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Electrophoretic deposition of zinc-substituted hydroxyapatite coatings
Materials Science and Engineering C 39 (2014) 67–72
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Electrophoretic deposition of zinc-substituted hydroxyapatite coatings Guangfei Sun a, Jun Ma a,b,⁎, Shengmin Zhang a,b a b
Advanced Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, Wuhan 430074, PR China Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, PR China
a r t i c l e
i n f o
Article history: Received 15 June 2013 Received in revised form 13 January 2014 Accepted 17 February 2014 Available online 23 February 2014 Keywords: Zinc substitution Hydroxyapatite Electrophoretic deposition n-Butanol Triethanolamine Coatings
a b s t r a c t Zinc-substituted hydroxyapatite nanoparticles synthesized by the co-precipitation method were used to coat stainless steel plates by electrophoretic deposition in n-butanol with triethanolamine as a dispersant. The effect of zinc concentration in the synthesis on the morphology and microstructure of coatings was investigated. It is found that the deposition current densities significantly increase with the increasing zinc concentration. The zinc-substituted hydroxyapatite coatings were analyzed by X-ray diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy. It is inferred that hydroxyapatite and triethanolamine predominate in the chemical composition of coatings. With the increasing Zn/Ca ratios, the contents of triethanolamine decrease in the final products. The triethanolamine can be burnt out by heat treatment. The tests of adhesive strength have confirmed good adhesion between the coatings and substrates. The formation of new apatite layer on the coatings has been observed after 7 days of immersion in a simulated body fluid. In summary, the results show that dense, uniform zinc-substituted hydroxyapatite coatings are obtained by electrophoretic deposition when the Zn/Ca ratio reaches 5%. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Due to good biocompatibility and osteoconductivity, hydroxyapatite (HAP) and calcium phosphate coatings on metallic biomedical implants are widely employed in orthopedic and dental applications, which can significantly improve bone integration and accelerate the formation of biological bonds between bones and metallic implants [1–6]. Among various coating techniques, electrophoretic deposition (EPD) of HAP has attracted lots of interest over the past decade [3,7,8]. Many conductive substrates can be modified by EPD of nanosized HAP, such as carbon rod [9], stainless steel [10,11], titanium [1,12,13] and magnesium alloy [5]. As a starting material, natural HAP extracted from bovine cortical bones has been used to coat stainless steel by EPD, and the resulting coatings are continuous and crack-free [10]. Nowadays, nanosized HAP particles can be facilely fabricated by the wet-chemical methods. The main advantage of using HAP nanoparticles during the EPD process is that the obtained coatings possess dense particle packing and homogeneous structures [3]. The in vivo experiments have demonstrated that little inflammatory reaction has occurred after implanting the HAPcoated titanium, which indicates better compatibility than bare titanium implants [14]. The shape and structure of metallic substrates can be processed before deposition in accordance with the designed requirements. With the aid of perfusion, EPD techniques can also be ⁎ Corresponding author at: Life Science Building, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, PR China. Tel.: + 86 27 87792216; fax: + 86 27 87792205. E-mail address: [email protected] (J. Ma).
http://dx.doi.org/10.1016/j.msec.2014.02.023 0928-4931/© 2014 Elsevier B.V. All rights reserved.
used to deposit calcium phosphate onto porous titanium scaffolds [15]. The porous titanium scaffolds modified by calcium phosphate coatings show high tensile strength and can support the growth of mesenchymal stem cells [2]. EPD techniques can be employed to modify the electrodes with surface patterns and only conductive areas are selectively coated by HAP nanoparticles [16]. Many parameters during EPD play important roles in the final properties of HAP coatings, such as dispersion medium, dispersant in the suspension [17,18] and applied electric field [13,19]. For instance, by varying the applied voltages, EPD can produce HAP coatings with different morphologies [13]. The crystal orientation of (002) plane is predominant on the HAP-coated titanium sheets, and (211) on the HAP-coated titanium rod samples [20]. Some nanoparticles can be inserted or embedded into the HAP coatings, such as carbon nanotubes [21] and Y2O3 nanoparticles [22], and therefore the reinforcement of the coatings is achieved. Electrodeposition is another method for HAP coatings, which is similar to EPD [2,14,23–26]. Electrochemical reactions occur during the electrodeposition process. The starting materials such as phosphate salts and calcium salts are dissolved and the supersaturated solution of calcium phosphate is used to deposit HAP [6]. Because of the mild conditions, some bioactive molecules are able to be embedded in the final coatings during electrodeposition. Via this method, the composite films containing heparin and HAP in the polypyrrole matrix have been fabricated [27]. However, due to the low stability of the supersaturated calcium phosphate solution, the electrodeposition method has its limitations. EPD is more suitable for deposition of biomaterials because EPD is mainly a physical process and is easy to be controlled during the coating process [3].
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It is well-known that natural HAP contains lots of trace elements which play very important roles in the biological process [28–31]. Functional element-substitutions in HAP can be easily fabricated by adding the related ions into the reactions, such as zinc [32], silicon [8] and strontium [4]. Using these element-substituted HAP nanoparticles, special functional HAP coatings can be obtained via the EPD method. The strontium-substituted hydroxyapatite coatings have been found to improve the cell adhesion and proliferation of osteoblast cells and the strontium incorporation greatly inhibits the osteoclast differentiation [4]. The composite coatings containing silicon substituted HAP and poly-(ε-caprolactone) have improved bonding strength and also have the ability to induce new bone formation [8]. Zinc plays very important roles in the bone formation and immune regulations [32,33]. Zinc substituted HAP (ZnHAP) nanoparticles containing 1.6 wt.% Zn have been found to enhance bioactivity to human adipose-derived mesenchymal stem cells and antimicrobial capability [34]. Another research also suggests that ZnHAP nanoparticles have antibacterial activity and are non-toxic to osteoprogenitor cells [35]. The aim of this study is to deposit ZnHAP nanoparticles onto stainless steel substrates by EPD. Microstructure, crystallinity and functional groups of ZnHAP coatings have been characterized. The effects of different concentrations of zinc used in the synthesis have been evaluated and discussed. 2. Materials and methods 2.1. Synthesis of ZnHAP nanoparticles
Fig. 1. Current density during the electrophoretic deposition of ZnHAP nanoparticles with different Zn/Ca ratios in the reaction.
2.3.2. Transmission electron microscopy The dried ZnHAP powders were dispersed in ethanol by sonication for 15 min. A drop of the suspension was applied on the carbon coated copper grids and dried in air. The samples were observed using a transmission electron microscope (TEM, Tecnai G2, FEI, Netherlands). The selected area electron diffraction (SAED) patterns of ZnHAP nanoparticles were studied.
The ZnHAP nanoparticles with different zinc concentrations were synthesized by co-precipitation [32,36,37]. Briefly, 270 ml of 0.11 M Ca(NO3)2 solution was mixed with 0.01 M Zn(NO3)2 solution. Then, 300 ml of 0.06 M (NH4)H2PO4 solution was added to the mixture during stirring. The Ca/P ratio was maintained at 1.65 and the Zn/Ca ratio was adjusted from 1% to 10% by adding different volumes of Zn(NO3)2 solution (30 ml, 150 ml and 300 ml). All the reactions were kept at 80 °C and pH was adjusted to about 9–10 using ammonium solution. After 3 h of aging, the obtained precipitates were separated from the solution by filtration and washed using distilled water. The ZnHAP nanoparticles were dried in 80 °C oven overnight. 2.2. Electrophoretic deposition Herein, 304 stainless steel plates were cut into 20 mm × 20 mm and used as cathode. The carbon rod was used as anode. The steel plates were washed with acetone and ethanol before usage. For EPD, about 0.6 g of ZnHAP powder was dispersed in 60 ml n-butanol containing about 2% triethanolamine (TEA). The suspensions were ultrasonicated for 5 min and aged overnight. The TEA adsorbed onto HAP nanoparticles and made them positively charged [17,18]. Before EPD, 2 drops of HCl were added to the suspension and ultrasonication was applied for 10 min to achieve homogenous dispersion [9]. The stainless steel plate as cathode and the carbon rod as anode were immersed in the ZnHAP suspension. The distance between the two electrodes was 10 mm. The deposition was performed at a constant voltage of 30 V. The currents were recorded during the EPD process. The coatings were gently rinsed with ethanol and dried in 60 °C oven overnight. 2.3. Characterization 2.3.1. X-ray diffraction The dried ZnHAP powders and coatings were analyzed using X-ray diffraction (XRD, D8 Advance, Bruker, Germany). The phases of the samples were determined using a Cu-Kα radiation (40 kV, 40 mA) over the 2θ range of 10°–70° with a step size of 0.1° at 0.1 s.
Fig. 2. X-ray diffraction patterns of (a) ZnHAP powders and (b) ZnHAP coatings on stainless steel plates.
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3. Results
Table 1 Cell parameters of ZnHAP powders. Zn/Ca ratio
a = b (Å)
c (Å)
V (Å3)
1% 5% 10%
9.404 9.392 9.393
6.876 6.868 6.879
526.5 524.7 525.6
2.3.3. Scanning electron microscopy All the samples were coated with a 10 nm Au films for better conductivity. The morphology of the coatings was examined using a scanning electron microscope (SEM, JSM-5610LV, JEOL, Japan). The Zn/Ca ratios of the coatings were measured using the energy dispersive spectrometer (EDS, Phoenix, USA) affiliated to SEM.
2.3.4. Fourier transform infrared spectroscopy For further compositional and structural investigations, Fourier transform infrared (FTIR) spectroscopy was employed running in reflected mode. The coatings of ZnHAP on stainless steel plates were analyzed directly using FTIR with an attenuated total reflectance (ATR) module. The measurements were performed using Nexus (Thermo Nicolet, USA) in the wavenumber range of 600–4000 cm−1 and a resolution of 0.5 cm−1.
2.4. Heat treatment and evaluation 2.4.1. Heat treatment The dried coatings were sintered at 550 °C for 2 h in air. The hot samples were cooled down slowly in the oven to reduce the stress. The obtained samples were examined using XRD and SEM.
2.4.2. In vitro apatite formation The ZnHAP coatings (Zn/Ca = 5%) after sintering were immersed in simulated body fluid (SBF) solution (pH 7.4) at 37 °C for 1 week [18]. The dried coatings without sintering were also treated by SBF. All of the samples were taken out and gently washed in water. The samples were dried in an oven of 60 °C overnight and observed using SEM.
2.4.3. Adhesive strength For the adhesive strength tests, an epoxy resin was used to glue the coatings to another piece of stainless steel plate [38]. After the resin was cured, the interfacial shear strength of the coatings on the substrates was evaluated using a universal testing machine (AG-100KN, Shimazu, Japan) with a 100KN load cell. The tensile speed was 1.0 mm/min. The bonding strength was calculated from the maximum drawing force over the fractured area.
3.1. Deposition current density The ZnHAP nanoparticles have been deposited directly from a stable colloidal suspension under a constant electric field (30 V/cm). After aging overnight, TEA is considered to adsorb on the surface of ZnHAP nanoparticles and the particles are positively charged in the acidic condition made by adding HCl. Driven by the electrophoretic force, the charged ZnHAP nanoparticles move towards the cathode and are packed onto the electrode surface. According to Fig. 1, with increasing zinc concentration, the deposition current density increases significantly. The current density of the sample Zn/Ca = 1% is about 1.1 mA/cm2. For the sample of Zn/Ca = 5%, the current density increases to about 1.5 mA/cm2. The sample of Zn/Ca = 10% has the highest current density which reaches 2.4 mA/cm2. It is noticed that all the currents decrease during the deposition process. It is suggested that the zinc incorporation increases surface charges of ZnHAP nanoparticles, and therefore the deposition currents significantly increase. On the other hand, the zinc substitution in HAP is suggested to be able to improve the efficiency of EPD.
3.2. Phase identification by XRD patterns Fig. 2(a) shows the XRD patterns of the ZnHAP powders with various zinc concentrations. All three samples result in the same diffraction peaks only corresponding to the hydroxyapatite phase (standard card of hydroxyapatite, JCPDS 09-0432). The zinc substitution limit is considered to be about 15% of the Zn/Ca ratio [36]. It is inferred that all the obtained powders in this study are predominantly HAP phase. The broadened peaks could be caused by the nanosized crystals and low crystallinity. With increasing zinc concentration, the XRD peaks do not change obviously. However, the previous results suggested that higher zinc concentration was found to lead to lower crystallinity and broader XRD peaks [36]. The difference is considered to be caused by the constant Ca/P ratios in the synthesis. According to the cell refinement results of the XRD patterns (Table 1), when the Zn/Ca ratio is no more than 5%, the crystal cell of ZnHAP continues to decrease compared to ZnHAP with low concentration substitution [37]. The coatings on the stainless steel plates are different to the powders in the XRD patterns, as shown in Fig. 2(b). The peaks assigned to the HAP phase have not changed after the nanoparticles have been deposited on the plates. The other peaks labeled by the + symbols are inferred to be attributed to the nitrilotriethanol hydrochloride (PDF: 09-0592), which is the reaction product of TEA and HCl. Some of those peaks overlapped with the peaks assigned to HAP, so the peaks at 26° and 31.8° are enhanced. It is observed that the peaks assigned to nitrilotriethanol hydrochloride become the weakest for Zn/Ca = 5% among the three samples. Meanwhile, the peaks at 21.4°, 24.4°, 29.3° and 36.6° have not been observed for the sample of Zn/Ca = 5%. It is considered that
Fig. 3. TEM images of the ZnHAP nanoparticles: (a) Zn/Ca = 1%, (b) Zn/Ca = 5%, and (c) Zn/Ca = 10%. The insets are the selected electronic area diffraction patterns.
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Fig. 4. SEM images of the ZnHAP coatings with different Zn/Ca ratios in the reaction (a, b) Zn/Ca = 1%, (c, d) Zn/Ca = 5%, and (e, f) Zn/Ca = 10%, the inset is the magnification.
the content of nitrilotriethanol hydrochloride decreases obviously in the ZnHAP coating sample of Zn/Ca = 5%. The peaks at 43.5° and 50.7° may also be attributed to the stainless steel substrates [11].
stainless steel plates. According to the magnified image of the inset in Fig. 4(f), the particle size of ZnHAP is smaller than 200 nm. According to our previous study, the resulted ZnHAP nanoparticles have rod-like shapes via the co-precipitation method [37]. The size of the particles
3.3. Morphology of nanoparticles and coatings Fig. 3(a)–(c) shows the morphology of the as-prepared ZnHAP nanoparticles with different zinc concentrations. For the sample of Zn/ Ca = 1%, the nanoparticles have plate-like shapes. When the Zn/Ca ratio increases to 5%, the particles become rod-like shapes with a high aspect ratio. However, the ZnHAP nanoparticles become smaller when the Zn/Ca ratio increases to 10% in the precipitation. The zinc ions are suggested to inhibit the growth of the crystals. In addition, the SEAD patterns of the nanoparticles all correspond to HAP, which are same as the above XRD results. The morphology of the ZnHAP coatings was studied by SEM, as shown in Fig. 4. For the coating sample of Zn/Ca = 1%, the ZnHAP nanoparticles aggregate into micro-particles and homogenously distribute on the surface. Due to the detection limitation, no zinc was found by EDS for the sample of Zn/Ca = 1%. When the Zn/Ca ratio increases to 5%, the aggregations decrease significantly and the coatings become more homogenous with higher packing density. The EDS results suggest that the Zn/Ca ratio reaches 5.7% similar to the ratio in the synthesis. For the sample of Zn/Ca = 10%, the packing density increases further and less aggregated particles have been observed on the surface of the
Fig. 5. FTIR spectra of the ZnHAP coatings with different Zn/Ca ratios in the reactions.
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Fig. 6. X-ray diffraction pattern of the ZnHAP (Zn/Ca = 5%) coating on the stainless steel plates after sintering.
decreases with the increase of Zn/Ca ratios [36]. However, the EDS measurements indicate that the Zn/Ca ratio is only 4.6% for the ZnHAP sample of Zn/Ca = 10%. It seems that the zinc concentration has reached the limit value in this condition. From the above results, it is obvious that higher Zn/Ca ratio results in higher packing density in the coatings of EPD and that the concentration of zinc substitution has been limited to about 5%. In addition, the packing density of ZnHAP using n-butanol as solvent seems higher than that of HAP using isopropanol [39]. 3.4. Functional groups by FTIR Fig. 5 shows the FTIR spectra of the ZnHAP coatings on the stainless steel plates. For the sample of Zn/Ca = 10%, the FTIR spectrum is almost the same to the spectrum of pure HAP (data not shown). The bands assigned to the hydroxyl stretching and bending modes are at 3570 and 633 cm−1, respectively, which are possibly caused by the hydroxyl groups in HAP or TEA [36,39]. The bands at 1092 cm−1 and 1030 cm−1 are attributed to the phosphate groups. The peak intensity at 1030 cm−1 increases when the zinc concentration increases. The band at 3311 cm − 1 is considered to be caused by adsorbed water. When zinc concentration increases, the band moves to 3309 cm − 1 and finally disappears when Zn/Ca = 10%. The bands at 2850–2940 cm−1 assigned to sp3 C–H stretching and the band at 917 cm−1 assigned to C–H bending disappear when zinc concentration increases. The bands at 1067 and 1080 cm− 1 may be caused by the TEA–HCl salts, which become weaker or even disappear when the zinc concentration increases. For Zn/Ca = 1%, the bands of carbonate groups are observed at 1458 and 1401 cm−1, which suggest that some of the phosphate groups are replaced by carbonate. For Zn/ Ca = 5%, the bands of the carbonate groups move to 1460 and 1403 cm−1. For higher zinc concentration (Zn/Ca = 10%), these bands
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disappear. The band at 1488 cm−1 assigned to the N–H bending mode also shows the similar trends. The incorporation of zinc into the HAP coatings changes the bands assigned to TEA. The results indicate that there are strong competitions between zinc ions and TEA, which decrease the related absorption bands. Combined with the XRD results, TEA forms nitrilotriethanol hydrochloride with HCl during EPD, which adsorbs on the surface of ZnHAP nanoparticles through the strong hydrogen bonding with the exposed P–OH groups [39]. It is observed the band at 962 cm− 1 assigned to the PO34 − symmetric stretching mode (ν1) becomes stronger due to the decreased hydrogen bonding with the HAP minerals. According to the SEM images, porous incompact surface is only observed on the ZnHAP coating sample of Zn/Ca = 1%. The other two samples are smooth and the nanosized particles are highly packed. The disappearance of peaks assigned to TEA is considered to be related to the morphology changes. Another possible reason for the disappearance is that the increased zinc concentration results in less P–OH groups on the surface, and as a result, less TEA molecules are needed to form bonds with the phosphate groups on the surface of ZnHAP. 3.5. Heat treatment and in vitro apatite formation The adhesive strength between the sintered ZnHAP coatings (Zn/Ca = 5%) and the stainless steel substrates is 16.9 ± 1.9 MPa. The adhesive strength of the un-sintered samples is 14.2 ± 1.0 MPa, which is a little lower than the sintered samples. Here, the temperature of heat treatment is lower than that used in the previous literatures [40]. After the heat treatment, there are no peaks attributed to the nitrilotriethanol hydrochloride salts shown in the XRD patterns (Fig. 6). All of the XRD peaks are assigned to the HAP phase. The peak at 44.48° becomes stronger which indicates that the (400) crystal plane becomes more on the surface of the coatings after the heat treatment. From the SEM images of the ZnHAP nanoparticles on the coatings (Fig. 7(a)), it is observed that the particles are still rod-like and some of them begin to fuse together. After 7 days of immersion in SBF solution at 37 °C, a new apatite coating was formed on the top surface of the coatings, as presented in Fig. 7(b). The apatite nanoparticles have needle-like shapes with a width of 20–40 nm. The formation of new apatite layer has been confirmed from the scratched position where the morphology is similar to that in Fig. 7(a). When the coatings were not sintered, the formation of apatite layer also occurred similarly on the surface, as shown in Fig. 7(c). The formation of the apatite layer indicates that the ZnHAP coatings may have good bioactivity, as described in previous publication [8]. 3.6. Formation mechanisms of the coatings Herein, EPD is performed in a two-electrode cell. As shown in Fig. 8, the TEA molecules adsorb on the surface of ZnHAP nanoparticles and the modified nanoparticles are positively charged with the help of HCl
Fig. 7. SEM images of the ZnHAP (Zn/Ca = 5%) coatings: (a) sintered samples, (b) sintered and immersed in SBF for 7 days, and (c) un-sintered and immersed in SBF for 7 days.
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functional ZnHAP coatings with uniform morphology, and high packing density, which would benefit future treatments and applications. Acknowledgements This work was supported by the National Basic Research Program of China (Grant No: 2012CB933601), the National Natural Science Foundation of China (Grant No: 51202075), the National High-Technology Research and Development Program of China (Grant No: 2012AA020503) and the National Key Technology Research and Development Program of China (Grant No: 2012BAI17B02). We are also grateful to the Analytical and Testing Center (HUST). References
Fig. 8. Scheme of the coating process by EPD.
in the solvent. During the EPD process, the nanoparticles are driven by the electrophoretic force and move toward the positive electrode. The formation of coatings is considered to be caused by particle coagulation on the electrode [3]. The size distribution and morphology of the nanoparticles play important roles in the formation of coatings. In this study, it is found that the nanoparticles with rod-like shapes and small size may help the formation of uniform coatings. The size reduction of the particles may also increase the stability of the colloidal solution and benefit the EPD process [3]. Herein, uniform dense coatings of ZnHAP with Zn/Ca = 5% have been prepared by EPD on stainless steel plates. Further studies are needed to improve the properties of the coatings, such as optimizing the TEA contents and combining biocompatible polymers. The TEA–HCl salts in the coatings can be removed by heat treatment or dissolution in water. The previous results suggested that TEA in the EPD coatings could improve the anti-corrosion behavior by decreasing corrosion currents [39]. Biocompatible polymers, such as poly(ε-caprolactone), could be co-deposited with HAP nanoparticles by EPD in n-butanol and chloroform mixture, which could increase the shear strength of the coatings [8]. 4. Conclusions Thick uniform ZnHAP coatings on stainless steel plates were obtained by EPD in n-butanol using TEA as a dispersant. The ZnHAP nanoparticles with different Zn/Ca ratios were prepared by the coprecipitation method. The XRD results reveal that the obtained particles are dominantly HAP phase and the coatings contain phases of HAP and TEA–HCl salts. After sintering at 550 °C in air, the TEA– HCl salts could be burnt out. Moreover, the zinc concentration in the reaction has significant effects on the final coatings due to the morphology changes of the ZnHAP nanoparticles. The results suggest that the synthesis condition of Zn/Ca = 5% can produce
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Electrophoretic deposition of zinc-substituted hydroxyapatite coatings Guangfei Sun a, Jun Ma a,b,⁎, Shengmin Zhang a,b a b
Advanced Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, Wuhan 430074, PR China Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, PR China
a r t i c l e
i n f o
Article history: Received 15 June 2013 Received in revised form 13 January 2014 Accepted 17 February 2014 Available online 23 February 2014 Keywords: Zinc substitution Hydroxyapatite Electrophoretic deposition n-Butanol Triethanolamine Coatings
a b s t r a c t Zinc-substituted hydroxyapatite nanoparticles synthesized by the co-precipitation method were used to coat stainless steel plates by electrophoretic deposition in n-butanol with triethanolamine as a dispersant. The effect of zinc concentration in the synthesis on the morphology and microstructure of coatings was investigated. It is found that the deposition current densities significantly increase with the increasing zinc concentration. The zinc-substituted hydroxyapatite coatings were analyzed by X-ray diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy. It is inferred that hydroxyapatite and triethanolamine predominate in the chemical composition of coatings. With the increasing Zn/Ca ratios, the contents of triethanolamine decrease in the final products. The triethanolamine can be burnt out by heat treatment. The tests of adhesive strength have confirmed good adhesion between the coatings and substrates. The formation of new apatite layer on the coatings has been observed after 7 days of immersion in a simulated body fluid. In summary, the results show that dense, uniform zinc-substituted hydroxyapatite coatings are obtained by electrophoretic deposition when the Zn/Ca ratio reaches 5%. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Due to good biocompatibility and osteoconductivity, hydroxyapatite (HAP) and calcium phosphate coatings on metallic biomedical implants are widely employed in orthopedic and dental applications, which can significantly improve bone integration and accelerate the formation of biological bonds between bones and metallic implants [1–6]. Among various coating techniques, electrophoretic deposition (EPD) of HAP has attracted lots of interest over the past decade [3,7,8]. Many conductive substrates can be modified by EPD of nanosized HAP, such as carbon rod [9], stainless steel [10,11], titanium [1,12,13] and magnesium alloy [5]. As a starting material, natural HAP extracted from bovine cortical bones has been used to coat stainless steel by EPD, and the resulting coatings are continuous and crack-free [10]. Nowadays, nanosized HAP particles can be facilely fabricated by the wet-chemical methods. The main advantage of using HAP nanoparticles during the EPD process is that the obtained coatings possess dense particle packing and homogeneous structures [3]. The in vivo experiments have demonstrated that little inflammatory reaction has occurred after implanting the HAPcoated titanium, which indicates better compatibility than bare titanium implants [14]. The shape and structure of metallic substrates can be processed before deposition in accordance with the designed requirements. With the aid of perfusion, EPD techniques can also be ⁎ Corresponding author at: Life Science Building, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, PR China. Tel.: + 86 27 87792216; fax: + 86 27 87792205. E-mail address: [email protected] (J. Ma).
http://dx.doi.org/10.1016/j.msec.2014.02.023 0928-4931/© 2014 Elsevier B.V. All rights reserved.
used to deposit calcium phosphate onto porous titanium scaffolds [15]. The porous titanium scaffolds modified by calcium phosphate coatings show high tensile strength and can support the growth of mesenchymal stem cells [2]. EPD techniques can be employed to modify the electrodes with surface patterns and only conductive areas are selectively coated by HAP nanoparticles [16]. Many parameters during EPD play important roles in the final properties of HAP coatings, such as dispersion medium, dispersant in the suspension [17,18] and applied electric field [13,19]. For instance, by varying the applied voltages, EPD can produce HAP coatings with different morphologies [13]. The crystal orientation of (002) plane is predominant on the HAP-coated titanium sheets, and (211) on the HAP-coated titanium rod samples [20]. Some nanoparticles can be inserted or embedded into the HAP coatings, such as carbon nanotubes [21] and Y2O3 nanoparticles [22], and therefore the reinforcement of the coatings is achieved. Electrodeposition is another method for HAP coatings, which is similar to EPD [2,14,23–26]. Electrochemical reactions occur during the electrodeposition process. The starting materials such as phosphate salts and calcium salts are dissolved and the supersaturated solution of calcium phosphate is used to deposit HAP [6]. Because of the mild conditions, some bioactive molecules are able to be embedded in the final coatings during electrodeposition. Via this method, the composite films containing heparin and HAP in the polypyrrole matrix have been fabricated [27]. However, due to the low stability of the supersaturated calcium phosphate solution, the electrodeposition method has its limitations. EPD is more suitable for deposition of biomaterials because EPD is mainly a physical process and is easy to be controlled during the coating process [3].
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It is well-known that natural HAP contains lots of trace elements which play very important roles in the biological process [28–31]. Functional element-substitutions in HAP can be easily fabricated by adding the related ions into the reactions, such as zinc [32], silicon [8] and strontium [4]. Using these element-substituted HAP nanoparticles, special functional HAP coatings can be obtained via the EPD method. The strontium-substituted hydroxyapatite coatings have been found to improve the cell adhesion and proliferation of osteoblast cells and the strontium incorporation greatly inhibits the osteoclast differentiation [4]. The composite coatings containing silicon substituted HAP and poly-(ε-caprolactone) have improved bonding strength and also have the ability to induce new bone formation [8]. Zinc plays very important roles in the bone formation and immune regulations [32,33]. Zinc substituted HAP (ZnHAP) nanoparticles containing 1.6 wt.% Zn have been found to enhance bioactivity to human adipose-derived mesenchymal stem cells and antimicrobial capability [34]. Another research also suggests that ZnHAP nanoparticles have antibacterial activity and are non-toxic to osteoprogenitor cells [35]. The aim of this study is to deposit ZnHAP nanoparticles onto stainless steel substrates by EPD. Microstructure, crystallinity and functional groups of ZnHAP coatings have been characterized. The effects of different concentrations of zinc used in the synthesis have been evaluated and discussed. 2. Materials and methods 2.1. Synthesis of ZnHAP nanoparticles
Fig. 1. Current density during the electrophoretic deposition of ZnHAP nanoparticles with different Zn/Ca ratios in the reaction.
2.3.2. Transmission electron microscopy The dried ZnHAP powders were dispersed in ethanol by sonication for 15 min. A drop of the suspension was applied on the carbon coated copper grids and dried in air. The samples were observed using a transmission electron microscope (TEM, Tecnai G2, FEI, Netherlands). The selected area electron diffraction (SAED) patterns of ZnHAP nanoparticles were studied.
The ZnHAP nanoparticles with different zinc concentrations were synthesized by co-precipitation [32,36,37]. Briefly, 270 ml of 0.11 M Ca(NO3)2 solution was mixed with 0.01 M Zn(NO3)2 solution. Then, 300 ml of 0.06 M (NH4)H2PO4 solution was added to the mixture during stirring. The Ca/P ratio was maintained at 1.65 and the Zn/Ca ratio was adjusted from 1% to 10% by adding different volumes of Zn(NO3)2 solution (30 ml, 150 ml and 300 ml). All the reactions were kept at 80 °C and pH was adjusted to about 9–10 using ammonium solution. After 3 h of aging, the obtained precipitates were separated from the solution by filtration and washed using distilled water. The ZnHAP nanoparticles were dried in 80 °C oven overnight. 2.2. Electrophoretic deposition Herein, 304 stainless steel plates were cut into 20 mm × 20 mm and used as cathode. The carbon rod was used as anode. The steel plates were washed with acetone and ethanol before usage. For EPD, about 0.6 g of ZnHAP powder was dispersed in 60 ml n-butanol containing about 2% triethanolamine (TEA). The suspensions were ultrasonicated for 5 min and aged overnight. The TEA adsorbed onto HAP nanoparticles and made them positively charged [17,18]. Before EPD, 2 drops of HCl were added to the suspension and ultrasonication was applied for 10 min to achieve homogenous dispersion [9]. The stainless steel plate as cathode and the carbon rod as anode were immersed in the ZnHAP suspension. The distance between the two electrodes was 10 mm. The deposition was performed at a constant voltage of 30 V. The currents were recorded during the EPD process. The coatings were gently rinsed with ethanol and dried in 60 °C oven overnight. 2.3. Characterization 2.3.1. X-ray diffraction The dried ZnHAP powders and coatings were analyzed using X-ray diffraction (XRD, D8 Advance, Bruker, Germany). The phases of the samples were determined using a Cu-Kα radiation (40 kV, 40 mA) over the 2θ range of 10°–70° with a step size of 0.1° at 0.1 s.
Fig. 2. X-ray diffraction patterns of (a) ZnHAP powders and (b) ZnHAP coatings on stainless steel plates.
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3. Results
Table 1 Cell parameters of ZnHAP powders. Zn/Ca ratio
a = b (Å)
c (Å)
V (Å3)
1% 5% 10%
9.404 9.392 9.393
6.876 6.868 6.879
526.5 524.7 525.6
2.3.3. Scanning electron microscopy All the samples were coated with a 10 nm Au films for better conductivity. The morphology of the coatings was examined using a scanning electron microscope (SEM, JSM-5610LV, JEOL, Japan). The Zn/Ca ratios of the coatings were measured using the energy dispersive spectrometer (EDS, Phoenix, USA) affiliated to SEM.
2.3.4. Fourier transform infrared spectroscopy For further compositional and structural investigations, Fourier transform infrared (FTIR) spectroscopy was employed running in reflected mode. The coatings of ZnHAP on stainless steel plates were analyzed directly using FTIR with an attenuated total reflectance (ATR) module. The measurements were performed using Nexus (Thermo Nicolet, USA) in the wavenumber range of 600–4000 cm−1 and a resolution of 0.5 cm−1.
2.4. Heat treatment and evaluation 2.4.1. Heat treatment The dried coatings were sintered at 550 °C for 2 h in air. The hot samples were cooled down slowly in the oven to reduce the stress. The obtained samples were examined using XRD and SEM.
2.4.2. In vitro apatite formation The ZnHAP coatings (Zn/Ca = 5%) after sintering were immersed in simulated body fluid (SBF) solution (pH 7.4) at 37 °C for 1 week [18]. The dried coatings without sintering were also treated by SBF. All of the samples were taken out and gently washed in water. The samples were dried in an oven of 60 °C overnight and observed using SEM.
2.4.3. Adhesive strength For the adhesive strength tests, an epoxy resin was used to glue the coatings to another piece of stainless steel plate [38]. After the resin was cured, the interfacial shear strength of the coatings on the substrates was evaluated using a universal testing machine (AG-100KN, Shimazu, Japan) with a 100KN load cell. The tensile speed was 1.0 mm/min. The bonding strength was calculated from the maximum drawing force over the fractured area.
3.1. Deposition current density The ZnHAP nanoparticles have been deposited directly from a stable colloidal suspension under a constant electric field (30 V/cm). After aging overnight, TEA is considered to adsorb on the surface of ZnHAP nanoparticles and the particles are positively charged in the acidic condition made by adding HCl. Driven by the electrophoretic force, the charged ZnHAP nanoparticles move towards the cathode and are packed onto the electrode surface. According to Fig. 1, with increasing zinc concentration, the deposition current density increases significantly. The current density of the sample Zn/Ca = 1% is about 1.1 mA/cm2. For the sample of Zn/Ca = 5%, the current density increases to about 1.5 mA/cm2. The sample of Zn/Ca = 10% has the highest current density which reaches 2.4 mA/cm2. It is noticed that all the currents decrease during the deposition process. It is suggested that the zinc incorporation increases surface charges of ZnHAP nanoparticles, and therefore the deposition currents significantly increase. On the other hand, the zinc substitution in HAP is suggested to be able to improve the efficiency of EPD.
3.2. Phase identification by XRD patterns Fig. 2(a) shows the XRD patterns of the ZnHAP powders with various zinc concentrations. All three samples result in the same diffraction peaks only corresponding to the hydroxyapatite phase (standard card of hydroxyapatite, JCPDS 09-0432). The zinc substitution limit is considered to be about 15% of the Zn/Ca ratio [36]. It is inferred that all the obtained powders in this study are predominantly HAP phase. The broadened peaks could be caused by the nanosized crystals and low crystallinity. With increasing zinc concentration, the XRD peaks do not change obviously. However, the previous results suggested that higher zinc concentration was found to lead to lower crystallinity and broader XRD peaks [36]. The difference is considered to be caused by the constant Ca/P ratios in the synthesis. According to the cell refinement results of the XRD patterns (Table 1), when the Zn/Ca ratio is no more than 5%, the crystal cell of ZnHAP continues to decrease compared to ZnHAP with low concentration substitution [37]. The coatings on the stainless steel plates are different to the powders in the XRD patterns, as shown in Fig. 2(b). The peaks assigned to the HAP phase have not changed after the nanoparticles have been deposited on the plates. The other peaks labeled by the + symbols are inferred to be attributed to the nitrilotriethanol hydrochloride (PDF: 09-0592), which is the reaction product of TEA and HCl. Some of those peaks overlapped with the peaks assigned to HAP, so the peaks at 26° and 31.8° are enhanced. It is observed that the peaks assigned to nitrilotriethanol hydrochloride become the weakest for Zn/Ca = 5% among the three samples. Meanwhile, the peaks at 21.4°, 24.4°, 29.3° and 36.6° have not been observed for the sample of Zn/Ca = 5%. It is considered that
Fig. 3. TEM images of the ZnHAP nanoparticles: (a) Zn/Ca = 1%, (b) Zn/Ca = 5%, and (c) Zn/Ca = 10%. The insets are the selected electronic area diffraction patterns.
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Fig. 4. SEM images of the ZnHAP coatings with different Zn/Ca ratios in the reaction (a, b) Zn/Ca = 1%, (c, d) Zn/Ca = 5%, and (e, f) Zn/Ca = 10%, the inset is the magnification.
the content of nitrilotriethanol hydrochloride decreases obviously in the ZnHAP coating sample of Zn/Ca = 5%. The peaks at 43.5° and 50.7° may also be attributed to the stainless steel substrates [11].
stainless steel plates. According to the magnified image of the inset in Fig. 4(f), the particle size of ZnHAP is smaller than 200 nm. According to our previous study, the resulted ZnHAP nanoparticles have rod-like shapes via the co-precipitation method [37]. The size of the particles
3.3. Morphology of nanoparticles and coatings Fig. 3(a)–(c) shows the morphology of the as-prepared ZnHAP nanoparticles with different zinc concentrations. For the sample of Zn/ Ca = 1%, the nanoparticles have plate-like shapes. When the Zn/Ca ratio increases to 5%, the particles become rod-like shapes with a high aspect ratio. However, the ZnHAP nanoparticles become smaller when the Zn/Ca ratio increases to 10% in the precipitation. The zinc ions are suggested to inhibit the growth of the crystals. In addition, the SEAD patterns of the nanoparticles all correspond to HAP, which are same as the above XRD results. The morphology of the ZnHAP coatings was studied by SEM, as shown in Fig. 4. For the coating sample of Zn/Ca = 1%, the ZnHAP nanoparticles aggregate into micro-particles and homogenously distribute on the surface. Due to the detection limitation, no zinc was found by EDS for the sample of Zn/Ca = 1%. When the Zn/Ca ratio increases to 5%, the aggregations decrease significantly and the coatings become more homogenous with higher packing density. The EDS results suggest that the Zn/Ca ratio reaches 5.7% similar to the ratio in the synthesis. For the sample of Zn/Ca = 10%, the packing density increases further and less aggregated particles have been observed on the surface of the
Fig. 5. FTIR spectra of the ZnHAP coatings with different Zn/Ca ratios in the reactions.
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Fig. 6. X-ray diffraction pattern of the ZnHAP (Zn/Ca = 5%) coating on the stainless steel plates after sintering.
decreases with the increase of Zn/Ca ratios [36]. However, the EDS measurements indicate that the Zn/Ca ratio is only 4.6% for the ZnHAP sample of Zn/Ca = 10%. It seems that the zinc concentration has reached the limit value in this condition. From the above results, it is obvious that higher Zn/Ca ratio results in higher packing density in the coatings of EPD and that the concentration of zinc substitution has been limited to about 5%. In addition, the packing density of ZnHAP using n-butanol as solvent seems higher than that of HAP using isopropanol [39]. 3.4. Functional groups by FTIR Fig. 5 shows the FTIR spectra of the ZnHAP coatings on the stainless steel plates. For the sample of Zn/Ca = 10%, the FTIR spectrum is almost the same to the spectrum of pure HAP (data not shown). The bands assigned to the hydroxyl stretching and bending modes are at 3570 and 633 cm−1, respectively, which are possibly caused by the hydroxyl groups in HAP or TEA [36,39]. The bands at 1092 cm−1 and 1030 cm−1 are attributed to the phosphate groups. The peak intensity at 1030 cm−1 increases when the zinc concentration increases. The band at 3311 cm − 1 is considered to be caused by adsorbed water. When zinc concentration increases, the band moves to 3309 cm − 1 and finally disappears when Zn/Ca = 10%. The bands at 2850–2940 cm−1 assigned to sp3 C–H stretching and the band at 917 cm−1 assigned to C–H bending disappear when zinc concentration increases. The bands at 1067 and 1080 cm− 1 may be caused by the TEA–HCl salts, which become weaker or even disappear when the zinc concentration increases. For Zn/Ca = 1%, the bands of carbonate groups are observed at 1458 and 1401 cm−1, which suggest that some of the phosphate groups are replaced by carbonate. For Zn/ Ca = 5%, the bands of the carbonate groups move to 1460 and 1403 cm−1. For higher zinc concentration (Zn/Ca = 10%), these bands
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disappear. The band at 1488 cm−1 assigned to the N–H bending mode also shows the similar trends. The incorporation of zinc into the HAP coatings changes the bands assigned to TEA. The results indicate that there are strong competitions between zinc ions and TEA, which decrease the related absorption bands. Combined with the XRD results, TEA forms nitrilotriethanol hydrochloride with HCl during EPD, which adsorbs on the surface of ZnHAP nanoparticles through the strong hydrogen bonding with the exposed P–OH groups [39]. It is observed the band at 962 cm− 1 assigned to the PO34 − symmetric stretching mode (ν1) becomes stronger due to the decreased hydrogen bonding with the HAP minerals. According to the SEM images, porous incompact surface is only observed on the ZnHAP coating sample of Zn/Ca = 1%. The other two samples are smooth and the nanosized particles are highly packed. The disappearance of peaks assigned to TEA is considered to be related to the morphology changes. Another possible reason for the disappearance is that the increased zinc concentration results in less P–OH groups on the surface, and as a result, less TEA molecules are needed to form bonds with the phosphate groups on the surface of ZnHAP. 3.5. Heat treatment and in vitro apatite formation The adhesive strength between the sintered ZnHAP coatings (Zn/Ca = 5%) and the stainless steel substrates is 16.9 ± 1.9 MPa. The adhesive strength of the un-sintered samples is 14.2 ± 1.0 MPa, which is a little lower than the sintered samples. Here, the temperature of heat treatment is lower than that used in the previous literatures [40]. After the heat treatment, there are no peaks attributed to the nitrilotriethanol hydrochloride salts shown in the XRD patterns (Fig. 6). All of the XRD peaks are assigned to the HAP phase. The peak at 44.48° becomes stronger which indicates that the (400) crystal plane becomes more on the surface of the coatings after the heat treatment. From the SEM images of the ZnHAP nanoparticles on the coatings (Fig. 7(a)), it is observed that the particles are still rod-like and some of them begin to fuse together. After 7 days of immersion in SBF solution at 37 °C, a new apatite coating was formed on the top surface of the coatings, as presented in Fig. 7(b). The apatite nanoparticles have needle-like shapes with a width of 20–40 nm. The formation of new apatite layer has been confirmed from the scratched position where the morphology is similar to that in Fig. 7(a). When the coatings were not sintered, the formation of apatite layer also occurred similarly on the surface, as shown in Fig. 7(c). The formation of the apatite layer indicates that the ZnHAP coatings may have good bioactivity, as described in previous publication [8]. 3.6. Formation mechanisms of the coatings Herein, EPD is performed in a two-electrode cell. As shown in Fig. 8, the TEA molecules adsorb on the surface of ZnHAP nanoparticles and the modified nanoparticles are positively charged with the help of HCl
Fig. 7. SEM images of the ZnHAP (Zn/Ca = 5%) coatings: (a) sintered samples, (b) sintered and immersed in SBF for 7 days, and (c) un-sintered and immersed in SBF for 7 days.
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functional ZnHAP coatings with uniform morphology, and high packing density, which would benefit future treatments and applications. Acknowledgements This work was supported by the National Basic Research Program of China (Grant No: 2012CB933601), the National Natural Science Foundation of China (Grant No: 51202075), the National High-Technology Research and Development Program of China (Grant No: 2012AA020503) and the National Key Technology Research and Development Program of China (Grant No: 2012BAI17B02). We are also grateful to the Analytical and Testing Center (HUST). References
Fig. 8. Scheme of the coating process by EPD.
in the solvent. During the EPD process, the nanoparticles are driven by the electrophoretic force and move toward the positive electrode. The formation of coatings is considered to be caused by particle coagulation on the electrode [3]. The size distribution and morphology of the nanoparticles play important roles in the formation of coatings. In this study, it is found that the nanoparticles with rod-like shapes and small size may help the formation of uniform coatings. The size reduction of the particles may also increase the stability of the colloidal solution and benefit the EPD process [3]. Herein, uniform dense coatings of ZnHAP with Zn/Ca = 5% have been prepared by EPD on stainless steel plates. Further studies are needed to improve the properties of the coatings, such as optimizing the TEA contents and combining biocompatible polymers. The TEA–HCl salts in the coatings can be removed by heat treatment or dissolution in water. The previous results suggested that TEA in the EPD coatings could improve the anti-corrosion behavior by decreasing corrosion currents [39]. Biocompatible polymers, such as poly(ε-caprolactone), could be co-deposited with HAP nanoparticles by EPD in n-butanol and chloroform mixture, which could increase the shear strength of the coatings [8]. 4. Conclusions Thick uniform ZnHAP coatings on stainless steel plates were obtained by EPD in n-butanol using TEA as a dispersant. The ZnHAP nanoparticles with different Zn/Ca ratios were prepared by the coprecipitation method. The XRD results reveal that the obtained particles are dominantly HAP phase and the coatings contain phases of HAP and TEA–HCl salts. After sintering at 550 °C in air, the TEA– HCl salts could be burnt out. Moreover, the zinc concentration in the reaction has significant effects on the final coatings due to the morphology changes of the ZnHAP nanoparticles. The results suggest that the synthesis condition of Zn/Ca = 5% can produce
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