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Electrodeposition of strontium apatite nanorod arrays and their cell compatibility
Author’s Accepted Manuscript Electrodeposition of strontium apatite nanorod arrays and their cell compatibility Shigeo Hori, Minoru Hirano, Riichiro Ohta
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To appear in: Ceramics International Received date: 20 March 2017 Revised date: 22 March 2017 Accepted date: 8 April 2017 Cite this article as: Shigeo Hori, Minoru Hirano and Riichiro Ohta, Electrodeposition of strontium apatite nanorod arrays and their cell compatibility, Ceramics International, http://dx.doi.org/10.1016/j.ceramint.2017.04.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Electrodeposition of strontium apatite nanorod arrays and their cell compatibility Shigeo Hori,*,† Minoru Hirano,*,† and Riichiro Ohta Toyota Central Research and Development Labs. Inc., 41-1 Yokomichi, Nagakute, Aichi 480-1192, Japan * Corresponding author E-mail: [email protected] (S. Hori), [email protected] (M. Hirano) † These authors contributed equally to this work.
Abstract Hydroxyapatites have a biocompatibility that is influenced by their composition and structure. We electrodeposited strontium hydroxyapatite (SrHAp) thin films on Ti substrate and evaluated their cell compatibility for the first time. Electrodeposited thin films have a uniform surface morphology on a scale of micrometers and comprise isolated nanorod-like grains with diameters of 100–200 nm standing on the substrate. High current densities during the electrodeposition were found to produce radial agglomerates of SrHAp rods on nanorod arrays. In comparison with the bare Ti substrate, the SrHAp thin film increased the proliferation and survival (reduced mortality) of the respiratory epithelial cell line. In addition, the levels of expression of bone
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differentiation markers such as runt-related transcription factor 2 (Runx2) and BGLAP genes in an osteoblast cell line significantly increased on the SrHAp thin film compared with those on the bare Ti substrate.
Keywords D: Apatite, A: Films, E: Biomedical applications, Electrodeposition
1. Introduction Hydroxyapatite [M10(PO4)6(OH)2; MHAp; where M = an alkali earth metal] is one of the phosphates with a hexagonal apatite crystal structure and is expected to be applied for phosphor, laser material, and biomaterial depending on its M elements [1]. Calcium hydroxyapatite (CaHAp) is a major inorganic material in human bones and teeth; thus, it has been studied as a biomaterial for bone filling, scaffolds, and surface coating of implants [2-6]. In addition, strontium (Sr)-substituted hydroxyapatite (CaSrHAp) has shown biocompatibility for these procedures to some extent [7,8]. However, to the best of our knowledge, there have been no reports on the synthesis and evaluation of Sr hydroxyapatite (SrHAp) containing no Ca element. At present, plasma spray deposition is the most popular commercialized method for synthesizing MHAp film coatings [3]. However, several problems associated with this process
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still remain. These include the production of deleterious secondary phase, damage to substrate material by plasma, and peeling and mechanical destruction of the film [9]. Precipitation using supersaturated solution [4], sol-gel [5], electrophoretic deposition [6], and electrodeposition has also been reported [10-12]. Among the above methods, electrodeposition is expected to be suitable for coatings on conductive substrates because of the following advantages: (1) reaction can be precisely controlled by electrode potential or current density in addition to common parameters (temperature, concentrations, etc.) and (2) thin films can be obtained with a thickness below a few micrometers and deposited on substrates with complicated surface shapes. In MHAp electrodeposition, electrochemically produced hydroxide ions (OH−) by reduction reaction of hydrogen peroxide [13,14], oxygen [15,16], and nitrate ions [17,18], increase the pH in the vicinity of the electrode, which leads to precipitation of MHAp, as shown in equations 1 and 2. H 2 O 2 2e 2OH
(1)
10M 2 6H2 PO4 14OH M10 PO4 6 OH2
(2)
Electrodeposited CaHAp films generally comprise plate, needle, or rod-like grains, and their inter-grain gaps have been up to several micrometers, resulting in a non-uniform surface morphology on a scale of micrometers, which is comparable to the cell size [12,19-21]. Surface morphology of thin films has been reported to affect biocompatibility [22,23]. Therefore,
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surface morphology and uniformity on a scale of micrometers are factors to be considered when evaluating biocompatibilities of thin films. In this report, SrHAp thin films with a uniform surface morphology on a scale of micrometers are electrodeposited on Ti substrate for the first time and their morphology, structure, deposition behavior, and cell compatibility have been explored.
2. Material and Methods 2.1. Electrodeposition of strontium hydroxyapatite thin films Ti or Ti-sputtered Si (Ti/Si) substrates were employed as working electrode and ultrasonically washed in acetone for 5 min before electrodeposition. Platinum (Pt) wires and potassium chloride (KCl)-saturated Ag/AgCl electrodes were used as counter and reference electrodes, respectively. As a supporting electrolyte, 0.1 M KCl, potassium nitrate (KNO3), tetramethylammonium chloride {[(CH3)4N]Cl; TMACl}, potassium acetate (CH3COOK; AcOK), or ammonium chloride (NH4Cl) were employed. The pH of the electrolyte solution was adjusted to 5.0–6.0 before electrodeposition using conjugated acid or base of each electrolyte. Electrodeposition of SrHAp was performed using the three-electrode system in 20 mL of electrolyte solution with magnetic stirring at 200 rpm. Temperatures of deposition bath (deposition temperature) varied between 65°C, 70°C, and 80°C. Electrode potentials of Ti or
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Ti/Si substrates were maintained at −1.2 V for 5 min and subsequently at −0.8 V for 22 min during SrHAp thin-film deposition. Furthermore, 0.2 mL of 0.5 M H2O2 solution and 0.42 mL of a solution mixture of 0.25 M strontium chloride (SrCl2) and 0.1 M ammonium dihydrogen phosphate (NH4H2PO4) were added to the deposition bath at 4 and 7 min, respectively, after electrodeposition had started. The consequent composition of the electrolyte solution was ca. 0.1 M supporting electrolyte, 5 mM H2O2, 5 mM SrCl2, and 2 mM NH4H2PO4. Obtained SrHAp thin films were analyzed using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), and Fourier transform infrared spectroscopy (FT-IR).
2.2. Cell compatibility evaluation of strontium hydroxyapatite thin films On each of the substrates, a normal respiratory epithelial cell line (BEAS-2B) was plated [(plating density: 1 × 104 cm−2; medium: RPMI-1640 containing 10% fetal bovine serum (FBS)], followed by medium replacement by RPMI-1640 containing 0.5% FBS the next day. Four days after cell plating, fluorescence images of live as well as dead cells were acquired using Live/Dead Cell Staining Kit II (PromoKine) and a fluorescence microscope BZ-9000 (Keyence) that automatically counted live/dead cells using its BZ-II analyzer software (Keyence).
2.3. Gene expression analysis
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On the bare Ti substrate and the substrate on which SrHAp thin films were electrodeposited, an osteoblast cell line (MC3T3-E1) was plated (plating density: 1 × 104 cm−2; medium: -MEM containing 10% FBS), followed by medium replacement by -MEM containing 0% FBS the next day. On the seventh day after the plating, the cells were subjected to total RNA isolation. RNA was used as a template for gene expression analysis using real-time RT-PCR (n = 3).
3. Results and Discussion 3.1. Electrodeposition of strontium hydroxyapatite thin films Figure 1 shows XRD patterns of SrHAp thin films electrodeposited on the Ti substrate at various deposition temperatures in the KCl electrolyte. Peaks assigned to the hexagonal apatite crystal structure appeared in the patterns for all deposition temperatures. Relatively high peak intensities of (00l) and (112) planes and indiscernible peaks of (hk0) planes suggest that the c-axis of SrHAp thin films electrodeposited at 65°C and 70°C tend to be perpendicular to the Ti substrate. In contrast, relatively large peaks of (hk0) planes for SrHAp thin films electrodeposited at 80°C indicate that this film partially contains structures with the c-axis parallel to the Ti substrate. IR spectrum for the SrHAp thin film electrodeposited at 70°C exhibited an absorption for PO43− at 900–1200 cm−1 [24], and a broad weak peak at 800–900 cm−1 (Fig. S1), which would be assigned to HPO42− [25]. Assignment of CO32− (872 cm−1) to the
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broad weak peak should be eliminated because a major peak of CO32− at around 1400–1600 cm−1 was not observed [24]. Morphologies of the SrHAp thin films were observed using SEM, as shown in Figure 2. Pointed nanorod arrays with no cracks were observed for all deposition temperatures. These nanorods have diameters of 100–200 nm and were isolated from each other. The axes of these rods tended to be perpendicular to the Ti surface. Combining with the consideration of XRD patterns of the SrHAp thin films, these rods could be SrHAp crystal grown in the c-axis direction anisotropically. Low-magnification SEM images revealed that SrHAp nanorod arrays have a uniform surface morphology on a scale of micrometers. The influence of variation of deposition temperature on morphologies of the nanorod arrays was not obvious. However, a number of radial agglomerates of thick SrHAp rods were observed on the arrays at a deposition temperature of 80°C. Because these radial agglomerates contained rods with their axes parallel to the substrate, large peak intensities of (hk0) planes in XRD pattern could result from the agglomerates. The composition of SrHAp thin films was evaluated using EDX analysis. Ratio of Sr and P (Sr/P) was determined to be ca. 1.6, which is close to the stoichiometric ratio of 1.67. However, the appearance of HPO42− peak in the IR spectrum indicates the presence of slight Sr deficiencies. Furthermore, a detection of small amount of Cl (< 1 %) suggests partial substitutions of OH− by Cl−. K was not detected.
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To investigate an electrodepositing behavior of SrHAp thin films, current–time relations for various deposition temperatures were compared, as shown in Figure 3. Times of each amperogram were shifted to set the points of minimum cathodic current to be zero. Current transients exhibited three features later by the addition of SrHAp sources (mixture solution of SrCl2 and NH4H2PO4) as follows: (i) rapid current decrease, (ii) peak formation by fast current increase, followed by moderate decrease, and (iii) plateau. These trends were observed for all deposition temperatures. Current transients similar to (ii) and (iii) have been observed for electrodeposition of CaHAp and ZnO, which have been considered to reflect the nucleation behavior [13,26]. The peak current formation in nucleation is generally ascribed to a current increase caused by an increase of reactive sites, i.e., surface area of growing nuclei and a subsequent current decrease due to an overlapping of the three dimensional diffusion zones of reactive material [27,28]. Therefore, a nucleation of SrHAp in our electrodeposition was possibly faster than that of conventional CaHAp electrodeposition, which requires few minutes to form the peak of current [11,12]. In addition, this fast nucleation would be the cause of the uniform surface structure because the fast nucleation will result in a high density nuclei and small differences in growth time of each grain. Based on the comparison of the peak currents among various deposition temperatures, times required to form the peaks were clipped with temperature, which suggests an acceleration of nucleation (inset in Fig. 3).
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A nucleation current transient for metal electrodeposition has been categorized into two types, instantaneous and progressive nucleation models [27]. Current transient for CaHAp electrodeposition has been reported to be an instantaneous behavior for current increase and progressive behavior for current decrease [21]. However, the current transient for SrHAp electrodeposition exhibited a complicated behavior. For a deposition temperature of 70°C, an increase and a decrease in current were accordant to progressive and instantaneous behaviors, respectively, whereas these relations were inverted for a temperature of 80°C (Fig. S2). We noted that there are some inapplicable points in the model for SrHAp electrodeposition; e.g., the model is based on metal deposition and hypothesizing diffusion limitation. However, interestingly, the production of radial agglomerates for deposition temperature of 80°C indicates that new SrHAp nuclei were generated on nanorod arrays over time; this corresponds to the progressive nucleation model. The abovementioned difference in the nucleation behavior caused by temperature is possibly explained by a magnitude of current density. Large current density for a deposition temperature of 80°C can lead a supersaturation of SrHAp high enough to generate nuclei on predeposited SrHAp grains with different crystallographic orientation. Furthermore, current for plateau was also high when progressive nucleation occurred. For electrodeposition of CaHAp, copresent ions (F−, KCl, etc.) in the deposition bath are generally known to affect the structure of CaHAp thin films [10,12,20]. Therefore, the structure
9
of SrHAp thin films were compared using various supporting electrolytes, KNO3, TMACl, AcOK, and NH4Cl, at 0.1 M. Deposition temperature was maintained at 70°C. Figure 4 shows the surface morphology of SrHAp thin films electrodeposited in each electrolyte solution. SrHAp nanorod arrays similar to those obtain in KCl solution were observed for the films obtained in KNO3 and TMACl solutions. In addition, plate-like particles with a width of 200– 300 nm were also found in the thin film obtained in the KNO3 solution. These plate-like particles had larger lengths than those of SrHAp nanorods. In AcOK solution, grain shapes were different from those obtained in other electrolytes. Spatulate grains with a width of ca. 300 nm mixed with nanorods were observed along with large agglomerates of rectangular rod-like grains with a length of ca. 5 μm. These plate-like grains obtained in KNO3 and large agglomerates in AcOK solutions are not expected to be SrHAp but secondary phases whose presence was assumed by unknown peaks that appeared in XRD patterns (Fig. S3). No thin films were obtained in NH4Cl solutions. From the above results, we conclude that the influence of electrolyte on the structure of thin films is due to two factors: grain shape of SrHAp and production of secondary phase. The origin of structure differences in SrHAp thin films caused by electrolyte species is difficult to be determined by the above data. However, the presence of halogen ions that can substitute OH− and differences in pH are possibilities. Halogen ions can act as alternatives to
10
OH− for producing apatite structures, which will reduce secondary phases that would be produced at a lower pH. KCl, TMACl, and KNO3 are expected to almost completely dissociate in solution because dissociation constants (pKa) of their conjugated acids, HCl and HNO3, are small. Therefore, the electrochemically produced OH− can increase the pH in the vicinity of electrodes, resulting in precipitation of SrHAp under higher pH as well as in supersaturation. In contrast, CH3COOH and NH4+ have large pKa of 4.76 and 9.25, respectively [29], indicating a presence of a considerable amount of undissociated acids, which will inhibit an increase in pH by neutralization with OH−. Such undissociated acid concentrations for AcOK and NH4Cl were calculated to be ca. 12 and 100 mM, respectively. Consequently, concentrations of undissociated acids comparable to those of H2O2 and H2PO4 could be expected to lead to the deposition of SrHAp thin films under lower pH in an AcOK solution. In contrast, the concentration of undissociated acid in NH4Cl solution is nearly 20-fold higher than that of H2O2, which can rule out both the increase in pH and deposition of SrHAp thin films.
3.2. Cell-compatible evaluation of strontium hydroxyapatite thin films To investigate biocompatibility of the prepared SrHAp thin films, cell assays using normal respiratory epithelial (BEAS-2B) and osteoblast (MC3T3-E1) cell lines were conducted. The SrHAp thin films used to culture BEAS-2B were prepared on a polished bare Ti substrate.
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Figure 5 shows fluorescence micrographs of live and dead BEAS-2B cells on the bare Ti substrate and SrHAp thin films. Figure 6 shows the results of automated separate counting of the numbers of live and dead cells through image analysis of each fluorescence micrograph. On the uncoated Ti substrate, the cells showed little proliferation even after 4 days of plating and mortality was approximately 6%, although they were homogeneously dispersed and engrafted. However, on the SrHAp thin films, the number of live cells was clearly higher than on the uncoated bare Ti substrate and the entire surface of the thin films was covered with cells. In particular, on the SrHAp thin films prepared using KCl and TMACl as supporting electrolytes, cells nearly 4 times as many as those on the uncoated bare Ti substrate were observed. In addition, not only did the cells proliferate but also their mortality was lower than that of the cells on the uncoated bare Ti substrate, suggesting an increased survival, and reduced mortality to around 1%–1.5%. Among KCl, TMACl, and AcOK, no significant difference in either the number of cells or mortality was detected, indicating that the difference in the structure of the SrHAp thin films used in this experiment had a small influence on the proliferation of BEAS-2B. In the gene expression analysis using MC3T3-E1 cell line, to make the control Ti surface smooth, a bare Ti/Si substrate and the substrate on which SrHAp thin films were electrodeposited were used. We confirmed that SrHAp prepared on the Ti/Si substrate has a
12
structure similar to that prepared on the Ti substrate (Fig. S4). Figure S5 shows fluorescence micrographs of live MC3T3-E1 cells on the bare Ti/Si substrate and the SrHAp thin films. A difference in the influence of FBS on the adhesion/proliferation potency of MC3T3-E1 was observed between cells on the bare Ti/Si substrate and those on the SrHAp thin films. On the bare Ti/Si substrate, the cells were visibly distributed homogeneously regardless of FBS concentration, whereas on the SrHAp thin films, the cells agglutinated mirroring increases in FBS concentration. This made accurate counting of live cells using the automated cell counting function impossible at 2% and 5% FBS because the cells agglutinated at high densities, making their outlines unclear on the SrHAp thin films. However, at 0.5% FBS, the number of live cells on the SrHAp thin films increased by approximately 2.5-fold compared to that on the bare Ti/Si substrate and the mortality also declined to approximately 60% (t-test, p < 0.05). Thus, the SrHAp thin film demonstrated a high biocompatibility (Fig. S6). Figure 7 shows the results of the gene expression analysis of MC3T3-E1 cells. Expression levels of Runx2, an early bone differentiation marker, and BGLAP, a final bone differentiation marker, increased by approximately 1.6- and 2.6-folds (t-test, p < 0.05), respectively. Thus, it was confirmed that induction of bone differentiation is facilitated on the SrHAp substrate.
4. Conclusions
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SrHAp thin films were electrodeposited on the Ti substrate and their film morphology, structures, deposition behavior, and cell compatibility were evaluated. Addition of the SrHAp source during H2O2 reduction resulted in SrHAp thin films with a uniform surface morphology on a scale of micrometers, which comprised isolated nanorod arrays with a diameter of 100–200 nm. These nanorod axes and the c-axis of SrHAp tended to be perpendicular to the Ti substrate. SrHAp thin-film structures were implied to be influenced by halogen ions and pH. A too high current density for nucleation was found to show current attenuation, which corresponded to the progressive nucleation behavior and the production of agglomerates on the uniform surface of thin films. The SrHAp-modified substrates were confirmed to have a high biocompatible effect because they facilitated adhesion/proliferation and reduced mortality of BEAS-2B, a normal respiratory epithelial cell line, and MC3T3-E1, an osteoblast cell line. Ti in itself used as a control substrate in this study has already been used as a biocompatible material for dental implants and other applications. This study demonstrated that modification of the Ti surface by SrHAp crystal structure
makes
the
substrate
usable
as
a
useful
material
that
facilitates
the
adhesion/proliferation of cells. It was also demonstrated that SrHAp modification induces the expression of bone differentiation markers by an osteoblast cell line, MC3T3-E1. Therefore, application of the SrHAp-modified substrates as surface materials of dental implants and
14
artificial joints may lead to the shortening of treatment period through facilitation of engraftment, spread, and differentiation of osteoblasts at the body site. We will confirm the usefulness of these findings through verifications using animal experiments and other applications.
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Fig. 1 XRD patterns of SrHAp thin films electrodeposited in 0.1 M KCl solution at (a) 65°C, (b) 70°C, and (c) 80°C.
20
Fig. 2 Top–bottom, SEM images of SrHAp thin films electrodeposited in 0.1 M KCl solution at (a,b) 65°C, (c,d) 70°C, and (e,f) 80°C.
21
Fig. 3 Current–time curves for electrodeposition of SrHAp thin films after adding SrHAp sources in 0.1 M KCl solution at (a) 65°C, (b) 70°C, and (c) 80°C. Times were shifted to set the points of minimum cathodic current to be zero. Inset is enlargement near the peaks.
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Fig. 4 Top–bottom, SEM images of SrHAp thin films electrodeposited in 0.1 M (a,b) KNO3, (c,d) TMACl, (e,f) AcOK at 70°C.
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Fig. 5 Images of live/dead cells of a normal respiratory epithelial cell line, BEAS-2B, on each substrate. a–d: live cells, e–h: dead cells, a and e: bare Ti substrate, b and f: SrHAp–KCl, c and g: SrHAp–TMACl, d and h: SrHAp–AcOK.
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Fig. 6 Number of live cells and mortality of a normal respiratory epithelial cell line, BEAS-2B, on each substrate.
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Fig. 7 Expression levels of bone differentiation markers in an osteoblast cell line, MC3T3-E1, on the bare Ti substrate and the SrHAp thin films (in comparison with Ti substrate). (a) Runt-related transcription factor 2 (Runx2), (b) BGLAP (osteocalcin). * p < 0.05 indicated significant difference based on t-test.
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S0272-8842(17)30652-1 http://dx.doi.org/10.1016/j.ceramint.2017.04.049 CERI15029
To appear in: Ceramics International Received date: 20 March 2017 Revised date: 22 March 2017 Accepted date: 8 April 2017 Cite this article as: Shigeo Hori, Minoru Hirano and Riichiro Ohta, Electrodeposition of strontium apatite nanorod arrays and their cell compatibility, Ceramics International, http://dx.doi.org/10.1016/j.ceramint.2017.04.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Electrodeposition of strontium apatite nanorod arrays and their cell compatibility Shigeo Hori,*,† Minoru Hirano,*,† and Riichiro Ohta Toyota Central Research and Development Labs. Inc., 41-1 Yokomichi, Nagakute, Aichi 480-1192, Japan * Corresponding author E-mail: [email protected] (S. Hori), [email protected] (M. Hirano) † These authors contributed equally to this work.
Abstract Hydroxyapatites have a biocompatibility that is influenced by their composition and structure. We electrodeposited strontium hydroxyapatite (SrHAp) thin films on Ti substrate and evaluated their cell compatibility for the first time. Electrodeposited thin films have a uniform surface morphology on a scale of micrometers and comprise isolated nanorod-like grains with diameters of 100–200 nm standing on the substrate. High current densities during the electrodeposition were found to produce radial agglomerates of SrHAp rods on nanorod arrays. In comparison with the bare Ti substrate, the SrHAp thin film increased the proliferation and survival (reduced mortality) of the respiratory epithelial cell line. In addition, the levels of expression of bone
1
differentiation markers such as runt-related transcription factor 2 (Runx2) and BGLAP genes in an osteoblast cell line significantly increased on the SrHAp thin film compared with those on the bare Ti substrate.
Keywords D: Apatite, A: Films, E: Biomedical applications, Electrodeposition
1. Introduction Hydroxyapatite [M10(PO4)6(OH)2; MHAp; where M = an alkali earth metal] is one of the phosphates with a hexagonal apatite crystal structure and is expected to be applied for phosphor, laser material, and biomaterial depending on its M elements [1]. Calcium hydroxyapatite (CaHAp) is a major inorganic material in human bones and teeth; thus, it has been studied as a biomaterial for bone filling, scaffolds, and surface coating of implants [2-6]. In addition, strontium (Sr)-substituted hydroxyapatite (CaSrHAp) has shown biocompatibility for these procedures to some extent [7,8]. However, to the best of our knowledge, there have been no reports on the synthesis and evaluation of Sr hydroxyapatite (SrHAp) containing no Ca element. At present, plasma spray deposition is the most popular commercialized method for synthesizing MHAp film coatings [3]. However, several problems associated with this process
2
still remain. These include the production of deleterious secondary phase, damage to substrate material by plasma, and peeling and mechanical destruction of the film [9]. Precipitation using supersaturated solution [4], sol-gel [5], electrophoretic deposition [6], and electrodeposition has also been reported [10-12]. Among the above methods, electrodeposition is expected to be suitable for coatings on conductive substrates because of the following advantages: (1) reaction can be precisely controlled by electrode potential or current density in addition to common parameters (temperature, concentrations, etc.) and (2) thin films can be obtained with a thickness below a few micrometers and deposited on substrates with complicated surface shapes. In MHAp electrodeposition, electrochemically produced hydroxide ions (OH−) by reduction reaction of hydrogen peroxide [13,14], oxygen [15,16], and nitrate ions [17,18], increase the pH in the vicinity of the electrode, which leads to precipitation of MHAp, as shown in equations 1 and 2. H 2 O 2 2e 2OH
(1)
10M 2 6H2 PO4 14OH M10 PO4 6 OH2
(2)
Electrodeposited CaHAp films generally comprise plate, needle, or rod-like grains, and their inter-grain gaps have been up to several micrometers, resulting in a non-uniform surface morphology on a scale of micrometers, which is comparable to the cell size [12,19-21]. Surface morphology of thin films has been reported to affect biocompatibility [22,23]. Therefore,
3
surface morphology and uniformity on a scale of micrometers are factors to be considered when evaluating biocompatibilities of thin films. In this report, SrHAp thin films with a uniform surface morphology on a scale of micrometers are electrodeposited on Ti substrate for the first time and their morphology, structure, deposition behavior, and cell compatibility have been explored.
2. Material and Methods 2.1. Electrodeposition of strontium hydroxyapatite thin films Ti or Ti-sputtered Si (Ti/Si) substrates were employed as working electrode and ultrasonically washed in acetone for 5 min before electrodeposition. Platinum (Pt) wires and potassium chloride (KCl)-saturated Ag/AgCl electrodes were used as counter and reference electrodes, respectively. As a supporting electrolyte, 0.1 M KCl, potassium nitrate (KNO3), tetramethylammonium chloride {[(CH3)4N]Cl; TMACl}, potassium acetate (CH3COOK; AcOK), or ammonium chloride (NH4Cl) were employed. The pH of the electrolyte solution was adjusted to 5.0–6.0 before electrodeposition using conjugated acid or base of each electrolyte. Electrodeposition of SrHAp was performed using the three-electrode system in 20 mL of electrolyte solution with magnetic stirring at 200 rpm. Temperatures of deposition bath (deposition temperature) varied between 65°C, 70°C, and 80°C. Electrode potentials of Ti or
4
Ti/Si substrates were maintained at −1.2 V for 5 min and subsequently at −0.8 V for 22 min during SrHAp thin-film deposition. Furthermore, 0.2 mL of 0.5 M H2O2 solution and 0.42 mL of a solution mixture of 0.25 M strontium chloride (SrCl2) and 0.1 M ammonium dihydrogen phosphate (NH4H2PO4) were added to the deposition bath at 4 and 7 min, respectively, after electrodeposition had started. The consequent composition of the electrolyte solution was ca. 0.1 M supporting electrolyte, 5 mM H2O2, 5 mM SrCl2, and 2 mM NH4H2PO4. Obtained SrHAp thin films were analyzed using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), and Fourier transform infrared spectroscopy (FT-IR).
2.2. Cell compatibility evaluation of strontium hydroxyapatite thin films On each of the substrates, a normal respiratory epithelial cell line (BEAS-2B) was plated [(plating density: 1 × 104 cm−2; medium: RPMI-1640 containing 10% fetal bovine serum (FBS)], followed by medium replacement by RPMI-1640 containing 0.5% FBS the next day. Four days after cell plating, fluorescence images of live as well as dead cells were acquired using Live/Dead Cell Staining Kit II (PromoKine) and a fluorescence microscope BZ-9000 (Keyence) that automatically counted live/dead cells using its BZ-II analyzer software (Keyence).
2.3. Gene expression analysis
5
On the bare Ti substrate and the substrate on which SrHAp thin films were electrodeposited, an osteoblast cell line (MC3T3-E1) was plated (plating density: 1 × 104 cm−2; medium: -MEM containing 10% FBS), followed by medium replacement by -MEM containing 0% FBS the next day. On the seventh day after the plating, the cells were subjected to total RNA isolation. RNA was used as a template for gene expression analysis using real-time RT-PCR (n = 3).
3. Results and Discussion 3.1. Electrodeposition of strontium hydroxyapatite thin films Figure 1 shows XRD patterns of SrHAp thin films electrodeposited on the Ti substrate at various deposition temperatures in the KCl electrolyte. Peaks assigned to the hexagonal apatite crystal structure appeared in the patterns for all deposition temperatures. Relatively high peak intensities of (00l) and (112) planes and indiscernible peaks of (hk0) planes suggest that the c-axis of SrHAp thin films electrodeposited at 65°C and 70°C tend to be perpendicular to the Ti substrate. In contrast, relatively large peaks of (hk0) planes for SrHAp thin films electrodeposited at 80°C indicate that this film partially contains structures with the c-axis parallel to the Ti substrate. IR spectrum for the SrHAp thin film electrodeposited at 70°C exhibited an absorption for PO43− at 900–1200 cm−1 [24], and a broad weak peak at 800–900 cm−1 (Fig. S1), which would be assigned to HPO42− [25]. Assignment of CO32− (872 cm−1) to the
6
broad weak peak should be eliminated because a major peak of CO32− at around 1400–1600 cm−1 was not observed [24]. Morphologies of the SrHAp thin films were observed using SEM, as shown in Figure 2. Pointed nanorod arrays with no cracks were observed for all deposition temperatures. These nanorods have diameters of 100–200 nm and were isolated from each other. The axes of these rods tended to be perpendicular to the Ti surface. Combining with the consideration of XRD patterns of the SrHAp thin films, these rods could be SrHAp crystal grown in the c-axis direction anisotropically. Low-magnification SEM images revealed that SrHAp nanorod arrays have a uniform surface morphology on a scale of micrometers. The influence of variation of deposition temperature on morphologies of the nanorod arrays was not obvious. However, a number of radial agglomerates of thick SrHAp rods were observed on the arrays at a deposition temperature of 80°C. Because these radial agglomerates contained rods with their axes parallel to the substrate, large peak intensities of (hk0) planes in XRD pattern could result from the agglomerates. The composition of SrHAp thin films was evaluated using EDX analysis. Ratio of Sr and P (Sr/P) was determined to be ca. 1.6, which is close to the stoichiometric ratio of 1.67. However, the appearance of HPO42− peak in the IR spectrum indicates the presence of slight Sr deficiencies. Furthermore, a detection of small amount of Cl (< 1 %) suggests partial substitutions of OH− by Cl−. K was not detected.
7
To investigate an electrodepositing behavior of SrHAp thin films, current–time relations for various deposition temperatures were compared, as shown in Figure 3. Times of each amperogram were shifted to set the points of minimum cathodic current to be zero. Current transients exhibited three features later by the addition of SrHAp sources (mixture solution of SrCl2 and NH4H2PO4) as follows: (i) rapid current decrease, (ii) peak formation by fast current increase, followed by moderate decrease, and (iii) plateau. These trends were observed for all deposition temperatures. Current transients similar to (ii) and (iii) have been observed for electrodeposition of CaHAp and ZnO, which have been considered to reflect the nucleation behavior [13,26]. The peak current formation in nucleation is generally ascribed to a current increase caused by an increase of reactive sites, i.e., surface area of growing nuclei and a subsequent current decrease due to an overlapping of the three dimensional diffusion zones of reactive material [27,28]. Therefore, a nucleation of SrHAp in our electrodeposition was possibly faster than that of conventional CaHAp electrodeposition, which requires few minutes to form the peak of current [11,12]. In addition, this fast nucleation would be the cause of the uniform surface structure because the fast nucleation will result in a high density nuclei and small differences in growth time of each grain. Based on the comparison of the peak currents among various deposition temperatures, times required to form the peaks were clipped with temperature, which suggests an acceleration of nucleation (inset in Fig. 3).
8
A nucleation current transient for metal electrodeposition has been categorized into two types, instantaneous and progressive nucleation models [27]. Current transient for CaHAp electrodeposition has been reported to be an instantaneous behavior for current increase and progressive behavior for current decrease [21]. However, the current transient for SrHAp electrodeposition exhibited a complicated behavior. For a deposition temperature of 70°C, an increase and a decrease in current were accordant to progressive and instantaneous behaviors, respectively, whereas these relations were inverted for a temperature of 80°C (Fig. S2). We noted that there are some inapplicable points in the model for SrHAp electrodeposition; e.g., the model is based on metal deposition and hypothesizing diffusion limitation. However, interestingly, the production of radial agglomerates for deposition temperature of 80°C indicates that new SrHAp nuclei were generated on nanorod arrays over time; this corresponds to the progressive nucleation model. The abovementioned difference in the nucleation behavior caused by temperature is possibly explained by a magnitude of current density. Large current density for a deposition temperature of 80°C can lead a supersaturation of SrHAp high enough to generate nuclei on predeposited SrHAp grains with different crystallographic orientation. Furthermore, current for plateau was also high when progressive nucleation occurred. For electrodeposition of CaHAp, copresent ions (F−, KCl, etc.) in the deposition bath are generally known to affect the structure of CaHAp thin films [10,12,20]. Therefore, the structure
9
of SrHAp thin films were compared using various supporting electrolytes, KNO3, TMACl, AcOK, and NH4Cl, at 0.1 M. Deposition temperature was maintained at 70°C. Figure 4 shows the surface morphology of SrHAp thin films electrodeposited in each electrolyte solution. SrHAp nanorod arrays similar to those obtain in KCl solution were observed for the films obtained in KNO3 and TMACl solutions. In addition, plate-like particles with a width of 200– 300 nm were also found in the thin film obtained in the KNO3 solution. These plate-like particles had larger lengths than those of SrHAp nanorods. In AcOK solution, grain shapes were different from those obtained in other electrolytes. Spatulate grains with a width of ca. 300 nm mixed with nanorods were observed along with large agglomerates of rectangular rod-like grains with a length of ca. 5 μm. These plate-like grains obtained in KNO3 and large agglomerates in AcOK solutions are not expected to be SrHAp but secondary phases whose presence was assumed by unknown peaks that appeared in XRD patterns (Fig. S3). No thin films were obtained in NH4Cl solutions. From the above results, we conclude that the influence of electrolyte on the structure of thin films is due to two factors: grain shape of SrHAp and production of secondary phase. The origin of structure differences in SrHAp thin films caused by electrolyte species is difficult to be determined by the above data. However, the presence of halogen ions that can substitute OH− and differences in pH are possibilities. Halogen ions can act as alternatives to
10
OH− for producing apatite structures, which will reduce secondary phases that would be produced at a lower pH. KCl, TMACl, and KNO3 are expected to almost completely dissociate in solution because dissociation constants (pKa) of their conjugated acids, HCl and HNO3, are small. Therefore, the electrochemically produced OH− can increase the pH in the vicinity of electrodes, resulting in precipitation of SrHAp under higher pH as well as in supersaturation. In contrast, CH3COOH and NH4+ have large pKa of 4.76 and 9.25, respectively [29], indicating a presence of a considerable amount of undissociated acids, which will inhibit an increase in pH by neutralization with OH−. Such undissociated acid concentrations for AcOK and NH4Cl were calculated to be ca. 12 and 100 mM, respectively. Consequently, concentrations of undissociated acids comparable to those of H2O2 and H2PO4 could be expected to lead to the deposition of SrHAp thin films under lower pH in an AcOK solution. In contrast, the concentration of undissociated acid in NH4Cl solution is nearly 20-fold higher than that of H2O2, which can rule out both the increase in pH and deposition of SrHAp thin films.
3.2. Cell-compatible evaluation of strontium hydroxyapatite thin films To investigate biocompatibility of the prepared SrHAp thin films, cell assays using normal respiratory epithelial (BEAS-2B) and osteoblast (MC3T3-E1) cell lines were conducted. The SrHAp thin films used to culture BEAS-2B were prepared on a polished bare Ti substrate.
11
Figure 5 shows fluorescence micrographs of live and dead BEAS-2B cells on the bare Ti substrate and SrHAp thin films. Figure 6 shows the results of automated separate counting of the numbers of live and dead cells through image analysis of each fluorescence micrograph. On the uncoated Ti substrate, the cells showed little proliferation even after 4 days of plating and mortality was approximately 6%, although they were homogeneously dispersed and engrafted. However, on the SrHAp thin films, the number of live cells was clearly higher than on the uncoated bare Ti substrate and the entire surface of the thin films was covered with cells. In particular, on the SrHAp thin films prepared using KCl and TMACl as supporting electrolytes, cells nearly 4 times as many as those on the uncoated bare Ti substrate were observed. In addition, not only did the cells proliferate but also their mortality was lower than that of the cells on the uncoated bare Ti substrate, suggesting an increased survival, and reduced mortality to around 1%–1.5%. Among KCl, TMACl, and AcOK, no significant difference in either the number of cells or mortality was detected, indicating that the difference in the structure of the SrHAp thin films used in this experiment had a small influence on the proliferation of BEAS-2B. In the gene expression analysis using MC3T3-E1 cell line, to make the control Ti surface smooth, a bare Ti/Si substrate and the substrate on which SrHAp thin films were electrodeposited were used. We confirmed that SrHAp prepared on the Ti/Si substrate has a
12
structure similar to that prepared on the Ti substrate (Fig. S4). Figure S5 shows fluorescence micrographs of live MC3T3-E1 cells on the bare Ti/Si substrate and the SrHAp thin films. A difference in the influence of FBS on the adhesion/proliferation potency of MC3T3-E1 was observed between cells on the bare Ti/Si substrate and those on the SrHAp thin films. On the bare Ti/Si substrate, the cells were visibly distributed homogeneously regardless of FBS concentration, whereas on the SrHAp thin films, the cells agglutinated mirroring increases in FBS concentration. This made accurate counting of live cells using the automated cell counting function impossible at 2% and 5% FBS because the cells agglutinated at high densities, making their outlines unclear on the SrHAp thin films. However, at 0.5% FBS, the number of live cells on the SrHAp thin films increased by approximately 2.5-fold compared to that on the bare Ti/Si substrate and the mortality also declined to approximately 60% (t-test, p < 0.05). Thus, the SrHAp thin film demonstrated a high biocompatibility (Fig. S6). Figure 7 shows the results of the gene expression analysis of MC3T3-E1 cells. Expression levels of Runx2, an early bone differentiation marker, and BGLAP, a final bone differentiation marker, increased by approximately 1.6- and 2.6-folds (t-test, p < 0.05), respectively. Thus, it was confirmed that induction of bone differentiation is facilitated on the SrHAp substrate.
4. Conclusions
13
SrHAp thin films were electrodeposited on the Ti substrate and their film morphology, structures, deposition behavior, and cell compatibility were evaluated. Addition of the SrHAp source during H2O2 reduction resulted in SrHAp thin films with a uniform surface morphology on a scale of micrometers, which comprised isolated nanorod arrays with a diameter of 100–200 nm. These nanorod axes and the c-axis of SrHAp tended to be perpendicular to the Ti substrate. SrHAp thin-film structures were implied to be influenced by halogen ions and pH. A too high current density for nucleation was found to show current attenuation, which corresponded to the progressive nucleation behavior and the production of agglomerates on the uniform surface of thin films. The SrHAp-modified substrates were confirmed to have a high biocompatible effect because they facilitated adhesion/proliferation and reduced mortality of BEAS-2B, a normal respiratory epithelial cell line, and MC3T3-E1, an osteoblast cell line. Ti in itself used as a control substrate in this study has already been used as a biocompatible material for dental implants and other applications. This study demonstrated that modification of the Ti surface by SrHAp crystal structure
makes
the
substrate
usable
as
a
useful
material
that
facilitates
the
adhesion/proliferation of cells. It was also demonstrated that SrHAp modification induces the expression of bone differentiation markers by an osteoblast cell line, MC3T3-E1. Therefore, application of the SrHAp-modified substrates as surface materials of dental implants and
14
artificial joints may lead to the shortening of treatment period through facilitation of engraftment, spread, and differentiation of osteoblasts at the body site. We will confirm the usefulness of these findings through verifications using animal experiments and other applications.
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Fig. 1 XRD patterns of SrHAp thin films electrodeposited in 0.1 M KCl solution at (a) 65°C, (b) 70°C, and (c) 80°C.
20
Fig. 2 Top–bottom, SEM images of SrHAp thin films electrodeposited in 0.1 M KCl solution at (a,b) 65°C, (c,d) 70°C, and (e,f) 80°C.
21
Fig. 3 Current–time curves for electrodeposition of SrHAp thin films after adding SrHAp sources in 0.1 M KCl solution at (a) 65°C, (b) 70°C, and (c) 80°C. Times were shifted to set the points of minimum cathodic current to be zero. Inset is enlargement near the peaks.
22
Fig. 4 Top–bottom, SEM images of SrHAp thin films electrodeposited in 0.1 M (a,b) KNO3, (c,d) TMACl, (e,f) AcOK at 70°C.
23
Fig. 5 Images of live/dead cells of a normal respiratory epithelial cell line, BEAS-2B, on each substrate. a–d: live cells, e–h: dead cells, a and e: bare Ti substrate, b and f: SrHAp–KCl, c and g: SrHAp–TMACl, d and h: SrHAp–AcOK.
24
Fig. 6 Number of live cells and mortality of a normal respiratory epithelial cell line, BEAS-2B, on each substrate.
25
Fig. 7 Expression levels of bone differentiation markers in an osteoblast cell line, MC3T3-E1, on the bare Ti substrate and the SrHAp thin films (in comparison with Ti substrate). (a) Runt-related transcription factor 2 (Runx2), (b) BGLAP (osteocalcin). * p < 0.05 indicated significant difference based on t-test.
26