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    Collds and Sustaces 8: Bointerfces 142 (2016) 20-29 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurtb ELSEVIER In vitro studies on silver implanted pure iron by metal vapor vacuum arc technique Dens Tao Huang*, Yan Cheng”, Yufeng Zheng** Department of Materials cence and Engineering Clee of Engineering Peking Univers, Beijing 100871, China * Centro Biomedical Maer and Tissue gineering Acadet for Advanced nerds Sus, Peking Univers, ling 10871 china ARTICLE INFO ABSTRACT Received 30 September 2015 ‘Accepted 30 January 2016 are ron has been verified as a promising biodegradable metal for absorbable cardiovascular cent usage However the degradation rate of pure iron is too slow. To accelerate the degradation of the surface of pure ton silver ions were implanted into pure iron by metal vapor vacuum are (MEA) source 2t an fxtracted voltage of 40 keV, The implanted influence was up t0 2 10" onslem, The composition and depth profiles corrosion behavior and biocompatibility of Ag ion implanted pre iron were investigated. = ‘The implantation depths of Ag was around 6Dnm. The element Ag existed as AgO in the outermost Peed layer then gradually transited to metal atoms in zero valent state with depth increase. The implantation Silver ion implantation of Agions accelerated the corrosion rate of pure iron matrix, ané exhibited much more uniform corrosion, Corres behavior. For cytotoxicity assessment, the implantation of Ap ions slightly decreased the viability ofall kinds of cell ines used in these tests. The hemolysis rate of Ag ion implanted pure ron was ower than 2% Which was acceptable. whereas the platelet adhesion tests indicated the implantation of Ag ions might increase the risk of thrombosis, (© 2016 Flsevier BY. Allrights reserved. 1. Introduction Currently approved stents are mainly made of metallic bioma- terials inchiding stainless steels, titanium and cobalt-chromiuim alloys [1]. These metallic biomaterials are used as permanent implants with high corrosion resistance, remaining in the vessel for a long time may cause serious complications, such as suba- ‘cute stent thrombosis [2] and in-stent restenosis [2], Two methods have been proposed to tackle these problems: drug eluting stents and biodegradable stents. However, present drug eluting stents are Still facing the problem of late stent thrombosis (4. Meanwhile, biodegradable stents are considered asthe ideal stents due to their potential to leave behind only a healed arterial vessel, preventing the problems of late stent thrombosis, in-stent restenosis and the prolonged antiplatelet therapy (5) Pure iron was considered as a potential candidate material of biodegradable stent, but its degradation rate is too slow [6]. It was ‘commonly believed that the ideal period for stents degradation is 12-24 months [7]. New variance had been introduced into pure iron to accelerate the degradation rate: (1) New kinds of alloys 7 corresponding autor. mal edressyahengep een {¥. Zheng pfx o.ong/102016) cls 2016.01.065 0929-7785}0 2006 seve BY Al including Fe-Mn (3-10), Fe-X (X=Min, Co, Al, W, Sn, B,C, $) [11], Fe-Mn-Si [12], Fe-Mn-C [13]. Fe-Mn-Pd [14] and Fe-Mn-C-{Pé) [15-17] were developed, and the results revealed that the acceler- ated effects were limited. (2) Pure iron powders were composited with W [15], CNT( 8], FeO [19], (20), Pe (20), HA. TCP and BCP [21] powders respectively, indicated faster degradation than that ‘of pure iron, yet they are difficult to be fabricated into rini-tube, which is the raw material for stent before laser cutting. (3) Some new preparation technologies such as electroforming 22] and 3D printing |23), showed enhanced mechanical strength, but are not applicable for stent. (4) For surface modification, the data shown in literatures (24-26) indicated that the degradation of pure iron, ‘were slowed down after implanting with 0, Nand La ‘Aghas good biocompatibility [2-25], as well as good antibacte- rial ability 30-32], Furchermore, the standard electrode potential of Ag (+0.7996V) is much higher than that of Fe (—0.44V). Since thereisno solubility of silver iniron 33}, silverions were implanted {nto pure iron in this work. [solated Ag elementary substance was expected to exist in the pure iron after implantation, then act as independent cathodes to accelerate the corrosion of pute iron, ‘matrix (as anodes), 1 Muang ea /Clllds and sujaces lief 142 (2018) 20-29, a 2, Materials and methods 2.1. Materials preperation Pure iron (purity, 99.9%) with size of 10x 10 x 0.5mm? were. mechanically polished up to 2000 grit and ultrasonically cleaned in anhydrous ethanol, then dried in the open air. Ag ion implan- tation was carried out with a metal vapor vacuum arc ion source (MEWA, Beijing Normal University, Beijing, China). The extracted voltage of silver ions was 45 KV, the vacuum level of the chamber ‘was 2 x 10-? Pa the implanted fluence was 2 « 10%? ions/cm. Dur~ ing implantation, the beam current density was 2 mAfem, and the ‘maximum temperature was lower than 200°C. 22. Surface characterization The chemical composition and distribution were analyzed by environment scanning electronic microscopy (ESEM, Quanta 200FEC), equipped with an energy dispersive spectrometer (EDS) attachment. AES (PHI-700 Auger Electron Spectroscopy, ULVAC- PHI, Japan) was adopted with the sputtering rate of SiOz at 31 nm/min. X-ray photoelectron spectroscopy (XPS, (Axis Ultra, KRATOS ANALYTICAL, Britain)) with Al Ka radiation was utilized {to measure the surface chemical composition and the elements valence. High resolution narrow scanning was conducted to deter~ ‘mine the binding state of Ag 34 23, Electrochemical measurements Electrochemical measurements were performed with a tradi tional three-electrode cell using an electrochemical work station (PGSTAT 302 N, Metrohm Autolab). The specimen, a saturated calomel electrode (SCE) and a platinum electrode were acted as the ‘working electrode, reference electrode and the auxiliary electrode, respectively. All the measurements were maintained at a tempera ‘ure of 37 £.0.5°C in Hank's solution |34] witha pH value of 7.4, The area of working electrode exposed to the solution was 0.3318 cm? The open circuit potential (OCP) measurement was set for 9000. Electrochemical impedance spectroscopy (EIS) was measured from 300Kdt2 to 10mHz at OCP value. The potentiodynamic polariza- ‘ion curves were carried out from (OCP value ~600) mV (vs, SCE) to (OCP value +600) mV (vs. SCE) at a scanning rate of0.33 mVs~".An average of atleast three measurements was Laken for each group, 2. Static immersion test In vitro static immersion test was performed in Hank's solu- ton for 3, 15 and 30 days, 50 mL Hank's solution was used for each sample following ASIM-G31-72 [35] at 37°C in water bath. After 3, 15 and 30 days respectively, the samples were removed from ‘he soaking solution, gently rinsed with distilled water and quickly dried in case of oxidation. Changes on the surface morphologies after immersion were characterized by ESEM (Quanta 200FEG), ‘equipped with an EDS attachment. The corrosion products on the surface of samples dissolved in the 10 mol/L NaOH solution before they were weighed. An average of three measurements was taken, for each group. The degradation rates were calculated based on the formula cae where CR (mgcm-2 day-1)is the corrosion rate, m (mg) is the mass lass, § (cm?) is the surface area of the specimen exposed to the solution and ¢ (day) is the immersion time. 25. Cytotoxicity test The cytotoxicity test was performed by an indirect contact ‘method. Mutine fibroblast cells (1-928), human vascular smooth ‘muscle cells (VSMC) and human umbilical vein endothelial ces, (EA. hy-926) were used to evaluate the cytotoxicity of the exper= imental Ag ion implanted pure iron. At frst, all the cell lines ‘were cultured in the Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100UmL-* penicillin and 100 xg mi" sterptomycin at 37°C in a humidified atmosphere of 5% COp. According to ISO 10993-12 [35], extraction medium was prepated using serum-free DMEM with a sutface areajextraction ‘mediim ratio of 1.25 em? mi=" ina humidified atmosphere withS% (COp at 37 °Cfor 72h, After the extracts were centrifuged, the super= natant 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 control. The concentrations of metallic ions in the extraction medium were measured by inductively coupled plasma atomic emission spectrometiy (ICP-AES) (Leeman, Profile). Cells were incubated in the 96-well plates at the density of approxi- ‘mately 5 x 10° cells per 100 j:L medium in each well and incubated {fot 24) to allow attachment, DMEM was then replaced by extrac- tion mediums. Then 10) serum was added to each well. Aver 1, 2 and 4 days incubation in the incubator respectively, the 95- well plates were observed under an optical microscope. Thereafter, 10 of cel counting kit (CCK-8) solution was added to each wel. The cells were incubated with CCK-8 for 3h, Then the absorbance of each well was tested by using microplate reader (Bio-RADS80) at the wavelength of 450.1m, Viability of cells (X) was calculated Using the following formula according to ISO 19003-5) 7] ops X= SpE x 100% Here OD is the mean absorbance of experimental sample groups and positive control group. OD» is the mean absorbance of negative control group. 26, Hemolysis test and platelet adhesion Healthy human blood (anticoagulant was 3.8wt citric acid sodium) extracted from volunteers was diluted by physiological saline according (0 volume ratio of 4:5. Untreated Pure iron and ‘Ag ion implanted pure iron were separately installed in centrifugal tubes with 10m physiological saline for 30min, emperature was kept at37°C. Then 0.2 mL diluted blood was added to each tube and incubated at 37°C for 6Omin, 10ml. deionized water with 0.2 mL diluted blood asthe positive control and 10 ml. physiological saline with 0.2 ml diluted blood as the negative control After completion of the above operations, samples were removed, and then these tubes were centrifuged at 800g for Smin. Supernatant was trans ferred to 96-well multiplates, the absorbance (OD) was determined by a microplate reader (Bio-RADSEO) at the wavelength of 545 nm, Hemolysis of samples was calculated by the formula: 00 (test) — OD (negative contra!) ‘DD positive control} — OD negative contraT) * Hemolysis 100% For platelet adhesion, whole blood from healthy human body was centrifuged at 1000./min for 10min. Platelet rich plasma (PRP) was obtained from the upper fluid. Samples after ultraviolet dis infection were moved to 24-well multiplates and 0.2m. PRP was added to each well, then incubated at 37°C for 1h, After gently rinsed by phosphate buffered saline (PBS), platelets on samples were fixed with 2.5% glutaraldehyde solution at room temper ature for 1h. Then dehydrated with gradient alcohol solution (50x, 60%, 70%, 80%, 90x, 95% and 100%), each concentration for 2 Tange a, /Callelds nd sufaces B: ointefces 1422018) 20-29, © “= (A) mf tery Biotag Hoey (@V) Fi. (EDS analysis of Agionimplace pure iron surface of Aion implanted pure ion ane) high-resolution XPS spect f Ag 36 10min, and finaly freeze-dried for 2 days. The morphologies of platelet adhered on the specimens were observed by ESEM (Quanta 200FEG). 3, Results 3.1. The composition and depth profiles of Ag ion implanted pure Fig. 1(a} shows the EDS analysis of Ag ion implanted pure iron. ‘Ag elements distributed uniformly on the surface of pure iron (as revealed by the element mapping image) and the content was round 1.5 wi.% at the surface. Fig. 1{b) shows the AES elements ‘depth profile, which reveals that the depth of A ions implantation layer was about 601m, and the highest content of Ag was 5at.% ‘On the surface, the content of oxygen was very high (over 60 ax), but it decreased drastically to only 5 at at the depth of 7nm. At the same time, the concentration of Ag ions increased. Fg. | (c) is the XPS whole range of the binding energy survey of the surface ‘of Ag ion implanted pure iron, There existed Fe, Ag, O and C ele- ments. As can be seen from high-resolution XPS spectra shown in ig. 1(@), the binding energy of Ag 3dsj» was 367 9eV and Ag 3432 ‘was 373.4 eV, which represented Ag)0 [38,39], Combining to the results of AES, Ag existed as Ag20 from the surface tothe depth of 7nm, thereafter Ag would exist as elementary substance, BindiogEaeray @V) 1 ements pth profes of Azions implanted pre ion.) the whole range of the binding energy survey ofthe 32, Corrosion properties of Ag on implanted pure iron 22.1. Electrochemical corrosion behavior Fig. 2 shows electrochemical test results of Ag ion implanted pure iron. Fig. 2(a) is the Tafel curves of Ag ion implanted pure ion, with untreated pure iron as control, The corresponding elec~ ‘tochemical parameters are listed in Table 1. It was found that the corrosion potential were slightly decreased and the corrosion cur- rent density almost doubled after the implantation of Ag ions into the surface of pure iron. Fig. 2(b) is the Nyquist plots of untreated pure iron and! Ag ion implanted pure iron. An equivalent circuit ‘mode! [Re(QuRe)] (inset, Fig. 2{b)) forall the experimental speci ‘mens was employed to fit the EIS data. The parameters R, and Ry represented electrolyte resistance and the transfer resistance, Qy ‘was a constant phase element (CPE), which was deemed asthe non= {deal capacitive behavior ofthe electric double layers, respectively. In the Nyquist plots (Fig. (b)), the diameters of the semicircles ‘ean be considered as the charge transfer resistance and the smaller transfer resistance means the faster corrosion rate [40]. There- fore, the corrosion rate of Ag ion implanted pure iron was faster than that of untreated pure iron. Fig. 2(¢) and (d) show the sur- face morphologies of specimens after electrochemical tess. The surface of untreated pure iron almost kept intact, except for a few big localized corrosion pits (Fg. 2(c)). By contrast, the corrosion on, the surface of Ag ion implanted pure iron was more uniform than that of untreated pure iron (Fig. 2(d)), Corrosion pits on the surface Muang ea /Clllds and sujaces 8: itterfces 142 (2018) 20-29, 2 (a) * (by i aN 400 & pire Fe +0. Potential vs SCE V) Fig. 2, Hecrochemvcl test results untreated pure ren and Ag implanted put rn in Hank’ sluin: a ptentledynamic polarization cure, (b) Nyquist pls.) and (are sutace morphology of unceated pure iron and Azion implanted pure ion afer eletrec emia ss, respecvly. 0.08: pure Fe |Ag-implanted 0.08: 0.04 0.03. 0.02. 001 - Corrosion rate (mg/em?-d) 0.00. 15 30 Immersion time (day) Fig. 3. Covtosion cates aeulated fom the weight ss of samples afer state mimes is Hank's sltion £63, 15 abd 30 days Muang eta /Clllds and Surfaces: olnerfces 142 (2018) 20-29, 3d 15d 30d Fig. SENG mags of samples setae morphology ater slaimmerson in Han alton fr 5,15 2nd 30a. table Average electrochemical parameters of Ag ion implanted pure tn (a-cast and assintere pure ton as conte ote: Corosion potential Ew), coroson caren ens (lw) and coronene Van) of Ag ion implanted pure iron were much smaller and distributed ‘continuously. 2.2.2. Static immersion corrosion behavior of Ag ion implanted pure iron shows the corrosion rates calculated from the weight loss after samples immersed in Hank’s solution for 3, 15 and 30 days, respectively. As can be seen from this figure, the Ag ion ‘implanted pure iron corroded faster than untreated pure iron. After 3 days immersion, the difference of corrosion rates between Ag ion ‘implanted pure iron and untreated pure iron was the largest. The ‘cotrasion rate of Ag ion implanted pure iron was 2.5 times as that ‘of untreated pure iron, But the gap narrowed with time. shows the surface morphologies of specimens after 3. 15, and 30 days immersion in Hank’s solution. AS can be seen from the Figure: (i) After 3 days immersion, the surface of untreated pure iron kept intact and only several salts deposited there, with no etch pit was found, On the contrary, small and shallow corro- sion pits distributed uniformly on the whole area of the surface of Ag ion implanted pure iron. (i) After 15 days immersion, obvi- fous corrosion can be found on the surface of untreated pure iron, ‘The corrosion seemed uniform except for several isolated deep cor rosion pits. As to the Ag ion implanted pure iron, comparing to 3 days results, corrosion pits on the surface of Ag ion implanted pure iron grew bigger with time, many of them combined to each other. (il) Afer 30 days immersion, grain boundary on the Muang eta /Clllds and sujaces 8 itterfces 142 (2018) 20-29, 2s wo 0 @ re) eae 1-929 ae BBR Positive is te % se di ° E ow: & oo. iz i jon 5 ™ ose. Ag ” = ‘pure Fe ‘Ag-implanted ° 1 2 4 incite ©] ee = EALhy-926 ©) oo] fmm nesrrunce VSMC es cy =" ? iC i 4 i = wo. = : i * 1 2 4 ‘Time in caltare ay) 1 2 4 ‘Tee in culture (day) Fig 5. lon concentration in experimental material extraction mediums 2) and cell wally after cultured in extraction mediums and positive control fr 1.2 ané 4 days: E529 bi. EA Hy and VSMC surface of untreated pure iron could be clearly observed. Corrosion in the interior of grain was relatively uniform, while there were some deep corrosion pits distributed along the grain boundaty. In terms of the Ag ion implanted pure iron, the corrosion pits on the surface continued growing bigger. As part of these corrosion pits ‘combined into bigger ones which corroded faster than the isolated ‘small pits, and the corrosion on the surface of Ag ion implanted pure iron started to transfer to asymmetrical as a whol. 3.2. Cytotoxicity of ion implanted pure iron Fig, 5 illustrates the ion concentrations of extractions (a) and the cell vibilties of (b) murine fibroblast cells L-928, (c) human ‘umbilical vein endothelial cells EA. hy-926 and (d) human VSMC expressed as a percentage of the viability of cells cultured in the negative control after 1,2 and 4 days incubation in experimental materials extraction mediums. There was no significant differ- cence between the iron ion concentration in extraction mediums of ‘untreated pure iton and Ag ion implanted pute iron. But the latter released a small amount of Ag ions into the extraction mediums. AS ‘ean be seen form Fig 5(b),L-929 cell viabilities of Ag ion implanted pure iron extractions were similar to that of untreated pure iron, ‘maintaining around 90% through the whole test petiod. As to EA, hhy-026 cells, the viabilites of EA. hy-826 cells remained over 80%in, untreated pure iron extractions and stayed in the range from 80% {to 85% in Ag ion implanted pure iron extractions. The viabilites fof VSMC in all extractions decreased with time, and after 4 days incubation, the VSMC cells viabilities of all the experimental com- posites decreased to lower than 70%. 3.4, Hemocompauibility of Ag ion implanted pure iron 3.4.1. Hemolysis ‘ASTM F756-08 required the hemolysis rate of medical devices which contact with blood must lower than 5% [41 Fig. 6(a) shows the hemolysis rate of Ag ion implanted pure iron. Although the hemolysis rate of Ag ion implanted pure iron was slightly higher than that of untreated pure iron, it still ower than 2%, revealing its good hemocompatibility 342, Platelet adhension ‘When implants contact blood, platelets will gradually adhere and gather on the surface of implants. Once the numberof platelets lover a certain value, coagulation will happen and thrombus appear [112), Based on Fis, 5(b), the number of platelets adhered on the sur- face of Ag ion implanted pure iron was more than two times as that onthe surface of untreated pureiton. fig. {e) and (d) show the mor= photogies of adhered platelets on the surface of untreated pure iron and Ag ion implanted pure iron, respectively. It can be seen from. these figures, the number of platelets adhered on the untreated. pure iron was lower than that on Ag ion implanted pure iron. In addition, most of the platelets adhered on the Ag ion implanted pure iron were activated and their pseudopods being stretched ‘The activation of platelets is the advance signal of thrombosis [43] ‘Therefore it should be cautioned during future application, Muang eta /Calllds and surfaces olaerfces 142 (2018) 20-29, => = = ere (b) ae Agimplated tan asnirica] “Pom iz mots percentage (6) Fi. (0) Hemeyss rats of Agion implanted pure to, the ube platelets adhered on the surface of usteated pute on and Asn planted spectively corrosion products: _ SOO ~ yay you? oxided layer Ag particle iron matrix een (a) Fig 7 iutration ofthe carason mechanism fr Ag implanted pre ian 1 Muang ea /Clllds and sujaces lief 142 (2018) 20-29, » 4, Discussion 4.1, Effect of Ag ion implantation on the corrosion behavior of pure iron Based on the results of electrochemical tests and static immer= sion tests, implantation of Ag ions exhibited an acceleration effect fn corrosion of pure iron, However, the implantation depth of ‘Ag ions was very shallow (about 60mm). Hence, the acceleration ceffect of Ag ions on corrosion was limited on the surface of pure iron. At the frst 3 days immersion, the acceleration effect of Ag ions on corrosion was most obvious. The corrosion rate of Ag ion ‘implanted pure iron was about 2,5 times as that of untreated pure iron. With time, the corrosion depth increased and the implan- tation layer fell off from the surface of pure iron along with corrosion products, then the acceleration effect of Ag ions on the corrosion of pure iron decreased. Therefore, the gap between the corrosion rate of Ag ion implanted pure iron and untreated one narrowed. The corrosion mechanism of Ag ion implanted pure iron was assumed and illustrated in Fig. 7, There is no solubility of silver in iron [33]. After implantation, the energy of Ag ions was very high. Moreover, the specific surface area of an isolated Ag atom. ‘was very large. so the surface energy of Ag atoms was significantly high. To decrease systemic energy. the implanted Ag ions were ‘end to get together, from atomic clusters to Ag particles in nano scale. Therefore, the Ag ion implanted pure iron can be ilustrated as Fig, (a). The outmost layer had high concentration of oxygen, it fell off rapidly after immersed into corrosion solution, Then Ag particles exposed to the corrosion solution. Due to the standard electrode potential of Ag (+0.7996 V) is much higher than that of Fe (~0.44V),Ag pattices (as cathodes} and iron matrix (as anodes) could form galvanic cells. Iron matrix would be oxidized into iron ions, showing in Eq, (1) Electrons generated from the dissolving fron matrix would transfer to the Ag particles (Fig. 7(b)).Then, they ‘would be consumed by dissolved oxygen, seeing Ba. (2). Fe — Fe +2e~(anode reaction) a 0 +210 + 4e~-+ 40H-(cathode reaction) @ Due to the solution alkalization near the Ag particles (Eq. (2), ron. hydroxide formed around them preferentially according to Eq, (2) Since the instability of ferrous hydroxide. it was easly oxidized to ferric hydroxide by dissolved oxygen (Fig. 7()), the reaction as Eq, (4) shows: Fe? 4. 20H" > Fe(OH), 8) AFe(OH); +0;42H,0 + 4Fe(OH)s a With corrosion continuing, the iron matrix surrounding Ag parti- cles was corroded. Ag particles fel off. meanwhile, the corrosion pits being eft and grew bigger with time, finally combined to each ‘other(Fig.7(d)). With the inerease of corrosion depth, new Ag parti cles exposed and the galvanic corrosion process repeated (Fig. (e)) The adhered iron oxides layer was found to be the main reason for the slow degradation of iron (44), After the iron matrix sur- rounding Ag particles corroded, Ag particles would separate from the matrix. Then the existence of Ag particles can break the contin ‘uous and integrity ofthe iron oxides layer, which can be considered as another reason for the accelerating effect of Ag particles on the corrosion rate of pure iron matrix. 42, Biocompatibilty concerning of Ag ion implanted pure iron Itis very difficult to oxidize Ag into ions in DMEM. Furthermore, from the discussion of corrosion mechanism of Ag ion implanted pure iron, Ag nanoparticles were moze likely released into the extractions rather than Ag” Therefore, Ag nanoparticles and iron fons should be taken into consideration to discuss the cytotoxi- city of Ag ion implanted pure iron. The good biocompatibility of pure iron has already been verified through many in vitro and in vivo tests (6,45~48], with an [C50 value of 303 wg mL~! for murine fbroblasts|49|. Iron ion concentration in untreated pure iron and ‘Agion implanted pure iron were 8.214 gmt! and 8.212 4g mL~! respectively, which were much lower than [C50. Hence, the viabil- ity of 1929 cells kept around 90% in both pure iron extraction and Ag {on implanted pure iron extraction. On the other hand, as reported by Zhu et al [50], there was almost no effect on the metabolic activity of endothelial cells when the concentration of iron ton was Tower than 50 gmL~!. Therefore, the viablities of EA.hy-926 in the extraction medium of untreated pure iron temained over 80% The viabilites of EA\hy-926 in the extraction medium of Ag ion implanted pure iron were similar to that of untreated pure iron However, the implantation of Ag ions slightly decreased the viabil- ity ofall inds of cell lines that used in these tests. Ag nanoparticles Inextractions should be responsible for these results. Ag nanoparti- cle isa widely used broad-spectrum antibacterial material |5|-53) Researches found that Ag nanoparticles can also exert toxicity on cells when using them to resistant bacteria, Shi etal (54 reported the toxicity of Ag nanoparticles on human umbilical vein endothe lial cells (HUVEC) and human umbilical atery smooth muscle cells (HUASMC). As results shown, the cytotoxicity on the HUVEC was larger than HUASMC when the concentration of Ag nanoparticles was bigh (0.125 mgml.~! and 025 mg mL), while cytotoxicity on. the HUVEC was smaller than HUASMC when the concentration of ‘Ag nanoparticles was low (<0.0525 mg mL") In this work, no sig- nificant difference between the cytotoxicity on the EA hy-826 cells, and VSMC cells was found. However, the viabilities of VSMC cells decreased obviously with ‘ime in both extraction of untreated pure iron andl Agion implanted fone. Muller etal [55] found that ferrous irons could exert adverse effect on the proliferation of VSMC cells, Aecording to the report of Schaffer et al, [55], Fe" and Fe" ions could repress the migration ‘of smooth muscle cells at the concentration of 1 mM. In the first several days, the concentration of released Ag par ticles would be relatively high, it might be dificult to endothelial cellsto adhere on the surface of Ag ion implanted pure iron because of the toxicity o Ag particles on cells. With time, the concentration of released Ag particles decreased gradually. then endothelial cells can begin to adhere on the surface of Ag ion implanted pure iron and finally complete endothelialization, Besides, in the long run, insoluble degradation products will, be produced and separate from pure iron, which may also exert toxicity on blood cells [57] 5. Conclusions Toimprove the corrosion behavior and biocompatibility of pure iron, Ag ions were implanted into the surface of pure ron through, MEVVA ion source. Its chemical composition, depth profil, corro- sion behavior and biocompatibility were investigated systemically. The implantation depth of Ag ions was about 6Onm. On the outer ‘implantation layer, Ag existed as Ags. With the increase of depth, ‘Ag gradually transfer from Agp0 to Ag elementary substance, Com- paring to untreated pure iron, Ag ion implanted pure icon exhibited faster and much more uniform corrosion, From the perspective of, biocompatibility, the implantation of Ag ions slightly decreased the viabilties of all the three experimental cells, including 1-929, EA, hy-926 cells and VSMC cells. The hemolysis percentage of Ag jon implanted pure iron was lower than 2%, revealed its good hhemocompatibiity. However, the results of platelets adhesion tests 2 Muang eta /Calllds and Surfaces: olaerfces 142 (2018) 20-29, ‘demonstrated that the implantation of Ag ions might increase the risk of thrombosis, and should be cautioned during future usage. ‘Acknowledgements This work was supported by the National Basic Research Pro- gram of China (973 Program) (Grant No. 2012CB619102 and 2012CB619100), National Science Fund for Distinguished Young ‘Scholars (Grant No. 51225101), National Natural Science Founda- tion of China (Grant No, 51431002 and 21170906), the NSFC/RGC Joint Research Scheme under Grant No. 51361165101, State Key Laboratory for Mechanical Behavior of Materials (Grant No. 20141615), Beijing Municipal Science and Technology Project (Z141100002814008). References (AC were ana ae Moan ecrtomed ons Sonar dept ts epoch nec [2] M.Movahed,) Vu. Ahsan, Simultaneous subacute stent thrombosis of two corte ne ser deena she cme bt tA senses ethene aa [4] AT. 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