Hydroxyapatite Thin Films with Giant Electrical Polarization

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Hydroxyapatite Thin Films with Giant Electrical Polarization Cong Fu,† Keith Savino,† Paul Gabrys,† Aibin Zeng,†,‡ Baohong Guan,†,§ Diana Olvera,⊥ Chenggong Wang,∥ Boao Song,† Hani Awad,⊥ Yongli Gao,∥ and Matthew Z. Yates*,† †

Department of Chemical Engineering and Laboratory for Laser Energetics, University of Rochester, Rochester, New York 14627, United States ‡ Department of Applied Engineering, Hangzhou Wanxiang Polytechnic, Hangzhou 310023, China § Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China ⊥ Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York 14627, United States ∥ Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, United States S Supporting Information *

ABSTRACT: It is demonstrated that hydroxyapatite, a type of calcium phosphate commonly found in bone tissue, retains surprisingly large stored charge when synthesized electrochemically from aqueous solution. Thin films of hydroxyapatite formed on titanium and stainless steel electrodes were found to display giant polarization with quasi-permanent stored charge in excess of 70 000 microcoulombs per square centimeter. The polarization of the hydroxyapatite film develops during synthesis as a result of fieldinduced changes in concentration of ionic reactants in the electrical double layer near the electrode surface. This novel mechanism of polarization during synthesis provides much larger stored charge than what is possible by postsynthesis poling of ferroelectric or electret materials. The polarized hydroxyapatite films on titanium are shown through in vitro experiments to hold promise in stimulating bone growth and may enable new applications in ion exchange separations, drug delivery, or energy storage.

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INTRODUCTION Many types of dielectric materials become permanently polarized after exposure to an electric field through the alignment of dipoles, ion transport, or accumulation of surface charge.1,2 Electrets are defined as materials that maintain quasipermanent electrical polarization, while ferroelectrics have permanent polarization that is easily reversed by an applied field. The switchable polarization of ferroelectrics has led to widespread use in a variety of microelectronic devices, including data storage, sensors, and actuators.2 The intrinsic permanent electric field in ferroelectric and electret materials opens the possibility of a number of novel applications, such as enhanced electron/hole separation in solar cells,3,4 energy harvesting devices,5−7 and photocatalysis.8 In ferroelectrics, the permanent polarization is thermodynamically determined by the molecular or crystal structure of the material. Electrets, on the other hand, can have polarization from kinetically trapped charges. As a result, the polarization of electrets can increase with increased poling field strength up to the point of dielectric breakdown of the material. The highest stored charge in ferroelectrics is around 150 μC/cm2 after polarization under a field strength ∼500 kV/cm.9 Common electrets typically have far lower stored charge than ferroelectrics, but recent reports show that naturally occurring biocomposites found in sea shells exhibit giant electret hysteresis with stored charge approaching 4000 μC/cm2 after polarization under applied field of ∼40 kV/cm.10 Hydrox© XXXX American Chemical Society

yapatite (HA), a mineral found in teeth and bones, is also an electret that exhibits stored charge as high as ∼4000 μC/cm2 after being polarized by an applied field of 2 kV/cm for 30 min at 350 °C.11 Hydroxyapatite is a crystalline form of calcium phosphate with either monoclinic or hexagonal crystal symmetry.12 The stoichiometric formula of HA is Ca5(PO4)3(OH), although the crystal structure can exist in a wide range of nonstoichiometric compositions. The chemical composition of HA can be widely varied by partial substitution of other ions at the calcium, phosphate, or hydroxyl positions in the crystal lattice. The hydroxyl groups form linear columns along the crystallographic c-axis that give rise to the electrical properties of HA, including high temperature proton conductivity, ferroelectricity, and electret behavior.11,13−15 Hydroxyapatite is the primary mineral component of bone tissue, and the osteoconductive properties of synthetic HA has led to its widespread use as a coating or additive in bone grafts, scaffolds, and orthopedic implants.16 It has been shown that electrical polarization of HA enhances bioactivity and osseointegration, both in vitro and in vivo.17−20 In previous studies, HA ceramics were polarized by ion transport at elevated temperature (>300 °C) driven by a strong applied electric field (>1 kV/cm), resulting in quasi-permanent Received: September 11, 2014 Revised: January 19, 2015

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electrochemically deposited sample was bombarded in situ by an argon ion beam accelerated to 5 keV. After each etching step, XPS spectra were recorded. The specific core level intensities of Ca and P were quantified, and Ca/P ratios were calculated. The Fourier transform infrared (FTIR) spectra were obtained using a model FTIR-8400S spectrophotometer (Shimadzu). The stored charge of the samples was determined by thermally stimulated depolarization current (TSDC) measurement. For TSDC measurements, samples were first heated to 300 °C in air for 1 h to remove any surface contamination. The upper surface of the HA coating was then sputter coated with ∼10 nm platinum. The sputter coated Pt covered the entire surface area of the HA film, and the HA film surface area (12.5 mm × 12.5 mm) was used in calculating the electrical surface charge density. Two Pt foils (12.5 mm × 12.5 mm) acting as current collectors were placed on the HA surface and Ti substrate. The Pt foils were attached to a picoammeter (Model 6487, Keithley Instruments) with Pt wires (0.5 mm in diameter). The Pt wires were fed through small ceramic tubes acting as insulators to avoid short circuiting inside the furnace. The sample was placed in the center of a tube furnace (Lindberg Blue M, Thermo Scientific), with the insulated Pt wires fed to the picoammeter outside the furnace. Then the sample was heated at a heating rate of 5 °C/min while the depolarization current was measured. Mineralization in Simulated Body Fluid. Solutions of 1.5× simulated body fluid (SBF) were prepared following the literature.22 Samples of titanium coated with HA were placed into vials containing 5 mL of 1.5× SBF at 37 °C with pH of 7.25. After 24 h, the samples were taken out of the solution, rinsed with deionized water, and placed in a desiccator to dry. To compare the effects of positive versus negative surface charge, a dense coating prepared hydrothermally was removed from the titanium substrate using a blade to fracture the coating. Pieces of the HA coating were placed in 5 mL of SBF as described above to compare mineralization onto the top and bottom surfaces of the HA layer. The mineralization was examined using SEM and XRD of the dried samples. Cell Culture on HA Substrates. Human bone marrow-derived mesenchymal stem cells were obtained from Lonza (Berkshire, U.K.) and cultured in vitro according to the manufacturer’s protocol. For cell nuclear density, sterilized substrates (n = 3/group) were placed into 48 well plates (BD Biosciences), one per well. The different surfaces were carefully seeded at a density of 10 000 cells/cm2 and incubated for 2 and 5 days under standard cell culture conditions. After each incubating period, cells were washed in Dulbecco’s phosphate-buffered saline, fixed in 4% paraformaldehyde, and then sequentially stained with mouse monoclonal primary antibodies against vinculin, F-actin, and nuclear To-Pro3. After immunostaining, cells were examined using an inverted confocal laser scanning microscope (Olympus FV1000 CLSM). Three samples of each substrate were analyzed for this study. The cell (nuclear) density of each substrate was analyzed using a 10× dry objective lens and a multiarea time lapse custom program (tiling). Quantitative cell attachment density results were obtained using custom image processing software. Parallel cell growth investigations were carried out on the polarized electrochemically synthesized HA layer as synthesized and the HA layer after depolarization by heating to 600 °C for 1 h.

stored charge as the ions are immobilized near room temperature.11,17,21 Here we report a novel polarization mechanism that occurs during electrochemical synthesis of HA thin films. The HA films are synthesized on a metal electrode surface from aqueous solution, and the applied potential driving the reaction affects the concentration of ionic reactants in the electrical double layer near the surface. As a result of a field-induced composition gradient in the HA film, giant polarization develops in the direction normal to the film surface. The electrochemically synthesized HA films have extremely large stored charge of ∼70 000 μC/cm2. Polarization of HA during electrochemical synthesis from aqueous solution enables far larger stored charge than is possible by postsynthesis poling techniques. The giant polarization may provide improved bioactivity in orthopedic applications and opens up the potential for new applications of HA in catalysis, ion exchange, and energy storage.

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MATERIALS AND METHODS

Materials. NaCl (≥99.0%), tris(hydroxymethyl)-amino methane (>99.8%), Ca(NO3)2·4H2O (99.0%), disodium ethylenediaminetetraacetatedihydrate (Na2EDTA·2H2O) (99.0−101.0%), K2HPO4 (99.99%), and CaCl2·2H2O (>99%) were all purchased from SigmaAldrich. Hydrochloric acid (37%) and ammonium hydroxide (28.0− 30.0%) were purchased from Mallinckrodt Chemicals. Urea (99.5%) and (NH4)2HPO4 (>99.0%) were obtained from Fluka and EMD, respectively. Titanium (Ti, 0.89 mm thick), stainless steel (type 304, 0.1 mm thick), and platinum (Pt, 0.127 mm thick) foils were obtained from Alfa Aesar. Sample Preparation. For electrochemical synthesis, a cleaned Ti (12.5 × 12.5 × 0.89 mm) or stainless steel (12.5 × 12.5 × 0.1 mm) plate was used as the cathode while a Pt foil (25 × 25 × 0.127 mm) was used as the anode. The electrolyte solution consisted of 138 mM NaCl, 50 mM tris(hydroxymethyl)-aminomethane, 1.3 mM CaCl2, and 0.84 mM K2HPO4. The solution was buffered to pH = 7.2 with 37% hydrochloric acid. The anode and cathode were immersed into the electrolyte solution and held apart by 1 cm. The solution was continuously stirred and heated to 95 °C using an oil bath. Upon reaching the reaction temperature of 95 °C, a constant current density of 12.5 mA/cm2 (relative to the Pt electrode surface area) was applied for 5 min. After the electrochemical reaction, the sample was taken out of the electrolyte solution, rinsed with deionized water several times, and dried in air. For hydrothermal crystallization, the synthesis solution consisted of 0.115 M Na2EDTA, 0.1 M Ca(NO3)2, 0.06 M (NH4)2HPO4, and 0.3 M urea. The pH of the solution was adjusted to 10.0 using ammonium hydroxide. The titanium substrate coated with electrochemically synthesized HA crystals was transferred to a Teflon-lined pressure vessel (Parr model 4744). The titanium was oriented in the vessel with the HA layer facing downward and oriented at an ∼45° angle relative to the bottom of the vessel. The vessel was placed in an oven at 200 °C for 10 h for the hydrothermal crystallization reaction. The reaction was repeated a total of 4 times. The sample was rinsed with deionized water and placed in fresh reagent solution between each reaction. After the final reaction, the sample was rinsed with deionized water several times and dried in air. Sample Characterization. The HA crystal structure was determined by X-ray diffraction (XRD, PW3020, Philips) with Cu Kα radiation (λ = 1.5418 Å). The product morphology was characterized using a field emission scanning electron microscope (SEM, DSM982, Zeiss-Leo). The chemical composition was analyzed by elemental analysis (CE-440, Exeter Analytical, Inc.). The surface composition was obtained with an XPS spectrometer (AXIS Ultra DLD, Kratos), equipped with a monochromatic Al anode X-ray gun (Kα = 1486.6 eV). The base pressure of the XPS system was 1 × 10−9 Torr. The spot size of the X-ray was chosen to be 1 mm by 1 mm, and the resolution selected for the energy analyzer was 0.1 eV. Argon ion etching was used to investigate the elemental depth profile. The

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RESULTS AND DISCUSSION Electrochemical growth of HA is a well-established method to produce uniform, crystalline coatings at relatively low temperature from aqueous electrolyte solutions.23−25 In this technique, the metal surface to be coated (often titanium or stainless steel) acts as the cathode and is separated from an anode (typically platinum or graphite) in a simulated body fluid electrolyte solution. When an electric potential is applied, the polarization of the electrodes causes the local ion concentration near the electrode surface to deviate from that in the bulk. Cations, such as Ca2+, are attracted to the cathode surface, while anions are repelled from the cathode and attracted to the anode. The B

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simplicity we refer to the calcium phosphate coating as “HA” for the remainder of the discussion. The FTIR spectrum of the dense hydrothermally synthesized HA coating (Supporting Information Figure S4) confirms that it contains carbonate groups substituted for a fraction of the phosphate groups (B-type substitution). Therefore, the stoichiometric formula Ca10−x(PO4)6−x(CO3)x(OH)2−x can be applied. With the help of elemental analysis, the carbon content was determined to be 1.22%, giving x ∼ 1 in the stoichiometric formula. The elemental analysis indicates a carbonate content of ∼6%. The carbonate composition is similar to that of natural apatite in bone that has 4−6% carbonate content.27 The stored electrical charge in the as-synthesized HA coating shown in Figure 1b was measured using the thermally stimulated depolarization current (TSDC) technique.13 Figure 2 shows the measured current density in microamps per square

voltage across the electrodes (typically 3−4 V) is sufficient to induce electrolysis of water and therefore cause OH− anions to form at the cathode surface. As a result of changes in pH and electrolyte concentration, HA becomes locally supersaturated and nucleates selectively on the cathode surface. Figure 1a

Figure 1. (a) Hydroxyapatite nanocrystals electrochemically synthesized on titanium. (b) Hydroxyapatite hydrothermally grown onto the seed crystals shown in (a).

shows HA nanocrystals formed on commercially pure titanium after a reaction time of 5 min at 95 °C. The crystals are needlelike with the long axis associated with the crystallographic c-axis. In the image, a section of the coating has been scraped away to show the underlying titanium surface, and preferential orientation of the rod-shaped crystals normal to the surface is apparent. The coating is approximately 500 nm thick. The Xray diffraction spectrum (Supporting Information Figure S1) confirms that the crystals are comprised of HA with no detectable secondary crystal phases. Enhanced intensity of the (002) peak in the XRD spectrum confirms preferential orientation of the c-axis normal to the substrate. The HA nanocrystals shown in Figure 1a were used as seeds to promote growth of additional HA on the surface during hydrothermal crystallization. Urea was added to promote the formation of a dense HA coating.26The urea undergoes thermal decomposition during the reaction, resulting in the release of carbonate ions that can substitute for a fraction of the phosphate groups in HA. Figure 1b shows a side view image of the dense HA coating after hydrothermal synthesis. The coating is approximately 10 μm thick. Hexagonal crystal facets are visible on the surface, indicating preferential orientation of the c-axis normal to the surface. X-ray diffraction (Supporting Information Figure S2) shows very large enhancement of the (002) and (004) peaks of HA, indicating near perfect alignment of the crystal domains with the c-axis normal to the surface. The XRD spectrum of a dense sample like the one shown in Figure 1b was also obtained after scraping the sample off of the titanium substrate and lightly grinding the sample in a mortar and pestle. Since there is less preferred crystal orientation, the pattern of the sample removed from the substrate (Supporting Information Figure S3) shows a number of additional peaks, all of which can be attributed to the apatite crystal structure. In every case, all peaks that appear on XRD spectra have been identified as originating from either the apatite crystal phase or the underlying titanium substrate. Therefore, hydroxyapatite is the only crystalline phase present in the thin film coating the titanium. It should be made clear, however, that even though the crystal domains have the HA structure, they most certainly do not have the stoichiometric HA composition. In addition, it is possible that other amorphous phases are present that are not identified by XRD. The amorphous material may have chemical composition varying greatly from that of stoichiometric HA. Even so, for

Figure 2. Thermally stimulated depolarization current for dense HA coating (similar to that shown in Figure 1b).

centimeter versus temperature for a sample that was heated at a rate of 5 °C per minute. A surprisingly high peak current density of 126 μA/cm2 was measured at 425 °C. The current density falls sharply to zero as the sample is heated above 425 °C, indicating that the sample was completely depolarized. The total stored charge density (Q) is obtained by integrating the current density, using the equation13 1 Q= J (T ) d T β (1)

∫

where β is the heating rate and J(T) is the current density at temperature T. The data in Figure 2 give a total stored charge of ∼70 000 μC/cm2. The TSDC measurement was repeated on three different samples, each giving similar results. The highest previously published stored charge for HA is ∼4000 μC/cm2 for a carbonated sample after being polarized under an electric field strength of 2 kV/cm for 30 min at 350 °C.11 The very large stored charge shown in the data of Figure 2 is unexpected since the sample was never heated under an applied field to induce polarization by ion transport. We hypothesize that the stored charge arises during the electrochemical synthesis of the HA seed crystals, as it is the only synthesis step in which an external field is applied. The nucleation and growth of the HA nanocrystals at the titanium surface involves the reaction of calcium, phosphate, and hydroxyl ions within the electrical double layer of the electrolyte solution at the titanium surface. The negatively charged titanium cathode attracts calcium ions, so that the material that initially nucleates on the surface is calcium-rich. As C

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ion conducting glass showed stored charge of 8500 μC/cm2 after poling in an external applied field of only 10 V/cm.30 In comparison, the measured electric field across the electrolyte solution during the electrochemical synthesis of HA was ∼3.5 V/cm. Although the measured stored charge in the electrochemically synthesized HA is higher than that measured for the ion-conducting glasses poled at similar field strength, the ion mobility in the liquid electrolyte solution is much greater than that in a solid glass phase. The results suggest that the electrochemical growth from aqueous electrolyte solution enables the creation of thin films with stored charge far greater than what is possible by postsynthesis electrical poling. It is believed that the key to high stored charge is the strong field gradients in the electrolyte solution immediately adjacent to the electrode surface. Factors that affect the field strength in the double layer include the electrode potential, ionic strength, ionic valence, and temperature. Future studies should investigate these parameters in detail to fully understand the polarization mechanism. It is expected that the stored charge in films deposited in potentiostatic mode will increase proportionately with increased applied field. Doping with trivalent cations may enable higher stored charge as the relative enhancement in ion concentration in the double layer increases with increasing valence. Supporting evidence of the postulated polarization mechanism was obtained from X-ray photoelectron spectroscopy (XPS) of the surface of HA films synthesized electrochemically for 5 min, as shown in Figure 4. The calcium/phosphorus ratio

the dielectric HA layer grows thicker, it partially shields the electric field so that the local field experienced by ions in the adjacent electrolyte solution decreases. As a result, the HA film has a gradient in concentration of positive calcium ions relative to negative phosphate and hydroxyl ions versus distance from the titanium surface, as illustrated in Figure 3. The ionic species

Figure 3. Illustration of the development of giant electrical polarization in HA during electrochemical synthesis. (a) Negatively charged titanium cathode. (b) Calcium rich HA nucleating onto the cathode. (c) As the HA grows, a composition gradient develops, with the surface becoming relatively more rich in negatively charged phosphate and hydroxyl groups. The arrow indicates the direction of the resulting dipole.

may become trapped within the nonstoichiometric HA crystal domains, in amorphous phases in the film, or at interfaces. The TSDC data (Figure 2) show that appreciable ion movement does not occur at temperatures below ∼225 °C. As a result, a quasi-permanent electrical dipole (illustrated by the arrow in Figure 3) is maintained in the HA film at lower temperatures. The proposed polarization mechanism is strongly related to the polarization of the freely moving ions in the electrical double layer near the charged electrode surface. There is a steep gradient in positive versus negative ionic concentration in the Helmholtz layer of electrolyte solutions near charged surfaces, resulting in local electric fields that can easily exceed 106 V/cm near the electrode surface even though the overall field strength across electrodes is rather low.28 The large energy density in the Helmholtz layer is the basis for energy storage in double-layer supercapacitor devices.28,29 Although not an exact relationship, the creation of remnant polarization during electrochemical synthesis of HA is somewhat analogous to repeatedly freezing the double layer as the film grows thicker. While we know of no direct comparison of the proposed polarization mechanism in other experimental systems, data from the literature support that it is plausible. Ion conducting glasses behave like electrolyte solutions at high temperatures, and these materials can have very large electret charge storage. Electrical poling of ion conducting glasses at high temperature results in field-induced displacement of ions that may then be kinetically trapped at lower temperatures. Studies of electrical poling of glasses have shown extremely strong local internal fields develop at the glass/ electrode interface due to field driven ion transport.30−32 Even though the overall field strength across the glass is low, the local field near the electrode surface may approach the limit of dielectric breakdown of the glass due to the double-layer-like distribution in concentration of ions.31 As a result, ion conducting glasses have extremely high values for stored charge with relatively low applied fields. For example, TSDC of one

Figure 4. Depth profile of the Ca/P ratio in an electrochemically synthesized HA sample similar to that shown in Figure 1a. The composition of the surface was repeatedly measured by XPS as the surface was etched by argon ions.

was repeatedly measured as the sample surface was removed by argon ion etching. The Ca/P ratio is 1.67 for stoichiometric HA. The data show that the sample surface is calcium deficient, with a Ca/P ratio near 1.3. As the sample is etched, the Ca/P ratio increases above 2, indicating calcium in excess of the stoichiometric ratio of HA near the titanium surface. Surface charge has been shown to affect the Ca/P ratio of hydroxyapatite deposited from simulated body fluid,33 although the magnitude of the change range in the Ca/P ratio was less than that shown in Figure 4. The XPS data show a gradient in the concentration of positive calcium ions relative to negative phosphate and hydroxide ions that is consistent with the mechanism illustrated in Figure 3. A high Ca/P ratio in solution D

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Chemistry of Materials can result in the formation of a mixture of calcium hydroxide and HA.34It is possible that the electrochemically synthesized sample has calcium hydroxide in addition to HA near the titanium surface. However, there is no direct evidence of phases other than HA from the XRD data. Unfortunately, attempts to directly measure stored charge in the electrochemically synthesized HA nanocrystal layer resulted in short circuiting due to the thin and porous morphology of the coating (Figure 1a). Good electrode contact for TSDC measurement requires a dense HA coating of relatively uniform thickness, as is obtained after hydrothermal crystal growth (Figure 1b). To further test the hypothesized mechanism of polarization, two different types of dense HA coatings were formed without electrochemical seeding of the titanium substrate. The first control sample was an ∼50 μm thick HA layer applied to titanium via plasma spray from a commercial supplier. In the orthopedic implant industry, plasma spraying is the most common method to apply HA coatings. The result is a dense HA coating without preferential alignment of crystal domains. An XRD spectrum and SEM image of the plasma sprayed sample is shown in Supporting Information Figure S5a,b. The second control sample was synthesized hydrothermally following the same procedure for the sample shown in Figure 1b except that seed crystals were deposited on titanium through evaporative deposition of a colloidal suspension of HA nanocrystals rather than electrochemically. The HA crystals in the hydrothermally synthesized control sample have preferential orientation of the c-axis, similar to that in the sample shown in Figure 1b, as evident by the strong enhancement of the (002) and (004) peaks on the XRD spectrum (Supporting Information Figure S5c) and hexagonal facets on the SEM image (Supporting Information Figure S5d). The TSDC spectra were recorded on the two control samples in comparison to the sample synthesized hydrothermally onto the electrochemically deposited seed layer, as shown in Figure 5. As expected, TSDC measurement on the

in the TSDC spectrum gave a total stored charge in the hydrothermal control sample of less than 1% of that measured for the hydrothermal sample synthesized on the electrochemically deposited seed layer. Therefore, we can assume that approximately >99% of the stored charge measured in Figure 2 is a result of the electrochemically synthesized nanocrystals. Since the hydrothermal control sample exhibits two distinct depolarization peaks, it indicates relaxation of two types of charges. Others have observed similar depolarization spectra for HA and have attributed the two peaks to ion transport and space charge polarization.35,36 We hypothesize that the relatively small stored charge observed in the hydrothermal control is a result of oriented crystal growth from the seeded substrate. It is known that nanocomposite apatite crystals can exhibit intrinsic polarization along the c-axis, even in the absence of an applied field during synthesis.37−40 In the case of apaptite nanocomposites, the intrinsic polarization is due to parallel orientation of protein fibrils during crystal growth.38 It is possible that intrinsic polarization develops in the hydrothermal control sample due to alignment of hydroxyl dipoles or through a gradient in ionic defects developing along the c-axis during anisotropic crystal growth from the seeded substrate. Further work is needed to explore this hypothesis. However, it is clear that the vast majority of the stored charge calculated from the data of Figure 2 is imparted during electrochemical synthesis step. The two negative controls described above gave very low or zero stored charge as expected. To prove that the high stored charge could be achieved on a different substrate, a third experiment was carried out as a positive control. In this experiment, the same procedure used to form the HA sample shown in Figure 1b was carried out on a type 304 stainless steel substrate instead of titanium. The stainless steel had the same surface area (12.5 × 12.5 mm) as the titanium used in previous experiments. Figure 6 shows a representative TSDC result from

Figure 5. TSDC data for carbonated HA grown hydrothermally onto an electrochemically seeded titanium substrate (i) in comparison to carbonated HA grown hydrothermally onto a nonelectrochemically seeded titanium substrate (ii) and HA deposited by plasma spray onto titanium (iii).

Figure 6. TSDC data for carbonated HA grown hydrothermally onto an electrochemically seeded stainless steel type 304 substrate.

the stainless steel substrate. Integration of the depolarization peak resulted in a measured stored charge of ∼60 000 μC/cm2. As with the data shown in Figure 2, the sample is completely depolarized when the temperature reaches ∼450 °C. The depolarization peak is broader on the stainless steel substrate than titanium. The data show that the titanium substrate is not responsible for the high measured stored charge. It does appear, however, that the substrate plays a minor role. The stainless steel foil was much thinner than the titanium used (0.1 mm

plasma sprayed HA film indicated zero stored charge. The hydrothermally synthesized control displayed two small depolarization peaks centered around 360 and 460 °C. It is somewhat surprising that the hydrothermal control exhibited any measurable stored charge since no potential was ever applied during its synthesis. Integration of the two small peaks E

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Chemistry of Materials versus 0.89 mm), and it is possible that the flexibility of the thin foil affected current collector contact during the TSDC measurement. Additional work is needed fully identify any role the substrate may play in the actual distribution and composition of trapped charges in the HA film. Supporting evidence of strong stored charge was obtained through measurement of in vitro growth of additional calcium phosphate onto the coating from simulated body fluid. It is known that negative charge on the surface of HA promotes deposition of calcium phosphate from simulated body fluid, while positive surface charge retards calcium phosphate deposition.17,18 Figure 7 shows the surface of the coatings

Figure 7b than in Figure 7d, which is consistent with the expected higher surface charge on electrochemically grown HA than on the hydrothermally grown HA. To explore the difference in growth onto positive versus negative charged surfaces, the dense hydrothermal HA coating was fractured and pieces carefully removed from the titanium substrate. Figure 7e shows the bottom surface of HA that was originally at the titanium/HA interface. No additional calcium phosphate was deposited onto this surface from simulated body fluid, as shown in Figure 7f. The result is consistent with the HA surface having strong positive charge at the titanium/HA interface, as positive surface charge is known to suppress calcium phosphate deposition from simulated body fluid.17,18 Negative surface charge on polarized HA has also been shown to enhance cell adhesion and proliferation.17,21,41,42 To explore the effect of polarization on cell growth, human bone marrow derived mesenchymal stem cells were cultured on electrochemically coated titanium substrates. Since cell adhesion and proliferation are strongly dependent on the surface roughness and composition,43 a depolarized coated sample was used as a negative control rather than uncoated titanium. For comparison, the cells were grown on the assynthesized coatings and coatings that were depolarized at 600 °C for 1 h. Figure 8 shows the number of cells per unit area

Figure 7. Hydroxyapatite coatings after exposure to simulated body fluid for 24 h. (a) and (b) are top and side views, respectively, of electrochemically synthesized coating. (c) and (d) are top and side views, respectively, of hydrothermally synthesized coating. Images in (e) and (f) are of the bottom of the hydrothermally synthesized coating before and after, respectively, exposure to simulated body fluid.

Figure 8. Intersurface comparison of cell (nuclear) density showing that the polarized HA layer has a higher potential of cell viability than the HA layer depolarized by heating to 600 °C for 1 h.

after 2 and 5 days on the two substrates. Cells were found to adhere more favorably to the as-synthesized HA coating than the depolarized coating. Figure 9 shows the SEM images of the cell morphology on the coatings on the two substrates after 2 and 5 days. The observation indicates that the polarized HA coating promoted better cell attachment after 2 days and higher growth rate by day 5.

after being placed in simulated body fluid for 24 h at 37 °C. A new porous calcium phosphate layer grew onto the surface of both the electrochemically synthesized HA nanocrystals and the dense HA coating synthesized hydrothermally on the electrochemically deposited seed layer. Figure 7a,b shows top and side views, respectively, of the new calcium phosphate deposited onto the electrochemically synthesized HA nanocrystals (shown in Figure 1a). The rod shaped seed crystals are still visible at the bottom of the coating in Figure 7b. Figure 7c,d shows the top and side views, respectively, of the new calcium phosphate deposited onto the hydrothermally synthesized HA coating (shown in Figure 1b). In Figure 7d, the interface between the hydrothermal HA coating and the new porous calcium phosphate layer is clearly visible. The porous morphology of the calcium phosphate deposited from simulated body fluid is similar for both samples. However, the new porous calcium phosphate layer is much thicker in

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CONCLUSIONS Electrochemical synthesis of hydroxyapatite results in giant electrical polarization normal to the surface of the coating. Polarization is enhanced along the c-axis of the oriented crystal domains and is a result of a field-induced composition gradient developing in the coating during electrochemical synthesis. The direction of polarization is such that the upper surface of the coating is negatively charged, which promotes biomineralization, cell adhesion, and cell proliferation. The unexpectedly large magnitude of polarization may enable new uses of F

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Chemistry of Materials

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Figure 9. SEM micrographs illustrating human mesenchymal stem cell spreading and growth after incubation of 2 days on (a) polarized HA seed layer, (b) seed layer HA depolarized by heating to 600 °C for 1 h and 5 days on (c) polarized HA seed layer, and (d) seed layer HA depolarized by heating to 600 °C for 1 h.

hydroxyapatite in areas such as field-enhanced catalysis, ionexchange separations, and energy storage.

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ASSOCIATED CONTENT

S Supporting Information *

X-ray diffraction patterns of all samples, an FTIR spectrum of hydrothermally synthesized hydroxyapatite, and SEM images of the control samples. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 1-585-273-2335. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge financial support from NSF (CMMI-0856128, DMR-1303742), the DOE through the Laboratory for Laser Energetics (DE-FC03-92SF19460), and the University of Rochester. We thank Transparent Materials, LLC, for donation of hydroxyapatite nanoparticle colloids. We also thank C. Pratt for assistance with XRD measurement.

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REFERENCES

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DOI: 10.1021/cm503364s Chem. Mater. XXXX, XXX, XXX−XXX

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DOI: 10.1021/cm503364s Chem. Mater. XXXX, XXX, XXX−XXX