Acta Biomaterialia 6 (2010) 1852–1860
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Fe–Mn alloys for metallic biodegradable stents: Degradation and cell viability studies q Hendra Hermawan a, Agung Purnama b, Dominique Dube a, Jacques Couet b, Diego Mantovani a,* a
Laboratory for Biomaterials and Bioengineering, Department of Mining, Metallurgy and Materials Engineering, Pav. Adrien-Pouliot, 1745-E, Laval University, 1065 Ave de la Médecine, Québec City, QC, Canada G1V 0A6 b Laval Hospital Research Center/Québec Heart Institute, Department of Medicine, Laval University, Québec, Canada
a r t i c l e
i n f o
Article history: Received 27 March 2009 Received in revised form 11 November 2009 Accepted 17 November 2009 Available online 23 November 2009 Keywords: Fe–Mn alloy Biodegradable stent Degradation Cell viability
a b s t r a c t Biodegradable stents have shown their potential to be a valid alternative for the treatment of coronary artery occlusion. This new class of stents requires materials having excellent mechanical properties and controllable degradation behaviour without inducing toxicological problems. The properties of the currently considered gold standard material for stents, stainless steel 316L, were approached by new Fe–Mn alloys. The degradation characteristics of these Fe–Mn alloys were investigated including in vitro cell viability. A speciﬁc test bench was used to investigate the degradation in ﬂow conditions simulating those of coronary artery. A water-soluble tetrazolium test method was used to study the effect of the alloy’s degradation product to the viability of ﬁbroblast cells. These tests have revealed the corrosion mechanism of the alloys. The degradation products consist of metal hydroxides and calcium/phosphorus layers. The alloys have shown low inhibition to ﬁbroblast cells’ metabolic activities. It is concluded that they demonstrate their potential to be developed as degradable metallic biomaterials. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Stents, tiny tubular mesh-like metallic structure implants, have proved their effectiveness in treating narrowed arteries [1,2]. Nevertheless, they present two remaining complications: subacute stent thrombosis  and in-stent restenosis . Two approaches have been proposed to deal with these limitations: the drug eluting and the biodegradable stents. The current technology of drug eluting stents is still facing the problem of late stent thrombosis . Meanwhile, the implantation of biodegradable stents hopefully will leave behind only a healed arterial vessel, preventing late stent thrombosis, in-stent restenosis and the prolonged antiplatelet therapy . They are expected to degrade within a reasonable period (12–24 months)  after the stented artery has been remodelled (6–12 months) [8,9]. Biodegradable stents made of metals were clinically implanted to treat congenital heart disease in babies [10–12] and to treat critical limb ischemia in adults . Recently, a non-randomized multi-center clinical trial on biodegradable magnesium stents was conducted with encouraging initial results .
q Part of the Thermec’2009 Biodegradable Metals Special Issue, edited by Professor Diego Mantovani and Professor Frank Witte. * Corresponding author. Tel.: +1 418 656 2131x6270; fax: +1 418 656 5343. E-mail address: Diego.Mantovani@gmn.ulaval.ca (D. Mantovani).
Two classes of materials have been used to prepare biodegradable stents: polymers, from the lactic acid, glycolic and caprolactone families [15–18], and metals, either magnesium alloys [13,19–25] or pure iron [26–28]. Metallic biodegradable stents were more developed than their polymeric counterparts. In fact, metals have superior mechanical properties than polymers for replicating the properties of stainless steel 316L (SS316L), the reference material for coronary stent . Nevertheless, improvements are needed mainly to decrease the degradation rate of magnesium alloys [14,19,23,25] or to accelerate that of iron-based stents [26,27]. Some attempts have been made by developing new magnesium alloys including Mg–Zn–Mn , Mg–Ca [31,32] and Fe–Mn alloys [33,34]. Fe–Mn alloys, containing between 20 and 35 wt.% manganese, exhibited mechanical properties comparable to those of SS316L alloy [33,34]. They possess a similar austenite (c) structure, even though the c forming elements are different as nickel was used for the SS316L and manganese for the Fe–Mn alloys. The presence of this austenitic phase reduces the magnetic susceptibility compared to SS316L alloy which will give an enhanced compatibility with the magnetic resonance imaging (MRI). From a biological point of view, the presence of an alloying element such as manganese for a biodegradable iron-based alloy appears more appropriate than nickel, the former being essential to human [35,36] the latter classiﬁed as toxic and carcinogenic . An overdosage of manganese could lead to intoxiﬁcation and neurotoxicity ; however, due to the extensive plasma
1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.11.025
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protein binding that counteract the effect of manganese toxicity, excess of manganese is not reported to be toxic in cardiovascular system [39,40]. Moreover, considering the very light weight of a stent which is 50–100 mg depending on design, the release of element from the alloy during a controlled degradation could be expected to be lower than their toxic level in the blood. In developing materials for biodegradable stents, studies on degradation behaviour and degradation products’ cytotoxicity have to be considered. The degradation study investigates the mechanisms of decomposition and rate. A speciﬁc test bench simulating conditions encountered in human coronary artery has been developed by Levesque et al. . The test bench was able to show the important role of shear stress on degradation behaviour of metals which cannot be assessed with the common immersion or electrochemical test methods . This so-called dynamic degradation test method was suggested to be used for evaluating materials proposed for biodegradable stents . The cytotoxicity study can assess the early signs for biocompatibility such as acute cytotoxicity. At present time, it is difﬁcult to ﬁnd literature on cytotoxicity study of degradable metals for cardiovascular applications. However, methods used in studies on magnesium alloys dedicated for bone implants could be transposed to assess the early sign of cytotoxicity for other metals proposed for biodegradable stents. Li et al. have conducted an evaluation of cytotoxicity of alkali heat treated pure magnesium using marrow cells . The same indirect contact cytotoxicity test method was carried out by Li et al.  on Mg–Ca alloys using L929 cells. The ISO 10993-5:1999 standard was used as a guide for preparing the extracts . Therefore, in the present study, the new iron-based alloys containing manganese (Fe–Mn) were assessed for their degradation characteristics and their early sign of cytotoxicity using a dynamic degradation test bench and using a cell viability test, respectively.
2. Materials and methods 2.1. Materials Materials used in this study were iron-based alloys containing 20–35 wt.% manganese denoted as Fe20Mn, Fe25Mn, Fe30Mn and Fe35Mn. The alloys were prepared through powder sintering process from high purity elemental powders of iron and manganese followed by a series of cold rolling and resintering cycles resulting in a highly dense material. The details about these alloys including fabrication process, structure and properties are described elsewhere [33,34]. The Fe20Mn and Fe25Mn are constituted of c + e phases, whereas Fe30Mn and Fe35Mn are composed of single c phase ; therefore for degradation tests, specimens of Fe25Mn and Fe35Mn alloys representing two different microstructure conditions were chosen. Specimens with an exposed surface area of 300 mm2 were mounted in acrylic resin. They were then polished using abrasive papers #1000, ultrasonically cleaned in 75% ethanol, air-dried and stored for 24 h in a desiccator prior to use. For cell viability tests, powders were used in order to provide an extreme condition of high surface area. Powders of Fe–Mn alloys with size of 53 lm were prepared by means of mechanical ﬁling and sieving. This size was chosen based on preliminary experiments with different powder size where it was found that 53 lm powders induced the most severe effect to the cells. Powders of commercial iron, manganese and SS316L were also used for comparison purposes (Atlantic Equipment Engineers, Bergenﬁeld, USA). The powders were sterilized with 75% ethanol and rinsed with phosphate buffered saline prior the tests. The Fe35Mn alloy
was considered for further tests as it contains the highest manganese content among the other Fe–Mn alloys under study. 2.2. Degradation test Test solution was prepared from modiﬁed Hank’s solution having ionic composition and concentration considerably similar to those of human blood plasma. A pseudo-physiological-like shear stress of 4 Pa was generated by a predetermined laminar ﬂowing solution in a test bench designed to mimic blood ﬂow condition in human coronary artery. More details on the test bench and test parameters are reported elsewhere . The specimens were taken out from the test bench after 1 week, 1 month and 3 months and were then characterized. The temperature of the test solution was kept at 37 °C and its pH was also recorded every 24 h. Solution samplings were carried out during the test and their concentration of iron and manganese was measured by a Perkin Elmer 3110 atomic absorption spectrometer (AAS). A Siemens D5000 X-ray diffractometer (XRD) with Cu Ka radiation at a scanning rate of 0.02°/1.2 s1 was employed to identify the structure of degradation products of the specimens. The microstructure was studied with an Olympus PME3 optical microscope (OM) and a Jeol JSM-840A scanning electron microscope (SEM). Images obtained from OM were analysed using a quantitative image analyser software (Clemex Vision) to measure corroded depth on at least 5 ﬁelds to obtain averages and standard deviations. The chemical composition of degradation products was analysed using X-ray photoelectron spectroscopy (XPS) in a Phi VersaProbe and using an energy dispersive spectrometer (EDS) which was coupled with the SEM. A Cameca SX100 electron probe micro-analyser (EPMA) was used to map iron, manganese and oxygen. Specimens for OM, SEM and EPMA were mounted in acrylic resin to expose their cross-section area and were then mechanically polished using abrasive papers up to #1200 and 0.1 lm diamond paste. 2.3. Cell viability test Cell viability tests were carried out by indirect contact with the 3T3 mouse ﬁbroblast cell line (3T3). They were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 U ml1 penicillin and 100 mg ml1 streptomycin at 37 °C in a humidiﬁed atmosphere of 5% CO2. The cells were incubated in 24-well tissue culture plates at the density of 50,000 cells/well for 24 h to allow attachment. Samples to be tested were then added into 3 lm tissue culture inserts in the same medium as for the cells. The content of samples in the medium was varied and expressed as concentration (mg ml1). At least 6 replicates were studied for each condition. After 48 h of incubation, the cell viability was assessed using water-soluble tetrazolium based assay (10% WST-1, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene-disulfonate) for 2 h. The dissolved degradation products were rinsed out as the medium was aspirated after the treatment with the samples. The absorbances of the solutions were measured spectrophotometrically at 440 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, USA) and were analysed using the Prism 5 software (GraphPad Software Inc., San Diego, USA). 3. Results 3.1. Degradation tests Fig. 1 shows cross-sectional proﬁles of Fe–Mn specimens before and after dynamic degradation test. The degradation (corrosion) took place over the entire surface and then went deeper into the
H. Hermawan et al. / Acta Biomaterialia 6 (2010) 1852–1860
Fig. 1. Cross-sectional proﬁle of polished Fe–Mn specimens: (a) before and (b and c) after 1 week and 3 months of degradation test respectively, and (d and e) etched Fe25Mn and Fe35Mn specimens after 3 months of degradation test respectively (etchant: Nital 2%).
bulk (Fig. 1a–c). The microstructure shows a preferential intergranular attack of the Fe25Mn alloy (Fig. 1d), while a more invasive attack is visible in Fe35Mn specimen (Fig. 1e). In Fig. 1b and c, specimens were polished with abrasive paper #1200 in order to prevent the edge smoothing effect and preserve the corroded proﬁle for measurement of the corroded depth (Fig. 2). Moreover, Fig. 2 shows a slightly deeper corrosion attack for Fe25Mn than in Fe35Mn specimens. The pH of the test solution was recorded as 7.4 at the beginning and never exceeded more than 7.8 until the end of the tests (after 3 months). Fig. 3 shows the evolution of the concentration of iron and manganese ions in solutions during 3 months of degradation test. There was no difference in ion concentration for both alloys up to 14 days, but then specimens of Fe25Mn alloy released slightly more ions than those of Fe35Mn alloy.
Fig. 3. Concentration of iron and manganese ions in test solution as a function of immersion time for specimens of Fe25Mn and Fe35Mn alloys measured by the AAS.
Fig. 2. Corroded depth as a function of test period for Fe25Mn and Fe35Mn alloys.
Fig. 4 shows microstructure and elemental mappings of the cross-section of Fe35Mn specimen after 3 months of degradation test. Fig. 4a illustrates a backscattered electron (BSE) image showing three regions with different atomic density: bulk, intermediate and top degradation layers. The intermediate layer was probably cracked during curing of the mounting resin or due to a dehydration event. Fig. 4b and c shows that iron and manganese were uniformly distributed in the intermediate layer and in the bulk, tiny dispersed manganese-rich inclusions being visible in the substratum. Somewhat less iron and manganese were found in intermediate layers than in the bulk. However in the intermediate layer, manganese was sometimes more visible than iron although its concentration was much lower than that of iron. (Fig. 4c). The concentration in oxygen was superior in the intermediate than in top layer and also oxygen penetrated locally in the bulk (Fig. 4d).
H. Hermawan et al. / Acta Biomaterialia 6 (2010) 1852â€“1860
Fig. 4. Images of cross-section area of Fe35Mn specimens after 3 months of degradation test: (a) BSE image, and (b, c and d) EPMA maps for iron, manganese and oxygen, respectively. The color represents the intensity of the mapped elements.
All the three elements demonstrated very low intensities in the top layer. The top layer of degradation product contained mainly carbon, oxygen, nitrogen, sodium and trace of phosphorus, chlorine and
sulphur as measured by the XPS (Fig. 5 insert). Meanwhile the XRD spectrum of the degradation layer (Fig. 5) showed a rather amorphous pattern with a very low intensity, but it approached the pattern of magnetite, Fe3O4. The presence of hydrogen could
Fig. 5. XRD pattern of degradation products and XPS pattern (insert) of top surface of degradation layer for Fe35Mn specimens after 3 months of degradation test.
H. Hermawan et al. / Acta Biomaterialia 6 (2010) 1852–1860
not be detected but it is expected as hydrated degradation products. The degradation products adhered to the surface and were not completely washed out by the ﬂowing solution in the test bench (Fig. 6a). The surface was covered by a ﬂat degradation layer and spreading agglomerates formed on it (Fig. 6b). At higher magniﬁcation, the porous agglomerates appear like a coral (mineral) structure (Fig. 6c and d). The EDS analysis shows a difference in the main detectable elements between the ﬂat layer and the agglomerates shown in Fig. 6 as presented in Table 1. The agglomerates contain more calcium and phosphorus compared to the ﬂat layer which contains more on oxygen and chlorine.
Table 1 Concentration of elements on the degradation surface of Fe35Mn specimen after 3 months of degradation test measured by the EDS. Element
Iron Manganese Oxygen Chlorine Calcium Phosphorus Sulphur
Concentration (wt.%) Flat layer
47.5 (0.6) 7.1 (0.4) 26.6 (0.6) 3.5 (0.1) 2.0 (0.1) 2.1 (0.1) 0.7 (0.1)
45.9 (0.5) 6.9 (0.3) 20.8 (0.6) 1.5 (0.1) 4.4 (0.1) 4.3 (0.1) 0.8 (0.1)
Standard deviation in parentheses.
3.2. Cell viability tests The WST-1 metabolic assay showed that manganese has the highest metabolic inhibition effect to the 3T3 ﬁbroblast cells compared to iron, SS316L and Fe35Mn alloy (Fig. 7). Its relative metabolic activities (RMA) declined to less than 80% when the cells were exposed to 0.01 mg ml1 manganese powder, and was undetectable when concentration reached 4 mg ml1. In contrast to manganese, cells treated with iron and SS316L showed higher RMA which was similar to the control along the tested concentrations. For these two powders, the RMA remained high, more than 95%, even when their concentration reached 16 mg ml1. Differently, when manganese is alloyed with iron to form Fe35Mn alloy, it showed less inhibition effect than that of pure manganese as demonstrated by higher RMA values. The alloy gave a distinct pattern compared to all tested metals in describing its metabolic inhibition (Fig. 7). Its 0% RMA was noted at the concentration of 16 mg ml1. Fig. 8 provides more evidence on the alloying effect in lowering metabolic inhibition. The 0.5 mg ml1 concentration of metals showed a good picture of inhibition whereas it was chosen since it gave 50% metabolic activity inhibition for manganese. The RMA
Fig. 7. Relative metabolic activity of 3T3 ﬁbroblast cells in presence of various metal powders as a function of concentration of the powders.
Fig. 6. Images of Fe35Mn surface after 3 months of degradation test: (a) macrograph showing the specimen mounted in resin, and (b, c and d) SEM images at different magniﬁcation.
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developed Fe–Mn alloys are presented in Table 2. Their microstructure is mainly composed of c phase with the appearance of e phase in alloys having a lower manganese content. As the manganese content increases, the maximum elongation increases while the yield strength decreases. The alloys possess an antiferromagnetic behaviour, giving the same non-magnetic nature as that of SS316L with best behaviour upon plastic deformation producing minimal magnetic susceptibility . 4.2. Degradation rate
Fig. 8. Relative metabolic activity of 3T3 ﬁbroblast cells in presence of various metal powders at a ﬁxed concentration of 0.5 mg ml1. Note: Fe + Mn = mixture of 65 wt.% of iron powder with 35 wt.% of manganese powder.
in the presence of SS316L, iron and Fe35Mn alloy resembled that of control. It was signiﬁcantly higher than those of manganese and the mixture of iron and manganese powders. Fig. 9 shows that there was nearly no difference in RMA demonstrated by the alloys containing different manganese content. 4. Discussion 4.1. Properties of Fe–Mn alloys Four alloys with manganese content ranging between 20 and 35 wt.% were developed via a powder metallurgy route followed by a series of cold rolling and resintering cycles [33,34]. The phase composition, magnetic and mechanical properties of these newly
The progress of degradation is clearly shown in Fig. 1a–c where the corrosion attack progressively went deeper and wider into the bulk of the material as a function of the degradation time. The corrosion attack in Fe25Mn was found to be slightly deeper than in Fe35Mn (Fig. 3). This could be related to the bi-phase composition in Fe25Mn where e-epsilon and c-austenite phases coexist. Therefore, it presents more micro-galvanic sites susceptible to corrosion initiation than in Fe35Mn, which has only c-austenite phase. It was also found that intergranular corrosion, which preferably attacks grain boundaries, was more evident in Fe25Mn than in Fe35Mn (Fig. 1d and e). The intergranular corrosion will mostly lead to a localized degradation  which should be avoided to prevent a premature failure of an implant. In this study, degradation rate was approached quantitatively by measuring the corroded depth as a function of degradation time (Fig. 2). During the 3 month test period, the total corroded depth of Fe25Mn specimens was 130 lm and that of Fe35Mn specimens was 110 lm, corresponding to average degradation rates of 520 and 440 lm year1, respectively. Compared to pure iron, which has a corrosion rate of 220–240 lm year1 , both Fe–Mn alloys have shown higher corrosion rates. This could imply a faster in vivo degradation rate than pure iron, which was not completely degraded in the aorta of New Zealand rabbits after 18 months . The in vivo degradation is known to be much slower than that of in vitro . The concentration of iron and manganese ions in the solution (Fig. 3) presents complementary data on the rate of ions release. The highest average concentration reached after 3 months of degradation test was 2 ppm for iron and 1.4 ppm for manganese in Fe25Mn alloy. This measured ion concentration is very low compared to the experiment on AM60B magnesium alloys that reached 50–100 ppm in 14 days . This could be related to the fact that the degradation products of Fe–Mn alloys which contain iron and manganese was not soluble. They mostly adhered to the surface of specimens (Figs. 4 and 6) and thus slowed down the ion exchange between the substrate and solution. 4.3. In vitro degradation mechanism
Fig. 9. Relative metabolic activity of 3T3 ﬁbroblast cells in presence of Fe–Mn alloys powders with different manganese content at a ﬁxed concentration of 1 mg ml1.
Fig. 10 illustrates the degradation mechanism of the Fe–Mn alloy during the dynamic degradation test in modiﬁed Hank’s solution. The mechanism can be divided into four steps:
Table 2 Properties of Fe–Mn alloys compared to SS316L (ASTM F138).
Nominal main composition (wt.%)
Phase at Troom
Magnetic susceptibilitya (lm3 kg1)
Yield (0.2%) strength (MPa)
Ultimate strength (MPa)
Maximum elongation (%)
Fe20Mn Fe25Mn Fe30Mn Fe35Mn SS316L
20 25 30 35 18
c +e c+e c c c
0.2 0.2 0.2 0.2 0.5
420 360 240 230 190
700 720 520 430 490
8 5 20 30 40
Mn Mn Mn Mn Cr, 14 Ni
(1.1) (0.2) (0.2) (0.2) (1.7)
The values in parenthesis are after the specimens were subjected to 20% of plastic deformation. Data compiled from [33,34] and ASTM F138-03 .
H. Hermawan et al. / Acta Biomaterialia 6 (2010) 1852–1860
Fig. 10. Illustration of the corrosion mechanisms for Fe–Mn alloys: (a) initial corrosion reaction, (b) formation of hydroxide layer, (c) formation of pits, and (d) formation of calcium/phosphorus layer.
1. Initial corrosion reaction (Fig. 10a) Immediately after immersion in the test solution, the alloy was oxidized to metal ions, in anodic spots following Eqs. (1) and (2). The electrons from the anodic reaction were consumed by a corresponding cathodic reaction and the reduction of oxygen dissolved in water, following Eq. (3). These reactions occurred randomly over the entire surface where a difference in potential existed, at grain boundaries and at the interface between different phases.
Fe ! Fe2þ þ 2e
Mn ! Mn2þ þ 2e
2H2 O þ O2 þ 4e ! 4OH
tom. The XRD analysis rather detected an amorphous pattern with weak peaks corresponding more to those of magnetite, Fe3O4 (Fig. 5). 3. Formation of pits (Fig. 10c) Since the hydroxide layers did not homogenously cover the surface, Cl ions from the solution penetrated to compensate the increase of metal ions beneath the hydroxide layer. The formed metal chloride was then hydrolysed by water to the hydroxide and free acid, Eq. (6), lowering the pH value in the pits while the bulk solution remains neutral. This autocatalytic reaction leads to the formation of pits  that grew wider and deeper as shown in Fig. 1.
Fe2þ þ 2Cl ! FeCl2 þ H2 O ! FeðOHÞ2 þ HCl
2. Formation of hydroxide layers (Fig. 10b) 4. Formation of calcium/phosphorus layer (Fig. 10d) The released metal ions then reacted with the hydroxyl ion (OH) released from the cathodic reaction to form insoluble hydroxides (hydrous metal oxides) according to Eqs. (4) and (5). Since iron was the main composition of the alloy, the equations are hereafter written for iron only.
2Fe2þ þ 4OH ! 2FeðOHÞ2 or2FeO:2H2 O
4FeðOHÞ2 þ O2 þ 2H2 O ! 4FeðOHÞ3 or2Fe2 O3 :6H2 O
The oxidized (corroded) surface layer of iron alloys normally consists of FeO.nH2O at the bottom, Fe3O4.nH2O in the middle and Fe2O3.nH2O on the top . Under visual observation on the Fe–Mn specimens, those hydroxides appeared as red–brown (Fe2O3) layer on the top and black (Fe3O4 and FeO) layer on the bot-
As the degradation process continued, there was a formation of a new layer (agglomerates) over the previously formed ﬂat degradation layer (Fig. 6). These agglomerates, which appeared like a coral (mineral) structure, contained signiﬁcant amount of calcium and phosphorus (Table 1). This ﬁnding is interesting since the presence of those two elements could lead to the formation of hydroxyapatite, which is considered biocompatible especially for bone implants. 4.4. The effect of Fe–Mn alloys on cells In most cases, metal toxicity arises only when reaction occurs between metals and body ﬂuids acting as electrolytes. The reaction
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is electrochemical in nature and it is the metal ion, formed by anodic reaction, e.g. Eqs. (1) and (2), that can provide toxic effects . This depends on the nature of the metal ions itself against cell metabolic activities. Fig. 7 shows that the powders of iron and SS316L at a concentration up to 16 mg ml1 demonstrated low inhibition to the 3T3 ﬁbroblast cells. In contrast, the powder of manganese showed high inhibition to the cells starting from very low concentration. Meanwhile, the powder of Fe35Mn alloy showed a moderate inhibition to the cells. Iron is an essential element with a high toxic level, i.e. 350– 500 lg dl1 in serum . Extracellular iron exclusively bound to transferrin, which maintains iron-soluble and non-toxic . Iron-loaded transferrin binds to its speciﬁc receptor on the cell surface, and undergoes endocytosis. The internalized excess of iron is detoxiﬁed by sequestration into ferritin, an iron storage protein . Those two characteristics of iron explained the low inhibition of metabolic activity when cells exposed to iron at the range of the tested concentration. Meanwhile, the low inhibition of SS316L was mainly due to the relatively inert behaviour conferred to this metal by its passive layer composed of chromium oxide (Cr2O3). On the other hand, despite its essential functions in human body, manganese shows a potential to be toxic, i.e. a level of 3–5.6 lg dl1 can cause neurologic symptoms . In tissues, manganese may exist primarily in the form of Mn2+  and may be oxidized to Mn3+, which is rather reactive and more toxic than Mn2+ . Manganese exerts its toxic effect by targeting mitochondria, causing a high level of lactic acid . This will decrease the cells’ ability to cleave WST-1 into soluble formazan, which is the parameter to measure the cell viability. This mechanism explained the result which showed that manganese has the highest inhibition effect to the 3T3 ﬁbroblast cells. Once iron is alloyed with manganese, i.e. at 35 wt.% of manganese, they formed a solid solution where the two atoms are homogenously arranged in a face-centered cubic (fcc) crystal structure known as the c phase. A structure that is different from those of the forming elements, i.e. body-centered cubic (bcc) for iron and simple cubic for manganese. As a consequence, the alloy has very different characteristics than its forming elements. Therefore, it is expected that the cytotoxic behaviour of the alloys is different from that of the forming elements, i.e. less toxic than manganese to cells. Fig. 8 clearly shows that alloying signiﬁcantly reduced the inhibition effect of manganese to the cells. This is related to slow (small) release of manganese ion during dynamic degradation test (Fig. 3). If the two elements – iron and manganese powders – were only mixed together without forming an alloy, the mixture, as expected, still has a high inhibition effect as manganese to the 3T3 ﬁbroblast cells (Fig. 8). Variations in manganese content in Fe–Mn alloys did not display signiﬁcant difference in RMA (Fig. 9). However, the inhibition effect of the alloys should be addressed to their degradation behaviour during the cell viability test period, which took only 48 h. It is shown in Fig. 3 that during the ﬁrst 48 h both alloys showed no difference in the quantity of the released manganese ions. There could be a slightly different concentration of manganese, but this was under the detection limit of the AAS. For longer degradation period, it is shown that Fe25Mn corroded slightly more severe and released more manganese ions than Fe35Mn (Figs. 2 and 3). Fig. 9 also shows that the optimum RMA was given by Fe30Mn, which could be the optimum composition of Fe–Mn alloys under study in giving compromise between inhibition effect and physical and mechanical properties. Finally, we would like to highlight some limitations of this work. The cell-based assays are simplistic and do not adequately test toxic effects. Cell viability is a single test of cellular metabolic activity, and it can be anticipated that a range of complementary assays could have been very appropriate. In particular, comple-
mentary assays devoted to compare potential for apoptosis, necrosis, DNA damage, mitochondrial membrane potential, and cytoskeletal rearrangement would greatly increase the power to compare effects on different toxic and functional endpoints in the cells. Second, the degradation studies were conducted in a salt solution that did not contain some of the normal sequestering proteins of serum. This more realistic solution would probably change both the ﬂow dynamics in the degradation assays and the proﬁle of metals on the surface of the degrading pieces. However, it is also known that tests carried out in solution tests containing serum proteins provide synergistic effects that complicated the analytical techniques for the characterization and the analysis of the results. In this context, mainly considering that these are pioneering works in the new ﬁeld of degradable metals, we decided to start from a simple model. Third, we are conscious that the use of indirect contact of the metal powders in the transwells might appear disputable. Certainly the degradation process of metal particles in the static transwell is probably greatly different from the processes that occur under ﬂow conditions. Vascular stents are in direct contact with the cells. However, again, we decided to start from a simple model. These directions will be the focus of future works. 5. Conclusion The in vitro degradation of Fe–Mn alloys is governed by the mechanism of corrosion involving the formation of pits over the entire surface which then went deeper and wider. The alloys were corroded at an average rate up to 520 lm year1 which is about two times faster than that of pure iron. The degradation products constituted of iron hydroxides and calcium/phosphorus containing layers. They adhered to the substrate and were not completely soluble in the test solution. The release of iron and manganese ions into the solution was limited due to the barrier effect of the insoluble degradation layer. The Fe–Mn alloys possess a low inhibition effect to 3T3 ﬁbroblast cells metabolic activities compared to pure manganese. The inhibition effect increases as the concentration of the alloys in cellular medium increases. Its 50% inhibition effect reached at concentration of 6 mg ml1 while its 100% inhibition effect was reached when the concentration exceeded 16 mg ml1. Finally, it can be concluded that the study demonstrates evidences of the potentiality of Fe–Mn alloys to be a biocompatible degradable biomaterial. Acknowledgements The authors would like to acknowledge the kind help and guidance of Elise Roussel from Laval Hospital during cell viability tests. Iron powder for the cell viability tests as well as for the Fe–Mn alloys development was kindly provided by the Quebec Metal Powders Inc. This work was partially supported by the Natural Science and Engineering Research Council (NSERC) of Canada, the Collaborative Health Research Projects of NSERC and the Canadian Institutes of Health Research (CIHR). Appendix A. Figures with essential colour discrimination. Certain ﬁgures in this article, particularly Figs. 3, 4, 7 and 10 are difﬁcult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio. 2009.11.025. References  Fischman DL, Leon MB, Baim DS, Schatz RA, Savage MP, Penn I, et al. A randomized comparison of coronary-stent placement and balloon angioplasty
   
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in the treatment of coronary artery disease. Stent restenosis study investigators. N Engl J Med 1994;331:496–501. Serruys PW, de Jaegere P, Kiemeneij F, Macaya C, Rutsch W, Heyndrickx G, et al. A comparison of balloon-expandable-stent implantation with balloon angioplasty in patients with coronary artery disease. Benestent study group. N Engl J Med 1994;331:489–95. Movahed MR, Vu J, Ahsan C. Simultaneous subacute stent thrombosis of two drug-eluting stents in the left anterior descending and the circumﬂex coronary arteries. Case report and review of the literature. J Invasive Cardiol 2006;18: E198–202. Mitra AK, Agrawal DK. In stent restenosis: bane of the stent era. J Clin Pathol 2006;59:232–9. Ong AT, McFadden EP, Regar E, de Jaegere PP, van Domburg RT, Serruys PW. Late angiographic stent thrombosis (LAST) events with drug-eluting stents. J Am Coll Cardiol 2005;45:2088–92. Waksman R. Update on bioabsorbable stents: from bench to clinical. J Interv Cardiol 2006;19:414–21. Serruys PW, Kutryk MJ, Ong AT. Coronary-artery stents. N Engl J Med 2006;354:483–95. El-Omar MM, Dangas G, Iakovou I, Mehran R. Update on in-stent restenosis. Curr Interv Cardiol Rep 2001;3:296–305. Schomig A, Kastrati A, Mudra H, Blasini R, Schuhlen H, Klauss V, et al. Four-year experience with Palmaz-Schatz stenting in coronary angioplasty complicated by dissection with threatened or present vessel closure. Circulation 1994;90: 2716–24. Zartner P, Cesnjevar R, Singer H, Weyand M. First successful implantation of a biodegradable metal stent into the left pulmonary artery of a preterm baby. Catheter Cardiovasc Interv 2005;66:590–4. Schranz D, Zartner P, Michel-Behnke I, Akinturk H. Bioabsorbable metal stents for percutaneous treatment of critical recoarctation of the aorta in a newborn. Catheter Cardiovasc Interv 2006;67:671–3. Zartner P, Buettner M, Singer H, Sigler M. First biodegradable metal stent in a child with congenital heart disease: evaluation of macro and histopathology. Catheter Cardiovasc Interv 2007;69:443–6. Peeters P, Bosiers M, Verbist J, Deloose K, Heublein B. Preliminary results after application of absorbable metal stents in patients with critical limb ischemia. J Endovasc Ther 2005;12:1–5. Erbel R, Di Mario C, Bartunek J, Bonnier J, de Bruyne B, Eberli FR, et al. Temporary scaffolding of coronary arteries with bioabsorbable magnesium stents: a prospective, non-randomised multicentre trial. Lancet 2007;369: 1869–75. Stack RS, Califf RM, Phillips HR, Pryor DB, Quigley PJ, Bauman RP, et al. Interventional cardiac catheterization at Duke Medical Center. Am J Cardiol 1988;62:3F–24F. Tamai H, Igaki K, Kyo E, Kosuga K, Kawashima A, Matsui S, et al. Initial and 6month results of biodegradable poly-l-lactic acid coronary stents in humans. Circulation 2000;102:399–404. Tsuji T, Tamai H, Igaki K, Kyo E, Kosuga K, Hata T, et al. Biodegradable polymeric stents. Curr Interv Cardiol Rep 2001;3:10–7. van der Giessen WJ, Lincoff AM, Schwartz RS, van Beusekom HM, Serruys PW, Holmes Jr DR, et al. Marked inﬂammatory sequelae to implantation of biodegradable and nonbiodegradable polymers in porcine coronary arteries. Circulation 1996;94:1690–7. Heublein B, Rohde R, Kaese V, Niemeyer M, Hartung W, Haverich A. Biocorrosion of magnesium alloys: a new principle in cardiovascular implant technology? Heart 2003;89:651–6. Levesque J, Dube D, Fiset M, Mantovani D. Investigation of corrosion behaviour of magnesium alloy AM60B-F under pseudo-physiological conditions. Mater Sci Forum 2003;426–432:521–6. Levesque J, Hermawan H, Dube D, Mantovani D. Design of a pseudophysiological test bench speciﬁc to the development of biodegradable metallic biomaterials. Acta Biomater 2008;4:284–95. Di Mario C, Grifﬁths H, Goktekin O, Peeters N, Verbist J, Bosiers M, et al. Drugeluting bioabsorbable magnesium stent. J Interv Cardiol 2004;17:391–5. Waksman R, Pakala R, Kuchulakanti PK, Baffour R, Hellinga D, Seabron R, et al. Safety and efﬁcacy of bioabsorbable magnesium alloy stents in porcine coronary arteries. Catheter Cardiovasc Interv 2006;68:606–17. Waksman R, Pakala R, Okabe T, Hellinga D, Chan R, Tio MO, et al. Efﬁcacy and safety of absorbable metallic stents with adjunct intracoronary beta radiation in porcine coronary arteries. J Interv Cardiol 2007;20:367–72. McMahon CJ, Oslizlok P, Walsh KP. Early restenosis following biodegradable stent implantation in an aortopulmonary collateral of a patient with pulmonary atresia and hypoplastic pulmonary arteries. Catheter Cardiovasc Interv 2007;69:735–8.
 Peuster M, Wohlsein P, Brugmann M, Ehlerding M, Seidler K, Fink C, et al. A novel approach to temporary stenting: degradable cardiovascular stents produced from corrodible metal-results 6–18 months after implantation into New Zealand white rabbits. Heart 2001;86:563–9.  Peuster M, Hesse C, Schloo T, Fink C, Beerbaum P, von Schnakenburg C. Longterm biocompatibility of a corrodible peripheral iron stent in the porcine descending aorta. Biomaterials 2006;27:4955–62.  Waksman R, Pakala R, Baffour R, Seabron R, Hellinga D, Tio FO. Short-term effects of biocorrodible iron stents in porcine coronary arteries. J Interv Cardiol 2008;21:15–20.  Balcon R, Beyar R, Chierchia S, De Scheerder I, Hugenholtz PG, Kiemeneij F, et al. Recommendations on stent manufacture, implantation and utilization. Eur Heart J 1997;18:1536–47.  Song G. Control of biodegradation of biocompatible magnesium alloys. Corr Sci 2007;49:1696–701.  Li Z, Gu X, Lou S, Zheng Y. The development of binary Mg–Ca alloys for use as biodegradable materials within bone. Biomaterials 2008;29:1329–44.  Kannan MB, Raman RKS. In vitro degradation and mechanical integrity of calcium-containing magnesium alloys in modiﬁed-simulated body ﬂuid. Biomaterials 2008;29:2306–14.  Hermawan H, Alamdari H, Mantovani D, Dubé D. Iron-manganese: New class of degradable metallic biomaterials prepared by powder metallurgy. Powder metall 2008;51:38–45.  Hermawan H, Dube D, Mantovani D. Degradable metallic biomaterials: design and development of Fe–Mn alloys for stents. J Biomed Mater Res A; in press: doi:10.1002/jbm.a.32224.  Emsley J. The elements. Oxford: Clarendon Press; 1998.  Keen CL, Ensunsa JL, Clegg MS. Manganese metabolism in animals and humans including the toxicity of manganese. In: Siegel A, Siegel H, editors. Manganese and its role in biological processes. New York: Marcel Dekker; 2000. p. 89–121.  McGregor DB, Baan RA, Partensky C, Rice JM, Wilbourn JD. Evaluation of the carcinogenic risks to humans associated with surgical implants and other foreign bodies – a report of an IARC Monographs Programme Meeting. Eur J Cancer 2000;36:307–13.  Aschner M, Guilarte TR, Schneider JS, Zheng W. Manganese: recent advances in understanding its transport and neurotoxicity. Toxicol Appl Pharmacol 2007;221:131–47.  Jynge P, Brurok H, Asplund A, Towart R, Refsum H, Karlsson JO. Cardiovascular safety of MnDPDP and MnCl2. Acta Radiol 1997;38:740–9.  Crossgrove J, Zheng W. Manganese toxicity upon overexposure. NMR Biomed 2004;17:544–53.  Li L, Gao J, Wang Y. Evaluation of cyto-toxicity and corrosion behavior of alkaliheat-treated magnesium in simulated body ﬂuid. Surf Coat Technol 2004;185:92–8.  ISO 10993-5. Biological evaluation of medical devices – part 5: tests for in vitro cytotoxicity. Arlington, VA: ANSI/AAMI; 1999.  ASTM F. 138: Standard speciﬁcation for wrought 18chromium-14nickel2.5molybdenum stainless steel bar and wire for surgical implants (UNS S31673). West Conshohocken: ASTM International; 2003.  Steigerwald R. Metallurgically inﬂuenced corrosion. In: Korb LJ, Olson DL, editors. ASM handbook volume 13: corrosion. Materials Park: ASM; 1987. p. 123–35.  Witte F, Fischer J, Nellesen J, Crostack H-A, Kaese V, Pisch A, et al. In vitro and in vivo corrosion measurements of magnesium alloys. Biomaterials 2006;27:1013–8.  Roberge PR. Handbook of corrosion engineering. Toronto: McGraw-Hill; 2000.  Shreir LL, Jarman RA, Burstein GT. Corrosion, metal/environment reactions. 3rd ed. Oxford: Butterworth-Heinemann; 2000.  Flint GN. A metallurgical approach to metal contact dermatitis. Cont Dermat 1998;39:213–21.  Frey RJ. Iron tests. Gale encyclopaedia of medicine. Gale Research; 1999.  Papanikolaou G, Pantopoulos K. Iron metabolism and toxicity. Toxicol Appl Pharmacol 2005;202:199–211.  Ellenhorn MJ, Schonwald S, Ordog G, Wasserberger J. Ellenhorn’s medical toxicology: diagnosis and treatment of human poisoning. Baltimore: Williams & Wilkins; 1997.  Harris RW, Chen Y. Electron paramagnetic resonance and difference ultraviolet studies of Mn2+ binding to serum transferrin. J Inorg Biochem 1994;54:1–19.  Chen JY, Tsao GC, Zhao Q, Zheng W. Differential cytotoxicity of Mn(II) and Mn(III): Special reference to mitochondrial [Fe–S] containing enzymes. Toxicol Appl Pharmacol 2001;175:160–8.  Hirata Y. Manganese-induced apoptosis in PC12 cells. Neurotoxicol Teratol 2002;24:639–53.