Superhydrophilic Vertically Aligned

Oct 20, 2010 - A method for the electrodeposition of hydroxyapatite films on superhydrophilic vertically aligned multiwalled carbon nanotubes is prese...
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Fast Preparation of Hydroxyapatite/Superhydrophilic Vertically Aligned Multiwalled Carbon Nanotube Composites for Bioactive Application Anderson O. Lobo,*,†,‡ Marcus A. F. Corat,§ Sandra C. Ramos,‡ Jorge T. Matsushima,‡ Alessandro E. C. Granato,‡,^ Cristina Pacheco-Soares,^ and Evaldo J. Corat†,‡ † Instituto Tecnologico de Aeronautica, Sao Jose dos Campos/SP, CEP: 12228-900, Brazil, ‡Laboratorio Associado de Sensores e Materiais, Instituto Nacional de Pesquisas Espaciais, CP 515, Sao Jose dos Campos/ SP, CEP: 12.245-970, Brazil, §Centro Multidisciplinar para Investigacao Biologica na Area da Ciencia em Animais de Laboratorio - CEMIB, Universidade Estadual de Campinas (UNICAMP), Campinas/SP, CEP:13083-877, CP 6095, Brazil, and ^Laboratorio de Dinamica de Compartimentos Celulares, Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraiba, Sao Jose dos Campos/SP, CEP: 12227-010, Brazil

Received August 31, 2010. Revised Manuscript Received September 24, 2010 A method for the electrodeposition of hydroxyapatite films on superhydrophilic vertically aligned multiwalled carbon nanotubes is presented. The formation of a thin homogeneous film with high crystallinity was observed without any thermal treatment and with bioactivity properties that accelerate the in vitro biomineralization process and osteoblast adhesion.

Introduction Hydroxyapatite (HA, Ca10(PO4)6(OH)2) is a form of calcium phosphate that bears close chemical resemblance with the mineral component of bones and teeth tissues.1 It promotes tissue adhesion and bone growth by spontaneously forming a biologically active bonelike apatite layer over its surface.2 Thus, HA is classified as one of the best biocompatible and bioactive materials, and HA coatings have been found in many biological applications such as dental or skeletal implants and bone repair scaffolds. Template-induced HA has broad prospects in the applied fields of regenerative medicine and bone repair. However, its poor mechanical properties such as brittleness and low wear resistance have limited the use of bulk HA coating in load-bearing and longterm implant applications. In order to create novel nano-biomaterials that mimic bone, it is essential to develop strategies to obtain better mechanical properties, besides of a crystalline structure that is favorable to increase the osteointegration due to bioactivity properties. Studies on the biomineralization mechanism have been aimed to develop a detailed understanding of interfacial interaction associated with biomineralization and template-directed crystallization.3 There is a resurgent interest in controlling HA crystal nucleation, crystallinity, and growth for assembling composite materials analogous to those produced by nature, involving the biomineralization process.4 It was recognized that the poor crystallinity can directly affect the factors governing the natural precipitation of the apatite formation to promote the natural osteointegration.5 With that, the development of *Corresponding author. Tel: 55-12-3208-6576. E-mail: [email protected]. (1) Chen, Y.; Gan, C.; Zhang, T.; Yu., G. Appl. Phys. Lett. 2005, 86, 251905– 251907. (2) Oyane, A.; Onuma, K.; Ito, A.; Kim, H. M.; Kokubo, T.; Nakamura, T. J. Biomed. Mater. Res., Part A 2003, 64A, 339–348. (3) Estroff, L. A.; Hamilton, A. D. Chem. Mater. 2001, 13, 3227. (4) Berman, A.; Ahn, D. J.; Lio, A.; Salmeron, M.; Reichert, A.; Charych, D. Science 1995, 269, 515. (5) Xu, T.; Zhang, N.; Nichols, H. L.; Shi, D. L.; Wen, X. J. Mater. Sci. Eng., C 2007, 27, 579.

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the crystalline nanobiomaterials is of particular interest in regenerative medicine. Among other nano-biomaterials, vertically aligned MWCNTs (VACNT) promise a great role for the study of tissues regeneration.6-10 The electronic structure, the surface morphology, and exceptional mechanical properties of VACNT are typical of graphite-like structures, but they can be distinguished by their tubular construction with nanometric diameters and high aspect ratio; i.e., they are considered a fibrous material.9-11 It has been recognized that hydrophilic surfaces are generally favorable to cellular adhesion, spreading, and proliferation.12 However, several studies have shown that as-grown CNTs are superhydrophobic,13,14 which may be a limitation for their application as nano-biomaterial.15 Recently, Lobo et al.16 have shown the much higher cell proliferation and adhesion on superhydrophilic VACNT. The wettability of VACNT may be controlled by several chemical and physical treatments for VACNT functionalization. As known, oxygen-containing functional groups are formed on the CNT surfaces by treatment with oxidation17 or acid treatment.18 The wettability for polar liquids, such as water, can be enhanced significantly in this way, leading to more reactive (6) Goldberg, M.; Langer, R.; Jia, X. J. Biomater. Sci. 2007, 18, 241–268. (7) Thomas, V.; Dean, D. R.; Vohra, Y. K. Curr. Nanosci. 2006, 2, 155. (8) Bhattacharyya, S.; Guillott, S.; Dabboue, H.; Tranchant, J. F.; Salvetat, J. P. Biomacromolecules 2008, 9, 505. (9) Cui, D. J. Nanosci. Nanotechnol. 2007, 7, 1298–1314. (10) Salvetat, J. P.; Bonard, J. M.; Thomson, N. H.; Kulik, A. J.; Forro, L.; Benoit, W.; Zuppiroli, L. Appl. Phys. A: Mater. Sci. Process. 1999, 69, 255. (11) Endo, M.; Strano, M. S.; Ajayan, P. M. Top. Appl. Phys. 2008, 111, 13. (12) Price, R. L.; Waid, M. C.; Haberstroh, K. M.; Webster, T. J. Biomaterials 2003, 24, 339. (13) Liu, H.; Zhai, J.; Jiang, L. Soft Matter 2006, 2, 811. (14) Sun, T.; Wang, G.; Liu, H.; Feng, L.; Jiang, L.; Zhu, D. J. Am. Chem. Soc. 2003, 125, 14996. (15) Monteiro-Riviere, N. A.; Nemanich, R. J.; Inman, A. O.; Wang, Y. Y.; Riviere, J. E. Toxicol. Lett. 2005, 3, 377. (16) Lobo, A. O.; Corat, M. A. F.; Antunes, E. F.; Ramos, S. C.; PachecoSoares, C.; Corat, E. J. Matter Sci. Eng. C 2010, DOI: 10.1016/j.msec.2010.08.010. (17) Priya, L.; Hossein, T.; Charles, P. Carbon 2004, 42, 2433. (18) Bhalchandra, K.; Vijayamohanan, P. J. Phys. Chem. C 2008, 112, 3183.

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VACNT surfaces.19 Compared to these methods, the exposure of VACNT to oxygen plasma is the most efficient way to introduce simultaneously polar functional groups (COH, OH, CdO, COOH) and roughness19 to the CNTs. Some investigations have been performed in the synthesis of HA over CNTs using various methods, such as simulated body fluid (SBF),20 composite coatings obtained by electrophoresis,21 aerosol,22 and sol-gel23 deposition. These methods consist of dispersing CNT in HA solution and show that thermal treatment was necessary to obtain crystalline HA and that several weeks are required to obtain sufficient mineralization soaked in SBF.20 Lifang et al.24 showed that VACNT were efficient templates to grown HA films using plasma-enhanced chemical vapor deposition and radio-frequency sputtering deposition, but clearly the authors showed that the Ca- and P-rich layer consists of a carbonate-containing HA with disordered structure and thus poor crystallinity. However, the rapid and direct electrodeposition of HA with a high crystallinity, reproducibility, and homogeneity on superhydrophilic VACNT has not been reported. In this paper we studied the use of superhydrophilic VACNT as template for HA growth. To improve film characteristics, the electrodeposition method was proposed. This method has the advantage of production of a thin, crystalline, homogeneous, and adherent film, besides to be rapid, low cost, and efficient process. In particular, the composition and control of the coating structure are possible due to the relatively low processing temperature. For the first time, the direct electrodeposition of HA with a high crystallinity, reproducibility, homogeneity, properties of biomineralization, and exceptional Human osteoblast cells adhesion was obtained. The use of the superhydrophobic VACNT was shown to be essential to obtain such exceptional characteristics. The unique high crystallinity of the HA obtained by the electrodeposition process appears to be responsible for all other properties observed. The HA crystal growth differences were evaluated by scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDX), X-ray diffraction (X-ray), and Raman spectroscopy.

Experimental Procedures The VACNTs were produced as thin film, using a microwave plasma chamber (2.45 GHz). The substrates were 10 mm titanium (Ti) squares, covered by a thin of Fe layer (7 nm), both deposited by an e-beam evaporator. The Fe layers were pretreated to promote nanocluster formation, which forms the catalyst for VACNTs growth. The pretreatment was carried out during 5 min in plasma of N2/H2 (10/90 sccm) at a substrate temperature around 760 C. After pretreatment, CH4 (14 sccm) was inserted in the chamber at a substrate temperature of 760 C for 2 min. The reactor was kept at a pressure of 30 Torr during the whole process.25 Functionalization of the VACNT tips by the incorporation of oxygen-containing groups to obtain the superhydrophilic character was performed in a pulsed-direct current plasma reactor with an oxygen flow rate of 1 sccm, at a pressure of 85 mTorr, -700 V, and with a frequency of 20 kHz.26 (19) Liu, M.; Yang, Y.; Zhu, T.; Liu, Z. Carbon 2005, 43, 1470. (20) Aryal, S.; Bhattaraia, S. R.; Remant, B. K. C.; Khil, M. S.; Lee Duck-Rae, H. Y.; Kim Mater. Sci. Eng., A 2006, 426, 202. (21) Balani, K.; Anderson, R.; Laha, T.; Andara, M.; Tercero, J.; Crumpler, E. Biomaterials 2007, 28, 618. (22) Hahna, B.-D.; Leea, J.-M.; Parka, D.-S.; Choia, J.-J.; Ryua, J.; Yoona, W.-H.; et al. Acta Biomater. 2009, 5, 3205. (23) Najafi, H.; Nemati, Z. A.; Sadeguian, Z. Ceram. Int. 2009, 35, 2987. (24) Lifang, N.; Kua, H.; Chua, D. H. C. Langmuir 2010, 26, 4069–4073. (25) Lobo, A. O.; Corat, M. A. F.; Antunes, E. F.; Palma, M. B. S.; PachecoSoares, C.; Garcia, E. E.; et al. Carbon 2010, 1, 245. (26) Ramos, S. C.; Vasconcelos, G.; Antunes, E. F.; Lobo, A. O.; Trava-Airoldi, V. J.; Corat, E. J. Diamond Relat. Mater. 2010, 19, 752.

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The electrodeposition of the HA crystals on the VACNT films was performed using 0.042 mol L-1 Ca(NO3)2 3 4H2O þ 0.025 mol L-1 (NH4) 3 2HPO4 electrolytes (pH=4.7). The electrochemical measurements were made using a three-electrode cell coupled to an Autolab PGSTAT 302 equipment. Superhydrophilic VACNT films were used as working electrode, and the geometric area in contact with electrolytic solution was 0.27 cm2. A platinum coil wire served as auxiliary electrode, and an Ag/AgCl electrode was used as reference electrode. The HA films were produced applying a constant potential of -2.0 V for 30 min, and the solution temperature was maintained at 70 C. The SBF (5) solution was used for in vitro bioactivity study.27 They were prepared by dissolution of NaCl, KCl, K2HPO4, CaCl2 3 2H2O, MgCl2 3 6H2O, NaHCO3, Na2SO4, and (Na2O) 3 SiO2;all of analytical purity in distilled and deionized water. The pH of all solutions was adjusted to 7.25 at 37 C with 1 N HCl and tris(hidroxymetil)aminomethane. The solutions were kept in closed polyethylene containers. Superhydrophilic HA/ VACNT composites were placed in a polyethylene recipient and were immersed in 15 mL of SBF. All the substrates in their respective recipient were put in an incubator at 37 C for 21 days. After this incubation period, the implants were immersed in distilled and deionized water and finally dried at room temperature. Human osteoblast cells were provided by Cell Line Bank of Rio de Janeiro/Brazil. The cells were maintained as subconfluent monolayer’s in low glucose Dulbeco’s modified essential medium (Sigma) with 1.5 mM L-glutamine adjusted to contain 2.2 g/L sodium bicarbonate 85%; fetal bovine serum (FBS) 15% (Gibco, BRL), 100 units/mL penicillin-streptomycin (Sigma), and 25 μg/mL L-ascorbic acid (Sigma). The incubation occurred within a CO2 (5%) atmosphere at 37 C. The capacities of cellular adhesion of Hob on HA/VACNT composites were evaluated after 7 days of incubation. After the incubation, the attached cells on the substrate were fixed with a 3% glutaraldehyde/0.1 M sodium cacodylate buffer for 1 h and dehydrated in a graded ethanol solution series (30%, 50%, 70%, 95%, 100%) for 10 min each. The drying stage used a 1:1 solution of ethanol with hexamethyldisilazane (HMDS), and the samples were dried with pure HMDS at room temperature. After deposition of a thin gold layer, the specimens were examined by SEM. The contact angle (CA) of deionized water drops (2 μL) with the VACNT films was measured by using the sessile drop method with a Kruss EasyDrop instrument (DSA 100). For each CA measurement was taken in five different values. It was performed immediately after it drops on surface in order to avoid evaporation process. SEM (field emission gun SEM, JEOL JSM-6330F) was used to observe the structure of the VACNTs and HA crystals morphologies. The incorporation of the polar groups on VACNT were monitored by X-ray photoelectron spectroscopy (XPS), using an equipment from VG Microtech (XR 705), operating at 1486.5 eV (Al KR). The elemental composition of the coating was investigated by energy-dispersive X-ray spectroscopy. The structural analysis of HA crystals was performed by X-ray diffractometry (X-Pert Philips) with Cu KR radiation generated at 40 kV and 50 mA. Crystal size of the HA phase (thkl) was calculated using Scherer’s formula. Raman spectroscopy (Renishaw micro-Raman model 2000 with Ar laser, λ = 514.5 nm) measurements were carried out to analyze the VACNT structural analysis, before and after the oxygen plasma functionalization and chemical composition of HA crystals. The structural analysis (X-ray and Raman spectroscopy) of NiTi alloys were used as control to evaluate the efficiency, crystallinity, and fast electrodeposition method of HA crystals on superhydrophilic VACNT. (27) Barrere, F.; Snel, M. M. E.; Blitterswijka, A.; de Klaas, G.; Layrolle, P. Biomaterials 2004, 25, 2901-2910.

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Figure 1. Effect of oxygen plasma functionalization on the VACNTs. Optical microscopy images of the contact angle between deionized water and VACNT before (a) and after (b) the oxygen plasma treatment (magnification 200). C 1s XPS peak analysis before (c) and after (d) the oxygen plasma treatment.

Results and Discussion Figure 1 shows the efficiency of the oxygen plasma treatment to transform VACNT surfaces from superhydrophobic to superhydrophilic behavior. The comparisons between as-grown VACNT and after the plasma treatment were studied using CA (a, b) and XPS (c, d) techniques. A significant change on the contact angle from ∼154 (Figure 1a) to ∼0 (Figure 1b) was achieved. To asses specific carboxylic groups attached on surface, the XPS spectra were deconvoluted at the C 1s. A deconvolution comparison between as-grown VACNT (Figure 1c) and after plasma functionalization (Figure 1d) was shown. All binding energies were referenced to C 1s at 284.5 eV. The spectra were deconvoluted by assuming a Lorentzian-Gaussian sum of functions (20% Lorentzian maximum contribution).28 The spectra were analyzed using Spectrum software XPS peak41. The C 1s peak was decomposed into four Gaussian components, referring to the bonds: CdC (∼284.5 eV), C-O (∼286.2 eV), CdO (286.8 eV), and -COO (287.9 eV).29-31 The intensity of the CdO and mainly the -COO peak increased after the oxidation. From these fits the full width at half-maximum (fwhm) shows an enlargement for all bands (data not shown) after oxygen plasma treatment (Figure 1d). In the quantitative analysis was observed that the oxygen content increased from 2.8% to 18.9%, after the oxygen plasma treatment. (28) Hueso, J. L.; Espino, J. P.; Caballero, A. J.; Cotrino, J.; Gonzalez-Elipe, A. R. Carbon 2007, 45, 89–96. (29) Chirila, V.; Marginean, G.; Brandl, W. Surf. Coat. Technol. 2005, 200, 548–551. (30) Xu, T.; Yang, J.; Liu, J.; Fu, Q. Appl. Surf. Sci. 2007, 253, 8945–8951. (31) Brandl, W.; Marginean, G. Thin Solid Films 2004, 447, 181–186.

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The use of oxygen plasma treatment on CNT surfaces has already been reported in the literature.28,29 All reported works showed only a partial change on the wettability of the CNT surface. Chirila et al.29 demonstrated that the wettability increased up to 68% after the treatment with microwave plasma and 20% with radio-frequency plasma as compared to the untreated CNT. Brandl and Marginean31 showed that the contact angle with water decreased from 88 to 58 after plasma treatment. The plasma conditions used in the present work shows a much higher efficiency as compared to previous reports. Figure 2 shows first-order Raman scattering spectrum from (a) as-grown and (b) oxygen plasma-treated VACNT films. These spectra show large differences in D (1350 cm-1) and G bands (1582 cm-1). The ID/IG ratio in both cases is around 1.7, indicating the similar structural defect densities. However, the bandwidth fwhm of the D band increased considerably, and it appeared a new band around 1525 cm-1 for the O2 plasma-treated sample. This change is characteristic of heavily functionalized VACNT.32 It was not possible to grow HA films on the as grown VACNT. Figure 3 shows the evolution of the cathodic current as a function of deposition time of the HA on the superhydrophilic VACNT films. Three different regions can be observed. A rapid increase of cathodic current due to electric double-layer charging (region 1) was observed. The current continues to increase until it reaches approximately a maximum value around 106 μA (region 2), which is associated with hydroxyl ion (OH-) generation process due to reduction of water and dissolved oxygen on nonuniform VACNT (32) Oswald, S.; Flahaut, E.; Ye, Y.; Gogotsi, Y. Chem. Phys. Lett. 2005, 402, 422–427.

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Figure 2. Raman scattering spectrum from (a) as-grown and (b) oxygen plasma-treated VACNT films.

Figure 3. Current-time profile during the deposition of HA films

on superhydrophilic VACNT films in 0.042 mol L-1 Ca(NO3)2 3 4H2O þ 0.025 mol L-1 (NH4)2HPO4 solution. Applied potential of -2.0 V (vs Ag/AgCl) and solution temperature kept at 70 C.

Figure 4. Energy-dispersive X-ray spectrum of HA electrodeposited on VACNT films.

surface. After this value, the current decreases to a limit value, indicating that the OH- ion generation process is limited by diffusion (region 3). In an electrochemical process, involving the formation of the HA, the OH- ion generation on the surface of interest is one of the fundamental parameters to control the HA characteristics due to acid-base reactions to form (PO4)3- and (HPO4)2-, which are responsible for leading calcium phosphate precipitation on superhydrophilic VACNT films.33 (33) Mahamid, J.; Sharir, A.; Addadi, L.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 12748.

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Figure 5. X-ray diffractograms of as-grown VACNT (a) and HA/ VACNT films (b).

The elemental composition of the coating was investigated by EDX analysis of the HA crystals grown on the VACNTs (Figure 4). The intense peak of Ca, P, and O only shows the presence of strong HA crystals coating over the substrate. The Ca/P ratio determined from the analysis was 1.64, a value near that of the stoichiometric HA (1.67) present in bone tissue.34 Figure 5 shows the X-ray diffraction pattern of the HA grown on superhydrophilic VACNT films. The diffraction peak of the substrate is also shown for comparison. Notice that the apatite formation is deduced from the presence of several characteristic X-ray reflection peaks in the diffraction pattern shown. The principal diffraction peaks of HA appear at 2θ values of 25.9 for reflection (002), at 31.9 (triplet) for reflections (211), (112), and (300), and at 34.0 for reflection (200).35 HA has a hexagonal space group p63/m with a = 0.9430 nm and c = 0.6891 nm. The O-H groups are ordered on the c-axis or (002) plane. For HA with a hexagonal structure, the [001] direction is the usual direction for preferred growth, along which crystal planes are most densely populated with atoms. So the (002) crystalline planes whose axes are the [001] direction would grow preferentially.36 The higher intensity of the (002) plane shows a standard HA growth pattern on superhydrophilic VACNT. The (34) Kale, S.; Biermann, S.; Edwards, C.; Tarnowski, C.; Morris, M.; Long, M. W. Nature Biotechnol. 2000, 18, 954. (35) Klug, H. P.; Alexander, L. E. X-ray diffraction procedures for polycrystalline and amorphous materials; Klug, H. P.; Alexander, L. E. Eds.; John Wiley and Sons, Inc., New York, (1954). 716 Pages. (36) Yousefpour, M.; Afshar, A.; Yang, X. D.; Li, X. D.; Yang, B. C.; Wu, Y. et al J. Electroanal. Chem. 2006, 589, 96.

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Figure 7. (a) SEM image of the cross section of HA film on top of the VACNT. (b-d) SEM images of direct electrodeposition of HA on VACNT films with increasing magnification. Figure 6. Raman spectra of HA obtained on the following conditions: (a) as grown on polished NiTi alloy, (b) as grown on anodized NiTi alloy,41 (c) after heat treatment for 24 h of sample shown in (b), and (d) as grown on the superhydrophilic VACNT films HA grown.

high frequency of these planes may be related to a platelike morphology, as evident in Figure 7c,d. A similar phenomenon of preferred orientation of apatite coatings on titanium substrates has been reported before,37-39 but they were obtained only after a thermal treatment around at 900 C. It is well-known that stoichiometric HA coatings are more crystalline, and therefore less soluble in vitro and in vivo, than other CaP coatings.33 The broadening of a diffraction peak can be related to the mean crystallite size via the Scherer equation (t = 0.89λ/B cos θB),40 where t is the mean crystallite size, B is the peak line width at halfmaximum (in radian), θB is the Bragg diffraction angle, and λ is the X-ray wavelength (Cu KR radiation in our case). The (002) reflection was chosen for analysis of the broadening of the Bragg line; the Gaussian symmetrical profile function was fitted to the Bragg peak for extraction of full width at half-maximum (fwhm). The mean crystallite size was then estimated to be 2.2 nm. The surface chemistry topography is another variable which strongly affects the process of electrodeposition. For instance, one might expect that the surface roughness generates charged regions, which are very susceptible to transfer electrons. Therefore, the structure and crystallinity of the coating would be significantly affected. For comparative studies, HA crystal electrodeposition was realized on NiTi alloys; more details are shown elsewhere.41 In a few words, to obtain crystalline HA crystals on NiTi alloys, it was necessary to apply an anodic current of 3.3 A/cm2 and a thermal treatment at 100 C for 24 h. The anodization process created a nanometric roughness on the NiTi surface. Figure 6 shows the Raman spectra of HA obtained on the following conditions: (a) as grown on polished NiTi alloy, (b) as (37) Eliaz, N.; Eliyahu, M. J. Biomed. Mater. Res., Part A 2007, 80, 621–634. (38) Vijayaraghavan, T. V.; Benesalem, A. J. Mater. Sci. Lett. 1994, 13, 1782– 1785. (39) Manso, M.; Jimenez, C.; Morant, C.; Herrero, P.; Martı´ nez-Duart, J. M. Biomaterials 2000, 21, 1755–1761. (40) Pezzatini, S.; Solito, R.; Morbidelli, L.; Lamponi, S.; Boanini, E.; Bigi, A.; Ziche, M. J. Biomed. Mater. Res. 2006, 76A, 656. (41) Lobo, A. O.; Otubo, J.; Matsushima, J. T.; Corat, E. J. J. Mater. Eng. Perform. DOI: 10.1007/s11665-010-9751-9.

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grown on anodized NiTi alloy,41 (c) after heat treatment for 24 h of sample shown in (b), and (d) as grown on the superhydrophilic VACNT films. The sharp band at 961 cm-1 is characteristic of crystalline HA.42 The sample grown on polished NiTi substrate does not show crystalline HA phase; it only show the peaks of amorphous CO3- and PO4-. Only after the introduction of a nanometric roughness by anodizing the NiTi substrate the HA crystalline phase appeared, but the Raman spectrum (Figure 6b) shows a large luminescent background and the 961 cm-1 band fwhm is around 20 cm-1. After thermal treatment of the HA film grown on the anodized NiTi substrate, the luminescent background decreased considerably, and the 961 cm-1 band fwhm decreased to 16 cm-1. For the HA grown on the superhydrophilic VACNT the higher crystallinity is evident due to the lower band fwhm of 12.5 cm-1, but also other bands of lower intensities were observed at ∼420, 580, and 780 cm-1. These bands are attributed to other forms of apatites such as octacalcium phosphate and dicalcium phosphate dehydrates. The 1030 cm-1 peak has been assigned to apatitic phosphate groups and is observed only in well-crystallized stoichiometric HA. The Raman band recorded at 1040-1045 cm-1 from a human bone ex vivo formed is assigned to P-O stretching.42 Figure 6d also shows the VACNT Raman bands. Figure 7a-d shows SEM of HA crystals formed on superhydrophilic VACNT films. Figure 7a reveals a homogeneous thin layer of HA crystals. Notice that thin HA crystal films (thickness of 3.5 μm) are grown without affecting the alignment of VACNT films. Figure 7b shows the SEM of surface morphology and structures of the as-deposited HA coating. The coating surface exhibited different crystal characteristics and orientation. Regular flakelike structures diverging from center toward periphery are shown. The lamellar and platelike crystal shapes do not show any regular orientation (Figure 7c). Details of the crystal (length of 1-3 μm and thickness of 1-3 nm) shape and orientation are shown (Figure 7d). Liao et al.43 suggested that dispersed CNT provides abundant sites for the nucleation of HA soaked in phosphate solution. They showed that the bamboo-like structure can be associated with nucleation sites for HA formation. The electrodeposition of HA (42) Kale, S.; Biermann, S.; Edwards, C.; Tarnowski, C.; Morris, M.; Long, M. W. Nature Biotechnol. 2000, 18, 954. (43) Liao, S.; Xu, G.; Wang, W.; Watari, F.; Cui, F.; Ramakrishna, S.; et al. Acta Biomater. 2007, 5, 669.

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shown here indicates the growth only on the top surface of the superhydrophilic VACNT, as shown in Figure 7a. This top surface is heavily attacked by the oxygen plasma, which is responsible for the grafting of oxygen groups on CNT and a further roughening of the already entangled characteristics of the VACNT surface. Both characteristics seem to be important to promote HA growth: some authors have already shown better HA growth on rough surfaces,1-5 and the mechanism of HA deposition depends on the liberation of OH- ions that may be enhanced by the presence of oxygen groups on surface.27,33,34 The platelet-like structure of the HA grown, as shown in Figure 7b-d, indicates a high crystallinity that is confirmed by Raman spectroscopy and X-ray analysis. The influence of surface roughness to obtain crystalline phases is clearly shown in the experiments with NiTi substrates. The crystalline phase is only observed on anodized samples that present a nanometric tubular structure, which are rough at the nanometric scale. Even though the HA grown on anodized NiTi surface shows some crystallinity, it is only enhanced by a thermal treatment after growth. The literature shows that further enhancement is only obtained with thermal treatment at high temperature (∼900 C for 24 h).1-5,27,33,34 In the case of superhydrophilic VACNT the thickness of the platelet-like structure is very close to the crystallite size obtained by Scherer formula from the X-ray diffratogram. This indicates that each platelet is a single HA crystal. This morphology is completely different from the HA obtained after heat treatment on NiTi substrate which shows a crystallite size of 4.1 nm. This larger value of the crystallite size may induce the wrong conclusion that this HA is more crystalline on NiTi, but the observation of the SEM image41 (not shown) clearly shows that the prolate paraboloid grains (typical dimensions of hundreds of nanometers) are polycrystalline while the platelets single crystals have dimensions in the order of a few micrometers.The results were absolutely reproducible, and the films were homogeneous through the entire deposition surface. This highly crystalline HA is obtained in very simple experiment and in only 20-30 min of deposition. To verify if the crystalline HA films obtained on superhydrophilic VACNT influence its bioactivity, we performed few experiments to observe biomineralization, bioactivity, and human osteoblast cell attachment. The mechanisms of the bonelike apatite formation on bioactive materials through in vitro incubation in SBF solution have been reported by a number of groups. Current literature has shown some investigations that reported several weeks required for sufficient mineralization.42-49 Akasaka et al.50 got a conclusion that the CNTs in biomineralization process may acts as an effective nucleation surface to induce the apatite formation. According to Liao et al.,43 there is no apatite formation with CNTs when standard SBF or phosphate bovine serum was used. However, fluor ions were added in order to increase the phosphate concentration. After this, the authors showed that a mixture of calcium, phosphate, and carbonated ions was necessary to obtain a mineralization after 1 day. However, the specific HA nucleation site and the influences of the biomineralization ambient conditions are ignored in their research. In fact, many factors, especially the surface chemistry, can greatly influence the growth rate of the apatite layer.44 Matsuda et al.45 found that the type of surface functional groups of the template had a great effect on apatite formation in the SBF solution, and the group -H2PO4 would (44) James, D. K.; Antonios, G. Tissue Eng. 2007, 13, 927–938. (45) Tanahashi, M.; Matsuda, T. J. Biomed. Mater. Res. 1997, 34, 305–315. (46) Boskey, A. L.; Posner, A. S. Calcif. Tissue Res. 1997, 23, 251–258.

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Figure 8. (a, b) SEM images of HA/VACNT composites after soaking in SBF for 21 days and (c, d) human osteoblast cells adhesion on platelet-like HA/VACNT composites after 7 days.

lead to the most satisfying apatite growth rate. Posner et al.46 further found that the presence of Ca-phospholipid-PO4 complexes could also cause the HA formation in vivo. Xiao et al.47 established that a prephosphorylation of CNT were necessary to improve the apatite crystals formation after soaked by 24 h with SBF. Biomineralization and bioactivity process of HA/VACNT composites were confirmed by SEM analysis after soaking in SBF for 21 days (Figure 8a,b). The top surface appears less rough due to the fusion of adjacent HA platelet to from a continuous film (Figure 8a). The cross-section image (Figure 8b) reveals HA film spreading down deep into the VACNT to form a consolidated composite with homogeneous apatite layer along the entire length of VACNT with an increase in thickness from ∼3.5 μm (Figure 7a) to ∼18 μm (Figure 8b). Biomineralization is a quit complicated but powerful approach for the synthesis of advanced materials.47 Niu et al.49 showed that HA/VACNT composites are template-induced HA formation and consequently accelerate in vitro biomineralization process. However, their coated apatite presented small crystallites, poor crystallinity, and defective structure. The initial composites obtained here shown that a high crystallinity and homogeneous surfaces were obtained (Figure 8a,b). Niu et al. showed that biomineralization process occurs after 35 days with homogeneous apatite layer along the entire length of the CNTs. In Figure 8a,b we show better characteristics and in only 21 days. Figure 8c,d shows the SEM images of human osteoblasts adhesion on the crystalline platelet-like HA surfaces after 7 days. The cells spread with no preferential direction, acquiring a flat roughly circular form over the surface. Figure 8c shows that cells proliferate and evolutes to tissue formation. Very healthy cell behavior is observed on HA/VACNT composites showing active formation of membrane projections all over the cell surfaces (Figure 8d). The cells already occupy a considerable area of HA/ VACNT composite surface. Further studies with in vitro and in vivo assays are under study to better characterize the platelet-like HA/VACNT composites bioactivity. (47) Xiao, Y.; Gong, T.; Zhou, S. Biomaterials 2010, 31, 5182–5190. (48) Xu, A. W.; Ma, Y.; C€olfen, H. J. Mater. Chem. 2007, 17, 415–449. (49) Niu, L.; Kua, H.; Chua, D. H. C. Langmuir 2010, 26, 4069–4073. (50) Akasaka, A.; Fumio, W.; Yoshinori, S.; Kazuyuki, T. Mater. Sci. Eng. C 2006, 26, 675–678.

DOI: 10.1021/la1034646

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Lobo et al.

Conclusion The great novelty of the current paper is the direct growth of crystalline HA on VACNT, which shows a high mineralization without any thermal treatment, in only 30 min, with bioactivity properties and exceptional cellular adhesion. These results also proved that the control of the initial stage of high crystallinity of HA nucleation due to the HA/VACNT composites. The high crystallinity influenced positively in the biomineralization by soaking in SBF, which shows an excellent bioactivity compared to similar experiments shown in the literature. Further studies on biomineralization are under way to establish better

18314 DOI: 10.1021/la1034646

conditions to decrease the biomineralization induction time. The consolidated composite obtained after biomineralization is under study to test its mechanical properties. This new method of HA formation may provide an alternative way for the design and preparation of bioactive nanomaterials with improved mechanical properties and tailored microstructure and macrodimension control. Note Added after ASAP Publication. This article was published on October 20, 2010 with an incorrect version of footnote 16. The corrected version was reposted October 26, 2010.

Langmuir 2010, 26(23), 18308–18314