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Characterization of silicon- substituted nano hydroxyapatite coating on magnesium alloy for biomaterial application
Materials Chemistry and Physics 203 (2018) 27e33
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Characterization of silicon- substituted nano hydroxyapatite coating on magnesium alloy for biomaterial application Changiz Dehghanian a, *, Neda Aboudzadeh a, Mohammad Ali Shokrgozar b a b
University of Tehran, College of Engineering, School of Metallurgy and Materials Engineering, Tehran, Iran National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
h i g h l i g h t s Uniform and dense coatings of Si-HA were deposited on Mg alloy by reverse pulse electrodeposition process. Coatings showed blade-shaped morphology and the blade width decreased as Si contents of coatings were increased. Increase in the amounts of Si in electrolyte decreased the coatings thickness. Addition of Si into HA coating increased its corrosion resistance up to 7 times. The cell experiment indicated that there was no toxicity for Si-HA coatings.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2017 Received in revised form 25 July 2017 Accepted 13 August 2017 Available online 22 September 2017
In this study, effects of substitution of Si in structure of HA (Si-HA) as coating on Mg alloy were investigated. The reverse pulse electrodeposition process with frequency of 10 and duty cycle of 0.1 was selected for coating. Four different electrolytes with different molars of SiO2 3 (0, 0.0025, 0.005 and 0.0075) were prepared in order to deposit Si-HA coatings on Mg alloy. The XRD and FTIR results showed that the main phase of deposited coatings was HA, and elemental analysis of coating confirmed the presence of Si in coatings. FE-SEM images of coatings indicated the presence of SiO2 3 in electrolyte caused a decrease in coating thickness and an increase in its compactness. The coating prepared by the electrolyte containing 0.005 mol/L SiO2 3 had higher corrosion resistance than that of HA coating as indicated by electrochemical tests. The cell experiment tests also showed that this high corrosion resistance coating had the nearest cell proliferation toward negative control. © 2017 Published by Elsevier B.V.
Keywords: Coating Si HA Biomaterials Pulse reverse electrodeposition
1. Introduction A new approach for repairing damaged bone tissue is done by tissue engineering. In tissue engineering, implant materials must be biocompatible and biodegradable which is gradually dissolved in human body during repairing bone tissue. In recent years, magnesium and its alloys with biocompatibility and biodegradability properties have been of interest in tissue engineering discipline and some researchers have focused on them to adjust their property with requirements in bone implantation [1,2]. Magnesium‘s primary concern is high degradation speed in human body fluid [3]. In response to this concern, different methods have been considered which among them, coating process may be a
* Corresponding author. E-mail address: [email protected] (C. Dehghanian). https://doi.org/10.1016/j.matchemphys.2017.08.020 0254-0584/© 2017 Published by Elsevier B.V.
relatively significant one. In fact, coatings prevent direct contact of Mg with corrosive environment and reduce its corrosion rate [4,5]. A coating material should be biodegradable and osteoconductive to be usable in bone tissue engineering. The calcium phosphate (CaP) family in particular hydroxyapatite (HA) are of special interest for Mg's coating [6]. HA (Ca10 (PO4)6(OH)2) has a hexagonal crystallographic lattice and a space group of P63/m together with a Ca:P ratio of 1.67 [7]. The biologic hydroxyapatite e the mineral phase of bone-mostly has various substituted ions such as Na, Mg, Zn, Sr and Si in its structure which is enhanced its bioactive behaviors [8e10]. Recently, many researchers have been interested in characterization of effects of these ions substitution in HA structure. It was shown that substitution of phosphate with silicate into HA structure enhances its osteoblast cell activity, attachment, and growth [11]. Moreover, silicon as one of the most important elements is known to be essential in the early stages of bone mineralization and
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2. Experimental procedures
2.8 mm2 was irradiated on the coatings samples placed in the vacuum chamber at 106 torr. The coating samples were placed at the angle of 90 with respect to the incident beam. The beam current was adjusted at about 3 nA in order to keep the counting rate below 1000 cps (count per second). Surface morphology, thickness and elemental analyses of coated samples were specified by field emission scanning electron microscope (FE-SEM: TSCAN-VEGA, China) and energy dispersive Xray spectroscopy (EDS: TSCAN-VEGA, China).
2.1. Preparation of substrate
2.4. Corrosion measurements
The Mg -5Zn- 0.3Ca (%wt.) alloy substrates were synthesized by powder metallurgy. Briefly, metal mixture from Mg (S5430817 002, Merck), Zn (K39617189 929, Merck) and Ca (S5101853 907, Merck) powders were prepared and mixed in ball mill for 6 min, then 0.8 g from metal mixture were pressed in shape of cylinder with the size of 10 mm in diameter and 5 mm in thickness and sintered at 550 C for 2 h in Argon atmosphere. After that, the surface of substrate were grinded with abrasive paper of 600e2000 grits, polished and degreased with acetone to be prepared for coating.
Corrosion properties of nanocomposites were evaluated by anodic potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) tests. The electrochemical tests were carried out in simulated body fluid (SBF) which was prepared according to Kokubo's protocol [15]. Electrochemical corrosion tests were carried out by using a conventional three-electrode cell with a sample as working electrode, saturated calomel electrode (SCE) as reference and platinum mesh as the counter.
soft tissue development [12,13]. In this study, the effects of Si substituted in HA structure as a coating on Mg alloy were investigated. Si-HA coatings with different percentages of Si were deposited on Mg-5Zn-0.3Ca alloy by pulse reverse current (PRC) process and the surface characteristics, corrosion and biocompatibility properties of these types of coatings were compared with the HA coating.
2.2. Electrochemical deposition Four different compositions of electrolytes were prepared according to Table 1. Ca (NO3)2$4H2O, NH4H2PO4, and Na2SiO3$9H2O salts were added as sources of Ca, P and Si in electrolyte, respectively, and NaNO3 (O.1 M/L) was added as conductive agent. The 2 molar ratio of Ca2þ/(PO3 4 þ SiO3 ) was constant at 1.67 for all 3 electrolytes compositions but the molar ratio of SiO2 3 /(PO4 þ 2 SiO3 ) was variable at 0, 0.1, 0.2 and 0.3 for each of electrolytes. The pH of electrolytes were adjusted to 5 by addition of dilute nitric acid HNO3 and ester (CH2OH)3CNH2. The pulse electrodeposition process was carried out by the electrochemical workstation (RST5200) with a two electrode cell [8,14]. Mg -5Zn- 0.3Ca alloy served as cathode and a graphite plate as an anode. The positive and reverse current densities were adjusted to 40 and -10 mA/cm2. The positive, delay and reverse plating time were adjusted to be 10, 90 and 5 ms, respectively for the duration of 30 min. Temperature and concentration of electrolyte were unified at 85 C by a water bath and magnetic stirrer. The coated samples in different electrolytes with different concentrations of SiO2 3 were classified as HA, HS1, HS2 and HS3. 2.3. Microstructural characterization Phase identification of coatings were identified by X-ray diffraction technique (XRD: JDX-8030, Jeol, Japan) with a Cu-Ka source and spectroscopic analysis of coatings was indicated with the fourier transform infrared spectroscopy (FTIR: 8400S, SHIMADZU, Japan). The elemental analysis of coatings were done by the method of proton induced X-ray emission (PIXE). The 3.0 MeV van-de-Graff electrostatic accelerator (High Voltage Company, USA) was used for PIXE analysis. A 2.0 MeV proton beam with the spite size of
2.4.1. Anodic potentiodynamic polarization test Anodic potentiodynamic polarization for coated samples were conducted from 200 to þ500 mV vs. SCE with a scan rate of 1 mV/ s after stabilizing open-circuit potential (OCP). The corrosion current densities and anodic and cathodic Tafel slopes were obtained from the polarization curves. The polarization resistance (Rp) of samples were also determined. 2.4.2. Electrochemical impedance spectroscopy test Electrochemical impedance spectroscopy (EIS) was carried out using an AC impedance analyzer (solartron, 1260) after reaching a stable OCP. The frequency was varied from 101e105 Hz. The software (ZSim version 3.4) was used to analyze the obtained EIS data and the best-fitted equivalent circuit model was found. 2.5. Cell experiment 2.5.1. MTT assay Indirect contact method was selected to investigate the toxicity of nanocomposites, so in compliance with ISO 10993-12, coated samples were sterilized by autoclave and each sample was separately incubated in Roswell Park Memorial Institute (RPMI/1640) media containing 10% fetal bovine serum (FBS; Gibco) for 36 h. After that the supernatant medium was withdrawn and collected. In the following, the MG63 cells were cultured for 24 h in cell culture plates and then their medium were replaced with extracts and incubated for the certain period of 1, 3 and 5 days. The cultured MG63 wells with no extracts were used as negative controls. Accuracy of results were ascertained by repetition of each test for 5 times. After different culture periods, the medium of each well was removed and solution of dimethylthiazol diphenyl tetrazolium bromide (MTT) (Sigma, USA) was added and incubated for 5 h, afterward MTT solution was detached and isopropanol (Sigma, Milwaukee, USA) was enhanced and incubated again for 10 min.
Table 1 The four different electrolytes compound for coatings. Abbreviation
3 2 SiO2 3 /(PO4 þSiO3 ) molar ratio
Na2SiO3$9H2O mol/L
NH4H2PO4 mol/L
Ca(No3)2.4H2O mol/L
NaNo3 mol/L
HA HS1 HS2 HS3
0 0.1 0.2 0.3
0 0.0025 0.005 0.0075
0.0251 0.0226 0.02 0.0176
0.042 0.042 0.042 0.042
0.1 0.1 0.1 0.1
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Absorbance measurements of wells as the indicator of cell growth were carried out by a multiwall microplate reader (ICN, Switzerland) at 570 nm [16]. The cell viabilities were determined by: Cell Viability (%) ¼ (OD
sample/OD negative control)
100%
The significant differences of cell viability in MTT assay were analyzed by the Analysis of Variance (ANOVA) technique in SPSS software (ver. 20.0) when the statistical significance value (r) was defined as 0.05. 2.5.2. Adhesion assay Direct cell adhesive assay was done for coated samples of HA, HS1, HS2, HS3. Initially, 1 mL of MG63 cell suspension with concentration of 3 104 cells ml1 was seeded in each sterilized sample and then incubated for 7 h. Afterward, the samples were soaked with PBS for three times and then fixed in 2.5% glutaraldehyde solution for 2 h at room temperature [17]. Finally, the adhesion of cells on surface of samples was observed by using SEM. 3. Results and discussion 3.1. Metallurgical characterization XRD pattern of Mg alloy with and without HS2 coating were shown in Fig. 1(a). Characteristic diffraction of HA were revealed in XRD pattern of HS2 in Fig. 1(a) which is suggested that HA is the main crystalline phase of the coating. The substrate peaks are strongly visible in XRD pattern of HS2 in Fig. 1(a) due to porosity and thinning of coating. According to HA standard diffraction pattern and stronger intensity of (002) diffraction, HA-Si crystals are preferentially oriented with [001] direction. The XRD patterns of HA, HS1, HS2 and HS3 coatings were shown in Fig. 1(a,b). Diffraction pattern of HA was specified in all coatings but since the amount of Si in coatings is low, no significant difference was distinguished between these four samples. As last studies
Fig. 1. The XRD patterns of (a) coated sample and substrate, (b) coating samples with different levels of Si.
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suggested the silicate tetrahedral were substituting for the phosphate tetrahedral in the HA structure [6,18]. Substitution of silicate in HA may change the lattice due to differences in the ionic radius of silicon compared to phosphorous, but the nearness of silicon and phosphorous in the periodic table makes these differences hard to be considered. Therefore, small amount of Si don't change the XRD patent of HA [19]. The FTIR spectrum of the coatings were shown in Fig. 2. Fig. 2 shows the main absorption bands of the ionic group of HA [5,6]; the bands at 1000e1200, 964 and 560 cm1 attributed to asymmetric stretching, symmetric stretching and asymmetric bending vibrations of PO3 groups, respectively; the bands at 4 3565 cm1corresponded to the stretching modes of the OH groups; and finally the bands at 1400e1500 and 875 cm1connected to asymmetric stretching and bending vibration of CO2 3 groups. Comparing the FTIR spectrum of coatings show that incorporation of silicon led to some changes in the FTIR spectra of 1 HA, PO3 seem to be merge and PO3 4 bands at 1020 and 1103 cm 4 1 bands at 964 and 564 cm are decreased. The OH absorption bands at 3565 cm1 were also decreased slightly with increasing silicon incorporation. So, the PO3 4 in the HA structure may be substituted with SiO44 in Si-HA coatings. The same results was reported by Li et al. [6]. Fig. 3 shows the PIXE results for HA, HS1, HS2 and HS3 coatings. The PIXE spectra for HA coatings indicated traces of Mg, P, Ca, Zn and Cl, Mn, Cu, Pb elements, which the last four elements were hardly seen and could be impurity. Mg and Zn are substrate element which are detected in PIXE spectra. Addition of these elements, the spectrum of HS1, HS2 and HS3 samples showed Si (1.9 Kev) peak. The results indicated 0.96, 1.24 and 1.51 (wt. %) for Si amount in coatings, respectively. The surface morphologies with three different magnifications and element compositions of coatings were presented in Fig. 4. The EDS results for coatings proved the presence of Si in HS1, HS2 and HS3 coatings. The SEM low magnification of coatings were shown the porous morphology in Figs. 4(a), 4(b), 4(c) and 4(d). These porous coatings may be excellent scaffold for culturing osteoblast cells and show the excellent bonding near the natural bone [20]. Porous coatings with greater surface area are expected to enhance formation of natural calcium phosphate precipitations in SBF solution and enhance implant's bonding with bone in vivo [21]. FE-SEM high magnification of coatings in Figs. 4(e), (f), 4(g) and 4(h) indicated that coatings had thin blade-shaped morphology. An increasing the amount of Si in the electrolyte caused a decreased the width of blades. According to Figs. 4(i) and 4(l), HA coatings had the blade morphology with about 35 nm thickness and 310 nm width, whereas HS3 coating shown the nano size blade
Fig. 2. FTIR spectra of scraped HA, HS1, HS2 and HS3 coatings.
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SEM images show that the compactness for the coatings increased up to HS2 coating but then were decreased for HS3 coating. HA coatings were deposited on Mg alloy according to Eqs. (1) and (2). The pH around the Mg alloy surface was increased by decomposition of H2O, when a voltage was applied. An increase in pH motivated Caþ and PO3 4 ions to react (Eq. (2)) and HA was deposited on surface of Mg alloy [23].
2H2 O þ 2e ¼ 2OH þ H2 [
(1)
10 Ca2þ þ 6PO3 4 þ 2OH / Ca10 ðPO4 Þ6 ðOHÞ2
(2)
However, when Na2SiO3$9H2O was added into the electrolyte, Na2SiO3$9H2O was initially reacted with H2O and produced H4SiO4 according to Eqs. (3) and (4) [8], and after applying the voltage, SiHA coatings were deposited on Mg alloy by Eq. (5).
Fig. 3. PIXE spectra of HA, HS1, HS2 and HS3 coatings.
morphology with about 10 nm thickness and 65 nm width. Kezhi et al. [22] were also detected Si-HA coating have a relatively smaller blade morphology in compare with HA. The SEM photomicrograph of coatings in Figs. 4(eeh) revealed that coatings compactness was also changed by increasing the Si concentrations in electrolyte. FE-
Na2 SiO3 þ H2 O ¼ Na2 H2 SiO4
(3)
Na2 H2 SiO4 þ H2 O ¼ H4 SiO4 þ NaOH
(4)
4 10 Ca2þ þ ð6 xÞPO3 4 þ xSiO4
þ ð2 xÞOH / Ca10 ðPO4 Þ6x ðSiO4 Þx ðOHÞ2x
(5) (PO3 4 )
It was assumed that the phosphate tetrahedral in the HA structure was substituted with the silicate tetrahedral (SiO44) in SiHA coatings [24]. Wang and et all [8] reported substitution of SiO4 4 instead of PO3 4 may occur on (100) surfaces and differences in charge between these ions made the negative charge on the surface
Fig. 4. SEM images in different magnification for (a)(e)(i) HA, (b)(f)(j) HS1, (c)(g)(k) HS2, (d)(h)(l) HS3.
C. Dehghanian et al. / Materials Chemistry and Physics 203 (2018) 27e33
and facilitated the secondary nuclei on (100) surface which led the morphology of Si-HA coatings to be nano size blades. The charge difference initiated from this substitution was compensated by losing OH [25,26] from HA structure, as evidenced in the FTIR spectra. The released OH ions near the Mg alloy surface caused an increase in pH and persuaded nucleation for new Si-HA crystals and increased the coating compactness (See Figs. 4 (l) and 4(g)). An increase in concentration of SiO44 ions in electrolyte caused an increase in the coating compactness up to HS2 coatings but further increase in concentration of SiO44 ions may restricted the access of Caþ and PO3 ions on Mg surface and 4 decreased HS3 coating compactness.
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Fig. 6. Polarization curves for (a) substrate (Mg-5Zn-0.3 Ca) and coated sample (HS2), (b) HA, HS1, HS2 and HS3 in SBF.
3.2. Thickness measurement The coating thickness of samples was shown in Fig. 5. Coating thickness was decreased form 7.15 mm to 3.92, 3.02 and 2.93 mm by increasing Na2SiO3$9H2O concentration from 0.0025 to 0.0075 mol/L in electrolyte for HS1, HS2 and HS3 coatings, respectively. As mention above, substitution of SiO44 in HA structure was accompanied by losing OH, as evidenced in the FTIR spectra, but the amount of OH which could be released from HA structure are limited. Thus a restricted amount of SiO44 ions could be incorporated into HA structure and the rest of SiO 44 ions were collected on surface. Collection of SiO4 4 ions on surface of substrate disturbed the access of other ions like Caþ and PO3 4 on surface which resulted in restriction of growth [25]. Therefore, an increase in Na2SiO3$9H2O concentration in electrolyte caused an increase in SiO4 4 ions on Mg alloy surface which resulted in coating thickness to be decreased. 3.3. Corrosion measurements Fig. 6 illustrates the electrochemical polarization curves for different samples in SBF solution at ambient conditions. The kinetic parameters were listed in Table 2. A significant change in corrosion potential (Ecorr) was observed by deposition of HS2 coating on Mg alloy (Fig. 6(a)). According to the electrochemical results, corrosion
current density (icorr) for substrate was decrease from 66 mA/cm2 to 24 mA/cm2 by deposition of HS2 coating on its surface. Therefore corrosion resistance of Mg alloy was improved after surface was modified by HS2 coating. Fig. 6(b) showed that the presence of Si in HA was improved the corrosion resistance of coating. The current density (icorr) for coatings were decreased from 52 mA/cm2 to 24 mA/cm2 by substitution of Si in coating structure (HA to HS2 sample) and their corrosion potential (Ecorr) were also improved to more noble potentials from 1.66 V to 1.5 V vs SCE, respectively. Impedance diagrams for HA, HS1, HS2 and HS3 samples were shown in Figs. 7(a), 7(b) and 7(c). Two capacitance loops were observed in the Nyquist plots for coated samples. One in high frequencies which is related to charge transfer and the other one in low frequencies which is related to coating on the surface [27]. A proposed equivalent circuit with two time constants for the EIS data was shown in Fig. 7(d). In this model, Rs is electrolyte resistance, Rcoat is coating resistance paralleled to coating constant phase element (CPEcoat), and RdL is the charge transfer resistance paralleled to an electric double layer capacitance (CdL). Kinetic parameters are listed in Table 3. An increase in concentration of Si in coating caused an increase in coating resistance (Rcoat). The coating resistance of HA was about ~349 (U cm2) whereas it was increased to about ~2608 (U cm2) for HS2. This indicated that an addition of 0.005 mol/L Si into electrolyte caused an increase of 7 times in corrosion resistance due to its higher compactness. In fact, Si-HA coating with higher density in comparison to that of HA coating may act as a resistive barrier for preventing the penetration of SBF to reach the Mg surface. This improves the corrosion resistance of Mg alloy.
3.4. Cell response
Fig. 5. Cross sectional SEM micrograph of (a) HA, (b) HS1, (c) HS2, (d) HS3.
3.4.1. MTT assay The cell viability results for MG63 cell proliferation in vicinity of different sample extractions were shown in Fig. 8. HS2 extracts showed no statistically significant difference (r > 0.05) in comparison to that of negative control during 1, 3 and 5days of culture. It indicated that HS2 coating samples extractions exhibit acceptable toxicity to MG63 cells with grade I toxicity. The cell viability for the HA, HS1, HS3 sample extracts showed no statistically significant difference (p > 0.05) during 1 day's culture, but indicated about 18% reduced (p < 0.05) on days 3 and 5. So HA, HS1 and HS3 displayed grade II toxicity, which may be attributed to the higher corrosion rate. High pH and ions concentration resulted from high corrosion rate leading to the osmolarity shock to the cells [28,29]. The pH values extractions for HA, HS1, HS2 and HS3coating samples were (8.73 ± 0.13), (8.68 ± 0.11), (8.21 ± 0.12) and (8.65 ± 0.13), respectively. Higher corrosion resistance for Si-HA coatings made their extract more suitable for cell proliferation and MG63 cells had
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C. Dehghanian et al. / Materials Chemistry and Physics 203 (2018) 27e33
Table 2 The kinetic parameters obtained from Fig. 5. Sample Mg-5Zn-0.3 Ca HA HS1 HS2 HS3
Anodic Slope ba(mv dec1)
bc(mv dec1)
Corrosion potential Ecorr(V vs SCE)
Current density icorr(mA/cm2)
75 52.61 72.26 84.642 96.122
74.6 442.77 94.36 330.39 136.22
1.72 1.66 1.66 1.5 1.67
66 52 36 24 23
Cathodic Slope
Fig. 7. (a) Nyquist, (b) bode, (c) Phase angel plots, and (d) A schematic equivalent circuit for coated samples of HA, HS1, HS2, HS3 in SBF.
Table 3 Kinetic parameters obtained from EIS plots for coated samples of HA, HS1, HS2, HS3. Sample
HA HS1 HS2 HS3
Rs
CPEcoat
(U cm2)
(U-1sn cm2)
23 23 23 23
1.7 2.2 1.5 1.1
105 105 105 105
ncoat
0.47 0.48 0.47 0.5
Rcoat
Cdl
Rt
(U cm2)
(mF cm2)
(U cm2)
349 1081 2608 1361
2.6 3.9 1.2 9.2
107 107 107 107
1453 972.3 2268 671
better proliferation in HS2 coating extract in comparison to that of HA coating extract. 3.4.2. Adhesion assay The morphology of MG63 cells on surface of HA, HS1, HS2 and HS3 samples after 7 h incubation was illustrated in Fig. 9. The cells on the surface of HA, HS1 and HS2 samples were sail-like, extended and thicker in the central area of the nucleus and flattened in the peripheral regions, and for HS3 sample, cells maintained a spindlelike and round morphology. The same results were reported by Zhang et al. [30] for Ca- P coating on Mg-Mn-Zn alloy. The SEM images of cells on samples were revealed that cells were attached into surface for all samples and some cells spread across the surface and contacted with each other. 4. Conclusion
Fig. 8. Absorbance results of MG63 proliferation in vicinity of HA, HS1, HS2 and HS3 extraction.
In this study uniform and dense coatings of Si-HA with different percentages of Si were deposited on Mg alloy. Substitution of SiO4 4 instead of PO3 4 ions in HA structure caused an increase in pH for substrate during electrodeposition process which provided an appropriate state for coating nucleation and an increase in density for Si-HA coatings. Moreover, it was also found that an increase in Na2SiO3$9H2O concentration in electrolyte increased accumulation of SiO4 4 ions on substrate surface and limited the Si-HA coatings growth and decreased its thickness. The electrochemical results revealed that addition of 0.005 mol/L Si into electrolyte caused 7 times increase in corrosion resistance due to its higher coating
C. Dehghanian et al. / Materials Chemistry and Physics 203 (2018) 27e33
Fig. 9. Morphology of MG63 cells on (a) HA, (b) HS1, (c) HS2 and (d) HS3 surface after 7 h incubation.
compactness. The MTT assay indicated that HS2 coatings had the nearest cell proliferation toward negative control during 5 days of culture and adhesion assay showed that MG63 cells were attached into surface for all samples coating after 7 h. References [1] C.F. Dunne, G.K. Levy, O. Hakimi, E. Aghion, B. Twomey, K.T. Stanton, Corrosion behavior of biodegradable magnesium alloys with hydroxyapatite coatings, Surf. Coatings Technol. 289 (2016) 37; [a] K. Abdelrazek Khalil; A new-developed nanostructured Mg/HAp nanocomposite by high frequency induction heat sintering process; Int. J. Electrochem. Sci., 7, (2012), 10698. [2] S. Shen, S. Cai, Y. Li, R. Ling, F. Zhang, G. Xu, F. Wang, Microwave aqueous synthesis of hydroxyapatite bilayer coating on magnesium alloy for orthopedic application, Chem. Eng. J. 309 (2017) 278. [3] H. Hornberger, S. Virtanen, A.R. Boccaccini, Biomedical coatings on magnesium alloys e a review, Acta Biomater. 8 (2012) 2442. [4] S. Shadanbaz, G.J. Dias, Calcium phosphate coatings on magnesium alloys for biomedical applications: a review, Acta Biomater. 8 (2012) 20. [5] J. Yang, X. Lu, C. Blawert, S. Di, M.L. Zheludkevich, Microstructure and corrosion behavior of Ca/P coatings prepared on magnesium by plasma electrolytic oxidation, Surf. Coatings Technol. 319 (2017) 359. [6] K. d. Groot, J. Wolke, J. Jansen, Calcium phosphate coatings for medical implants, Proc. Institution Mech. Eng. H 212 (1998) 137. [7] E.S. Thian, J. Huang, M.E. Vickers, S.M. Best, Z.H. Barber, W. Bonfield, Siliconsubstituted hydroxyapatite (SiHA): a novel calcium phosphate coating for biomedical applications, J. Mater. Sci. 41 (2006) 709. [8] D. Li, J. Lin, D. Lin, X. Wang, Synthesized silicon-substituted hydroxyapatite coating on titanium substrate by electrochemical deposition, J. Mater. Sci. Mater. Med. 22 (2011) 1205.
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[9] Y. Cai, S. Zhang, X. Zeng, M. Qian, D. Sun, W. Weng, Interfacial study of magnesium-containing fluoridated hydroxyapatite coatings, Thin Solid Films 519 (2011) 4629. [10] A.E. Porter, N. Patel, J.N. Skepper, S.M. best, W. Bonfield, Comparison of in vivo dissolution processes in hydroxyapatite and silicon- substituted hydroxyapatite bioceramics, Biomaterials 24 (2003) 4609. [11] I. Gibson, J.A. Hing, S. Best, W. Bonfield, Enhanced in vitro cell activity and surface apatite layer formation on novel silicon substituted hydroxyapatites, Bioceramics 12 (1999) 191. [12] E.M. Carlisle, Biochemical and morphological changes associated with long bone abnormalities in silicon deficiency, J. Nutr. 110 (5) (1980) 1046. [13] E.M. Carlisle, Silicon a possible factor in bone calcification, Science 167 (1970) 279. [14] P. Wan, X. Qiu, L. Tan, X. Fan, K. Yang, The effects of pulse electrodeposition parameters on morphology and formation of dual-layer Si doped calcium phosphate coating on AZ31alloy, Ceram. Int. 41 (2015) 787. [15] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterial 27 (2006) 2907. [16] H. Yang, K. Xia, T. Wang, J. Niu, Y. Song, Z. Xiong, K. Zheng, S. Wei, W. Lu, Growth, in vitro biodegradation and cytocompatibility properties of nanohydroxyapatite coatings on biodegradable magnesium alloys, J. Alloys Compd. 672 (2016) 366. pez-Alvarez, lez, J. Serra, B. Leo n, A.P. Marques, [17] M. Lo E.L. Solla, P. Gonza R.L. Reis, Siliconehydroxyapatite bioactive coatings (SieHA) from diatomaceous earth and silica. Study of adhesion and proliferation of osteoblast-like cells, J. Mater. Sci. Mater. Med. 20 (2009) 1131. [18] E.S. Thian, J. Huang, S.M. Best, Z.H. Barber, W. Bonfield, Silicon-substituted hydroxyapatite: the next generation of bioactive coatings, Mater. Sci. Eng. C 27 (2007) 251. [19] G. Graziani, M. Bianchi, E. Sassoni, A. Russo, M. Marcacci, Ion-substituted calcium phosphate coatings deposited by plasma-assisted techniques: a review, Mater. Sci. Eng. C 74 (2017) 219. [20] H.R. Bakhsheshi-Rad, E. Hamzah, M. Daroonparvar, M.A.M. Yajid, M. Medraj, Fabrication and corrosion behavior of Si/HA nano-composite coatings on biodegradable MgeZneMneCa alloy, Surf. Coatings Technol. 258 (2014) 1090. [21] J. Park, Y. Lee, K. Kim, K. Kim, Bioactive calcium phosphate coating prepared on H2O2-treated titanium substrate by electrodeposition, Surf. Coatings Technol. 195 (2005) 252. [22] L. Kezhi, G. Qian, Z. Leilei, Z. Yulei, L. Shoujie, G. Kebing, L. Shaoxian, Synthesis and characterization of Si-substituted hydroxyapatite bioactive coating for SiC-coated carbon/carbon composites, Ceram. Int. 43 (2017) 1410. [23] D. Lin, X. Wang, Electrodeposition of hydroxyapatite coating on CoNiCrMo substrate in dilute solution, Surf. Coatings Technol. 204 (2010) 3205. [24] S. Zou, J. Huang, S. Best, W. Bonfield, Crystal imperfection studies of pure and silicon substituted hydroxyapatite using Raman and XRD, J. Mater. Sci. Mater. Med. 16 (2005) 1143. [25] X. Tang, X. Xiao, R. Liu, Structural characterization of silicon substituted hydroxyapatite synthesized by a hydrothermal method, Mater. Lett. 59 (2005) 3841. [26] E. Kim, Y. Jeong, H. Choe, W. Brantley, Preparation of silicon-substituted hydroxyapatite coatings on Tie30NbexTa alloys using cyclic electrochemical deposition method, Thin Solid Films 572 (2014) 99. [27] Y. Zheng, X. Gu, Y. Xi, D. Chai, In vitro degradation and cytotoxicity of Mg/Ca composites produced by powder metallurgy, Acta Biomater. 6 (2010) 1783. [28] X.N. Gu, N. Li, Y.F. Zheng, F. Kang, J.T. Wang, L. Ruan, In vitro study on equal channel angular pressing AZ31 magnesium alloy with and without back pressure, Mater. Sci. Eng. B 15 (2011) 1802. [29] F. Witte, N. Hort, C. Vogt, S. Cohen, K.U. Kainer, R. Willumeit, F. Feryerabend, Degradable biomaterials based on magnesium corrosion, Curr. Opin. Solid State Mater. Sci. 12 (2008) 63. [30] L. Xu, F. Pan, G. Yu, L. Yang, E. Zhang, Ke Yang, In vitro and in vivo evaluation of the surface bioactivity of a calcium phosphate coated magnesium alloy, Biomaterials 30 (2009) 1512.
Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Characterization of silicon- substituted nano hydroxyapatite coating on magnesium alloy for biomaterial application Changiz Dehghanian a, *, Neda Aboudzadeh a, Mohammad Ali Shokrgozar b a b
University of Tehran, College of Engineering, School of Metallurgy and Materials Engineering, Tehran, Iran National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
h i g h l i g h t s Uniform and dense coatings of Si-HA were deposited on Mg alloy by reverse pulse electrodeposition process. Coatings showed blade-shaped morphology and the blade width decreased as Si contents of coatings were increased. Increase in the amounts of Si in electrolyte decreased the coatings thickness. Addition of Si into HA coating increased its corrosion resistance up to 7 times. The cell experiment indicated that there was no toxicity for Si-HA coatings.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2017 Received in revised form 25 July 2017 Accepted 13 August 2017 Available online 22 September 2017
In this study, effects of substitution of Si in structure of HA (Si-HA) as coating on Mg alloy were investigated. The reverse pulse electrodeposition process with frequency of 10 and duty cycle of 0.1 was selected for coating. Four different electrolytes with different molars of SiO2 3 (0, 0.0025, 0.005 and 0.0075) were prepared in order to deposit Si-HA coatings on Mg alloy. The XRD and FTIR results showed that the main phase of deposited coatings was HA, and elemental analysis of coating confirmed the presence of Si in coatings. FE-SEM images of coatings indicated the presence of SiO2 3 in electrolyte caused a decrease in coating thickness and an increase in its compactness. The coating prepared by the electrolyte containing 0.005 mol/L SiO2 3 had higher corrosion resistance than that of HA coating as indicated by electrochemical tests. The cell experiment tests also showed that this high corrosion resistance coating had the nearest cell proliferation toward negative control. © 2017 Published by Elsevier B.V.
Keywords: Coating Si HA Biomaterials Pulse reverse electrodeposition
1. Introduction A new approach for repairing damaged bone tissue is done by tissue engineering. In tissue engineering, implant materials must be biocompatible and biodegradable which is gradually dissolved in human body during repairing bone tissue. In recent years, magnesium and its alloys with biocompatibility and biodegradability properties have been of interest in tissue engineering discipline and some researchers have focused on them to adjust their property with requirements in bone implantation [1,2]. Magnesium‘s primary concern is high degradation speed in human body fluid [3]. In response to this concern, different methods have been considered which among them, coating process may be a
* Corresponding author. E-mail address: [email protected] (C. Dehghanian). https://doi.org/10.1016/j.matchemphys.2017.08.020 0254-0584/© 2017 Published by Elsevier B.V.
relatively significant one. In fact, coatings prevent direct contact of Mg with corrosive environment and reduce its corrosion rate [4,5]. A coating material should be biodegradable and osteoconductive to be usable in bone tissue engineering. The calcium phosphate (CaP) family in particular hydroxyapatite (HA) are of special interest for Mg's coating [6]. HA (Ca10 (PO4)6(OH)2) has a hexagonal crystallographic lattice and a space group of P63/m together with a Ca:P ratio of 1.67 [7]. The biologic hydroxyapatite e the mineral phase of bone-mostly has various substituted ions such as Na, Mg, Zn, Sr and Si in its structure which is enhanced its bioactive behaviors [8e10]. Recently, many researchers have been interested in characterization of effects of these ions substitution in HA structure. It was shown that substitution of phosphate with silicate into HA structure enhances its osteoblast cell activity, attachment, and growth [11]. Moreover, silicon as one of the most important elements is known to be essential in the early stages of bone mineralization and
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2. Experimental procedures
2.8 mm2 was irradiated on the coatings samples placed in the vacuum chamber at 106 torr. The coating samples were placed at the angle of 90 with respect to the incident beam. The beam current was adjusted at about 3 nA in order to keep the counting rate below 1000 cps (count per second). Surface morphology, thickness and elemental analyses of coated samples were specified by field emission scanning electron microscope (FE-SEM: TSCAN-VEGA, China) and energy dispersive Xray spectroscopy (EDS: TSCAN-VEGA, China).
2.1. Preparation of substrate
2.4. Corrosion measurements
The Mg -5Zn- 0.3Ca (%wt.) alloy substrates were synthesized by powder metallurgy. Briefly, metal mixture from Mg (S5430817 002, Merck), Zn (K39617189 929, Merck) and Ca (S5101853 907, Merck) powders were prepared and mixed in ball mill for 6 min, then 0.8 g from metal mixture were pressed in shape of cylinder with the size of 10 mm in diameter and 5 mm in thickness and sintered at 550 C for 2 h in Argon atmosphere. After that, the surface of substrate were grinded with abrasive paper of 600e2000 grits, polished and degreased with acetone to be prepared for coating.
Corrosion properties of nanocomposites were evaluated by anodic potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) tests. The electrochemical tests were carried out in simulated body fluid (SBF) which was prepared according to Kokubo's protocol [15]. Electrochemical corrosion tests were carried out by using a conventional three-electrode cell with a sample as working electrode, saturated calomel electrode (SCE) as reference and platinum mesh as the counter.
soft tissue development [12,13]. In this study, the effects of Si substituted in HA structure as a coating on Mg alloy were investigated. Si-HA coatings with different percentages of Si were deposited on Mg-5Zn-0.3Ca alloy by pulse reverse current (PRC) process and the surface characteristics, corrosion and biocompatibility properties of these types of coatings were compared with the HA coating.
2.2. Electrochemical deposition Four different compositions of electrolytes were prepared according to Table 1. Ca (NO3)2$4H2O, NH4H2PO4, and Na2SiO3$9H2O salts were added as sources of Ca, P and Si in electrolyte, respectively, and NaNO3 (O.1 M/L) was added as conductive agent. The 2 molar ratio of Ca2þ/(PO3 4 þ SiO3 ) was constant at 1.67 for all 3 electrolytes compositions but the molar ratio of SiO2 3 /(PO4 þ 2 SiO3 ) was variable at 0, 0.1, 0.2 and 0.3 for each of electrolytes. The pH of electrolytes were adjusted to 5 by addition of dilute nitric acid HNO3 and ester (CH2OH)3CNH2. The pulse electrodeposition process was carried out by the electrochemical workstation (RST5200) with a two electrode cell [8,14]. Mg -5Zn- 0.3Ca alloy served as cathode and a graphite plate as an anode. The positive and reverse current densities were adjusted to 40 and -10 mA/cm2. The positive, delay and reverse plating time were adjusted to be 10, 90 and 5 ms, respectively for the duration of 30 min. Temperature and concentration of electrolyte were unified at 85 C by a water bath and magnetic stirrer. The coated samples in different electrolytes with different concentrations of SiO2 3 were classified as HA, HS1, HS2 and HS3. 2.3. Microstructural characterization Phase identification of coatings were identified by X-ray diffraction technique (XRD: JDX-8030, Jeol, Japan) with a Cu-Ka source and spectroscopic analysis of coatings was indicated with the fourier transform infrared spectroscopy (FTIR: 8400S, SHIMADZU, Japan). The elemental analysis of coatings were done by the method of proton induced X-ray emission (PIXE). The 3.0 MeV van-de-Graff electrostatic accelerator (High Voltage Company, USA) was used for PIXE analysis. A 2.0 MeV proton beam with the spite size of
2.4.1. Anodic potentiodynamic polarization test Anodic potentiodynamic polarization for coated samples were conducted from 200 to þ500 mV vs. SCE with a scan rate of 1 mV/ s after stabilizing open-circuit potential (OCP). The corrosion current densities and anodic and cathodic Tafel slopes were obtained from the polarization curves. The polarization resistance (Rp) of samples were also determined. 2.4.2. Electrochemical impedance spectroscopy test Electrochemical impedance spectroscopy (EIS) was carried out using an AC impedance analyzer (solartron, 1260) after reaching a stable OCP. The frequency was varied from 101e105 Hz. The software (ZSim version 3.4) was used to analyze the obtained EIS data and the best-fitted equivalent circuit model was found. 2.5. Cell experiment 2.5.1. MTT assay Indirect contact method was selected to investigate the toxicity of nanocomposites, so in compliance with ISO 10993-12, coated samples were sterilized by autoclave and each sample was separately incubated in Roswell Park Memorial Institute (RPMI/1640) media containing 10% fetal bovine serum (FBS; Gibco) for 36 h. After that the supernatant medium was withdrawn and collected. In the following, the MG63 cells were cultured for 24 h in cell culture plates and then their medium were replaced with extracts and incubated for the certain period of 1, 3 and 5 days. The cultured MG63 wells with no extracts were used as negative controls. Accuracy of results were ascertained by repetition of each test for 5 times. After different culture periods, the medium of each well was removed and solution of dimethylthiazol diphenyl tetrazolium bromide (MTT) (Sigma, USA) was added and incubated for 5 h, afterward MTT solution was detached and isopropanol (Sigma, Milwaukee, USA) was enhanced and incubated again for 10 min.
Table 1 The four different electrolytes compound for coatings. Abbreviation
3 2 SiO2 3 /(PO4 þSiO3 ) molar ratio
Na2SiO3$9H2O mol/L
NH4H2PO4 mol/L
Ca(No3)2.4H2O mol/L
NaNo3 mol/L
HA HS1 HS2 HS3
0 0.1 0.2 0.3
0 0.0025 0.005 0.0075
0.0251 0.0226 0.02 0.0176
0.042 0.042 0.042 0.042
0.1 0.1 0.1 0.1
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Absorbance measurements of wells as the indicator of cell growth were carried out by a multiwall microplate reader (ICN, Switzerland) at 570 nm [16]. The cell viabilities were determined by: Cell Viability (%) ¼ (OD
sample/OD negative control)
100%
The significant differences of cell viability in MTT assay were analyzed by the Analysis of Variance (ANOVA) technique in SPSS software (ver. 20.0) when the statistical significance value (r) was defined as 0.05. 2.5.2. Adhesion assay Direct cell adhesive assay was done for coated samples of HA, HS1, HS2, HS3. Initially, 1 mL of MG63 cell suspension with concentration of 3 104 cells ml1 was seeded in each sterilized sample and then incubated for 7 h. Afterward, the samples were soaked with PBS for three times and then fixed in 2.5% glutaraldehyde solution for 2 h at room temperature [17]. Finally, the adhesion of cells on surface of samples was observed by using SEM. 3. Results and discussion 3.1. Metallurgical characterization XRD pattern of Mg alloy with and without HS2 coating were shown in Fig. 1(a). Characteristic diffraction of HA were revealed in XRD pattern of HS2 in Fig. 1(a) which is suggested that HA is the main crystalline phase of the coating. The substrate peaks are strongly visible in XRD pattern of HS2 in Fig. 1(a) due to porosity and thinning of coating. According to HA standard diffraction pattern and stronger intensity of (002) diffraction, HA-Si crystals are preferentially oriented with [001] direction. The XRD patterns of HA, HS1, HS2 and HS3 coatings were shown in Fig. 1(a,b). Diffraction pattern of HA was specified in all coatings but since the amount of Si in coatings is low, no significant difference was distinguished between these four samples. As last studies
Fig. 1. The XRD patterns of (a) coated sample and substrate, (b) coating samples with different levels of Si.
29
suggested the silicate tetrahedral were substituting for the phosphate tetrahedral in the HA structure [6,18]. Substitution of silicate in HA may change the lattice due to differences in the ionic radius of silicon compared to phosphorous, but the nearness of silicon and phosphorous in the periodic table makes these differences hard to be considered. Therefore, small amount of Si don't change the XRD patent of HA [19]. The FTIR spectrum of the coatings were shown in Fig. 2. Fig. 2 shows the main absorption bands of the ionic group of HA [5,6]; the bands at 1000e1200, 964 and 560 cm1 attributed to asymmetric stretching, symmetric stretching and asymmetric bending vibrations of PO3 groups, respectively; the bands at 4 3565 cm1corresponded to the stretching modes of the OH groups; and finally the bands at 1400e1500 and 875 cm1connected to asymmetric stretching and bending vibration of CO2 3 groups. Comparing the FTIR spectrum of coatings show that incorporation of silicon led to some changes in the FTIR spectra of 1 HA, PO3 seem to be merge and PO3 4 bands at 1020 and 1103 cm 4 1 bands at 964 and 564 cm are decreased. The OH absorption bands at 3565 cm1 were also decreased slightly with increasing silicon incorporation. So, the PO3 4 in the HA structure may be substituted with SiO44 in Si-HA coatings. The same results was reported by Li et al. [6]. Fig. 3 shows the PIXE results for HA, HS1, HS2 and HS3 coatings. The PIXE spectra for HA coatings indicated traces of Mg, P, Ca, Zn and Cl, Mn, Cu, Pb elements, which the last four elements were hardly seen and could be impurity. Mg and Zn are substrate element which are detected in PIXE spectra. Addition of these elements, the spectrum of HS1, HS2 and HS3 samples showed Si (1.9 Kev) peak. The results indicated 0.96, 1.24 and 1.51 (wt. %) for Si amount in coatings, respectively. The surface morphologies with three different magnifications and element compositions of coatings were presented in Fig. 4. The EDS results for coatings proved the presence of Si in HS1, HS2 and HS3 coatings. The SEM low magnification of coatings were shown the porous morphology in Figs. 4(a), 4(b), 4(c) and 4(d). These porous coatings may be excellent scaffold for culturing osteoblast cells and show the excellent bonding near the natural bone [20]. Porous coatings with greater surface area are expected to enhance formation of natural calcium phosphate precipitations in SBF solution and enhance implant's bonding with bone in vivo [21]. FE-SEM high magnification of coatings in Figs. 4(e), (f), 4(g) and 4(h) indicated that coatings had thin blade-shaped morphology. An increasing the amount of Si in the electrolyte caused a decreased the width of blades. According to Figs. 4(i) and 4(l), HA coatings had the blade morphology with about 35 nm thickness and 310 nm width, whereas HS3 coating shown the nano size blade
Fig. 2. FTIR spectra of scraped HA, HS1, HS2 and HS3 coatings.
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SEM images show that the compactness for the coatings increased up to HS2 coating but then were decreased for HS3 coating. HA coatings were deposited on Mg alloy according to Eqs. (1) and (2). The pH around the Mg alloy surface was increased by decomposition of H2O, when a voltage was applied. An increase in pH motivated Caþ and PO3 4 ions to react (Eq. (2)) and HA was deposited on surface of Mg alloy [23].
2H2 O þ 2e ¼ 2OH þ H2 [
(1)
10 Ca2þ þ 6PO3 4 þ 2OH / Ca10 ðPO4 Þ6 ðOHÞ2
(2)
However, when Na2SiO3$9H2O was added into the electrolyte, Na2SiO3$9H2O was initially reacted with H2O and produced H4SiO4 according to Eqs. (3) and (4) [8], and after applying the voltage, SiHA coatings were deposited on Mg alloy by Eq. (5).
Fig. 3. PIXE spectra of HA, HS1, HS2 and HS3 coatings.
morphology with about 10 nm thickness and 65 nm width. Kezhi et al. [22] were also detected Si-HA coating have a relatively smaller blade morphology in compare with HA. The SEM photomicrograph of coatings in Figs. 4(eeh) revealed that coatings compactness was also changed by increasing the Si concentrations in electrolyte. FE-
Na2 SiO3 þ H2 O ¼ Na2 H2 SiO4
(3)
Na2 H2 SiO4 þ H2 O ¼ H4 SiO4 þ NaOH
(4)
4 10 Ca2þ þ ð6 xÞPO3 4 þ xSiO4
þ ð2 xÞOH / Ca10 ðPO4 Þ6x ðSiO4 Þx ðOHÞ2x
(5) (PO3 4 )
It was assumed that the phosphate tetrahedral in the HA structure was substituted with the silicate tetrahedral (SiO44) in SiHA coatings [24]. Wang and et all [8] reported substitution of SiO4 4 instead of PO3 4 may occur on (100) surfaces and differences in charge between these ions made the negative charge on the surface
Fig. 4. SEM images in different magnification for (a)(e)(i) HA, (b)(f)(j) HS1, (c)(g)(k) HS2, (d)(h)(l) HS3.
C. Dehghanian et al. / Materials Chemistry and Physics 203 (2018) 27e33
and facilitated the secondary nuclei on (100) surface which led the morphology of Si-HA coatings to be nano size blades. The charge difference initiated from this substitution was compensated by losing OH [25,26] from HA structure, as evidenced in the FTIR spectra. The released OH ions near the Mg alloy surface caused an increase in pH and persuaded nucleation for new Si-HA crystals and increased the coating compactness (See Figs. 4 (l) and 4(g)). An increase in concentration of SiO44 ions in electrolyte caused an increase in the coating compactness up to HS2 coatings but further increase in concentration of SiO44 ions may restricted the access of Caþ and PO3 ions on Mg surface and 4 decreased HS3 coating compactness.
31
Fig. 6. Polarization curves for (a) substrate (Mg-5Zn-0.3 Ca) and coated sample (HS2), (b) HA, HS1, HS2 and HS3 in SBF.
3.2. Thickness measurement The coating thickness of samples was shown in Fig. 5. Coating thickness was decreased form 7.15 mm to 3.92, 3.02 and 2.93 mm by increasing Na2SiO3$9H2O concentration from 0.0025 to 0.0075 mol/L in electrolyte for HS1, HS2 and HS3 coatings, respectively. As mention above, substitution of SiO44 in HA structure was accompanied by losing OH, as evidenced in the FTIR spectra, but the amount of OH which could be released from HA structure are limited. Thus a restricted amount of SiO44 ions could be incorporated into HA structure and the rest of SiO 44 ions were collected on surface. Collection of SiO4 4 ions on surface of substrate disturbed the access of other ions like Caþ and PO3 4 on surface which resulted in restriction of growth [25]. Therefore, an increase in Na2SiO3$9H2O concentration in electrolyte caused an increase in SiO4 4 ions on Mg alloy surface which resulted in coating thickness to be decreased. 3.3. Corrosion measurements Fig. 6 illustrates the electrochemical polarization curves for different samples in SBF solution at ambient conditions. The kinetic parameters were listed in Table 2. A significant change in corrosion potential (Ecorr) was observed by deposition of HS2 coating on Mg alloy (Fig. 6(a)). According to the electrochemical results, corrosion
current density (icorr) for substrate was decrease from 66 mA/cm2 to 24 mA/cm2 by deposition of HS2 coating on its surface. Therefore corrosion resistance of Mg alloy was improved after surface was modified by HS2 coating. Fig. 6(b) showed that the presence of Si in HA was improved the corrosion resistance of coating. The current density (icorr) for coatings were decreased from 52 mA/cm2 to 24 mA/cm2 by substitution of Si in coating structure (HA to HS2 sample) and their corrosion potential (Ecorr) were also improved to more noble potentials from 1.66 V to 1.5 V vs SCE, respectively. Impedance diagrams for HA, HS1, HS2 and HS3 samples were shown in Figs. 7(a), 7(b) and 7(c). Two capacitance loops were observed in the Nyquist plots for coated samples. One in high frequencies which is related to charge transfer and the other one in low frequencies which is related to coating on the surface [27]. A proposed equivalent circuit with two time constants for the EIS data was shown in Fig. 7(d). In this model, Rs is electrolyte resistance, Rcoat is coating resistance paralleled to coating constant phase element (CPEcoat), and RdL is the charge transfer resistance paralleled to an electric double layer capacitance (CdL). Kinetic parameters are listed in Table 3. An increase in concentration of Si in coating caused an increase in coating resistance (Rcoat). The coating resistance of HA was about ~349 (U cm2) whereas it was increased to about ~2608 (U cm2) for HS2. This indicated that an addition of 0.005 mol/L Si into electrolyte caused an increase of 7 times in corrosion resistance due to its higher compactness. In fact, Si-HA coating with higher density in comparison to that of HA coating may act as a resistive barrier for preventing the penetration of SBF to reach the Mg surface. This improves the corrosion resistance of Mg alloy.
3.4. Cell response
Fig. 5. Cross sectional SEM micrograph of (a) HA, (b) HS1, (c) HS2, (d) HS3.
3.4.1. MTT assay The cell viability results for MG63 cell proliferation in vicinity of different sample extractions were shown in Fig. 8. HS2 extracts showed no statistically significant difference (r > 0.05) in comparison to that of negative control during 1, 3 and 5days of culture. It indicated that HS2 coating samples extractions exhibit acceptable toxicity to MG63 cells with grade I toxicity. The cell viability for the HA, HS1, HS3 sample extracts showed no statistically significant difference (p > 0.05) during 1 day's culture, but indicated about 18% reduced (p < 0.05) on days 3 and 5. So HA, HS1 and HS3 displayed grade II toxicity, which may be attributed to the higher corrosion rate. High pH and ions concentration resulted from high corrosion rate leading to the osmolarity shock to the cells [28,29]. The pH values extractions for HA, HS1, HS2 and HS3coating samples were (8.73 ± 0.13), (8.68 ± 0.11), (8.21 ± 0.12) and (8.65 ± 0.13), respectively. Higher corrosion resistance for Si-HA coatings made their extract more suitable for cell proliferation and MG63 cells had
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C. Dehghanian et al. / Materials Chemistry and Physics 203 (2018) 27e33
Table 2 The kinetic parameters obtained from Fig. 5. Sample Mg-5Zn-0.3 Ca HA HS1 HS2 HS3
Anodic Slope ba(mv dec1)
bc(mv dec1)
Corrosion potential Ecorr(V vs SCE)
Current density icorr(mA/cm2)
75 52.61 72.26 84.642 96.122
74.6 442.77 94.36 330.39 136.22
1.72 1.66 1.66 1.5 1.67
66 52 36 24 23
Cathodic Slope
Fig. 7. (a) Nyquist, (b) bode, (c) Phase angel plots, and (d) A schematic equivalent circuit for coated samples of HA, HS1, HS2, HS3 in SBF.
Table 3 Kinetic parameters obtained from EIS plots for coated samples of HA, HS1, HS2, HS3. Sample
HA HS1 HS2 HS3
Rs
CPEcoat
(U cm2)
(U-1sn cm2)
23 23 23 23
1.7 2.2 1.5 1.1
105 105 105 105
ncoat
0.47 0.48 0.47 0.5
Rcoat
Cdl
Rt
(U cm2)
(mF cm2)
(U cm2)
349 1081 2608 1361
2.6 3.9 1.2 9.2
107 107 107 107
1453 972.3 2268 671
better proliferation in HS2 coating extract in comparison to that of HA coating extract. 3.4.2. Adhesion assay The morphology of MG63 cells on surface of HA, HS1, HS2 and HS3 samples after 7 h incubation was illustrated in Fig. 9. The cells on the surface of HA, HS1 and HS2 samples were sail-like, extended and thicker in the central area of the nucleus and flattened in the peripheral regions, and for HS3 sample, cells maintained a spindlelike and round morphology. The same results were reported by Zhang et al. [30] for Ca- P coating on Mg-Mn-Zn alloy. The SEM images of cells on samples were revealed that cells were attached into surface for all samples and some cells spread across the surface and contacted with each other. 4. Conclusion
Fig. 8. Absorbance results of MG63 proliferation in vicinity of HA, HS1, HS2 and HS3 extraction.
In this study uniform and dense coatings of Si-HA with different percentages of Si were deposited on Mg alloy. Substitution of SiO4 4 instead of PO3 4 ions in HA structure caused an increase in pH for substrate during electrodeposition process which provided an appropriate state for coating nucleation and an increase in density for Si-HA coatings. Moreover, it was also found that an increase in Na2SiO3$9H2O concentration in electrolyte increased accumulation of SiO4 4 ions on substrate surface and limited the Si-HA coatings growth and decreased its thickness. The electrochemical results revealed that addition of 0.005 mol/L Si into electrolyte caused 7 times increase in corrosion resistance due to its higher coating
C. Dehghanian et al. / Materials Chemistry and Physics 203 (2018) 27e33
Fig. 9. Morphology of MG63 cells on (a) HA, (b) HS1, (c) HS2 and (d) HS3 surface after 7 h incubation.
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