksman, R. Pakala, R. Baffour, R. Seabron, D. Hellinga, F.O. Tio, Short-term effects of biocorrodible iron stents in porcine coronary arteries, J. Interv. Cardiol. 21 (2008) 15–20. [12] M. Peuster, C. Hesse, T. Schloo, C. Fink, P. Beerbaum, C. von Schnakenburg, Long-term biocompatibility of a corrodible peripheral iron stent in the porcine descending aorta, Biomaterials 27 (2006) 4955–4962. [13] B. Heublein, R. Rohde, V. Kaese, M. Niemeyer, W. Hartung, A. Haverich, Biocorrosion of magnesium alloys: a new principle in cardiovascular implant technology? Heart 89 (2003) 651–656. [14] P. Peeters, M. Bosiers, J. Verbist, K. Deloose, B. Heublein, Preliminary results after application of absorbable metal stents in patients with critical limb ischemia, J. Endovasc. Ther. 12 (2005) 1–5. [15] R. Waksman, R. Pakala, P.K. Kuchulakanti, R. Baffour, D. Hellinga, R. Seabron, F.O. Tio, E. Wittchow, S. Hartwig, C. Harder, R. Rohde, B. Heublein, A. Andreae, K.H. Waldmann, A. Haverich, Safety and efficacy of bioabsorbable magnesium alloy stents in porcine coronary arteries, Catheter. Cardiovasc. Interv. 68 (2006) 607–617.
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In vitro corrosion and biocompatibility study of phytic acid modified WE43 magnesium alloy C.H. Ye a , Y.F. Zheng a,b , S.Q. Wang c , T.F. Xi a,c,∗ , Y.D. Li d a
Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China School of Information & Engineering, Wenzhou Medical College, Zhejiang 325035, China d DongGuan EONTEC Co., Ltd., Guangdong 523000, China b c
a r t i c l e
i n f o
Article history: Received 2 October 2011 Received in revised form 15 November 2011 Accepted 17 November 2011 Available online 26 November 2011 Keywords: Magnesium alloy Phytic acid Corrosion Biocompatibility
a b s t r a c t Phytic acid (PA) conversion coating on WE43 magnesium alloy was prepared by the method of immersion. The influences of phytic acid solution with different pH on the microstructure, properties of the conversion coating and the corrosion resistance were investigated by SEM, FTIR and potentiodynamic polarization method. Furthermore, the biocompatibility of different pH phytic acid solution modified WE43 magnesium alloys was evaluated by MTT and hemolysis test. The results show that PA can enhance the corrosion resistance of WE43 magnesium especially when the pH value of modified solution is 5 and the cytotoxicity of the PA coated WE43 magnesium alloy is much better than that of the bare WE43 magnesium alloy. Moreover, all the hemolysis rates of the PA coated WE43 Mg alloy were lower than 5%, indicating that the modified Mg alloy met the hemolysis standard of biomaterials. Therefore, PA coating is a good candidate to improve the biocompatibility of WE43 magnesium alloy. © 2012 Published by Elsevier B.V.
1. Introduction As we all know coronary stent placement has replaced balloon angioplasty as the standard of care for percutaneous coronary intervention. But at the same time in-stent restenosis (ISR) has become a widespread problem because of the permanent nature of the metallic stents, they posed the risk of a continuous interaction between the stent and the surrounding tissue, leading to long-term endothelial dysfunction or chronic inflammatory reaction. Although the usage of rapamycin or paclitaxel can reduce late in-stent restenosis rates [1], thrombosis or the inflammatory reaction caused by the long-time implanted stent or the polymer restricted the usage of drug eluting stent still exist. Stack et al. [2] have taken the lead in developing biodegradable stent (BDS) in 1988, and the animal experiments revealed that it can significantly reduce the in-stent restenosis rates. The BDSs under investigation mainly include (1): biodegradable magnesium alloy [3–6]; (2): biodegradable polymer, for example, PLA/PGA [7,8], PHBV [9], PCL [10] etc.; (3): biodegradable iron alloy [11,12].
∗ Corresponding author at: Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. Tel.: +86 10 6275 3404; fax: +86 10 6275 3404. E-mail address: [email protected] (T.F. Xi). 0169-4332/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.apsusc.2011.11.087
For biodegradable magnesium alloy, the current research mainly focused on AE21 [13], WE43 [14,15], AZ91 [16], etc. Di Mario et al. [17] studied the in vivo corrosion of magnesium stents made of WE43 alloy implanted in minipigs. Their animal and clinical experiment results showed that the WE43 alloy stents possessed sufficient anti-proliferative properties and drastically reduced the restenosis rate in comparison to conventional stainless steel stents. Loos et al. [18] also found that reduced WE43 magnesium alloy will reduce the viability and proliferation of smooth muscle cells compared with clinically applied steel stent (316L). However, the magnesium alloys corrodes too quickly in the physiological environment. To improve the corrosion resistance of the Mg alloys, various surface modification techniques, including alkaline treatment [19], heat treatment [20], microarcoxidation [21] and electrodeposition [22] have been developed recently. One surface treatment technique, phytic acid (PA) treatment has been investigated as a simple and effective methods for Mg alloy [23–25]. Firstly, phytic acid (C6 H18 O24 P6 ) is a natural and nontoxic, organic large molecular compound which present in most legumes, including corn, soy beans and nuts [26,27]. On the other hand, recent studies have shown its ability of alteration in signal transduction [28], stimulation of genes toward greater cell differentiation [29], antioxidant as food additive [30,27], and anticancer [31,32]. Even so, how is about the corrosion
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Fig. 1. The FTIR characteristic bands of phytic acid and phytic acid modified WE43 Mg alloy with different pH.
resistance of phytic acid modified magnesium alloy in simulated body fluid and the biocompatibility of phytic acid are still unknown. In this paper, in vitro corrosion and biocompatibility of phytic acid modified WE43 magnesium alloy was studied. The chemical nature of this conversion coating was investigated by Fourier transform infrared spectroscopy (FTIR) and ESEM equipped with the energy dispersive spectrum (EDS). The corrosion resistance was examined by potentiodynamic polarization method and the immersion test was performed in simulated body fluid (SBF). Furthermore, the biocompatibility of different pH phytic acid solution modified WE43 magnesium alloys was evaluated by MTT and hemolysis test.
2. Material and methods 2.1. Preparation of specimens The as-extruded WE43 alloy (91.35 wt.% Mg, 4.16 wt.% Y, 3.80 wt.% RE, 0.36 wt.% Zr, 0.20 wt.% Zn, 0.13 wt.% Mn) provided by Changchun Zhong-Ke-Xi-Mei Magnesium Alloy Co. Ltd., China, was cut into specimens with diameter of 8 mm and the height of 1.5 mm, then polished successively with 800#, 1200#, and 2000# grit SiC paper, pretreated with acetone and deionized water and dried with hot air, and treated in phytic acid solution. Phytic acid is chemical reagent, purity > 75%, and the other materials are all analytical reagent, purity ≥ 99%. The conversion coatings were prepared by immerging the substrate in the phytic acid solutions with different pH (pH 3, 5, 8 and 10, adjusted by ammonia water) at 40 ◦ C. After immersion, the samples were thoroughly washed with deionized water, dried overnight at 50 ◦ C and the untreated WE43 Mg alloy sample is taken as control. 2.2. Microstructural analysis of the specimens The microstructure was characterized using environmental scanning electron microscope equipping with the energy dispersive spectrum and Fourier transform infrared spectroscopy spectrophotometer (Nicolet 750, Nicolet Co.) in the spectral range 650–4000 cm−1 using attenuated total reflectance mode.
2.3. Immersion test The immersion test was identical to the hydrogen evolution method according to Lin [33]. The samples were put into the center of the beaker filled with SBF solution, and the hydrogen formed in the process of magnesium alloy corrosion was collected in the above volume of the funnel which was vertically laid above the sample surface. The corrosion resistance of samples could be evaluated based on the change of the rate of hydrogen evolved at interval time. 2.4. Electrochemical test The electrochemical measurement was carried out in the SBF solution and the potentiodynamic polarization curves of the modified WE43 magnesium alloy samples were obtained on a three-electrode system and the immersion tests were carried out in SBF buffer. The working electrode is WE43 Mg alloy samples with the exposed area of 1 cm2 . The temperature was kept to be 37 ± 0.5 ◦ C using a water bath. 2.5. Biocompatibility evaluation The biocompatibility of the WE43 magnesium alloy with or without phytic acid modified was evaluated by cytotoxicity experiment and blood hemolysis experiment. L929 cells were used to evaluate the cytotoxicity of the magnesium alloy with or without phytic acid modified samples by the indirect assay. The extracts were prepared using Dulbecco’s Modified Eagle’s Medium (DMEM) serum free medium with the surface area of extraction medium ratio 1.5 ml/cm2 in a humidified atmosphere with 5% CO2 at 37 ◦ C for 72 h. The supernatant fluid was withdrawn after centrifugation and then refrigerated at 4 ◦ C. L929 cells were incubated in 96-well culture plates at 1 × 104 /ml for 24 h to allow attachment and the medium was then replaced with 100 l extract. After 1, 2 and 4 days culture, 10 l MTT was added to each well cultured for 4 h and then 100 ml of the supernatant was measured spectrophotometrically at 570 nm by Elx800 (BioTek instruments). The background with MTT results of extracts was subtracted. In order to evaluate the blood compatibility of the samples, the samples were soaked in 10 ml normal physiological saline in a tube
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Fig. 2. SEM micrographs and the EDS spectrum of the phytic acid modified WE43 magnesium alloy with the solution of (a) pH 3, (b) pH 5, (c) pH 8, (d) pH 10.
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and kept at 37 ◦ C for 30 min. Eight milliliter arterial blood of the volunteer was diluted by 10 ml normal physiological saline that contained 0.5 ml potassium oxalate (20 g/l) decoagulant. After that 0.2 ml diluted blood was added into the tube and kept at 37 ◦ C for 60 min. Then the tube was centrifuged at 2500 rpm for 5 min. At last, the optical density (OD) was obtained by a spectrophotometer (722, Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China) at 545 nm wave length. The negative and positive groups were 10 ml normal physiological saline with 0.2 ml diluted blood and 10 ml distilled water with 0.2 ml diluted blood, respectively. The hemolysis ratio (HR) was calculated according to the equation: HR = [(ODt − ODn)/(ODp − ODn)] × 100%. The ODt means the OD value of tested sample. The ODn and ODp were OD value of negative and positive groups, respectively. 3. Results 3.1. Microstructure of the specimens The characteristic bonds of pure phytic acid and phytic acid with different pH modified WE43 Mg alloy are shown in Fig. 1. The characteristic bands of phytic acid centers at 995 cm−1 , 1462 cm−1 and 3302 cm−1 , corresponding to PO4 3− , HPO4 2− and OH− respectively. PO4 3− can be identified from the FTIR spectra of phytic acid conversion coating, which was shown at about 1200–940 cm−1 . Though some bands have shifted a little, the functional groups of conversion coatings formed in different pH of phytic acid solution are much close to the constituent of phytic acid. But as the conversion coating was dried at 50 ◦ C overnight, OH– (at about 3400 cm−1 ) was not obvious on the phytic acid treated WE43 magnesium alloy samples by FTIR method. Fig. 2 shows the morphology and the element compositions of the coatings formed in different pH of phytic acid solutions. It can be seen that the coatings presents bulk feature, and plenty of microcracks exist on the surface of the film. But the cracks are much bigger in Fig. 2(a) and (b) which corresponding the pH 3 and pH 5 modification solutions than that in Fig. 2(c) and (d), while the film of the latter groups are smoother. The chemical composition of the film was analyzed by EDS as shown on the right of the morphology picture. From the element compositions we can see that the coatings was mainly composed of Mg, O, P, and C elements, and the presence of P showed that phytic acid was successfully coated on the Mg alloy surface, in the formation of the coatings. But its content changed greatly, with the pH of the coating solution changed from 3, 5, 8 to 10, P element content (wt.%) changed from 11.47%, 11.67%, 7.22%, to 5.87% correspondingly, which illustrated that more phytic acid were coated on the pH 3 and pH 5 solution, while the solution of pH 8 and pH 10 formed less phytic acid coatings. 3.2. Immersion test Fig. 3 shows the hydrogen evolution rate of the bare WE43 magnesium alloy and the modified WE43 magnesium alloy with phytic acid solution in SBF solution for about 120 h. The overall corrosion reaction of magnesium at its free corrosion potential can be expressed as follows: Mg + 2H2 O = Mg2+ + 2OH− + H2 ↑
(1)
One mole hydrogen evolution corresponding to 1 mole WE43 magnesium alloy has been corroded, which can be taken as a good indicator to evaluate the corrosiveness of Mg alloy. The results showed that (1) the sample modified in pH 10 group exhibit faster corrosion rate than the other groups; (2) the samples modified in pH 8 and pH 3 groups have the similar corrosion rate with the control; (3) pH 5 group has the lowest corrosion rate.
Fig. 3. The hydrogen evolution volumes of the phytic acid modified WE43 Mg with different pH and the control samples as a function of the immersion time in SBF.
Fig. 4. Potentiodynamic polarization curves of the phytic acid modified WE43 Mg samples and the control in SBF solutions at 37 ◦ C.
Table 1 The average electrochemical parameters for the electrochemical polarization curves of different samples immersed in SBF solutions at 37 ◦ C.
pH 3 pH 5 pH 8 pH 10 Control
Potential (V)
Logi (A cm−2 )
−1.84 −1.79 −1.85 −2.08 −1.85
5.26 × 10−4 8.08 × 10−4 5.08 × 10−4 3.07 × 10−3 5.09 × 10−4
3.3. Electrochemical test Fig. 4 presents the electrochemical polarization curves of different samples immersed in SBF solutions at 37 ◦ C. The average electrochemical parameters are also show in Table 1. The results showed that (1) the order of the corrosion rate is similar to the hydrogen results; (2) the pH 5 group has the best corrosion resistance, while the pH 10 group has the fast corrosion rate.
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Fig. 5. L-929 cell viability expressed as a percentage of the viability of cells in the control and phytic acid modified WE43 Mg extraction solution after 1, 2 and 4 days of culture.
solution pH has a great effect on the modification effect. Especially when the reaction solution’s pH 5, the modified Mg alloy has the best corrosion resistance and biocompatibility. The FTIR analysis (Fig. 1) indicated that phytic acid could modify WE43 Mg alloy whether the solution is in acidic or basic conditions. Cui [34] has given the simple mechanism of the chelate process of the phytic acid on magnesium alloy. In short, a phytic has 12 hydroxyl groups and 6 phosphate carboxyl groups (Fig. 7), which makes it have a powerful capability of chelating with many metal ions in a wide pH scope, for example, copper [35], magnesium [34], zinc [36]. In aqueous solution, the phosphate group of the phytic acid will ionize to form hydronium ion, phytic acid ion with different number of phosphate ion (PO4 3− ) and hydrogen phosphate ion (HPO4 2− ) according to the different pH condition. At the same time, a large number of magnesium ions are ionized at the incipient stage when the WE43 magnesium alloy is immersed in phytic acid solution. These magnesium ions will combine with phytic acid ion to form phytic–Mg phytate complex, which then deposited on the surface of the WE43 magnesium alloy substrate (Fig. 8(a)). As the phytic acid have 12 hydroxyl groups, one phytic acid can chelate with different number of magnesium ions at different solution conditions which will result in different modification effect. The reaction can be referred as the following equation [38]: −iMgn+ + Hj Phy(12−j)− = Mgi Hj (12−2i−j)− Phy
Fig. 6. Hemolysis percentage of the WE43 Mg alloy and the phytic acid modified WE43 Mg alloy.
3.4. Biocompatibility evaluation Fig. 5 illustrates the viability of L-929 expressed as a percentage of the viability of cells cultured in negative control after cultured in DMEM and the phytic acid modified Mg alloys extraction medium solutions for different periods (1, 2 and 4 days). It means that all the modified groups have a better cell viability than the unmodified control. Among the modified groups, the pH 5 group has the highest viability in the three time point ranging from 60% to 75% while the control group’s cell viability is only from 28% to 34%. For the hemolysis test, compared with the control group, the rate of hemolysis decreased (the hemolysis of modified WE43 magnesium alloy changed from 9.27% to about 2% as shown in Fig. 6) after the WE43 Mg alloy immersed in the PA solution with different pH values. All the hemolysis rates of the PA coated WE43 magnesium alloys were lower than 5%, indicating that the modified WE43 magnesium alloy met the hemolysis standard of biomedical materials. 4. Discussion Our study has demonstrated that phytic acid modified WE43 Mg alloy has a good corrosion resistance and biocompatibility compared with the bare WE43 Mg alloy. In addition, the aqueous
(2)
in which Phy referred as phytic acid ions, i and j referred as the different reaction content, and this reaction was influenced by the following factors [37–40]: (1) the pH of the solutions; (2) the concentration of magnesium ions; (3) the conformation of phytic acid. In brief, the more Mg2+ and more phytic acid ions will accelerate this reaction to form the chelate complex’s, but the complex was not stable in a too low pH or a too high pH solution [38], though this condition will produce more Mg2+ or phytic acid radical ion compare with the moderate solution. So for a given pH solution and the magnesium alloy, the conformation of the phytic acid and the concentration of Mg2+ were the only two factors that strongly influence the reaction. But on the other hand, the magnesium ions can obtained only by the WE43 Mg alloy corrosion, from the Eq. (1) we know that the magnesium alloy corrosion rate was controlled by the pH and is increased with the decrease of solution pH, Ambat et al. [41] have also proved this conclusion. In Fig. 2 we can see in pH 3 and pH 5 solution, the coating has a higher content of P which suggests the higher level of Phy–Mg complex on the surface of the Mg alloy, in contrast the pH 8 and pH 10 solution have the less content of this complex. This can be explained that in pH 3 solution the strong acid environment can produce more Mg2+ , but Davidsson et al. [40] have proved that the Phy–Mg complex was partly soluble in pH 3 solution, so under this condition, there will be a little chelate complex on the surface of the WE43 Mg alloy. But for the pH 5 the acid of the solution condition was not so strong, this condition can give rise to enough Mg2+ to react with the deionizated hydroxyl groups in phytic acid, and the complex–Mg complex is stable in this pH condition, so we can see the more P content in pH 5 group. But in pH 8 solution, the basic condition will slow down Mg alloy corrosion rate which will inhibit the Mg2+ release though the phytic acid can produce many hydroxyl ions groups, which is similar with the solution in pH 10, so in pH 8 group, there were less Phy–Mg complex on the coating, the corrosion resistance only slightly higher (Fig. 2) compared with the two acid groups. The difference between the pH 10 solution and the other experimental groups is that the conformation of the phytic acid will change to 5 ax/1 eq from 1 ax/5 eq when the pH changed from below 9.0 to above 9.0 [39] (‘ax’ is the axial bond which is the bond between a carbon and a substituent that is projected vertically up or down on a ring conformation. ‘eq’ is the equatorial band which is the bond between a carbon and a
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Fig. 7. The structure of the phytic acid.
Fig. 8. (a) The mechanism of the Phy–Mg complex formation; (b) the conformation of the phytic acid in different solution condition.
substituent that extends out of a ring of carbons in a ring conformation). As the eq band is unstable, this conformation will change back to 1 ax/5 eq when the solution pH below 9.0, so in the reaction solution, the deprotonated Phy will react with the trace amounts of Mg2+ to form Phy–Mg complex, but when the modified alloy was placed into the SBF testing solution, the conformation of the Phy which was deposited onto the surface of the alloy will quickly reverts to 1 ax/5 eq conformation. During this change, the chelate bond between Mg2+ with phosphate groups, especially the phosphate group in the meta position will break, which will result in the migration of phytic acid to the SBF solution, and because the SBF testing solution has more Ca2+ than Mg2+ (2.5 mM vs 1.5 Mm) and the stability of the calcium–Phy is higher than the magnesium–Phy [37], the Phy after the bond cleavage will react with the calcium in the SBF testing solution to form Ca–Phy complex. But as the phytic acid resolved from the coating is very little, so little phytic acid chelate complex will precipitate on the surface, during this balance reaction, the Phy–Mg chelate complex resolved to phytic acid and Mg2+ would be the main form, which will accelerate Mg alloy corrosion. From Figs. 2 and 3 we can see the samples modified in the pH 5 has the best corrosion resistance, but samples modified in the pH 10 has the fastest corrosion rate.
As the phytic acid is widely present in nature (plants, animals and soils), mainly as calcium, magnesium and potassium mixed salts (also called phytines) and its important biological activity and the high number of applications, since the last century it has been the subject of investigation for many scientists in different fields, for example, enhanced immunity [42], anticancer including breast [43], colon [44], liver [45,46], prostate [47], rhabdomyosarcoma [48], antioxidant properties [49], etc. Without a doubt, the biocompatibility of the phytic acid must be very good, which is also proved by the present results of the MTT test and hymolysis. 5. Conclusions The present study investigated the corrosion resistance and the biocompatibility of the phytic acid modified WE43 magnesium alloy. The hydrogen evolution and the potentiodynamic polarization results show an improved corrosion resistance for phytic acid treated WE43 magnesium alloy in SBF solution compared with the untreated sample. For the SEM and EDS results, we found that the different solution coating will form a film with different P contents, as the P element was only formed though the phytic acid,
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so in the four modified groups, samples treated with phytic acid solution with pH 3 and pH 5 have high P contents which corresponding less phytic acid coating, though the surfaces have big crack compared with the pH 8 and pH 10 groups. But from the hydrogen evolution and the potentiodynamic polarization results we know that the corrosion rate of the phytic acid treated samples followed the ranking order pH 5 < pH 3 < pH 8 < pH 10. These results also indicate the more phytic–Mg complex on the surface of the magnesium alloy, the more better corrosion resistance will be obtained in SBF solution. The cytotoxicity evaluation results show that the biocompatibility of the phytic acid treated WE43 Mg alloys is much better than the blank WE43 magnesium alloy. Among the modified groups, the pH 5 solution modified has the best cell viability which suggests that the modified samples with more Phy–Mg complex will have a better biocompatibility. The hemolysis results also show that the pH 5 group has the smallest hemolysis rate, which proved once more that the phytic acid have a good biocompatibility. Therefore, phytic acid treatment might be a promising technique to improve the corrosion resistance and biocompatibility of biomedical WE43 alloy. Acknowledgements This work was supported by the National Basic Research Program of China (973 Program) (no. 2012CB619102), National High Technology Research and Development Program of China (863 Program) under grant number 2011AA030103 and Guangdong innovation R&D team project (no. 201001C0104669453). References [1] L.M. Khachigian, H.C. Lowe, S.N. Oesterle, Coronary in-stent restenosis: current status and future strategies, J. Am. Coll. Cardiol. 39 (2002) 183–193. [2] R.S. Stack, R.M. Califf, H.R. Phillips, D.B. Pryor, P.J. Quigley, R.P. Bauman, J.E. Tcheng, J.C. Greenfield, Interventional cardiac-catheterization at DukeMedical-Center, Am. J. Cardiol. 62 (1988) F3–F24. [3] H. Eggebrecht, J. Rodermann, P. Hunold, A. Schmermund, D. Bose, M. Haude, R. Erbel, Novel magnetic resonance-compatible coronary stent-The absorbable magnesium-alloy stent, Circulation. 112 (2005) E303–E304. [4] G. Ghimire, J.R. Spiro, R.K. Kharbanda, M. Roughton, et al., Evidence for the return of coronary vasoreactivity following absorption of a bioabsorbable magnesium alloy coronary stent, Am J Cardiol. 100 (2007) 146l. [5] M. Maeng, L.O. Jensen, L. Thuesen, Constrictive vascular remodeling after implantation of a bioabsorbable magnesium alloy stent in porcine coronary arteries, J Am Coll Cardiol. 51 (2008) B91. [6] R. Waksman, R. Pakala, D. Hellinga, R. Baffour, P. Kuchulakanti, R. Seabron, F. Tio, Effect of bioabsorbable magnesium alloy stent on neointimal formation in a porcine coronary model, Eur Heart J. 26 (2005) 417. [7] T.C. Robey, T. Valimaa, H.S. Murphy, P. Tormala, D.J. Mooney, R.A. Weatherly, Use of internal bioabsorbable PLGA finger-type stents in a rabbit tracheal reconstruction model, Arch. Otolaryngol. 126 (2000) 985–991. [8] S.S. Venkatraman, L.P. Tan, J.F.D. Joso, Y.C.F. Boey, X.T. Wang, Biodegradable stents with elastic memory, Biomaterials 27 (2006) 1573–1578. [9] Z. Li, L.A. Xue, S.Y. Dai, Biodegradable shape-memory block co-polymers for fast self-expandable stents, Biomaterials 31 (2010) 8132–8140. [10] J.W. Jukema, N.M.M. Pires, B.L. van der Hoeven, M.R. de Vries, L.M. Havekes, B.J. van Vlijmen, W.E. Hennink, P.H.A. Quax, Local perivascular delivery of antirestenotic agents from a drug-eluting poly(epsilon-caprolactone) stent cuff, Biomaterials 26 (2005) 5386–5394. [11] R. Waksman, R. Pakala, R. Baffour, R. Seabron, D. Hellinga, F.O. Tio, Short-term effects of biocorrodible iron stents in porcine coronary arteries, J. Interv. Cardiol. 21 (2008) 15–20. [12] M. Peuster, C. Hesse, T. Schloo, C. Fink, P. Beerbaum, C. von Schnakenburg, Long-term biocompatibility of a corrodible peripheral iron stent in the porcine descending aorta, Biomaterials 27 (2006) 4955–4962. [13] B. Heublein, R. Rohde, V. Kaese, M. Niemeyer, W. Hartung, A. Haverich, Biocorrosion of magnesium alloys: a new principle in cardiovascular implant technology? Heart 89 (2003) 651–656. [14] P. Peeters, M. Bosiers, J. Verbist, K. Deloose, B. Heublein, Preliminary results after application of absorbable metal stents in patients with critical limb ischemia, J. Endovasc. Ther. 12 (2005) 1–5. [15] R. Waksman, R. Pakala, P.K. Kuchulakanti, R. Baffour, D. Hellinga, R. Seabron, F.O. Tio, E. Wittchow, S. Hartwig, C. Harder, R. Rohde, B. Heublein, A. Andreae, K.H. Waldmann, A. Haverich, Safety and efficacy of bioabsorbable magnesium alloy stents in porcine coronary arteries, Catheter. Cardiovasc. Interv. 68 (2006) 607–617.
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