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Influence of EDTA-2Na on the hydroxyapatite coating deposited by hydrothermal-electrochemical method on Ti6Al4V surface
Surface & Coatings Technology xxx (xxxx) xxx–xxx
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Influence of EDTA-2Na on the hydroxyapatite coating deposited by hydrothermal-electrochemical method on Ti6Al4V surface ⁎
Daihua He , Jing Du, Ping Liu, Xinkuan Liu, Xiaohong Chen, Wei Li, Ke Zhang, Fengcang Ma School of Materials Science and Engineering, University of Shanghai for Science and Technology, No. 516 Jungong Road, Shanghai 200093, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Ti6Al4V Ethylenediamine Tetraacetic Acid Disodium salt (EDTA-2Na) Hydroxyapatite coating Hydrothermal-electrochemical
The influences of the Ethylenediamine Tetraacetic Acid Disodium salt (EDTA-2Na) on the hydroxyapatite (HA) coating deposited by hydrothermal-electrochemical methods on the Ti6Al4V surface were investigated. The morphology of the HA crystals of the first layer changed from needle- or rod-like to flowerlike. The HA crystal became wider and the first layer became denser gradually, whereas the second layer became sparser. The thickness of the HA coating gradually decreased with the increase of EDTA-2Na concentration in the electrolyte. The bonding strength between coating and substrate reached the maximum of 16.8 MPa when the EDTA-2Na concentration was 7.5 × 10−4 mol/L. Cell-culture test indicated that the HA coating with 7.5 × 10−4 mol/L EDTA-2Na benefits the adhesion of cell onto the HA surface.
1. Introduction Titanium and its alloys have been widely used as biomedical implant materials due to their low density, good mechanical properties, superior corrosion resistance and biocompatibility compared with those of other metallic biomaterials such as CoeCr alloys and stainless steels [1–4]. However, the biological activity of titanium and its alloy is inadequate, and they display low stability after being implanted in the human body. The use of surface modification to improve their bioactivity and biocompatibility has been paid considerable attention [5–6]. Titanium alloy with hydroxyapatite (HA) coating is a bone implant material that combines the good mechanical properties of titanium alloy and the excellent bioactivity and biocompatibility of synthetic HA [7]. HA coating can be deposited on titanium or titanium alloy through various methods, such as plasma spray, sol-gel method, microarc oxidation, pulsed laser deposition, electrophoresis, hydrothermal-electrochemical method, and so on. The hydrothermal-electrochemical methods is developed on the basis of electrochemical deposition and hydrothermal method [8]. This method offers many advantages: 1) The experimental temperature of this method is higher than that of the hydrothermal method and significantly lower than that of plasma spraying method. 2) The HA coating has a high crystallinity, and no subsequent heat treatment is needed to improve the crystallinity. 3) This method can coat a complex-shaped substrate with a uniform thickness. A series of researches were done by our group about the HA coating deposited by hydrothermal chemical method on titanium alloy
⁎
surface [9–12]. Nonetheless, the uniformity and compactness of the coating should be further improved. EDTA-2Na is a good chelating agent that has a wide range of coordination properties, and it can form stable chelates with nearly all metal ions [13–14]. In the references [15–18] were shown that the EDTA or EDTA-2Na can control the shape and size, and improve the crystallinity of HA. In the present paper, EDTA-2Na was added to the electrolyte, which was applied to deposit the HA coating on the Ti6Al4V surface by the hydrothermal-electrochemical methods and its effect on the coating was intensively investigated. 2. Experimental 2.1. Preparation of titanium alloy samples Commercially available medical Ti6Al4V sheets with dimensions of 20mm × 20mm × 2mm were used. The samples were polished firstly by wet sandpapers, and then cleaned ultrasonically with acetone, ethanol and distilled water in sequence for 10min. 2.2. Deposition of HA coating The hydrothermal–electrochemical deposition experiments were performed in a stainless-steel autoclave with a Teflon liner. The electrolyte contained 0.025mol/L Ca(NO3)2·4H2O、0.015mol/L NH4H2PO4, 0.1 mol/L NaNO3, and different concentrations of EDTA-
Corresponding author. E-mail address: [email protected] (D. He).
https://doi.org/10.1016/j.surfcoat.2018.10.065 Received 4 April 2018; Received in revised form 30 September 2018; Accepted 24 October 2018 0257-8972/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: He, D., Surface & Coatings Technology, https://doi.org/10.1016/j.surfcoat.2018.10.065
Surface & Coatings Technology xxx (xxxx) xxx–xxx
D. He et al.
Fig. 1. SEM micrographs of HA coatings deposited with different EDTA-2Na concentration in the electrolyte (a) 0 mol/L, (a1) × 2000, (a2) × 10,000 (b) 2.5 × 10−4 mol/L, (b1) × 2000, (b2) × 10,000 (c) 5.0 × 10−4 mol/L, (c1) × 2000, (c2) × 10,000 (d) 7.5 × 10−4 mol/L, (d1) × 2000, (d2) × 10,000 (e) 1 × 10−3mol/L, (e1) × 2000, (e2) × 10,000.
(a2)
(a1)
50μm
10μm a (b2)
(b1)
50μm
10μm b
(c1)
(c2)
Fig. 2. XRD patterns of HA coating deposited with different proportioning electrolytes (a) 0 mol/L, (b) 2.5 × 10−4 mol/L, (c) 5 × 10−4 mol/L, (d) 7.5 × 10−4 mol/L, (e) 1 × 10−3 mol/L.
50μm
10μm
2Na solution (0, 2.5 × 10−4, 5 × 10−4, 7.5 × 10−4, and 1 × 10−3 mol/L). According to the references [19–20], the pH value of the electrolyte was adjusted to 4.5. The Ti6Al4V plate and a platinum plate served as the cathode and the anode, respectively. The values of electrolyte temperature, deposition time, current density, and the stirring speed were set to 120°C, 120 min, 1.25mA/cm2, and 100r/min, respectively.
c (d2)
(d1)
2.3. Cell culture and adhesion assay
50μm
MC3T3-E1 osteoblast cells (Cell Bank, The Chinese Academy of Sciences) were selected as well-characterized models for morphological analysis. Osteoblasts are critical in regulating bone metabolism during osteogenesis, differentiation, and normal physiology. The cells were cultured in an alpha-MEM cell culture medium containing 10% fetal bovine serum (FBS; Gibco), penicillin (100 U/mL; Gibco), and streptomycin (100 mg/mL; Gibco) at 37 °C under 5% CO2. The cells were detached using 0.25% trypsin/1 mmol/L EDTA·4Na (Gibco) to prevent contact inhibition when the cells reached 80% confluence. The Ti6Al4V-HA coating samples were sterilized and then placed in the 24- well plate. A total of 1 × 104 MC3T3-E1 cells in 100 μL was placed on the surface of each sample. After incubating for 24 h, the samples were rinsed thrice with 1 mL of PBS buffer to remove the excess water. The cells were fixed for 3 h with 2.5% glutaraldehyde solution. And then the samples were washed with different concentrations of ethanol solution (30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%) for 2 × 15min.
10μm d
(e1)
(e2)
50μm
10μm e
2.4. Surface characterization A Quanta FEG-450 field emission scanning electron microscope 2
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tensile tests were observed using FESEM with an energy dispersive spectrometer (EDS).
Table 1 Crystallinity vs. EDTA-2Na concentration. Concentration (x10−4 mol/L)
0
2.5
5.0
7.5
10
Crystallinity (%)
62.16
63.39
81.97
82.76
61.41
3. Results and discussion Fig. 1 displays the microstructures of the HA coatings deposited with different EDTA-2Na concentration in the electrolyte. The results reveal that all the HA coatings exhibit layered structure and the microstructures are obviously dependent on the EDTA-2Na. In the absence of EDTA-2Na, the HA crystals of the first layer show needle- or rod-like shape, not excessively dense, and upward perpendicular to the substrate, as shown in Fig. 1(a). The HA crystals of the first layer, in Figs. 1(b)-(e), are flower-like and no longer grew preferentially along the C axis. The HA coating gradually became denser and the HA crystal became wider with the increase of EDTA-2NA concentration. By contrast, the second layer became sparser, and the microstructure of the second layer only slightly changed. The HA crystal has two growth units, namely, Ca-P6O24 as a growth unit along c axis and OH-Ca6 as the growth unit along the a and b faces [21]. HA belongs to hexagol structure. The spiral structure along the c axis can be easily formed. The reason is that the growth element along its c axis showed a circular distribution, which is characteristic of the screw dislocation growth pattern. However, the smooth surface growth pattern will appear when the OH-Ca6 growth unit does not own the required spiral growth ring structure. When the supersaturation of electrolyte solution is lower, the growth rate of the screw dislocation growth pattern is faster than that of the smooth surface growth pattern, resulting in needle- or rod-like shape HA crystals. Therefore, the HA crystals obtained in the absence of EDTA-2Na are rod-like shape. After the addition of EDTA-2Na, given its chelating effect on Ca2+, the supersaturation of the electrolyte solution increased, and leading to the increase of the OH-Ca6 unit growth rate and the reduction of the CaP6O24 unit growth rate. The reduction in preferential growth along the c axis caused the HA to achieve proportionable growth on all crystal faces, as shown in the Fig. 1(d) and (e). The higher the concentration of EDTA-2Na in the electrolyte is, the more Ca-EDTA chelates form between EDTA-2Na and Ca2+, which results in the decrease of free Ca2+ concentration in the solution and inhibits further growth of HA crystals. Thus, bonding effect between the ion and growth unit of the HA crystal face will be influenced by this. According to the reference [22], the binding ability of EDTA-2Na varies in different crystal surfaces. It has strong binding capacity with the (100) or (010) faces, and a weak one with the (001) face. Thus, a change in the concentration of Ca2+ supersaturation and ion energy deviation of the crystal plane affects the growth rate ratio of the (100) and (001) faces changed, resulting in a fibrous HA crystal. Fig. 2 depicts the XRD patterns of the HA coatings deposited by hydrothermal-electrochemical deposition with different proportioning electrolytes. The patterns indicated that HA coating is obtained referring to the JCPDS cards No. 09-0432 [23]. Comparison of the Fig. 2(a)–(e) revealed that the intensities of the (002) plane decrease with the increase of the EDTA-2Na contents. Moreover, the peaks of the three crystal planes, namely, (211), (112) and (300), were very sharp, which displays the HA coating with high crystallinity [24]. The reference [25] stated that the high crystallinity coating showed the higher shear strengths and remained integration, whereas the separation of the coating fragments was clearly observed in the coating with low crystallinity. The reference [26] stated that low crystallinity coating exhibited a faster degradation rate. The degradation products of the coating stimulate the reaction of some cells and proteins in vivo, and induce more new bone forms. So that the right crystallinity is needed to the HA coating. The HA crsytallinity, dependent upon the arrangement of the ions deposited on the substrate, was evaluated using the following relationship (1) [27]:
Fig. 3. Thickness of HA coating vs. EDTA-2Na concentration (a) 0 mol/L, (b) 2.5 × 10−4 mol/L, (c) 5 × 10−4 mol/L, (d) 7.5 × 10−4 mol/L, (e) 1 × 10−3 mol/L.
Fig. 4. Bonding strength between the HA coating and the substrates vs. EDTA2Na concentration.
(FESEM, Quatan 450, FEI, Holland) was used to observe the microstructures of the HA coating deposited by hydrothermal-electrochemical deposition method, and the MC3T3-E1 osteoblast cell. An Xray diffractometer (XRD, D8-Advanced, Bruker, Germany) was utilized to examine the phase composition of the coating. The thickness of the HA coating was measured by a step profiler (AMBIOS Technology Inc., XP-1). The bonding strength between the substrate and the HA coating was tested by performing the standard tensile adhesion test (GB 23101.4-2008/ISO 13779-4:2002) with a 50 kN universal testing machine (Zwick, USA). The SEM micrographs of HA coating after adhesive
X C ≈ 1 − (V112/300/I300) 3
(1)
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Fig. 5. Fractographs and EDS patterns of the sample after bonding strength test (a) and (b) EDS on the different parts.
by the data in Fig. 1. That is, the needle- or rod-like HA were upward perpendicular to the substrate when the electrolyte solution lacked EDTA-2Na. Thus, the thickness of the coating increased rapidly. With the increase of the EDTA-2Na content, the microstructure and the density changed, and the HA became more compact. In the other words, an equal growth probabilities is present for the different crystal plane. Therefore, the growth area on the matrix increased, the film tended to spread parallel to matrix, and the thickness decreased. When the EDTA2Na content was excessively large, which resulted in a high solution saturation, the free ions in the solution formed the precursor HA by precipitating spontaneously before they were exposed to the surface of the substrate. The calcium and phosphate ion concentration of the electrode surface decreased, and the deposition rate also decreased. Consequently, the HA coating became thinner. Fig. 4 shows the bonding strength between the HA coating and the
In Eq. (1), I300 is the intensity of the (300) reflection peak, and V112/ is the intensity of the hollow area between the (112) and (300) reflection peaks, which completely disappears in non-crystalline samples. As shown in Table 1, the crystallinity of the HA coating was obviously dependent on EDTA-2Na concentration. The HA deposited with 7.5 × 10−4 mol/L in the electrolyte had the highest crystallinity, which is 82.76. When the EDTA-2Na was 10 × 10−4 mol/L, the crystallinity decreased. The reason maybe is that the excessive concentration of complexing agent has, the more stable the complex formed, resulting in the lower concentration of Ca2+ in the solution, thus resulting in a lower crystallinity of HA. Fig. 3 exhibits the relationship of the thickness of HA coating vs. the EDTA-2Na concentration. The thickness gradually decreased with increased EDTA-2Na concentration. This phenomenon can be explained 300
4
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20μm
20μm
20μm
20μm
20μm Fig. 6. SEM micrographs of HA coating after cell adhesion test (a) 0 mol/L, (b) 2.5 × 10−4 mol/L, (c) 5 × 10−4 mol/L, (d) 7.5 × 10−4 mol/L, (e) 1 × 10−3 mol/L.
deposited with or without the EDTA-2Na in the electrolyte had good biocompatibility. Comparison of the five images reveals that the cell in image (a) is smaller than those of in the other four images, indicating that the HA coating deposited with the EDTA-2Na in the electrolyte owns better biocompatibility. The cell in image (d) is polygonal and the largest, and the pseudo-foot is filamentous and flaky, implying that the samples deposited with 7.5 × 10−4 mol/L EDTA-2Na in the electrolyte own the best biocompatibility. The cell culture test indicated that the HA coating microstructure shown in Fig. 1(d) benefitted the adhesion of cell onto the HA surface.
substrates. As shown, the bonding strength increased with the increased EDTA-2Na concentration, reaching the maximum value of 16.8 MPa when the EDTA-2Na content was 7.5 × 10−4 mol/L. In comparison, the value for the HA coatings deposited without EDTA-2Na was about 4 MPa. As shown in Fig. 1(c) and (d), the obtained coating was uniform and compact, thus, the bonding strength increased. When the EDTA2Na concentration increased to more than 7.5 × 10−4 mol/L, the bonding strength decreased. The reason is that an excessive EDTA-2Na concentration resulted in more stable complexes with Ca2+. In addition, the concentration of Ca2+ in the solution decreased during the deposition process, changing the HA crystal morphology and reducing the bonding strength. In summary, the addition of EDTA-2Na in the appropriate concentration range can improve the uniformity of the HA coating and its bonding strength with the titanium substrates. Fig. 5 displays the fractographs and the EDS patterns of the HA sample deposited with 7.5 × 10−4 mol/L EDTA-2Na in the electrolyte. The results revealed a mixed failure mode with adhesive and cohesive. Adhesive failure occurred in the HA coating and the substrate interface, whereas cohesive failure occurred in the HA coatings. This finding indicates that the measured bonding strength was a combination of adhesive and cohesive strength. In generally, adhesive conditions may be affected by the coating structure, residual stress and surface roughness of substrate. A smaller area that fails cohesively results in a higher bonding strength of the HA coating. Cohesive strength is mainly determined by the coating structure, such as porosity, crack and microscopic shape, etc. In the research, all of the HA coatings were deposited on the substrates with the same treatment, suggesting that the main factors affecting the bonding strengths were the parameters during the hydrothermal electrochemical deposition process. The results of bonding strength test imply that the sample deposited with 7.5 × 10−4 mol/L EDTA-2Na in the electrolyte displayed higher cohesive and adhesive strengths. Fig. 6 shows the SEM images of cells culture on the samples for 3 days. The results showed that the cells had different morphologies and sizes on the surfaces of the HA coatings, indicating that all HA coating
4. Conclusions The results of the research displayed that the EDTA-2Na concentration in the electrolyte effected the microstructure, the crystallinity, the thickness of the HA coating, and the bonding strength between the substrate and the HA coating. The specific conclusions of the study are as follows: (1) The addition of EDTA-2Na to the electrolyte did not change the phase composition of the HA coating deposited by hydrothermalelectrochemical deposition method. The chelating effect of EDTA2Na on Ca2+ changed the supersaturation of the electrolyte solution, such that the HA crystal morphologies change obviously. (2) The HA deposited with 7.5 × 10−4 mol/L in the electrolyte had the highest crystallinity, which is 82.76. (3) The thickness of the HA coating decreased gradually with increased EDTA-2Na concentration. (4) The bonding strength between HA coating and the substrate initially increased with the increase of EDTA-2Na concentration and subsequently decreased, reaching the maximum value with 16.8 MPa when the EDTA-2Na content was 7.5 × 10−4 mol/L. The cell culture and adhesion test showed that the samples deposited with 7.5 × 10−4 mol/L EDTA-2Na in the electrolyte exhibited the best biocompatibility. 5
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Acknowledgements
461–467. [13] P. Mani, K. Manikandan, J.J. Prince, Influence of molar concentration on ethylene diamine tetra acetic acid (EDTA) added to tin sulfide (SnS) thin films grown by silar method, J. Mater. Sci. Mater. Electron. 28 (2017) 1–11. [14] C. Shang, W. Hong, Y. Guo, J. Wang, E. Wang, Water-based synthesis of palladium trigonal bipyramidal/tetrahedral nanocrystals with enhanced electrocatalytic oxidation activity [J], Chemistry 23 (2017). [15] H.B. Zhang, K.C. Zhou, Z.Y. Li, S.P. Huang, Y.Z. Zhao, Morphologies of hydroxyapatite nanoparticles adjusted by organic additives in hydrothermal synthesis, J. Cent. S. Univ. Technol. 16 (2009) 871–875. [16] D.D. Zhao, R.X. Sun, K.Z. Chen, Influence of different chelating agents on corrosion performance of microstructured hydroxyapatite coatings on AZ91D magnesium alloy, J. Wuhan Univ. Technol. 32 (2017) 179–185. [17] N. Ohtsu, S. Hiromoto, M. Yamane, K. Satoh, M. Tomozawa, Chemical and crystallographic characterizations of hydroxyapatite- and octacalcium phosphate coatings on magnesium synthesized by chemical solution deposition using XPS and XRD, Surf. Coat. Technol. 218 (2013) 114–118. [18] M.L. Montero, A. Sáenz, J.G. Rodríguez, J. Arenas, V.M. Castaño, Electrochemical synthesis of nanosized hydroxyapatite, J. Mater. Sci. 41 (2006) 141–2144. [19] C.B. Mao, H.D. Li, F.Z. Cui, Q.L. Feng, C.L. Ma, The functionalization of titanium with EDTA to induce biomimetic mineralization of hydroxyapatite, J. Mater. Chem. 9 (1999) 2573–2582. [20] X.L. Jia, C.J. He, R. Xiong, X.F. Pang, Effect of disodium ethylenediaminetetraacetate on hydrOxyapatite electrodeposition on nickel-titanium alloy surface, Electroplating & Finishing 31 (2012) 76–78. [21] H.B. Zhang, Study on Controlled Synthesis of Hydroxyapatitie Crystal/particle under Hydrothermal Condition [D], Central South University, 2011. [22] Y. Jiang, Effect of Organic Modifiers on the Electrochemical Deposition of Nanohydroxyapatite [D], Zhejiang University, 2009. [23] L.C. Zhao, C.X. Cui, X. Wang, S.J. Liu, S.J. Bu, Q.Z. Wang, Y.M. Qi, Corrosion resistance and calcium — phosphorus precipitation of micro-arc oxidized magnesium for biomedical applications [J], Appl. Surf. Sci. 330 (2015) 431–438. [24] Y.J. Wang, C.Y. Ning, Z.Z. Zhao, Phase transition and structural stability of hydroxyapatite in bioactive gradient coatings [J], J. Chin. Ceram. Soc. 26 (1998) 655–661. [25] W.C. Xue, S.Y. Tao, X.Y. Liu, In vivo evaluation of plasma sprayed hydroxyapatite coatings having different crystallinity, Biomaterials 25 (2004) 415–421. [26] M. Nagano, T. Nakamura, T. Kokubo, M. Tanahashi, M. Ogawa, Differences of bone bonding ability and degradation behaviour in vivo between amorphous calcium phosphate and highly crystalline hydroxyapatite coating, Biomaterials 17 (1996) 1771–1777. [27] E. Landi, A. Tampieri, G. Celotti, S. Sprio, Densification behavior and mechanisms of synthetic hydroxyapatites, J. Eur. Ceram. Soc. 20 (2000) 2377–2387.
The authors would like to acknowledge the financial supports provided by the “Shanghai Municipal Natural Science Foundation” under Grant No. 15ZR1428300 sponsored by Shanghai Municipal Science and Technology Commission. References [1] M. Niinomi, C.J. Boehlert, Titanium alloys for biomedical applications, in: M. Niinomi, T. Narushima, M. Nakai (Eds.), Advances in Metallic Biomaterials. Springer Series in Biomaterials Science and Engineering, 3 Springer, Berlin, Heidelberg, 2015. [2] T. Yamamuro, Patterns of osteogenesis in relation to various biomaterials, J Jpn Soc Biomater 7 (1989) 19–23. [3] M. Niinomi, M. Nakai, J. Hieda, Development of new metallic alloys for biomedical applications, Acta Biomater. 8 (2012) 3888–3903. [4] R.I. Asri, W.S. Harun, M.A. Hassan, S.A. Ghani, Z. Buyong, A review of hydroxyapatite-based coating techniques: sol-gel and electrochemical depositions on biocompatible metals, J. Mech. Behav. Biomed. Mater. 57 (2016) 95–108. [5] A.C. Hee, Wear and Corrosion Resistance of Tantalum Coating on Titanium Alloys for Biomedical Implant Applications [D], University of Wollongong, 2017. [6] K. Savino, M.Z. Yates, Thermal stability of electrochemical-hydrothermal hydroxyapatite coatings, Ceram. Int. 41 (2015) 8568–8577. [7] S.R. Paital, N.B. Dahotre, Calcium phosphate coatings for bio-implant applications: materials, performance factors, and methodologies, Mater. Sci. Eng. R 66 (2009) 1–70. [8] S. Bana, J. Hasegawa, Morphological regulation and crystal growth of hydrothermal electrochemically deposited apatite, Biomaterials 23 (2002) 2965–2972. [9] D.H. He, P. Liu, X.K. Liu, F.C. Ma, X.H. Chen, W. Li, J.D. Du, P. Wang, J. Zhao, Characterization of hydroxyapatite coatings deposited by hydrothermal electrochemical method on NaOH immersed Ti6Al4V, J. Alloys Compd. 672 (3) (2016) 336–343. [10] J.D. Du, X.K. Liu, D.H. He, P. Liu, F.C. Ma, Q. Li, N.N. Feng, Influence of alkali treatment on Ti6Al4V alloy and the HA coating deposited by hydrothermal-electrochemical methods, Rare Metal Mater. Eng. 43 (2014) 0830–0835. [11] D.H. He, P. Wang, P. Liu, X.K. Liu, F.C. Ma, J. Zhao, HA coating fabricated by electrochemical deposition on modified Ti6Al4V alloy, Surf. Coat. Technol. 277 (1) (2015) 203–209. [12] D.H. He, P. Wang, P. Liu, X.K. Liu, F.C. Ma, W. Li, X.H. Chen, J. Zhao, H. Ye, Preparation of hydroxyapatite-titanium dioxide coating on Ti6Al4V substrates using hydrothermal-electrochemical method, J. Wuhan Univ. Technol. 31 (2016)
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Contents lists available at ScienceDirect
Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat
Influence of EDTA-2Na on the hydroxyapatite coating deposited by hydrothermal-electrochemical method on Ti6Al4V surface ⁎
Daihua He , Jing Du, Ping Liu, Xinkuan Liu, Xiaohong Chen, Wei Li, Ke Zhang, Fengcang Ma School of Materials Science and Engineering, University of Shanghai for Science and Technology, No. 516 Jungong Road, Shanghai 200093, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Ti6Al4V Ethylenediamine Tetraacetic Acid Disodium salt (EDTA-2Na) Hydroxyapatite coating Hydrothermal-electrochemical
The influences of the Ethylenediamine Tetraacetic Acid Disodium salt (EDTA-2Na) on the hydroxyapatite (HA) coating deposited by hydrothermal-electrochemical methods on the Ti6Al4V surface were investigated. The morphology of the HA crystals of the first layer changed from needle- or rod-like to flowerlike. The HA crystal became wider and the first layer became denser gradually, whereas the second layer became sparser. The thickness of the HA coating gradually decreased with the increase of EDTA-2Na concentration in the electrolyte. The bonding strength between coating and substrate reached the maximum of 16.8 MPa when the EDTA-2Na concentration was 7.5 × 10−4 mol/L. Cell-culture test indicated that the HA coating with 7.5 × 10−4 mol/L EDTA-2Na benefits the adhesion of cell onto the HA surface.
1. Introduction Titanium and its alloys have been widely used as biomedical implant materials due to their low density, good mechanical properties, superior corrosion resistance and biocompatibility compared with those of other metallic biomaterials such as CoeCr alloys and stainless steels [1–4]. However, the biological activity of titanium and its alloy is inadequate, and they display low stability after being implanted in the human body. The use of surface modification to improve their bioactivity and biocompatibility has been paid considerable attention [5–6]. Titanium alloy with hydroxyapatite (HA) coating is a bone implant material that combines the good mechanical properties of titanium alloy and the excellent bioactivity and biocompatibility of synthetic HA [7]. HA coating can be deposited on titanium or titanium alloy through various methods, such as plasma spray, sol-gel method, microarc oxidation, pulsed laser deposition, electrophoresis, hydrothermal-electrochemical method, and so on. The hydrothermal-electrochemical methods is developed on the basis of electrochemical deposition and hydrothermal method [8]. This method offers many advantages: 1) The experimental temperature of this method is higher than that of the hydrothermal method and significantly lower than that of plasma spraying method. 2) The HA coating has a high crystallinity, and no subsequent heat treatment is needed to improve the crystallinity. 3) This method can coat a complex-shaped substrate with a uniform thickness. A series of researches were done by our group about the HA coating deposited by hydrothermal chemical method on titanium alloy
⁎
surface [9–12]. Nonetheless, the uniformity and compactness of the coating should be further improved. EDTA-2Na is a good chelating agent that has a wide range of coordination properties, and it can form stable chelates with nearly all metal ions [13–14]. In the references [15–18] were shown that the EDTA or EDTA-2Na can control the shape and size, and improve the crystallinity of HA. In the present paper, EDTA-2Na was added to the electrolyte, which was applied to deposit the HA coating on the Ti6Al4V surface by the hydrothermal-electrochemical methods and its effect on the coating was intensively investigated. 2. Experimental 2.1. Preparation of titanium alloy samples Commercially available medical Ti6Al4V sheets with dimensions of 20mm × 20mm × 2mm were used. The samples were polished firstly by wet sandpapers, and then cleaned ultrasonically with acetone, ethanol and distilled water in sequence for 10min. 2.2. Deposition of HA coating The hydrothermal–electrochemical deposition experiments were performed in a stainless-steel autoclave with a Teflon liner. The electrolyte contained 0.025mol/L Ca(NO3)2·4H2O、0.015mol/L NH4H2PO4, 0.1 mol/L NaNO3, and different concentrations of EDTA-
Corresponding author. E-mail address: [email protected] (D. He).
https://doi.org/10.1016/j.surfcoat.2018.10.065 Received 4 April 2018; Received in revised form 30 September 2018; Accepted 24 October 2018 0257-8972/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: He, D., Surface & Coatings Technology, https://doi.org/10.1016/j.surfcoat.2018.10.065
Surface & Coatings Technology xxx (xxxx) xxx–xxx
D. He et al.
Fig. 1. SEM micrographs of HA coatings deposited with different EDTA-2Na concentration in the electrolyte (a) 0 mol/L, (a1) × 2000, (a2) × 10,000 (b) 2.5 × 10−4 mol/L, (b1) × 2000, (b2) × 10,000 (c) 5.0 × 10−4 mol/L, (c1) × 2000, (c2) × 10,000 (d) 7.5 × 10−4 mol/L, (d1) × 2000, (d2) × 10,000 (e) 1 × 10−3mol/L, (e1) × 2000, (e2) × 10,000.
(a2)
(a1)
50μm
10μm a (b2)
(b1)
50μm
10μm b
(c1)
(c2)
Fig. 2. XRD patterns of HA coating deposited with different proportioning electrolytes (a) 0 mol/L, (b) 2.5 × 10−4 mol/L, (c) 5 × 10−4 mol/L, (d) 7.5 × 10−4 mol/L, (e) 1 × 10−3 mol/L.
50μm
10μm
2Na solution (0, 2.5 × 10−4, 5 × 10−4, 7.5 × 10−4, and 1 × 10−3 mol/L). According to the references [19–20], the pH value of the electrolyte was adjusted to 4.5. The Ti6Al4V plate and a platinum plate served as the cathode and the anode, respectively. The values of electrolyte temperature, deposition time, current density, and the stirring speed were set to 120°C, 120 min, 1.25mA/cm2, and 100r/min, respectively.
c (d2)
(d1)
2.3. Cell culture and adhesion assay
50μm
MC3T3-E1 osteoblast cells (Cell Bank, The Chinese Academy of Sciences) were selected as well-characterized models for morphological analysis. Osteoblasts are critical in regulating bone metabolism during osteogenesis, differentiation, and normal physiology. The cells were cultured in an alpha-MEM cell culture medium containing 10% fetal bovine serum (FBS; Gibco), penicillin (100 U/mL; Gibco), and streptomycin (100 mg/mL; Gibco) at 37 °C under 5% CO2. The cells were detached using 0.25% trypsin/1 mmol/L EDTA·4Na (Gibco) to prevent contact inhibition when the cells reached 80% confluence. The Ti6Al4V-HA coating samples were sterilized and then placed in the 24- well plate. A total of 1 × 104 MC3T3-E1 cells in 100 μL was placed on the surface of each sample. After incubating for 24 h, the samples were rinsed thrice with 1 mL of PBS buffer to remove the excess water. The cells were fixed for 3 h with 2.5% glutaraldehyde solution. And then the samples were washed with different concentrations of ethanol solution (30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%) for 2 × 15min.
10μm d
(e1)
(e2)
50μm
10μm e
2.4. Surface characterization A Quanta FEG-450 field emission scanning electron microscope 2
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tensile tests were observed using FESEM with an energy dispersive spectrometer (EDS).
Table 1 Crystallinity vs. EDTA-2Na concentration. Concentration (x10−4 mol/L)
0
2.5
5.0
7.5
10
Crystallinity (%)
62.16
63.39
81.97
82.76
61.41
3. Results and discussion Fig. 1 displays the microstructures of the HA coatings deposited with different EDTA-2Na concentration in the electrolyte. The results reveal that all the HA coatings exhibit layered structure and the microstructures are obviously dependent on the EDTA-2Na. In the absence of EDTA-2Na, the HA crystals of the first layer show needle- or rod-like shape, not excessively dense, and upward perpendicular to the substrate, as shown in Fig. 1(a). The HA crystals of the first layer, in Figs. 1(b)-(e), are flower-like and no longer grew preferentially along the C axis. The HA coating gradually became denser and the HA crystal became wider with the increase of EDTA-2NA concentration. By contrast, the second layer became sparser, and the microstructure of the second layer only slightly changed. The HA crystal has two growth units, namely, Ca-P6O24 as a growth unit along c axis and OH-Ca6 as the growth unit along the a and b faces [21]. HA belongs to hexagol structure. The spiral structure along the c axis can be easily formed. The reason is that the growth element along its c axis showed a circular distribution, which is characteristic of the screw dislocation growth pattern. However, the smooth surface growth pattern will appear when the OH-Ca6 growth unit does not own the required spiral growth ring structure. When the supersaturation of electrolyte solution is lower, the growth rate of the screw dislocation growth pattern is faster than that of the smooth surface growth pattern, resulting in needle- or rod-like shape HA crystals. Therefore, the HA crystals obtained in the absence of EDTA-2Na are rod-like shape. After the addition of EDTA-2Na, given its chelating effect on Ca2+, the supersaturation of the electrolyte solution increased, and leading to the increase of the OH-Ca6 unit growth rate and the reduction of the CaP6O24 unit growth rate. The reduction in preferential growth along the c axis caused the HA to achieve proportionable growth on all crystal faces, as shown in the Fig. 1(d) and (e). The higher the concentration of EDTA-2Na in the electrolyte is, the more Ca-EDTA chelates form between EDTA-2Na and Ca2+, which results in the decrease of free Ca2+ concentration in the solution and inhibits further growth of HA crystals. Thus, bonding effect between the ion and growth unit of the HA crystal face will be influenced by this. According to the reference [22], the binding ability of EDTA-2Na varies in different crystal surfaces. It has strong binding capacity with the (100) or (010) faces, and a weak one with the (001) face. Thus, a change in the concentration of Ca2+ supersaturation and ion energy deviation of the crystal plane affects the growth rate ratio of the (100) and (001) faces changed, resulting in a fibrous HA crystal. Fig. 2 depicts the XRD patterns of the HA coatings deposited by hydrothermal-electrochemical deposition with different proportioning electrolytes. The patterns indicated that HA coating is obtained referring to the JCPDS cards No. 09-0432 [23]. Comparison of the Fig. 2(a)–(e) revealed that the intensities of the (002) plane decrease with the increase of the EDTA-2Na contents. Moreover, the peaks of the three crystal planes, namely, (211), (112) and (300), were very sharp, which displays the HA coating with high crystallinity [24]. The reference [25] stated that the high crystallinity coating showed the higher shear strengths and remained integration, whereas the separation of the coating fragments was clearly observed in the coating with low crystallinity. The reference [26] stated that low crystallinity coating exhibited a faster degradation rate. The degradation products of the coating stimulate the reaction of some cells and proteins in vivo, and induce more new bone forms. So that the right crystallinity is needed to the HA coating. The HA crsytallinity, dependent upon the arrangement of the ions deposited on the substrate, was evaluated using the following relationship (1) [27]:
Fig. 3. Thickness of HA coating vs. EDTA-2Na concentration (a) 0 mol/L, (b) 2.5 × 10−4 mol/L, (c) 5 × 10−4 mol/L, (d) 7.5 × 10−4 mol/L, (e) 1 × 10−3 mol/L.
Fig. 4. Bonding strength between the HA coating and the substrates vs. EDTA2Na concentration.
(FESEM, Quatan 450, FEI, Holland) was used to observe the microstructures of the HA coating deposited by hydrothermal-electrochemical deposition method, and the MC3T3-E1 osteoblast cell. An Xray diffractometer (XRD, D8-Advanced, Bruker, Germany) was utilized to examine the phase composition of the coating. The thickness of the HA coating was measured by a step profiler (AMBIOS Technology Inc., XP-1). The bonding strength between the substrate and the HA coating was tested by performing the standard tensile adhesion test (GB 23101.4-2008/ISO 13779-4:2002) with a 50 kN universal testing machine (Zwick, USA). The SEM micrographs of HA coating after adhesive
X C ≈ 1 − (V112/300/I300) 3
(1)
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Fig. 5. Fractographs and EDS patterns of the sample after bonding strength test (a) and (b) EDS on the different parts.
by the data in Fig. 1. That is, the needle- or rod-like HA were upward perpendicular to the substrate when the electrolyte solution lacked EDTA-2Na. Thus, the thickness of the coating increased rapidly. With the increase of the EDTA-2Na content, the microstructure and the density changed, and the HA became more compact. In the other words, an equal growth probabilities is present for the different crystal plane. Therefore, the growth area on the matrix increased, the film tended to spread parallel to matrix, and the thickness decreased. When the EDTA2Na content was excessively large, which resulted in a high solution saturation, the free ions in the solution formed the precursor HA by precipitating spontaneously before they were exposed to the surface of the substrate. The calcium and phosphate ion concentration of the electrode surface decreased, and the deposition rate also decreased. Consequently, the HA coating became thinner. Fig. 4 shows the bonding strength between the HA coating and the
In Eq. (1), I300 is the intensity of the (300) reflection peak, and V112/ is the intensity of the hollow area between the (112) and (300) reflection peaks, which completely disappears in non-crystalline samples. As shown in Table 1, the crystallinity of the HA coating was obviously dependent on EDTA-2Na concentration. The HA deposited with 7.5 × 10−4 mol/L in the electrolyte had the highest crystallinity, which is 82.76. When the EDTA-2Na was 10 × 10−4 mol/L, the crystallinity decreased. The reason maybe is that the excessive concentration of complexing agent has, the more stable the complex formed, resulting in the lower concentration of Ca2+ in the solution, thus resulting in a lower crystallinity of HA. Fig. 3 exhibits the relationship of the thickness of HA coating vs. the EDTA-2Na concentration. The thickness gradually decreased with increased EDTA-2Na concentration. This phenomenon can be explained 300
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20μm
20μm
20μm
20μm
20μm Fig. 6. SEM micrographs of HA coating after cell adhesion test (a) 0 mol/L, (b) 2.5 × 10−4 mol/L, (c) 5 × 10−4 mol/L, (d) 7.5 × 10−4 mol/L, (e) 1 × 10−3 mol/L.
deposited with or without the EDTA-2Na in the electrolyte had good biocompatibility. Comparison of the five images reveals that the cell in image (a) is smaller than those of in the other four images, indicating that the HA coating deposited with the EDTA-2Na in the electrolyte owns better biocompatibility. The cell in image (d) is polygonal and the largest, and the pseudo-foot is filamentous and flaky, implying that the samples deposited with 7.5 × 10−4 mol/L EDTA-2Na in the electrolyte own the best biocompatibility. The cell culture test indicated that the HA coating microstructure shown in Fig. 1(d) benefitted the adhesion of cell onto the HA surface.
substrates. As shown, the bonding strength increased with the increased EDTA-2Na concentration, reaching the maximum value of 16.8 MPa when the EDTA-2Na content was 7.5 × 10−4 mol/L. In comparison, the value for the HA coatings deposited without EDTA-2Na was about 4 MPa. As shown in Fig. 1(c) and (d), the obtained coating was uniform and compact, thus, the bonding strength increased. When the EDTA2Na concentration increased to more than 7.5 × 10−4 mol/L, the bonding strength decreased. The reason is that an excessive EDTA-2Na concentration resulted in more stable complexes with Ca2+. In addition, the concentration of Ca2+ in the solution decreased during the deposition process, changing the HA crystal morphology and reducing the bonding strength. In summary, the addition of EDTA-2Na in the appropriate concentration range can improve the uniformity of the HA coating and its bonding strength with the titanium substrates. Fig. 5 displays the fractographs and the EDS patterns of the HA sample deposited with 7.5 × 10−4 mol/L EDTA-2Na in the electrolyte. The results revealed a mixed failure mode with adhesive and cohesive. Adhesive failure occurred in the HA coating and the substrate interface, whereas cohesive failure occurred in the HA coatings. This finding indicates that the measured bonding strength was a combination of adhesive and cohesive strength. In generally, adhesive conditions may be affected by the coating structure, residual stress and surface roughness of substrate. A smaller area that fails cohesively results in a higher bonding strength of the HA coating. Cohesive strength is mainly determined by the coating structure, such as porosity, crack and microscopic shape, etc. In the research, all of the HA coatings were deposited on the substrates with the same treatment, suggesting that the main factors affecting the bonding strengths were the parameters during the hydrothermal electrochemical deposition process. The results of bonding strength test imply that the sample deposited with 7.5 × 10−4 mol/L EDTA-2Na in the electrolyte displayed higher cohesive and adhesive strengths. Fig. 6 shows the SEM images of cells culture on the samples for 3 days. The results showed that the cells had different morphologies and sizes on the surfaces of the HA coatings, indicating that all HA coating
4. Conclusions The results of the research displayed that the EDTA-2Na concentration in the electrolyte effected the microstructure, the crystallinity, the thickness of the HA coating, and the bonding strength between the substrate and the HA coating. The specific conclusions of the study are as follows: (1) The addition of EDTA-2Na to the electrolyte did not change the phase composition of the HA coating deposited by hydrothermalelectrochemical deposition method. The chelating effect of EDTA2Na on Ca2+ changed the supersaturation of the electrolyte solution, such that the HA crystal morphologies change obviously. (2) The HA deposited with 7.5 × 10−4 mol/L in the electrolyte had the highest crystallinity, which is 82.76. (3) The thickness of the HA coating decreased gradually with increased EDTA-2Na concentration. (4) The bonding strength between HA coating and the substrate initially increased with the increase of EDTA-2Na concentration and subsequently decreased, reaching the maximum value with 16.8 MPa when the EDTA-2Na content was 7.5 × 10−4 mol/L. The cell culture and adhesion test showed that the samples deposited with 7.5 × 10−4 mol/L EDTA-2Na in the electrolyte exhibited the best biocompatibility. 5
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Acknowledgements
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