materials science and engineering c - lbmd
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In vitro degradation and biocompatibility of Fe–Pd and Fe–Pt
composites fabricated by spark plasma sinteringContents lists
available at ScienceDirect
Materials Science and Engineering C
j ourna l homepage: www.e lsev ie r .com/ locate /msec
In vitro degradation and biocompatibility of Fe–Pd and Fe–Pt composites fabricated by spark plasma sintering
T. Huang a,b, J. Cheng c, Y.F. Zheng a,b,c, a State Key Laboratory for Turbulence and Complex System, College of Engineering, Peking University, Beijing 100871, China b Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China c Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
Corresponding author at: Department ofMaterials Sci Engineering, Peking University, Beijing 100871, China. Tel
E-mail address: [email protected] (Y.F. Zheng).
0928-4931/$ – see front matter © 2013 Elsevier B.V. All ri http://dx.doi.org/10.1016/j.msec.2013.10.023
a b s t r a c t
a r t i c l e i n f o
Article history: Received 21 June 2013 Received in revised form 12 October 2013 Accepted 21 October 2013 Available online 31 October 2013
Keywords: Biodegradable metal Fe–Pt composite Fe–Pd composite Corrosion Biocompatibility
In order to obtain biodegradable Fe-basedmaterialswith similarmechanical properties as 316L stainless steel and faster degradation rate than pure iron, Fe-5 wt.%Pd and Fe-5 wt.%Pt composites were prepared by spark plasma sinteringwith powders of pure Fe and Pd/Pt, respectively. The grain size of Fe-5 wt.%Pd and Fe-5 wt.%Pt compos- ites wasmuch smaller than that of as-cast pure iron. The metallic elements Pd and Pt were uniformly distributed in the matrix and the mechanical properties of these materials were improved. Uniform corrosion of Fe–Pd and Fe–Pt composites was observed in both electrochemical tests and immersion tests, and the degradation rates of Fe–Pd and Fe–Pt composites were much faster than that of pure iron. It was found that viabilities of mouse fibro- blast L-929 cells and human umbilical vein endothelial cells (ECV304) cultured in extraction mediums of Fe–Pd and Fe–Pt composites were close to that of pure iron. After 4 days' culture, the viabilities of L-929 and ECV304 cells in extraction medium of experimental materials were about 80%. The result of direct contact cytotoxicity also indicated that experimental materials exhibited no inhibition on vascular endothelial process. Meanwhile, iron ions released from experimental materials could inhibit proliferation of vascular smooth muscle cells (VSMC), which may be beneficial for hindering vascular restenosis. Furthermore, compared with that of as-cast pure iron, the hemolysis rates of Fe–Pd and Fe–Pt composites were slightly higher, but still within the range of 5%, which is the criteria for good blood compatibility. The numbers of platelet adhered on the surface of Fe–Pd and Fe–Pt composites were lower than that of pure iron, and the morphology of platelets kept spherical. To sum up, the Fe-5wt.%Pd and Fe-5wt.%Pt composites exhibited good mechanical properties and degradation be- havior, closely approaching the requirements for biodegradable metallic stents.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Biodegradable stents are currently considered as ideal stents. They are expected to effectively avoid the late stent thrombosis which is often caused by permanent stents. Thematerial for biodegradable stents is required to have good biocompatibility and it should be kept intact for several months before completion of vascular healing [1]. Two typical classes of metallic materials including Mg-based [2–9] and Fe-based [10–18] alloys have been researched for this application. Compared with Mg-based metals, Fe-based metals possess more attractive me- chanical properties [10]. Iron is an essential nutrient element in human body and plays an important role in vital biochemical activities, such as oxygen sensing and transport, electron transfer and catalysis [19]. Pure iron also has goodmechanical property [10], biocompatibility and hemocompatibility [20,21] close to 316L stainless steel, which serves as the golden standard for stent material. Animal experiments demonstrated that degradable iron stents can be safely implanted
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without significant vascular restenosis caused by inflammation, neointi- mal proliferation or thrombotic events, and there was no evidence for local or systemic toxicity [20,22–24]. A clinical outcome also showed that there was no association between iron status and the incidence of major adverse cardiac events or coronary restenosis in human body [25]. Therefore, iron-based materials are considered as suitable mate- rials for further development of novel biodegradable coronary artery stent. However, a faster degradation rate of iron is desired to apply to stents [20,22].
To obtain iron-based materials with appropriate properties for clin- ical applications, researchers have focused on the development of new kinds of Fe-based materials by modifying the chemical composition and microstructure. Hermawan et al. [10,26] were the first to study the effect of alloying elements on the performance of biodegradable iron-based materials. They found that alloying with Mn increased the strength and degradation rate of pure iron. Liu et al. [27] discovered that the addition of 6 wt.% of Si in Fe-30 wt.%Mn alloy established the shape memory effect. Xu et al. [28] reported that Fe-30Mn-1C alloy showed lowermagnetic susceptibility and bettermechanical properties than Fe-30Mn alloy. Schinhammer et al. [29] added 1 wt.% of Pd into Fe- 10Mn alloy that sped up the degradation rate ten times faster than that
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of low carbon steel. They also did a research for Fe–Mn–C(–Pd) alloys. The Fe–Mn–C–Pd alloys were characterized by an increased degrada- tion rate compared to pure iron [16]. The effects of alloying elements (Mn, Co, Al, W, Sn, B, C, and S) on the in vitro degradation behavior and biocompatibility of pure iron were also investigated by Liu and Zheng [30]. The results showed that the addition of all alloying elements except for Sn improved the mechanical properties of iron after rolling. New fabricating methods were also tried by researchers, such as pow- der metallurgy [31], electroforming [32–34], equal channel angular pressing (ECAP) technique [35] and SPS sintering [36]. Although the predecessors made a lot of efforts for the improvement of Fe-basedma- terials, the corrosion rate of Fe-based materials still could not meet the clinical application demand.
Palladium is a common alloying element in dental alloys. It has rela- tively low price and density when compared to gold and platinum. Pal- ladium possesses a good range of solubility in several metals and could improve the mechanical properties of the matrix metals, too [37]. There have also been reports of using Pd-based organometallic com- pounds as antineoplastic agents [38]. Platinum has good mechanical property, low corrosivity and high biocompatibility [39,40]. It is used as essential components for many medical devices including implant- able defibrillators, catheters, stents, pacemakers, neuromodulation device and upper-lid implant [41,42]. So palladium and platinum them- selves are both excellent biomedical materials with good biocompatibil- ity. In addition, the standard electrode potential of palladium and platinum (Pd is about −0.126 V, Pt is about 1.2 V) is higher than that of iron (about −0.44 V) [43], and galvanic corrosion may occur in the placewhere two kinds of metals contact, then accelerating the degrada- tion of pure iron. Dispersion of platinum and palladium into pure iron matrixmay alsomake the corrosion of pure ironmore uniform. Further- more, the effect of dispersion strengthening caused by the addition of platinum and palladiumwas expected to improve themechanical prop- erties of pure iron. The objective of the presentwork is to investigate the feasibility of adding palladium and platinum to pure iron for the prepa- ration of biodegradable iron-based composites.
2. Materials and methods
2.1. Material preparation
Pure iron powder (99.0%, −300 mesh), pure palladium powder (99.99%, −200 mesh) and pure platinum powder (99.99%, −150 mesh), whichwere provided by ChinaMetalMaterials Technology Com- pany, were used as raw materials. Iron powder mixed with 5 wt.% of palladium and platinum powder were prepared, respectively. Then the mixed powders were put into a 20-mm diameter graphite die and sintered under vacuum by SPS-1050 system (Sumitomo Coal Mining Company, Ltd.) with sintering temperature of 1000 °C and holding time of 5 min. Pure iron (99.9%) prepared by casting served as control.
The as-sintered Fe–Pd and Fe–Pt composite specimens (Φ20 × 7 mm) and as-cast pure iron were cut into square pieces (10 × 10 × 1.5 mm3) and cylinders (Φ2 × 5 mm), respectively. Square pieces were used for density measurements, microstructure characterization and series tests of microhardness, electrochemical properties, corrosion behavior, cytotoxicity, hemocompatibility and platelet adhesiveness. Each sample was mechanically polished to 2000 grit, then ultrasonically cleaned in anhydrous ethanol and dried in the open air. Before cytotoxicity test, the samples were sterilized with ultraviolet radiation for at least 2 h. Cylinder speci- mens were used for compressive test.
2.2. Microstructure characterization
Specimens were polished by 0.15 μm diamond paste, further ultra- sonically cleaned in anhydrous ethanol and dried in the open air. Optical microscope (Olympus BX51M)was used to observe themetallographic
structure after the specimens were etched with a 4% HNO3/alcohol solution. X-ray diffractometer (XRD, Rigaku DMAX 2400) using CuKα radiation was adopted to identify the constituent phases of Fe–Pd and Fe–Pt composites with scanning range from 10° to 100° and scan rate of 6°/min. Energy dispersive spectrometer (EDS) was used for the analysis of chemical composition.
2.3. Mechanical test
Themechanical properties of experimental composites and pure iron were determined by microhardness test and compressive test. Micro- hardness tests were carried out by a microhardness tester (SHIMADZU HMV-2t) measuring Vickers hardness with loading force of 0.2 kg and dwell time of 10 s. Center point and other four points uniformly distrib- uted around the center point on the surface of each sample were select- ed for microhardness test. An average of five measurements was taken for eachmaterial. Instron 5969 universal testmachinewasused for com- pressive tests according to ASTME9-89a [44]. The specimenswere in the form of cylinder 2.5 mm in diameter and 5 mm in length. Compression strain rate was 2 × 10−4/s. An average of at least three measurements was taken for each group.
2.4. Electrochemical measurements
Electrochemical measurements were performed using three- electrode system at an electrochemical work station (CHI660C, China). The specimen, a platinum electrode and a saturated calomel electrode (SCE) were set as the working electrode, auxiliary electrode and the ref- erence electrode, respectively. All themeasurementswere carried out at a temperature of 37 ± 0.5 °C in Hank's solution. The area of working electrode exposed to the solution was 1 cm2. The open circuit potential (OCP) measurement was set for 7200 s. Electrochemical impedance spectroscopy (EIS) was measured from 100 kHz to 10 mHz at OCP value after 2 h immersion in Hank's solution. The potentiodynamic po- larization curves were carried out from −1000 mV (vs. SCE) to 0 mV (vs. SCE) at a scanning rate of 0.33 mV·s−1.
2.5. Static immersion test
In vitro static immersion test was performed in Hank's solution, 50 ml for each sample following ASTM-G31-72 [45] at 37 °C in water bath. After 3, 10 and 30 days, the samples were removed from the soaking solution, gently rinsed with distilled water and quickly dried in case of oxidation. Changes on the surface morphologies of the speci- mens after immersion were characterized by ESEM (Quanta 200FEG), equipped with an EDS attachment. Then, weighing after the corrosion products on the surface of samples dissolved in the 10 mol/l NaOH solu- tion, an average of three measurements was taken for each group. The degradation rate was calculated based on the formula:
CR ¼ m St
where CR (mg·cm−2·day−1) is the corrosion rate, m (mg) is the mass loss, S (cm2) is the surface area of the specimen and t (day) is the time.
2.6. Dynamic immersion test
Dynamic immersion test was carried out using a dynamic corrosion test bench which was built in our previous research work [11]. Dis- solved oxygen is an important factor that influences degradation of iron in a neutral medium, so the dissolved oxygen concentration in Hank's solution was regulated to better simulate the blood environ- ment. In this bench the wall shear stress (0.68 Pa), temperature (36.0–37.1 °C), pH value (7.35–7.45), and dissolved oxygen (2.8– 3.2 mg−1) were controlled to approach the common values in the
Fig. 1.Metallographs of experimentalmaterials: (a) pure iron and (b) Fe–Pd and (c) Fe–Pt composites. The corresponding grain size distribution estimated by themetallographs: (d) pure iron; (e) Fe–Pd and (f) Fe–Pt composites.
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human coronary artery. Plate specimens were mounted in paraffin and then placed in a specimen holder. All specimens were exposed to the dynamic fluid flow (Hank's solution) for 30 days (considering the gen- erally bare metallic coronary stents would be covered by endothelial cells after 30 days in vivo implantation and no longer contact with blood directly) [11]. After the test, the samples were taken out, then the macroscopic surface morphology was characterized using a 2.5 di- mensional precision image measuring instrument (HLEO, VACD-1010, China). After that the specimens were cleaned by deionized water and ethanol in turn, they were dried in the air. Changes on the surface mor- phology were characterized by ESEM (Quanta 200FEG), equipped with
Fig. 2. X-ray diffraction patterns of pure iron and Fe–Pd and Fe–Pt composites.
an EDS attachment. The corrosion rate was calculated based on the weight loss of specimens. Corrosion layers on the surface of samples were peeled off at first. The residual corrosion products were dissolved by 10 mol/l NaOH and rinsed by deionized water and alcohol in turn, then samples were weighed after they were dried in the open air. Cor- rosion rates were calculated by the same formula in static immersion test.
2.7. Contact angle measurement
Water contact angle of iron-basedmaterials wasmeasured by a con- tact angle analyzer (Kino SL200B); for each sample the measurements were carried out three times.
2.8. Cytotoxicity test
The cytotoxicity test was performed by an indirect contact method. Murine fibroblast cells (L-929), human vascular smooth muscle cells (VSMC) and umbilical vein endothelial cells (ECV304) were used to evaluate the cytotoxicity of experimental Fe–Pd and Fe–Pt composites. At first, all the cell lineswere cultured in theDulbecco'smodified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100U·ml−1 pen- icillin and 100 μg·ml−1 streptomycin at 37 °C in a humidified atmo- sphere of 5% CO2. According to ISO 10993-12 [46], extraction medium was prepared using DMEM with a surface area/extraction medium ratio of 1.25 cm2·ml−1 in a humidified atmosphere with 5% CO2 at 37 °C for 72 h. After the extracts were centrifuged, the supernatant fluid was withdrawn and stored at 4 °C before cytotoxicity test. The control groups involved DMEM medium as the negative control and DMEM including 10% dimethyl sulfoxide (DMSO) as the positive con- trol. The metallic concentrations of ions in the extraction medium were measured by inductively coupled plasma atomic emission spectrometry (ICP-AES) (Leeman, Profile). Cellswere incubated in the 96-well plates at the density of approximately 5 × 103 cells per 100 μl medium in each well and incubated for 24 h to allow attachment. DMEM was then sucked out and replaced by extraction medium. Then 10 μl serum was
Fig. 3. ESEM images of surface morphology and energy spectrum analysis of (a) Fe–Pd and (b) Fe–Pt composites.
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added to each well. After 1, 2 and 4 days' incubation in the incubator re- spectively, the 96-well plates were observed under an optical micro- scope. Thereafter, 10 μl of MTT was added to each well. The cells were incubatedwithMTT for 4 h. Then 100 μl of formazan solubilization solu- tion (10% sodium dodecyl sulfate in 0.01 MHCl) was added to each well and incubated for 10 h. The absorbance of each well was tested by a mi- croplate reader (Bio-RAD680) at 570 nmwith a referencewavelength of
Table 1 Density of Fe–Pd and Fe–Pt composites with as-cast pure iron as the control at room temperature.
Materials Density (g/cm3) Relative density
Pure iron 7.83 (0.01)a \ Fe–Pd 7.15 (0.09)a 88.9% Fe–Pt 7.04 (0.03)a 86.3%
a Number in the brackets represents the standard deviation. Fig. 4. Compressive stress–strain curves of pure iron and Fe–Pd and Fe–Pt composites.
Fig. 5. Electrochemical test results of pure iron and Fe–Pd and Fe–Pt composites in Hank's solution: (a) Potentiodynamic polarization curve, (b) Nyquist plots.
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630 nm. Viability of cells (X) was calculated using the following formula according to ISO 19003-5 [47]:
X ¼ OD1
OD2 100%:
Here OD1 is themean absorbance of the experimental sample group and positive control group. OD2 is the mean absorbance of the negative con- trol group.
Direct contact cytotoxicity of ECV304 cells was also carried out ac- cording to ISO 10993-5 [47]. 600 μl cell suspension was respectively added to each sterilized sample at a cell density of 6 × 103/ml. After being cultured in the incubator for 24 h, the medium was removed. Then the samples were gently washed by phosphate buffered saline (PBS); cells on the samples were fixed with 2.5% glutaraldehyde solu- tion at room temperature for 2 h, then dewateredwith gradient alcohol solution (50%, 60%, 70%, 80%, 90%, 95% and 100%) for 10 min, and finally freeze-dried for 2 days. The morphology of cells adhered on the speci- mens was observed by ESEM (Quanta 200FEG).
2.9. Hemolysis test and platelet adhesion
Healthy human blood (anticoagulant was 3.8 wt.% citric acid sodi- um) extracted from volunteers was diluted by physiological saline according to volume ratio 4:5. Pure iron and Fe–Pd and Fe–Pt compos- ites were installed in centrifugal tubes with 10 ml physiological saline. Temperature was kept at 37 °C for 30 min. Then 0.2 ml diluted blood was added to each tube and incubated at 37 °C for 60 min, 10 ml deion- ized water with 0.2 ml diluted blood as the positive control and 10 ml physiological saline with 0.2 ml diluted blood as the negative control. Upon completion of the above operations, samples were removed, and then these tubes were centrifuged at 800 g for 5 min. Supernatant was transferred to 96-well multiplates, the absorbance (OD) was
Table 2 Average electrochemical parameters of Fe–Pd and Fe–Pt composites (as-cast pure iron as control).
Materials Vcorr (V) Icorr (μA/cm2)
Pure iron −0.324 (0.020) 0.871 (0.040) Fe–Pd −0.471 (0.010) 1.550 (0.120) Fe–Pt −0.545 (0.008) 6.698 (0.370)
Corrosion potential (Vcorr), corrosion current (Icorr).
determined by a microplate reader (Bio-RAD680) at a wavelength of 545 nm. Hemolysis of samples was calculated by the formula:
Hemolysis ¼ OD testð Þ−OD negative controlð Þ OD positive controlð Þ−OD negative controlð Þ 100%:
For platelet adhesion, whole blood from healthy human body was centrifuged at 1000 r/min for 10 min. Platelet rich plasma (PRP) was obtained from the upper fluid. Samples after ultraviolet disinfection were moved to 24-well multiplates and 0.2 ml PRP was added to each well, incubated at 37 °C for 1 h. After gently flushed by phosphate buff- ered saline (PBS), platelets on samples were fixed with 2.5% glutaralde- hyde solution at room temperature for 1 h, then dewatered with gradient alcohol solution (50%, 60%, 70%, 80%, 90%, 95% and 100%) for 10 min, and finally freeze-dried for 2 days. The morphology of platelet adhered on the specimens was observed by ESEM (Quanta 200FEG).
3. Results and discussion
3.1. Microstructures
Fig. 1 shows the metallographs of pure iron and Fe–Pd and Fe–Pt composites and the corresponding grain size distribution measured frommetallographs. The average grain sizes of these two kinds of exper- imental composites are quite close, both about 20 ± 8 μm, which are much smaller than that of as-cast pure iron (180 ± 90 μm). It may be caused by the different preparation methods. For spark plasma sintering, lower temperature and shorter holding time could sinter powders to crystal with little grain growth [48]. The second phase par- ticles inhibiting grain growth should also be taken into consideration. The decrease of the grain size could contribute to the improvement of mechanical properties.
Fig. 2 displays XRD patterns of experimental composites, as-cast pure iron as control. It can be found that the Fe-5wt.%Pd and Fe- 5wt.%Pt composites both consist of α-Fe phase and FeO phase, but pure Pd or Pt phase was not found. PDF CARDS recorded that the peaks of Fe9.7Pd0.3 and Fe9.7Pt0.3 phases almost overlap with that of α-Fe phase in X-ray diffraction spectrum. As the contents of both Pd and Pt in experimental composites are less than 3 at.%, there might be quite a part of Pd and Pt solute in the surrounding pure iron matrix forming Fe9.7Pd0.3 and Fe9.7Pt0.3 phases in the sintering process, so the content of Pd or Pt phase was too low to be detected by X-ray dif- fraction. Fig. 3 showed that Pd and Pt were uniformly distributed in the iron matrix. The dark areas mainly consisted of Fe and O, which
Fig. 6. Macrographs of surface morphology of experimental materials static immersed in Hank's solution for 10 and 30 days.
Fig. 7. SEM images of samples' surfacemorphology after statically immersed inHank's solution for different periods and the corresponding X-ray diffraction patterns of corrosion products on the surface of pure iron.
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Fig. 8. Corrosion rates calculated from the weight loss of samples after static or dynamic immersion in Hank's solution for 30 days.
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should be FeO phase, as revealed by the XRD results. Table 1 shows the density of Fe–Pd and Fe–Pt composites with as-cast pure iron as control at room temperature. Relative density of both Fe–Pd and Fe–Pt compos- ites were less than 90%, which was probably due to the relatively low sintering temperature and pressure, as well as the existence of FeO in the composites.
3.2. Mechanical properties
Fig. 4 shows the compression stress–strain curves of Fe–Pd and Fe– Pt composites, with pure iron as control. The yield strengths (YS) of Fe–Pd and Fe–Pt composites were both about three times as that of pure iron. The ultimate compressive strength (UCS) of Fe–Pt composite was slightly higher than that of Fe–Pd composite. The rigidity of Fe–Pd and Fe–Pt composites was also much larger than that of pure iron. Ac- cording to the binary phase diagramof Fe–Pd [49] and Fe–Pt [50] alloys, a small quantity of Pd and Pt could dissolve into iron matrix. However, the atomic radii of Fe (1.40 ), Pd (1.40 ) and Pt (1.35 ) are very close [51], so the lattice distortion of α-Fe was negligible. Then the
Fig. 9. SEM images of samples' surfacemorphology after dynamically immersed in Hank's soluti samples' surface morphology after removed corrosion products: (d) pure iron and (e) Fe–Pd a
solution strengthening effect in these composites could not be signifi- cant. Therefore, the strength enhancement of the composites should be mainly attributed to the second phase strengthening effects. More- over, the decrease of the grain size could make considerable contribu- tions to the strength improvement of these composites [52]. However, the Fe–Pd and Fe–Pt composites exhibited low ductility, which can be explained mainly by the relatively low density (Table 1) [53].
The microhardness of Fe–Pd (184 ± 10 HV0.2/10) and Fe–Pt (309 ± 8 HV0.2/10) composites was much higher than that of pure iron (119 ± 3 HV0.2/10), especially for Fe–Pt composite, which was nearly three times as that of pure iron. Obviously, the addition of Pd or Pt greatly enhanced the strength of iron matrix.
3.3. Corrosion properties of Fe–Pd and Fe–Pt Composites
3.3.1. Electrochemical measurements Fig. 5 shows the potentiodynamic polarization curves (Fig. 5(a)) and
Nyquist plots (Fig. 5(b)) of Fe–Pd and Fe–Pt composites immersed in Hank's solution, with as-cast pure iron as the control. The average elec- trochemical parameters were listed in Table 2. Itwas found that the cor- rosion potential was largely decreased and the corrosion current densities were increased after adding Pd and Pt into the iron matrix. As the Nyquist plots shown, plotted the variation in the excitation frequency impedance imaginary part (z") as a function of the imped- ance real part (z′) which obtained a series of semicircles. The diameters of semicircles obtained from Fe–Pd and Fe–Pt composites were smaller than that of pure iron, revealing their worse corrosion resistance. Since the diameter of high frequency capacitive loop can be considered as the charge transfer resistance and smaller charge transfer resistance corre- sponds to faster corrosion rate [54].
3.3.2. Static immersion corrosion behavior Samples after 3 days' immersion in Hank's solution exhibited no
conspicuous change on the surface. After 10 days' immersion, localized corrosion mainly happened at the edge of pure iron sample, and the productswere reddish brown.However, uniform corrosion could be ob- served on the surfaces of Fe–Pd and Fe–Pt composites, with claybank corrosion products (Fig. 6).When the sampleswere immersed inHank's solution for 30 days, most of corrosion products fell off, as shown in Fig. 6. Different orientations of the grains showed different colors.
on for 30 days: (a) pure iron and (b) Fe–Pd and (c) Fe–Pt composites. SEM images of these nd (f) Fe–Pt composites.
Fig. 10.Water contact angles of (a) pure iron and (b) Fe–Pd and (c) Fe–Pt composites.
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Local distribution of black etch pits could also be found on the surface of pure iron, indicating localized corrosion feature of pure iron. However, a large number of corrosion pits were distributed uniformly on the sur- face of Fe–Pd and Fe–Pt composites, representing their macroscopically uniform corrosion behavior. Fig. 7 shows the SEM images of surface morphology of experimental samples, as can be seen from the figure:
(1) After 3 days' immersion, all the samples' surfaces almost kept intact.
(2) After 10 days' immersion, most area on the surface of pure iron remained intact, and only several corrosion pits were locally dis- tributed on the surface. The surface of Fe–Pd composite was
Fig. 11. (a) Ion concentration in extraction mediums of experimental materials and cell viabilit (c) ECV304 and (d) VSMC.
covered by a thin corrosion layer, and this layer didn't closely ad- here to thematrix. Fe–Pt composites corrodedmost severely and a lot of deep corrosion pits were uniformly distributed on the surface.
(3) After 30 days' immersion, because the most of the corrosion products fell off from the surface of samples, grain boundary ob- viously presented on the surface of pure iron. Fe–Pd and Fe–Pt composites corroded much more seriously than pure iron. The iron substrate surrounded Pd- and Pt-rich areas corroded more seriously. This should be attributed to the galvanic corrosion oc- curred at the contact place of Fe and Pd or Pt. Pd- and Pt-rich areas that have relatively high corrosion potential as cathode
y after cultured in extraction mediums and positive control for 1, 2 and 4 days; (b) L-929,
Fig. 12. The morphologies of ECV 304 cells directly cultured on (a) pure iron and (b) Fe–Pd and (c) Fe–Pt composites for 24 h.
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sites, iron as anode sites, then formed a lot of tiny galvanic cells in the compositeswhen immersed inHank's solution, so as to accel- erate the corrosion rate of iron.
(4) The XRD spectrum of corrosion products on the surface of as-cast pure ironwhich had immersed inHank's solution for 10 days indi- cated that the corrosion products were mainly the Fe + 3O(OH) and Fe(OH)3. Corrosion products on the surface of Fe–Pd and Fe– Pt composites were the same as that on pure iron.
Fig. 8 shows the corrosion rates thatwere calculated from theweight loss after samples immersed in Hank's solution for 30 days. The corro- sion rates of both Fe–Pd and Fe–Pt composites were faster than that of as-cast pure iron. The corrosion rate of Fe–Pt composite was the fastest, about 2.73 times as that of as-cast pure iron. Compared to the corrosion rate of Fe-5wt.%W(about 1.15 times as that of as-cast pure iron) and Fe- 0.5wt.%CNT composites (about 1.96 times as that of as-cast pure iron) [36], the addition of Pd and Pt increased the corrosion rate of pure iron much more significantly. In addition to the galvanic corrosion, FeO contained in Fe–Pd and Fe–Pt composites which could be easily oxidized to oxidation products was also an innegligible reason for increasing corrosion rate.
3.3.3. Dynamic immersion corrosion behavior Fig. 9 panels (a)–(c) were SEM images showing surfacemorphology
of experimental materials after corroding in dynamic environments. As can be seen from these figures, the numbers of corrosion products on the surface of Fe–Pd and Fe–Pt composites are much more than that on the surface of pure iron.
Fig. 13. (a) Hemolysis rates of pure iron and Fe–Pd and Fe–Pt composites. (b) The number of p
After the corrosion product layers were removed, surface morphol- ogies of the substrates appeared, as shown in Fig. 9(d)–(f). Grain boundaries could be observed obviously on the surface of pure iron. The surface of Fe–Pd composite was much smoother. Fe–Pt composite corrodedmost seriously, with a lot of deep corrosion pits on the surface.
The corrosion rates calculated from weight loss of samples after dynamic immersion tests were also shown in Fig. 8. Compared with as- cast pure iron, both the Fe–Pd and Fe–Pt composites corroded much faster, and the Fe–Pt composite performed the fastest corrosion behavior. This result was in good consistencewith the results from electrochemical tests and static immersion tests. Furthermore, corrosion rate of samples in dynamic immersion tests was much faster than that in static immersion tests. It was because in the dynamic immersion tests, 2.8–3.2 mg·l−1 ox- ygen was dissolved in Hank's solution to simulate blood environment, which was much higher than that in static immersion tests. In addition, continuous flow of Hank's solution could impede deposition of ferrous ion and wash away part of the corrosion products, so the contact time of materials and Hank's solution was prolonged.
3.4. Biocompatibility
3.4.1. Contact angle tests of Fe–Pd and Fe–Pt composites The contact angles of Fe–Pd and Fe–Pt composites were shown
in Fig. 10, with pure iron as control. The contact angles of pure iron and Fe–Pd and Fe–Pt compositeswere 51.73°, 64.74° and 51.83°, respec- tively. Generally, the smaller the contact angle is, the better the hydro- philicity will be and much easier the cell adhesion becomes. So, it could be a preliminary judgment that the biocompatibility of as-cast pure iron and Fe–Pt composite was superior to that of Fe–Pd composite.
latelets per unit area adhered on the surface of pure iron and Fe–Pd and Fe–Pt composites.
Fig. 14.Morphology of platelets adhered on the surface of pure iron and Fe–Pd and Fe–Pt composites.
52 T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
3.4.2. Cytotoxicity tests of Fe–Pd and Fe–Pt composites Fig. 11 illustrates the ion concentration in the extraction mediums
(Fig. 11(a)) and the cell viabilities of L929, ECV304 and VSMC expressed as a percentage of the viability of cells cultured in the negative control after 1, 2, and 4 days' incubation in pure iron and Fe–Pd and Fe–Pt com- posite extraction mediums (Fig. 11(b) to (d)). It can be seen that the order of iron ion concentration in extraction mediums was: Fe–Pt composite N Fe–Pd composite N as-cast pure iron, which matched well with the results of in vitro corrosion tests. Pd and Pt ion concentra- tions were very low. Firstly, the contents of these two kinds of metals added to the composites were low. A more important reason was that these two kinds ofmetals have high chemical stability andwere difficult to be corroded. Based on Fig. 11(b) to (d), the cell viability slightly de- creased after being cultured with Fe–Pd and Fe–Pt composite extracts compared with pure iron group. This may be related to the concentra- tion of leaching solution as higher iron ion concentration led to lower cell viability value. As for palladium and platinum ions, studies showed that palladium ion has a slight toxicity to eukaryotic cells and it is con- sidered to be tolerated by human body [37,55], but Pt ion has high cyto- toxicity [56]. However, the ion concentration of both palladium and platinum was extremely low, so their effects on cell reproduction should be very weak.
For L-929 and ECV304 cells, values of cell viability in experimental materials' extracts were nearly 100% or more than 100% after cultured for 1 day. However, viabilities decreased to around 80% compared with that of negative control after 2 or 4 days' incubation. According to ISO 19003-5 [47], the cell viabilities of L-929 and ECV304 cells for ex- perimental materials were both more than 70%, indicating Fe–Pd and Fe–Pt composites' slight cell toxicity to L929 and ECV304 cells. Zhu's work [57] also demonstrated that iron ions almost have no effect onme- tabolism of ECV304 cells. For VSMC, the cell viability in extracts of experimental materials was just about 60% compared with that of neg- ative control after 4 days' incubation. According to Mueller's work [58], ferrous iron could reduce the proliferation rate of VSMC by influencing growth-related gene expression. The cell toxicity for VSMC was good for antagonizing restenosis in vivo [58].
Endothelial process is very important for stents. Thrombogenicity would decrease as the stent surface is covered by regrowth of endothe- lium [59]. So, direct contact cytotoxicity of ECV304 cells was also per- formed. The morphology of ECV304 cells after 24 h cultivation on the pure iron and Fe–Pd and Fe–Pt composites is shown in Fig. 12. ECV304 cells spread out on the surface of experimental materials in good condi- tion, indicating that the experimental materials exhibited no inhibition on vascular endothelial process.
3.4.3. Hemocompatibility tests of Fe–Pd and Fe–Pt composites The hemolysis rates of experimental materials were shown in
Fig. 13(a). Hemolysis rates of Fe–Pd and Fe–Pt composites were slightly higher than that of pure iron. Nevertheless, the hemolysis rates of all
materials were still below 5%, the judging criterion for excellent blood compatibility in ASTM F756-08 [60].
Fig. 13(b) illustrates the number of platelets per unit area adhered on the surface of samples. The number of platelets adhered to the Fe– Pd composite was the least, about 50% as that of pure iron. Platelets ad- hered to the Fe–Pt composite was slightly less than that to pure iron. Combined with the result of contact angle test, platelet adhesion might be associated with materials' hydrophilicity. Materials with poor hydrophilicity resist platelet adhesion. The morphologies of ad- hered human platelets on the experimental materials were shown in Fig. 14. Although a lot of corrosion products can be observed on the sur- faces of Fe–Pd and Fe–Pt composites, the platelets adhered on these sur- faces stayed round. However, the platelets adhered on the as-cast pure iron stretched a small amount of pseudopods. The generation of pseu- dopods is an obvious sign of platelet activation [61]. Therefore, com- pared to pure iron, the thrombosis of both Fe–Pd and Fe–Pt composites would decrease.
4. Conclusions
In order to meet the clinical application demands of biodegradable coronary stent, Fe-5wt.%Pd and Fe-5wt.%Pt composites were prepared by spark plasma sintering. The mechanical properties, corrosion behav- iors andbiocompatibility of these compositeswere systematically inves- tigated by XRD, EDS, ESEM, ICP-AES and some other characterization techniques. The main conclusions were listed as follows:
(1) Pd and Pt were uniformly distributed in iron matrix. The grain size of Fe-5wt.%Pd and Fe-5wt.%Pt composites wasmuch smaller than that of as-cast pure iron. The addition of Pd or Pt improved the yield strength, rigidity and microhardness of pure iron.
(2) Fe-5wt.%Pd and Fe-5wt.%Pt uniformly corrode inHank's solution. The addition of Pd or Pt greatly accelerates the degradation rate of pure iron, especially for the Fe–Pt composite.
(3) Fe-5wt.%Pd and Fe-5wt.%Pt composites exhibited slight cytotox- icity to L-929 and ECV304 cells. At the same time, Fe–Pd and Fe– Pt composites indicate an inhibitory potential to VSMC, which is beneficial to prevent vascular restenosis.
(4) The hemolysis of both Fe-5wt.%Pd and Fe-5wt.%Pt composites is slightly higher than that of as-cast pure iron, but still less than 5%. Furthermore, the number of platelets adhered to Fe–Pd and Fe–Pt composites is less than that of as-cast pure iron, and the platelets adhered on the specimenswere round, without apparent induced thrombosis.
In summary, iron-based composites reinforced by Pd or Pt are prom- ising biodegradable biomaterials for vascular stentswith good combina- tion of mechanical properties and biocompatibility as well as a proper degradation rate.
53T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
Acknowledgments
This work was supported by the National Basic Research Program of China (973 Program) (Grant Nos. 2012CB619102 and 2012CB619100), National Science Fund for Distinguished Young Scholars (Grant No. 51225101), Research Fund for the Doctoral Program of Higher Educa- tion under Grant No. 20100001110011, and National Natural Science Foundation of China (No. 31170909).
References
[1] S. Saito, Catheter. Cardiovasc. Interv. 66 (2005) 595–596. [2] B. Heublein, R. Rohde, V. Kaese, M. Niemeyer, W. Hartung, A. Haverich, Heart 89
(2003) 651–656. [3] C. Di Mario, H. Griffiths, O. Goktekin, N. Peeters, J. Verbist, M. Bosiers, K. Deloose, B.
Heublein, R. Rohde, V. Kasese, J. Interv. Cardiol. 17 (2004) 391–395. [4] P. Peeters,M. Bosiers, J. Verbist, K.Deloose, B.Heublein, J. Endovasc. Ther. 12 (2005) 1–5. [5] M. Bosiers, Cardiovasc. Intervent. Radiol 32 (2009) 424–435. [6] P. Zartner, R. Cesnjevar, H. Singer, M. Weyand, Catheter. Cardiovasc. Interv. 66
(2005) 590–594. [7] R. Waksman, R. Pakala, P.K. Kuchulakanti, R. Baffour, D. Hellinga, R. Seabron, F.O. Tio,
E. Wittchow, S. Hartwig, C. Harder, Catheter. Cardiovasc. Interv. 68 (2006) 607–617. [8] R. Erbel, C. Di Mario, J. Bartunek, J. Bonnier, B. de Bruyne, F.R. Eberli, P. Erne, M.
Haude, B. Heublein, M. Horrigan, Lancet 369 (2007) 1869–1875. [9] X.N. Gu, Y.F. Zheng, Front. Mater. Sci. Chin. 4 (2010) 111–115.
[10] H. Hermawan, D. Dubé, D. Mantovani, J. Biomed. Mater. Res. A 93 (2010) 1–11. [11] B. Liu, Y. Zheng, Acta Biomater. 7 (2011) 1407–1420. [12] J. Lévesque, H. Hermawan, D. Dubé, D. Mantovani, Acta Biomater. 4 (2008) 284–295. [13] M. Moravej, A. Purnama, M. Fiset, J. Couet, D. Mantovani, Acta Biomater. 6 (2010)
1843–1851. [14] P.P. Mueller, S. Arnold, M. Badar, D. Bormann, F.W. Bach, A. Drynda, A. Meyer
Lindenberg, H. Hauser, M. Peuster, J. Biomed. Mater. Res. A 100 (2012) 2881–2889. [15] M. Schinhammer, I. Gerber, A.C. Hänzi, P.J. Uggowitzer, Mater. Sci. Eng. C 33 (2012)
782–789. [16] M. Schinhammer, P. Steiger, F. Moszner, J.F. Löffler, P.J. Uggowitzer, Mater. Sci. Eng. C
33 (2012) 1882–1893. [17] D.-T. Chou, D. Wells, D. Hong, B. Lee, H. Kuhn, P.N. Kumta, Acta Biomater. 9 (2013)
8593–8603. [18] Q. Feng, D. Zhang, C. Xin, X. Liu, W. Lin, W. Zhang, S. Chen, K. Sun, J. Mater. Sci. Mater.
Med. (2013) 1–12. [19] G. Papanikolaou, K. Pantopoulos, Toxicol. Appl. Pharmacol. 202 (2005) 199–211. [20] M. Peuster, C. Hesse, T. Schloo, C. Fink, P. Beerbaum, C. von Schnakenburg, Biomate-
rials 27 (2006) 4955–4962. [21] M.-M. Song, W.-J. Song, H. Bi, J. Wang, W.-L. Wu, J. Sun, M. Yu, Biomaterials 31
(2010) 1509–1517. [22] M. Peuster, P. Wohlsein, M. Brügmann, M. Ehlerding, K. Seidler, C. Fink, H. Brauer, A.
Fischer, G. Hausdorf, Heart 86 (2001) 563–569. [23] R. Waksman, R. Pakala, R. Baffour, R. Seabron, D. Hellinga, F.O. Tio, J. Interv. Cardiol.
21 (2008) 15–20. [24] C. Wu, X. Hu, H. Qiu, Y. Ruan, Y. Tang, A. Wu, Y. Tian, P. Peng, Y. Chu, X. Xu, J. Am.
Coll. Cardiol. 60 (2012).
[25] G. Ndrepepa, S. Braun, A. Dibra, J. Mehilli, W. Vogt, A. Schömig, A. Kastrati, Nutr. Metab. Cardiovasc. Dis. 15 (2005) 418–425.
[26] M. Fiset, D. Mantovani, D. Dubé, H. Hermawan, M. Moravej, Adv. Mater. Res. 15 (2007) 113–118.
[27] B. Liu, Y. Zheng, L. Ruan, Mater. Lett. 65 (2011) 540–543. [28] W.L. Xu, X. Lu, L.L. Tan, K. Yang, Acta Metall. Sin. 47 (2011) 1342–1347. [29] M. Schinhammer, A.C. Hänzi, J.F. Löffler, P.J. Uggowitzer, Acta Biomater. 6 (2010)
1705–1713. [30] B. Liu, Y.F. Zheng, Acta Biomater. 7 (2011) 1407–1420. [31] B. Wegener, B. Sievers, S. Utzschneider, P. Müller, V. Jansson, S. Rößler, B. Nies, G.
Stephani, B. Kieback, P. Quadbeck, Mater. Sci. Eng. B 176 (2011) 1789–1796. [32] M. Moravej, S. Amira, F. Prima, A. Rahem, M. Fiset, D. Mantovani, Mater. Sci. Eng. B
176 (2011) 1812–1822. [33] M. Moravej, F. Prima, M. Fiset, D. Mantovani, Acta Biomater. 6 (2010) 1726–1735. [34] M. Moravej, A. Purnama, M. Fiset, J. Couet, D. Mantovani, Acta Biomater. 6 (2010)
1843–1851. [35] F.L. Nie, Y.F. Zheng, S.C. Wei, C. Hu, G. Yang, Biomed. Mater. 5 (2010) 065015. [36] J. Cheng, Y. Zheng, J. Biomed. Mater. Res. B Appl. Biomater. (2013) 485–497. [37] W. Geurtsen, Crit. Rev. Oral Biol. Med. 13 (2002) 71–84. [38] J. Wataha, C. Hanks, J. Oral Rehabil. 23 (1996) 309–320. [39] W. Agnew, T. Yuen, D. McCreery, L. Bullara, Exp. Neurol. 92 (1986) 162–185. [40] S. Brummer, M. Turner, IEEE Trans. Biomed. Eng. (1977) 440–443. [41] A. Cowley, B. Woodward, Platin. Met. Rev. 55 (2011) 98–107. [42] A. Berghaus, K. Neumann, T. Schrom, Arch. Facial Plast. Surg. 5 (2003)
166–170. [43] P. Vanýsek, Electrochemical series, in: D.R. Lide (Ed.), CRC Handbook of Chemistry
and Physics, CRC Press, Boca Raton, FL, 2005, pp. 20–29. [44] ASTM E. E9-89a, Standard Test Methods of Compression Testing of Metallic Mate-
rials at Room Temperature, Annual Book of ASTM Standards, 2000. [45] ASTM G. G 31–72, Standard Practice for Laboratory Immersion Corrosion Testing of
Metals, ASTM Book of Standards, 2004. [46] E. ISO, German Version: DIN EN ISO, 2008. (10993-10912). [47] E. Iso, Tests for In Vitro Cytotoxicity, 2009. [48] Z. Munir, U. Anselmi-Tamburini, M. Ohyanagi, J. Mater. Sci. 41 (2006) 763–777. [49] H. Okamoto, Phase Diagrams of Binary Iron Alloys, ASM International Materials Park,
OH, 1993. [50] H. Okamoto, J. Phase Equilib. Diffus. 25 (2004) 395-395. [51] J.C. Slater, J. Chem. Phys. 41 (1964) 3199. [52] R. Armstrong, Metall. Mater. Trans. 1 (1970) 1169–1176. [53] C. Ramesh, M. Safiulla, Wear 263 (2007) 629–635. [54] X. Liu, S. Chen, H. Ma, G. Liu, L. Shen, Appl. Surf. Sci. 253 (2006) 814–820. [55] J.C. Wataha, C. Hanks, R.G. Craig, J. Biomed. Mater. Res. 25 (1991) 1133–1149. [56] A. Biesiekierski, J. Wang, M. Abdel-Hady Gepreel, C. Wen, Acta Biomater. 8 (2012)
1661–1669. [57] S. Zhu, N. Huang, L. Xu, Y. Zhang, H. Liu, H. Sun, Y. Leng, Mater. Sci. Eng. C 29 (2009)
1589–1592. [58] P.P. Mueller, T. May, A. Perz, H. Hauser, M. Peuster, Biomaterials 27 (2006)
2193–2200. [59] D.A. Dichek, R. Neville, J. Zwiebel, S. Freeman, M. Leon, W. Anderson, Circulation 80
(1989) 1347–1353. [60] F756-00, A., Standard Practice for Assessment of Hemolytic Properties of Materials,
2000, ASTM International West Conshohocken, PA, USA, 2000. [61] D.A. Armitage, T.L. Parker, D.M. Grant, J. Biomed. Mater. Res. A 66 (2003)
129–137.
1. Introduction
3. Results and discussion
3.3. Corrosion properties of Fe–Pd and Fe–Pt Composites
3.3.1. Electrochemical measurements
3.4. Biocompatibility
3.4.1. Contact angle tests of Fe–Pd and Fe–Pt composites
3.4.2. Cytotoxicity tests of Fe–Pd and Fe–Pt composites
3.4.3. Hemocompatibility tests of Fe–Pd and Fe–Pt composites
4. Conclusions
Materials Science and Engineering C
j ourna l homepage: www.e lsev ie r .com/ locate /msec
In vitro degradation and biocompatibility of Fe–Pd and Fe–Pt composites fabricated by spark plasma sintering
T. Huang a,b, J. Cheng c, Y.F. Zheng a,b,c, a State Key Laboratory for Turbulence and Complex System, College of Engineering, Peking University, Beijing 100871, China b Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China c Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
Corresponding author at: Department ofMaterials Sci Engineering, Peking University, Beijing 100871, China. Tel
E-mail address: [email protected] (Y.F. Zheng).
0928-4931/$ – see front matter © 2013 Elsevier B.V. All ri http://dx.doi.org/10.1016/j.msec.2013.10.023
a b s t r a c t
a r t i c l e i n f o
Article history: Received 21 June 2013 Received in revised form 12 October 2013 Accepted 21 October 2013 Available online 31 October 2013
Keywords: Biodegradable metal Fe–Pt composite Fe–Pd composite Corrosion Biocompatibility
In order to obtain biodegradable Fe-basedmaterialswith similarmechanical properties as 316L stainless steel and faster degradation rate than pure iron, Fe-5 wt.%Pd and Fe-5 wt.%Pt composites were prepared by spark plasma sinteringwith powders of pure Fe and Pd/Pt, respectively. The grain size of Fe-5 wt.%Pd and Fe-5 wt.%Pt compos- ites wasmuch smaller than that of as-cast pure iron. The metallic elements Pd and Pt were uniformly distributed in the matrix and the mechanical properties of these materials were improved. Uniform corrosion of Fe–Pd and Fe–Pt composites was observed in both electrochemical tests and immersion tests, and the degradation rates of Fe–Pd and Fe–Pt composites were much faster than that of pure iron. It was found that viabilities of mouse fibro- blast L-929 cells and human umbilical vein endothelial cells (ECV304) cultured in extraction mediums of Fe–Pd and Fe–Pt composites were close to that of pure iron. After 4 days' culture, the viabilities of L-929 and ECV304 cells in extraction medium of experimental materials were about 80%. The result of direct contact cytotoxicity also indicated that experimental materials exhibited no inhibition on vascular endothelial process. Meanwhile, iron ions released from experimental materials could inhibit proliferation of vascular smooth muscle cells (VSMC), which may be beneficial for hindering vascular restenosis. Furthermore, compared with that of as-cast pure iron, the hemolysis rates of Fe–Pd and Fe–Pt composites were slightly higher, but still within the range of 5%, which is the criteria for good blood compatibility. The numbers of platelet adhered on the surface of Fe–Pd and Fe–Pt composites were lower than that of pure iron, and the morphology of platelets kept spherical. To sum up, the Fe-5wt.%Pd and Fe-5wt.%Pt composites exhibited good mechanical properties and degradation be- havior, closely approaching the requirements for biodegradable metallic stents.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Biodegradable stents are currently considered as ideal stents. They are expected to effectively avoid the late stent thrombosis which is often caused by permanent stents. Thematerial for biodegradable stents is required to have good biocompatibility and it should be kept intact for several months before completion of vascular healing [1]. Two typical classes of metallic materials including Mg-based [2–9] and Fe-based [10–18] alloys have been researched for this application. Compared with Mg-based metals, Fe-based metals possess more attractive me- chanical properties [10]. Iron is an essential nutrient element in human body and plays an important role in vital biochemical activities, such as oxygen sensing and transport, electron transfer and catalysis [19]. Pure iron also has goodmechanical property [10], biocompatibility and hemocompatibility [20,21] close to 316L stainless steel, which serves as the golden standard for stent material. Animal experiments demonstrated that degradable iron stents can be safely implanted
ence and Engineering, College of ./fax: +86 10 62767411.
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without significant vascular restenosis caused by inflammation, neointi- mal proliferation or thrombotic events, and there was no evidence for local or systemic toxicity [20,22–24]. A clinical outcome also showed that there was no association between iron status and the incidence of major adverse cardiac events or coronary restenosis in human body [25]. Therefore, iron-based materials are considered as suitable mate- rials for further development of novel biodegradable coronary artery stent. However, a faster degradation rate of iron is desired to apply to stents [20,22].
To obtain iron-based materials with appropriate properties for clin- ical applications, researchers have focused on the development of new kinds of Fe-based materials by modifying the chemical composition and microstructure. Hermawan et al. [10,26] were the first to study the effect of alloying elements on the performance of biodegradable iron-based materials. They found that alloying with Mn increased the strength and degradation rate of pure iron. Liu et al. [27] discovered that the addition of 6 wt.% of Si in Fe-30 wt.%Mn alloy established the shape memory effect. Xu et al. [28] reported that Fe-30Mn-1C alloy showed lowermagnetic susceptibility and bettermechanical properties than Fe-30Mn alloy. Schinhammer et al. [29] added 1 wt.% of Pd into Fe- 10Mn alloy that sped up the degradation rate ten times faster than that
44 T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
of low carbon steel. They also did a research for Fe–Mn–C(–Pd) alloys. The Fe–Mn–C–Pd alloys were characterized by an increased degrada- tion rate compared to pure iron [16]. The effects of alloying elements (Mn, Co, Al, W, Sn, B, C, and S) on the in vitro degradation behavior and biocompatibility of pure iron were also investigated by Liu and Zheng [30]. The results showed that the addition of all alloying elements except for Sn improved the mechanical properties of iron after rolling. New fabricating methods were also tried by researchers, such as pow- der metallurgy [31], electroforming [32–34], equal channel angular pressing (ECAP) technique [35] and SPS sintering [36]. Although the predecessors made a lot of efforts for the improvement of Fe-basedma- terials, the corrosion rate of Fe-based materials still could not meet the clinical application demand.
Palladium is a common alloying element in dental alloys. It has rela- tively low price and density when compared to gold and platinum. Pal- ladium possesses a good range of solubility in several metals and could improve the mechanical properties of the matrix metals, too [37]. There have also been reports of using Pd-based organometallic com- pounds as antineoplastic agents [38]. Platinum has good mechanical property, low corrosivity and high biocompatibility [39,40]. It is used as essential components for many medical devices including implant- able defibrillators, catheters, stents, pacemakers, neuromodulation device and upper-lid implant [41,42]. So palladium and platinum them- selves are both excellent biomedical materials with good biocompatibil- ity. In addition, the standard electrode potential of palladium and platinum (Pd is about −0.126 V, Pt is about 1.2 V) is higher than that of iron (about −0.44 V) [43], and galvanic corrosion may occur in the placewhere two kinds of metals contact, then accelerating the degrada- tion of pure iron. Dispersion of platinum and palladium into pure iron matrixmay alsomake the corrosion of pure ironmore uniform. Further- more, the effect of dispersion strengthening caused by the addition of platinum and palladiumwas expected to improve themechanical prop- erties of pure iron. The objective of the presentwork is to investigate the feasibility of adding palladium and platinum to pure iron for the prepa- ration of biodegradable iron-based composites.
2. Materials and methods
2.1. Material preparation
Pure iron powder (99.0%, −300 mesh), pure palladium powder (99.99%, −200 mesh) and pure platinum powder (99.99%, −150 mesh), whichwere provided by ChinaMetalMaterials Technology Com- pany, were used as raw materials. Iron powder mixed with 5 wt.% of palladium and platinum powder were prepared, respectively. Then the mixed powders were put into a 20-mm diameter graphite die and sintered under vacuum by SPS-1050 system (Sumitomo Coal Mining Company, Ltd.) with sintering temperature of 1000 °C and holding time of 5 min. Pure iron (99.9%) prepared by casting served as control.
The as-sintered Fe–Pd and Fe–Pt composite specimens (Φ20 × 7 mm) and as-cast pure iron were cut into square pieces (10 × 10 × 1.5 mm3) and cylinders (Φ2 × 5 mm), respectively. Square pieces were used for density measurements, microstructure characterization and series tests of microhardness, electrochemical properties, corrosion behavior, cytotoxicity, hemocompatibility and platelet adhesiveness. Each sample was mechanically polished to 2000 grit, then ultrasonically cleaned in anhydrous ethanol and dried in the open air. Before cytotoxicity test, the samples were sterilized with ultraviolet radiation for at least 2 h. Cylinder speci- mens were used for compressive test.
2.2. Microstructure characterization
Specimens were polished by 0.15 μm diamond paste, further ultra- sonically cleaned in anhydrous ethanol and dried in the open air. Optical microscope (Olympus BX51M)was used to observe themetallographic
structure after the specimens were etched with a 4% HNO3/alcohol solution. X-ray diffractometer (XRD, Rigaku DMAX 2400) using CuKα radiation was adopted to identify the constituent phases of Fe–Pd and Fe–Pt composites with scanning range from 10° to 100° and scan rate of 6°/min. Energy dispersive spectrometer (EDS) was used for the analysis of chemical composition.
2.3. Mechanical test
Themechanical properties of experimental composites and pure iron were determined by microhardness test and compressive test. Micro- hardness tests were carried out by a microhardness tester (SHIMADZU HMV-2t) measuring Vickers hardness with loading force of 0.2 kg and dwell time of 10 s. Center point and other four points uniformly distrib- uted around the center point on the surface of each sample were select- ed for microhardness test. An average of five measurements was taken for eachmaterial. Instron 5969 universal testmachinewasused for com- pressive tests according to ASTME9-89a [44]. The specimenswere in the form of cylinder 2.5 mm in diameter and 5 mm in length. Compression strain rate was 2 × 10−4/s. An average of at least three measurements was taken for each group.
2.4. Electrochemical measurements
Electrochemical measurements were performed using three- electrode system at an electrochemical work station (CHI660C, China). The specimen, a platinum electrode and a saturated calomel electrode (SCE) were set as the working electrode, auxiliary electrode and the ref- erence electrode, respectively. All themeasurementswere carried out at a temperature of 37 ± 0.5 °C in Hank's solution. The area of working electrode exposed to the solution was 1 cm2. The open circuit potential (OCP) measurement was set for 7200 s. Electrochemical impedance spectroscopy (EIS) was measured from 100 kHz to 10 mHz at OCP value after 2 h immersion in Hank's solution. The potentiodynamic po- larization curves were carried out from −1000 mV (vs. SCE) to 0 mV (vs. SCE) at a scanning rate of 0.33 mV·s−1.
2.5. Static immersion test
In vitro static immersion test was performed in Hank's solution, 50 ml for each sample following ASTM-G31-72 [45] at 37 °C in water bath. After 3, 10 and 30 days, the samples were removed from the soaking solution, gently rinsed with distilled water and quickly dried in case of oxidation. Changes on the surface morphologies of the speci- mens after immersion were characterized by ESEM (Quanta 200FEG), equipped with an EDS attachment. Then, weighing after the corrosion products on the surface of samples dissolved in the 10 mol/l NaOH solu- tion, an average of three measurements was taken for each group. The degradation rate was calculated based on the formula:
CR ¼ m St
where CR (mg·cm−2·day−1) is the corrosion rate, m (mg) is the mass loss, S (cm2) is the surface area of the specimen and t (day) is the time.
2.6. Dynamic immersion test
Dynamic immersion test was carried out using a dynamic corrosion test bench which was built in our previous research work [11]. Dis- solved oxygen is an important factor that influences degradation of iron in a neutral medium, so the dissolved oxygen concentration in Hank's solution was regulated to better simulate the blood environ- ment. In this bench the wall shear stress (0.68 Pa), temperature (36.0–37.1 °C), pH value (7.35–7.45), and dissolved oxygen (2.8– 3.2 mg−1) were controlled to approach the common values in the
Fig. 1.Metallographs of experimentalmaterials: (a) pure iron and (b) Fe–Pd and (c) Fe–Pt composites. The corresponding grain size distribution estimated by themetallographs: (d) pure iron; (e) Fe–Pd and (f) Fe–Pt composites.
45T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
human coronary artery. Plate specimens were mounted in paraffin and then placed in a specimen holder. All specimens were exposed to the dynamic fluid flow (Hank's solution) for 30 days (considering the gen- erally bare metallic coronary stents would be covered by endothelial cells after 30 days in vivo implantation and no longer contact with blood directly) [11]. After the test, the samples were taken out, then the macroscopic surface morphology was characterized using a 2.5 di- mensional precision image measuring instrument (HLEO, VACD-1010, China). After that the specimens were cleaned by deionized water and ethanol in turn, they were dried in the air. Changes on the surface mor- phology were characterized by ESEM (Quanta 200FEG), equipped with
Fig. 2. X-ray diffraction patterns of pure iron and Fe–Pd and Fe–Pt composites.
an EDS attachment. The corrosion rate was calculated based on the weight loss of specimens. Corrosion layers on the surface of samples were peeled off at first. The residual corrosion products were dissolved by 10 mol/l NaOH and rinsed by deionized water and alcohol in turn, then samples were weighed after they were dried in the open air. Cor- rosion rates were calculated by the same formula in static immersion test.
2.7. Contact angle measurement
Water contact angle of iron-basedmaterials wasmeasured by a con- tact angle analyzer (Kino SL200B); for each sample the measurements were carried out three times.
2.8. Cytotoxicity test
The cytotoxicity test was performed by an indirect contact method. Murine fibroblast cells (L-929), human vascular smooth muscle cells (VSMC) and umbilical vein endothelial cells (ECV304) were used to evaluate the cytotoxicity of experimental Fe–Pd and Fe–Pt composites. At first, all the cell lineswere cultured in theDulbecco'smodified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100U·ml−1 pen- icillin and 100 μg·ml−1 streptomycin at 37 °C in a humidified atmo- sphere of 5% CO2. According to ISO 10993-12 [46], extraction medium was prepared using DMEM with a surface area/extraction medium ratio of 1.25 cm2·ml−1 in a humidified atmosphere with 5% CO2 at 37 °C for 72 h. After the extracts were centrifuged, the supernatant fluid was withdrawn and stored at 4 °C before cytotoxicity test. The control groups involved DMEM medium as the negative control and DMEM including 10% dimethyl sulfoxide (DMSO) as the positive con- trol. The metallic concentrations of ions in the extraction medium were measured by inductively coupled plasma atomic emission spectrometry (ICP-AES) (Leeman, Profile). Cellswere incubated in the 96-well plates at the density of approximately 5 × 103 cells per 100 μl medium in each well and incubated for 24 h to allow attachment. DMEM was then sucked out and replaced by extraction medium. Then 10 μl serum was
Fig. 3. ESEM images of surface morphology and energy spectrum analysis of (a) Fe–Pd and (b) Fe–Pt composites.
46 T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
added to each well. After 1, 2 and 4 days' incubation in the incubator re- spectively, the 96-well plates were observed under an optical micro- scope. Thereafter, 10 μl of MTT was added to each well. The cells were incubatedwithMTT for 4 h. Then 100 μl of formazan solubilization solu- tion (10% sodium dodecyl sulfate in 0.01 MHCl) was added to each well and incubated for 10 h. The absorbance of each well was tested by a mi- croplate reader (Bio-RAD680) at 570 nmwith a referencewavelength of
Table 1 Density of Fe–Pd and Fe–Pt composites with as-cast pure iron as the control at room temperature.
Materials Density (g/cm3) Relative density
Pure iron 7.83 (0.01)a \ Fe–Pd 7.15 (0.09)a 88.9% Fe–Pt 7.04 (0.03)a 86.3%
a Number in the brackets represents the standard deviation. Fig. 4. Compressive stress–strain curves of pure iron and Fe–Pd and Fe–Pt composites.
Fig. 5. Electrochemical test results of pure iron and Fe–Pd and Fe–Pt composites in Hank's solution: (a) Potentiodynamic polarization curve, (b) Nyquist plots.
47T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
630 nm. Viability of cells (X) was calculated using the following formula according to ISO 19003-5 [47]:
X ¼ OD1
OD2 100%:
Here OD1 is themean absorbance of the experimental sample group and positive control group. OD2 is the mean absorbance of the negative con- trol group.
Direct contact cytotoxicity of ECV304 cells was also carried out ac- cording to ISO 10993-5 [47]. 600 μl cell suspension was respectively added to each sterilized sample at a cell density of 6 × 103/ml. After being cultured in the incubator for 24 h, the medium was removed. Then the samples were gently washed by phosphate buffered saline (PBS); cells on the samples were fixed with 2.5% glutaraldehyde solu- tion at room temperature for 2 h, then dewateredwith gradient alcohol solution (50%, 60%, 70%, 80%, 90%, 95% and 100%) for 10 min, and finally freeze-dried for 2 days. The morphology of cells adhered on the speci- mens was observed by ESEM (Quanta 200FEG).
2.9. Hemolysis test and platelet adhesion
Healthy human blood (anticoagulant was 3.8 wt.% citric acid sodi- um) extracted from volunteers was diluted by physiological saline according to volume ratio 4:5. Pure iron and Fe–Pd and Fe–Pt compos- ites were installed in centrifugal tubes with 10 ml physiological saline. Temperature was kept at 37 °C for 30 min. Then 0.2 ml diluted blood was added to each tube and incubated at 37 °C for 60 min, 10 ml deion- ized water with 0.2 ml diluted blood as the positive control and 10 ml physiological saline with 0.2 ml diluted blood as the negative control. Upon completion of the above operations, samples were removed, and then these tubes were centrifuged at 800 g for 5 min. Supernatant was transferred to 96-well multiplates, the absorbance (OD) was
Table 2 Average electrochemical parameters of Fe–Pd and Fe–Pt composites (as-cast pure iron as control).
Materials Vcorr (V) Icorr (μA/cm2)
Pure iron −0.324 (0.020) 0.871 (0.040) Fe–Pd −0.471 (0.010) 1.550 (0.120) Fe–Pt −0.545 (0.008) 6.698 (0.370)
Corrosion potential (Vcorr), corrosion current (Icorr).
determined by a microplate reader (Bio-RAD680) at a wavelength of 545 nm. Hemolysis of samples was calculated by the formula:
Hemolysis ¼ OD testð Þ−OD negative controlð Þ OD positive controlð Þ−OD negative controlð Þ 100%:
For platelet adhesion, whole blood from healthy human body was centrifuged at 1000 r/min for 10 min. Platelet rich plasma (PRP) was obtained from the upper fluid. Samples after ultraviolet disinfection were moved to 24-well multiplates and 0.2 ml PRP was added to each well, incubated at 37 °C for 1 h. After gently flushed by phosphate buff- ered saline (PBS), platelets on samples were fixed with 2.5% glutaralde- hyde solution at room temperature for 1 h, then dewatered with gradient alcohol solution (50%, 60%, 70%, 80%, 90%, 95% and 100%) for 10 min, and finally freeze-dried for 2 days. The morphology of platelet adhered on the specimens was observed by ESEM (Quanta 200FEG).
3. Results and discussion
3.1. Microstructures
Fig. 1 shows the metallographs of pure iron and Fe–Pd and Fe–Pt composites and the corresponding grain size distribution measured frommetallographs. The average grain sizes of these two kinds of exper- imental composites are quite close, both about 20 ± 8 μm, which are much smaller than that of as-cast pure iron (180 ± 90 μm). It may be caused by the different preparation methods. For spark plasma sintering, lower temperature and shorter holding time could sinter powders to crystal with little grain growth [48]. The second phase par- ticles inhibiting grain growth should also be taken into consideration. The decrease of the grain size could contribute to the improvement of mechanical properties.
Fig. 2 displays XRD patterns of experimental composites, as-cast pure iron as control. It can be found that the Fe-5wt.%Pd and Fe- 5wt.%Pt composites both consist of α-Fe phase and FeO phase, but pure Pd or Pt phase was not found. PDF CARDS recorded that the peaks of Fe9.7Pd0.3 and Fe9.7Pt0.3 phases almost overlap with that of α-Fe phase in X-ray diffraction spectrum. As the contents of both Pd and Pt in experimental composites are less than 3 at.%, there might be quite a part of Pd and Pt solute in the surrounding pure iron matrix forming Fe9.7Pd0.3 and Fe9.7Pt0.3 phases in the sintering process, so the content of Pd or Pt phase was too low to be detected by X-ray dif- fraction. Fig. 3 showed that Pd and Pt were uniformly distributed in the iron matrix. The dark areas mainly consisted of Fe and O, which
Fig. 6. Macrographs of surface morphology of experimental materials static immersed in Hank's solution for 10 and 30 days.
Fig. 7. SEM images of samples' surfacemorphology after statically immersed inHank's solution for different periods and the corresponding X-ray diffraction patterns of corrosion products on the surface of pure iron.
48 T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
Fig. 8. Corrosion rates calculated from the weight loss of samples after static or dynamic immersion in Hank's solution for 30 days.
49T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
should be FeO phase, as revealed by the XRD results. Table 1 shows the density of Fe–Pd and Fe–Pt composites with as-cast pure iron as control at room temperature. Relative density of both Fe–Pd and Fe–Pt compos- ites were less than 90%, which was probably due to the relatively low sintering temperature and pressure, as well as the existence of FeO in the composites.
3.2. Mechanical properties
Fig. 4 shows the compression stress–strain curves of Fe–Pd and Fe– Pt composites, with pure iron as control. The yield strengths (YS) of Fe–Pd and Fe–Pt composites were both about three times as that of pure iron. The ultimate compressive strength (UCS) of Fe–Pt composite was slightly higher than that of Fe–Pd composite. The rigidity of Fe–Pd and Fe–Pt composites was also much larger than that of pure iron. Ac- cording to the binary phase diagramof Fe–Pd [49] and Fe–Pt [50] alloys, a small quantity of Pd and Pt could dissolve into iron matrix. However, the atomic radii of Fe (1.40 ), Pd (1.40 ) and Pt (1.35 ) are very close [51], so the lattice distortion of α-Fe was negligible. Then the
Fig. 9. SEM images of samples' surfacemorphology after dynamically immersed in Hank's soluti samples' surface morphology after removed corrosion products: (d) pure iron and (e) Fe–Pd a
solution strengthening effect in these composites could not be signifi- cant. Therefore, the strength enhancement of the composites should be mainly attributed to the second phase strengthening effects. More- over, the decrease of the grain size could make considerable contribu- tions to the strength improvement of these composites [52]. However, the Fe–Pd and Fe–Pt composites exhibited low ductility, which can be explained mainly by the relatively low density (Table 1) [53].
The microhardness of Fe–Pd (184 ± 10 HV0.2/10) and Fe–Pt (309 ± 8 HV0.2/10) composites was much higher than that of pure iron (119 ± 3 HV0.2/10), especially for Fe–Pt composite, which was nearly three times as that of pure iron. Obviously, the addition of Pd or Pt greatly enhanced the strength of iron matrix.
3.3. Corrosion properties of Fe–Pd and Fe–Pt Composites
3.3.1. Electrochemical measurements Fig. 5 shows the potentiodynamic polarization curves (Fig. 5(a)) and
Nyquist plots (Fig. 5(b)) of Fe–Pd and Fe–Pt composites immersed in Hank's solution, with as-cast pure iron as the control. The average elec- trochemical parameters were listed in Table 2. Itwas found that the cor- rosion potential was largely decreased and the corrosion current densities were increased after adding Pd and Pt into the iron matrix. As the Nyquist plots shown, plotted the variation in the excitation frequency impedance imaginary part (z") as a function of the imped- ance real part (z′) which obtained a series of semicircles. The diameters of semicircles obtained from Fe–Pd and Fe–Pt composites were smaller than that of pure iron, revealing their worse corrosion resistance. Since the diameter of high frequency capacitive loop can be considered as the charge transfer resistance and smaller charge transfer resistance corre- sponds to faster corrosion rate [54].
3.3.2. Static immersion corrosion behavior Samples after 3 days' immersion in Hank's solution exhibited no
conspicuous change on the surface. After 10 days' immersion, localized corrosion mainly happened at the edge of pure iron sample, and the productswere reddish brown.However, uniform corrosion could be ob- served on the surfaces of Fe–Pd and Fe–Pt composites, with claybank corrosion products (Fig. 6).When the sampleswere immersed inHank's solution for 30 days, most of corrosion products fell off, as shown in Fig. 6. Different orientations of the grains showed different colors.
on for 30 days: (a) pure iron and (b) Fe–Pd and (c) Fe–Pt composites. SEM images of these nd (f) Fe–Pt composites.
Fig. 10.Water contact angles of (a) pure iron and (b) Fe–Pd and (c) Fe–Pt composites.
50 T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
Local distribution of black etch pits could also be found on the surface of pure iron, indicating localized corrosion feature of pure iron. However, a large number of corrosion pits were distributed uniformly on the sur- face of Fe–Pd and Fe–Pt composites, representing their macroscopically uniform corrosion behavior. Fig. 7 shows the SEM images of surface morphology of experimental samples, as can be seen from the figure:
(1) After 3 days' immersion, all the samples' surfaces almost kept intact.
(2) After 10 days' immersion, most area on the surface of pure iron remained intact, and only several corrosion pits were locally dis- tributed on the surface. The surface of Fe–Pd composite was
Fig. 11. (a) Ion concentration in extraction mediums of experimental materials and cell viabilit (c) ECV304 and (d) VSMC.
covered by a thin corrosion layer, and this layer didn't closely ad- here to thematrix. Fe–Pt composites corrodedmost severely and a lot of deep corrosion pits were uniformly distributed on the surface.
(3) After 30 days' immersion, because the most of the corrosion products fell off from the surface of samples, grain boundary ob- viously presented on the surface of pure iron. Fe–Pd and Fe–Pt composites corroded much more seriously than pure iron. The iron substrate surrounded Pd- and Pt-rich areas corroded more seriously. This should be attributed to the galvanic corrosion oc- curred at the contact place of Fe and Pd or Pt. Pd- and Pt-rich areas that have relatively high corrosion potential as cathode
y after cultured in extraction mediums and positive control for 1, 2 and 4 days; (b) L-929,
Fig. 12. The morphologies of ECV 304 cells directly cultured on (a) pure iron and (b) Fe–Pd and (c) Fe–Pt composites for 24 h.
51T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
sites, iron as anode sites, then formed a lot of tiny galvanic cells in the compositeswhen immersed inHank's solution, so as to accel- erate the corrosion rate of iron.
(4) The XRD spectrum of corrosion products on the surface of as-cast pure ironwhich had immersed inHank's solution for 10 days indi- cated that the corrosion products were mainly the Fe + 3O(OH) and Fe(OH)3. Corrosion products on the surface of Fe–Pd and Fe– Pt composites were the same as that on pure iron.
Fig. 8 shows the corrosion rates thatwere calculated from theweight loss after samples immersed in Hank's solution for 30 days. The corro- sion rates of both Fe–Pd and Fe–Pt composites were faster than that of as-cast pure iron. The corrosion rate of Fe–Pt composite was the fastest, about 2.73 times as that of as-cast pure iron. Compared to the corrosion rate of Fe-5wt.%W(about 1.15 times as that of as-cast pure iron) and Fe- 0.5wt.%CNT composites (about 1.96 times as that of as-cast pure iron) [36], the addition of Pd and Pt increased the corrosion rate of pure iron much more significantly. In addition to the galvanic corrosion, FeO contained in Fe–Pd and Fe–Pt composites which could be easily oxidized to oxidation products was also an innegligible reason for increasing corrosion rate.
3.3.3. Dynamic immersion corrosion behavior Fig. 9 panels (a)–(c) were SEM images showing surfacemorphology
of experimental materials after corroding in dynamic environments. As can be seen from these figures, the numbers of corrosion products on the surface of Fe–Pd and Fe–Pt composites are much more than that on the surface of pure iron.
Fig. 13. (a) Hemolysis rates of pure iron and Fe–Pd and Fe–Pt composites. (b) The number of p
After the corrosion product layers were removed, surface morphol- ogies of the substrates appeared, as shown in Fig. 9(d)–(f). Grain boundaries could be observed obviously on the surface of pure iron. The surface of Fe–Pd composite was much smoother. Fe–Pt composite corrodedmost seriously, with a lot of deep corrosion pits on the surface.
The corrosion rates calculated from weight loss of samples after dynamic immersion tests were also shown in Fig. 8. Compared with as- cast pure iron, both the Fe–Pd and Fe–Pt composites corroded much faster, and the Fe–Pt composite performed the fastest corrosion behavior. This result was in good consistencewith the results from electrochemical tests and static immersion tests. Furthermore, corrosion rate of samples in dynamic immersion tests was much faster than that in static immersion tests. It was because in the dynamic immersion tests, 2.8–3.2 mg·l−1 ox- ygen was dissolved in Hank's solution to simulate blood environment, which was much higher than that in static immersion tests. In addition, continuous flow of Hank's solution could impede deposition of ferrous ion and wash away part of the corrosion products, so the contact time of materials and Hank's solution was prolonged.
3.4. Biocompatibility
3.4.1. Contact angle tests of Fe–Pd and Fe–Pt composites The contact angles of Fe–Pd and Fe–Pt composites were shown
in Fig. 10, with pure iron as control. The contact angles of pure iron and Fe–Pd and Fe–Pt compositeswere 51.73°, 64.74° and 51.83°, respec- tively. Generally, the smaller the contact angle is, the better the hydro- philicity will be and much easier the cell adhesion becomes. So, it could be a preliminary judgment that the biocompatibility of as-cast pure iron and Fe–Pt composite was superior to that of Fe–Pd composite.
latelets per unit area adhered on the surface of pure iron and Fe–Pd and Fe–Pt composites.
Fig. 14.Morphology of platelets adhered on the surface of pure iron and Fe–Pd and Fe–Pt composites.
52 T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
3.4.2. Cytotoxicity tests of Fe–Pd and Fe–Pt composites Fig. 11 illustrates the ion concentration in the extraction mediums
(Fig. 11(a)) and the cell viabilities of L929, ECV304 and VSMC expressed as a percentage of the viability of cells cultured in the negative control after 1, 2, and 4 days' incubation in pure iron and Fe–Pd and Fe–Pt com- posite extraction mediums (Fig. 11(b) to (d)). It can be seen that the order of iron ion concentration in extraction mediums was: Fe–Pt composite N Fe–Pd composite N as-cast pure iron, which matched well with the results of in vitro corrosion tests. Pd and Pt ion concentra- tions were very low. Firstly, the contents of these two kinds of metals added to the composites were low. A more important reason was that these two kinds ofmetals have high chemical stability andwere difficult to be corroded. Based on Fig. 11(b) to (d), the cell viability slightly de- creased after being cultured with Fe–Pd and Fe–Pt composite extracts compared with pure iron group. This may be related to the concentra- tion of leaching solution as higher iron ion concentration led to lower cell viability value. As for palladium and platinum ions, studies showed that palladium ion has a slight toxicity to eukaryotic cells and it is con- sidered to be tolerated by human body [37,55], but Pt ion has high cyto- toxicity [56]. However, the ion concentration of both palladium and platinum was extremely low, so their effects on cell reproduction should be very weak.
For L-929 and ECV304 cells, values of cell viability in experimental materials' extracts were nearly 100% or more than 100% after cultured for 1 day. However, viabilities decreased to around 80% compared with that of negative control after 2 or 4 days' incubation. According to ISO 19003-5 [47], the cell viabilities of L-929 and ECV304 cells for ex- perimental materials were both more than 70%, indicating Fe–Pd and Fe–Pt composites' slight cell toxicity to L929 and ECV304 cells. Zhu's work [57] also demonstrated that iron ions almost have no effect onme- tabolism of ECV304 cells. For VSMC, the cell viability in extracts of experimental materials was just about 60% compared with that of neg- ative control after 4 days' incubation. According to Mueller's work [58], ferrous iron could reduce the proliferation rate of VSMC by influencing growth-related gene expression. The cell toxicity for VSMC was good for antagonizing restenosis in vivo [58].
Endothelial process is very important for stents. Thrombogenicity would decrease as the stent surface is covered by regrowth of endothe- lium [59]. So, direct contact cytotoxicity of ECV304 cells was also per- formed. The morphology of ECV304 cells after 24 h cultivation on the pure iron and Fe–Pd and Fe–Pt composites is shown in Fig. 12. ECV304 cells spread out on the surface of experimental materials in good condi- tion, indicating that the experimental materials exhibited no inhibition on vascular endothelial process.
3.4.3. Hemocompatibility tests of Fe–Pd and Fe–Pt composites The hemolysis rates of experimental materials were shown in
Fig. 13(a). Hemolysis rates of Fe–Pd and Fe–Pt composites were slightly higher than that of pure iron. Nevertheless, the hemolysis rates of all
materials were still below 5%, the judging criterion for excellent blood compatibility in ASTM F756-08 [60].
Fig. 13(b) illustrates the number of platelets per unit area adhered on the surface of samples. The number of platelets adhered to the Fe– Pd composite was the least, about 50% as that of pure iron. Platelets ad- hered to the Fe–Pt composite was slightly less than that to pure iron. Combined with the result of contact angle test, platelet adhesion might be associated with materials' hydrophilicity. Materials with poor hydrophilicity resist platelet adhesion. The morphologies of ad- hered human platelets on the experimental materials were shown in Fig. 14. Although a lot of corrosion products can be observed on the sur- faces of Fe–Pd and Fe–Pt composites, the platelets adhered on these sur- faces stayed round. However, the platelets adhered on the as-cast pure iron stretched a small amount of pseudopods. The generation of pseu- dopods is an obvious sign of platelet activation [61]. Therefore, com- pared to pure iron, the thrombosis of both Fe–Pd and Fe–Pt composites would decrease.
4. Conclusions
In order to meet the clinical application demands of biodegradable coronary stent, Fe-5wt.%Pd and Fe-5wt.%Pt composites were prepared by spark plasma sintering. The mechanical properties, corrosion behav- iors andbiocompatibility of these compositeswere systematically inves- tigated by XRD, EDS, ESEM, ICP-AES and some other characterization techniques. The main conclusions were listed as follows:
(1) Pd and Pt were uniformly distributed in iron matrix. The grain size of Fe-5wt.%Pd and Fe-5wt.%Pt composites wasmuch smaller than that of as-cast pure iron. The addition of Pd or Pt improved the yield strength, rigidity and microhardness of pure iron.
(2) Fe-5wt.%Pd and Fe-5wt.%Pt uniformly corrode inHank's solution. The addition of Pd or Pt greatly accelerates the degradation rate of pure iron, especially for the Fe–Pt composite.
(3) Fe-5wt.%Pd and Fe-5wt.%Pt composites exhibited slight cytotox- icity to L-929 and ECV304 cells. At the same time, Fe–Pd and Fe– Pt composites indicate an inhibitory potential to VSMC, which is beneficial to prevent vascular restenosis.
(4) The hemolysis of both Fe-5wt.%Pd and Fe-5wt.%Pt composites is slightly higher than that of as-cast pure iron, but still less than 5%. Furthermore, the number of platelets adhered to Fe–Pd and Fe–Pt composites is less than that of as-cast pure iron, and the platelets adhered on the specimenswere round, without apparent induced thrombosis.
In summary, iron-based composites reinforced by Pd or Pt are prom- ising biodegradable biomaterials for vascular stentswith good combina- tion of mechanical properties and biocompatibility as well as a proper degradation rate.
53T. Huang et al. / Materials Science and Engineering C 35 (2014) 43–53
Acknowledgments
This work was supported by the National Basic Research Program of China (973 Program) (Grant Nos. 2012CB619102 and 2012CB619100), National Science Fund for Distinguished Young Scholars (Grant No. 51225101), Research Fund for the Doctoral Program of Higher Educa- tion under Grant No. 20100001110011, and National Natural Science Foundation of China (No. 31170909).
References
[1] S. Saito, Catheter. Cardiovasc. Interv. 66 (2005) 595–596. [2] B. Heublein, R. Rohde, V. Kaese, M. Niemeyer, W. Hartung, A. Haverich, Heart 89
(2003) 651–656. [3] C. Di Mario, H. Griffiths, O. Goktekin, N. Peeters, J. Verbist, M. Bosiers, K. Deloose, B.
Heublein, R. Rohde, V. Kasese, J. Interv. Cardiol. 17 (2004) 391–395. [4] P. Peeters,M. Bosiers, J. Verbist, K.Deloose, B.Heublein, J. Endovasc. Ther. 12 (2005) 1–5. [5] M. Bosiers, Cardiovasc. Intervent. Radiol 32 (2009) 424–435. [6] P. Zartner, R. Cesnjevar, H. Singer, M. Weyand, Catheter. Cardiovasc. Interv. 66
(2005) 590–594. [7] R. Waksman, R. Pakala, P.K. Kuchulakanti, R. Baffour, D. Hellinga, R. Seabron, F.O. Tio,
E. Wittchow, S. Hartwig, C. Harder, Catheter. Cardiovasc. Interv. 68 (2006) 607–617. [8] R. Erbel, C. Di Mario, J. Bartunek, J. Bonnier, B. de Bruyne, F.R. Eberli, P. Erne, M.
Haude, B. Heublein, M. Horrigan, Lancet 369 (2007) 1869–1875. [9] X.N. Gu, Y.F. Zheng, Front. Mater. Sci. Chin. 4 (2010) 111–115.
[10] H. Hermawan, D. Dubé, D. Mantovani, J. Biomed. Mater. Res. A 93 (2010) 1–11. [11] B. Liu, Y. Zheng, Acta Biomater. 7 (2011) 1407–1420. [12] J. Lévesque, H. Hermawan, D. Dubé, D. Mantovani, Acta Biomater. 4 (2008) 284–295. [13] M. Moravej, A. Purnama, M. Fiset, J. Couet, D. Mantovani, Acta Biomater. 6 (2010)
1843–1851. [14] P.P. Mueller, S. Arnold, M. Badar, D. Bormann, F.W. Bach, A. Drynda, A. Meyer
Lindenberg, H. Hauser, M. Peuster, J. Biomed. Mater. Res. A 100 (2012) 2881–2889. [15] M. Schinhammer, I. Gerber, A.C. Hänzi, P.J. Uggowitzer, Mater. Sci. Eng. C 33 (2012)
782–789. [16] M. Schinhammer, P. Steiger, F. Moszner, J.F. Löffler, P.J. Uggowitzer, Mater. Sci. Eng. C
33 (2012) 1882–1893. [17] D.-T. Chou, D. Wells, D. Hong, B. Lee, H. Kuhn, P.N. Kumta, Acta Biomater. 9 (2013)
8593–8603. [18] Q. Feng, D. Zhang, C. Xin, X. Liu, W. Lin, W. Zhang, S. Chen, K. Sun, J. Mater. Sci. Mater.
Med. (2013) 1–12. [19] G. Papanikolaou, K. Pantopoulos, Toxicol. Appl. Pharmacol. 202 (2005) 199–211. [20] M. Peuster, C. Hesse, T. Schloo, C. Fink, P. Beerbaum, C. von Schnakenburg, Biomate-
rials 27 (2006) 4955–4962. [21] M.-M. Song, W.-J. Song, H. Bi, J. Wang, W.-L. Wu, J. Sun, M. Yu, Biomaterials 31
(2010) 1509–1517. [22] M. Peuster, P. Wohlsein, M. Brügmann, M. Ehlerding, K. Seidler, C. Fink, H. Brauer, A.
Fischer, G. Hausdorf, Heart 86 (2001) 563–569. [23] R. Waksman, R. Pakala, R. Baffour, R. Seabron, D. Hellinga, F.O. Tio, J. Interv. Cardiol.
21 (2008) 15–20. [24] C. Wu, X. Hu, H. Qiu, Y. Ruan, Y. Tang, A. Wu, Y. Tian, P. Peng, Y. Chu, X. Xu, J. Am.
Coll. Cardiol. 60 (2012).
[25] G. Ndrepepa, S. Braun, A. Dibra, J. Mehilli, W. Vogt, A. Schömig, A. Kastrati, Nutr. Metab. Cardiovasc. Dis. 15 (2005) 418–425.
[26] M. Fiset, D. Mantovani, D. Dubé, H. Hermawan, M. Moravej, Adv. Mater. Res. 15 (2007) 113–118.
[27] B. Liu, Y. Zheng, L. Ruan, Mater. Lett. 65 (2011) 540–543. [28] W.L. Xu, X. Lu, L.L. Tan, K. Yang, Acta Metall. Sin. 47 (2011) 1342–1347. [29] M. Schinhammer, A.C. Hänzi, J.F. Löffler, P.J. Uggowitzer, Acta Biomater. 6 (2010)
1705–1713. [30] B. Liu, Y.F. Zheng, Acta Biomater. 7 (2011) 1407–1420. [31] B. Wegener, B. Sievers, S. Utzschneider, P. Müller, V. Jansson, S. Rößler, B. Nies, G.
Stephani, B. Kieback, P. Quadbeck, Mater. Sci. Eng. B 176 (2011) 1789–1796. [32] M. Moravej, S. Amira, F. Prima, A. Rahem, M. Fiset, D. Mantovani, Mater. Sci. Eng. B
176 (2011) 1812–1822. [33] M. Moravej, F. Prima, M. Fiset, D. Mantovani, Acta Biomater. 6 (2010) 1726–1735. [34] M. Moravej, A. Purnama, M. Fiset, J. Couet, D. Mantovani, Acta Biomater. 6 (2010)
1843–1851. [35] F.L. Nie, Y.F. Zheng, S.C. Wei, C. Hu, G. Yang, Biomed. Mater. 5 (2010) 065015. [36] J. Cheng, Y. Zheng, J. Biomed. Mater. Res. B Appl. Biomater. (2013) 485–497. [37] W. Geurtsen, Crit. Rev. Oral Biol. Med. 13 (2002) 71–84. [38] J. Wataha, C. Hanks, J. Oral Rehabil. 23 (1996) 309–320. [39] W. Agnew, T. Yuen, D. McCreery, L. Bullara, Exp. Neurol. 92 (1986) 162–185. [40] S. Brummer, M. Turner, IEEE Trans. Biomed. Eng. (1977) 440–443. [41] A. Cowley, B. Woodward, Platin. Met. Rev. 55 (2011) 98–107. [42] A. Berghaus, K. Neumann, T. Schrom, Arch. Facial Plast. Surg. 5 (2003)
166–170. [43] P. Vanýsek, Electrochemical series, in: D.R. Lide (Ed.), CRC Handbook of Chemistry
and Physics, CRC Press, Boca Raton, FL, 2005, pp. 20–29. [44] ASTM E. E9-89a, Standard Test Methods of Compression Testing of Metallic Mate-
rials at Room Temperature, Annual Book of ASTM Standards, 2000. [45] ASTM G. G 31–72, Standard Practice for Laboratory Immersion Corrosion Testing of
Metals, ASTM Book of Standards, 2004. [46] E. ISO, German Version: DIN EN ISO, 2008. (10993-10912). [47] E. Iso, Tests for In Vitro Cytotoxicity, 2009. [48] Z. Munir, U. Anselmi-Tamburini, M. Ohyanagi, J. Mater. Sci. 41 (2006) 763–777. [49] H. Okamoto, Phase Diagrams of Binary Iron Alloys, ASM International Materials Park,
OH, 1993. [50] H. Okamoto, J. Phase Equilib. Diffus. 25 (2004) 395-395. [51] J.C. Slater, J. Chem. Phys. 41 (1964) 3199. [52] R. Armstrong, Metall. Mater. Trans. 1 (1970) 1169–1176. [53] C. Ramesh, M. Safiulla, Wear 263 (2007) 629–635. [54] X. Liu, S. Chen, H. Ma, G. Liu, L. Shen, Appl. Surf. Sci. 253 (2006) 814–820. [55] J.C. Wataha, C. Hanks, R.G. Craig, J. Biomed. Mater. Res. 25 (1991) 1133–1149. [56] A. Biesiekierski, J. Wang, M. Abdel-Hady Gepreel, C. Wen, Acta Biomater. 8 (2012)
1661–1669. [57] S. Zhu, N. Huang, L. Xu, Y. Zhang, H. Liu, H. Sun, Y. Leng, Mater. Sci. Eng. C 29 (2009)
1589–1592. [58] P.P. Mueller, T. May, A. Perz, H. Hauser, M. Peuster, Biomaterials 27 (2006)
2193–2200. [59] D.A. Dichek, R. Neville, J. Zwiebel, S. Freeman, M. Leon, W. Anderson, Circulation 80
(1989) 1347–1353. [60] F756-00, A., Standard Practice for Assessment of Hemolytic Properties of Materials,
2000, ASTM International West Conshohocken, PA, USA, 2000. [61] D.A. Armitage, T.L. Parker, D.M. Grant, J. Biomed. Mater. Res. A 66 (2003)
129–137.
1. Introduction
3. Results and discussion
3.3. Corrosion properties of Fe–Pd and Fe–Pt Composites
3.3.1. Electrochemical measurements
3.4. Biocompatibility
3.4.1. Contact angle tests of Fe–Pd and Fe–Pt composites
3.4.2. Cytotoxicity tests of Fe–Pd and Fe–Pt composites
3.4.3. Hemocompatibility tests of Fe–Pd and Fe–Pt composites
4. Conclusions