Biomineralization of Superhydrophilic Vertically Aligned Carbon

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Biomineralization of Superhydrophilic Vertically Aligned Carbon Nanotubes Teresa Cristina O. Marsi,† Tiago G. Santos,† Cristina Pacheco-Soares,‡ Evaldo J. Corat,§ Fernanda R. Marciano,† and Anderson O. Lobo*,† †

Laboratory of Biomedical Nanotechnology, ‡Laboratory of Cellular and Tissue Biology, Development Research Institute (IP&D), University of Vale do Paraiba (Univap), Av. Shishima Hifumi, 2911 - São José dos Campos, 12244-000, SP, Brazil § Associated Laboratory for Sensors and Materials (LAS), National Institute for Space Research (INPE), Av. dos Astronautas 1758, São José dos Campos, 12227-010, SP, Brazil

ABSTRACT: Vertically aligned carbon nanotubes (VACNT) promise a great role for the study of tissue regeneration. In this paper, we introduce a new biomimetic mineralization routine employing superhydrophilic VACNT films as highly stable template materials. The biomineralization was obtained after VACNT soaking in simulated body fluid solution. Detailed structural analysis reveals that the polycrystalline biological apatites formed due to the −COOH terminations attached to VACNT tips after oxygen plasma etching. Our approach not only provides a novel route for nanostructured materials, but also suggests that COOH termination sites can play a significant role in biomimetic mineralization. These new nanocomposites are very promising as nanobiomaterials due to the excellent human osteoblast adhesion.

1. INTRODUCTION Biomineralization is a natural self-assembly that generates complex mineral organization at organic template surfaces.1 It is also a natural process in humans and animals, resulting in the formation of bones and teeth. Until now, more than 60 species of biominerals have been reported and produced with desired complex architectures.2 In general, biomineralization occurs in an environmentally benign process in aqueous solution at neutral pH at room pressure and temperature.3 Furthermore, unlike conventional vapor phase deposition, biomineralization enables low cost, rapid mineral deposition with low energy consumption. Nevertheless, biomineralization has only partially succeeded in creating high performance composites thus far. The poor strength and low thermal or chemical stability of organic templates, such as peptides or synthetic nitrogen containing polymers, deteriorate the ultimate strength and sustainability of biomineralized structures.4 Among other nanobiomaterials, vertically aligned carbon nanotubes (VACNT) promise a great role for the study of tissue regeneration. The CNTs have diameters around 10−100 nm, similar to the physical dimensions of extracellular matrix proteins. Hence, CNTs are particularly promising for tissue regeneration.5,6 The electronic structure, surface morphology, and exceptional mechanical properties of carbon nanotubes (CNT) are typical of graphite-like structures, but they can be distinguished by their tubular construction with nanometric © 2012 American Chemical Society

diameters and high aspect ratio; i.e., they are considered a fibrous material.7 It has been recognized that hydrophilic surfaces are generally favorable to cellular adhesion, spreading, and proliferation.8 However, several studies have shown that as-grown CNTs are superhydrophobic,9 which may be a limitation for their application as nanobiomaterials. Recently, Lobo et al.10,11 showed that superhydrophilic VACNT provided higher cell proliferation and adhesion. The wettability of VACNT may be controlled by several chemical and physical treatments for their functionalization. As known, oxygen-containing functional groups are formed on the CNT surfaces by oxidation12 or acid treatment.13 The wettability for polar liquids, such as water, can be enhanced significantly in this way, leading to more reactive VACNT surfaces. Compared to these methods, the exposure of VACNT to oxygen plasma is the most efficient way to simultaneously introduce polar functional groups (COH, OH, CdO, COOH) and roughness10,11,14 to the CNT. Recently, we showed a new and fast method to functionalize VACNT films using oxygen DC-pulsed plasma.15 Here, we introduce a new biomimetic mineralization routine employing Received: January 12, 2012 Revised: February 9, 2012 Published: February 9, 2012 4413

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Figure 1. SEM images, of the as-grown (a) and (c) superhydrophilic VACNT films obtained by Fe catalysis. TEM images of bamboo-like structure of (b) as-grown and (d) superhydrophilic VACNT after the oxygen plasma etching. mTorr, −700 V, and a frequency of 20 kHz.14 The total time of the plasma etching was 120 s. The superhydrophilic properties are given elsewhere.21 The incorporation of the polar groups was monitored by X-ray photoelectron spectroscopy (XPS), using an instrument from VG Microtech (XR 705) operating at 486.5 eV (Al Kα). The curve fitting and data analysis software Fityk 0.9.8 assigned the peak locations and corresponding fitting of XPS spectra (http://www.unipress.waw. pl/fityk/). 2.3. Bioactivity Analyses of Superhydrophilic VACNTs. A simulated body fluid (SBF) (5×) solution was used for in vitro bioactivity study.22 It was prepared by dissolution of NaCl, KCl, K2HPO4, CaCl2 32H2O, MgCl2 36H2O, 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 M HCl and tris(hidroxymetil)aminomethane. The solutions were kept in closed polyethylene containers. Superhydrophilic VACNT films 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 superhydrophilic VACNTs were immersed in distilled and deionized water and finally dried at room temperature. 2.3.1. Biological Apatite Characterization. Surface chemical compositions of the biological apatites were investigated by Fourier transform infrared attenuated total reflection spectroscopy (FT-IR ATR: Spectrum Spotlight-400, Perkin-Elmer) and Raman spectroscopy (Renishaw micro-Raman model 2000 with an Ar ion laser, λ = 514.5 nm). The structural analysis of biological apatites on superhydrophilic VACNT were performed at room temperature by X-ray diffractometry using X-Pert Philips with Cu Kα radiation (λ = 0.154 056 nm) from 10° to 70° in 2θ with the following conditions: voltage of 40 kV and current of 30 mA, step size of 0.02° and counting time of 2 s per step.22 Semiquantitative elemental analyses of calcium (Ca) and phosphorus (P) were carried out by a micro X-ray energy-dispersive fluorescence spectrometer (μ-EDX 1300, Shimadzu, Kyoto, Japan), equipped with a rhodium X-ray tube and a Si (Li) detector cooled by liquid nitrogen (N2). The equipment was coupled to a computer system for data processing. The energy range of scans was from 0.0 to 40.0 eV. The voltage in the tube was set at 15 kV, with automatic current adjustment. The analyses of Ca and P characteristic emissions

superhydrophilic VACNT films as highly stable template materials. The biomineralization was obtained after VACNT soaking in a synthetic body fluid (SBF) know to favor the calcification process. The superhydrophilic VACNT calcification were unambiguously characterized and discussed by Raman spectroscopy, Fourier transform infrared spectrophotometry (FTIR), micro X-ray fluorescence energy-dispersive (μEDX) spectrometry and scanning electron microscopy (SEM). These new biomineralized VACNT nanocomposites show excellent bioactivity and human osteoblast adhesion. Owing to the superior material properties of biomineralized superhydrophilic VACNT nanocomposites and straightforward processing via biomimetic mineralization using SBF, our approach offers a new opportunity for carbon-based mineral nanocomposites for bone tissue regeneration.

2. MATERIALS AND METHODS 2.1. Synthesis of VACNTs. The VACNT films were produced as thin films using a microwave plasma chamber equipped with a 2.45 GHz microwave generator (MWCVD).16−20 The substrates were 10 mm titanium (Ti) squares covered by a thin Fe layer (10 nm) deposited by an e-beam evaporator. The Fe layers were pretreated to promote nanocluster formation, which forms the catalyst for VACNT growth. The pretreatment was carried out during 5 min in plasma of N2/H2 (10/90 sccm) with a substrate temperature of around 760 °C. After pretreatment, CH4 (14 sccm) was inserted in the chamber at a substrate temperature of 800 °C during 2 min. The reactor was kept at a pressure of 30 Torr during the whole process. More details about morphological and structural analyses are given elsewhere.15−20 Scanning electron microscopy (SEM) was used to observe the alignment of the VACNTs. Transmission electron microscopy (TEM) was used to analyze internal bamboo-like structure. 2.2. VACNT Functionalization by Polar Groups. A Krüss Easy Drop system in sessile drop method measured the CA at room temperature to evaluate the wettability of as-grown and superhydrophilic VACNT films. The functionalization of the nanotube tips by the incorporation of oxygen-containing groups was performed in a pulsed-direct current plasma reactor with an oxygen flow rate of 1 sccm, at a pressure of 85 4414

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Figure 2. 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×). C1s XPS peak analysis before (c) and after (d) the oxygen plasma treatment. O1s XPS peak analysis before (e) and after (f) the oxygen plasma treatment. were taken longitudinally on the sample surfaces, with incident beam diameter of 50 μm. The stepping mapping was taken using 40 × 30 points with a step of 20 μm along the biological apatites formed on superhydrophilic VACNT films. Consequently, the analysis was performed in a line 100 μm long and 50 μm thick. The scans were performed with a count rate of 10 s per point (live time) and a dead time of 25%. The equipment was adjusted using a certified commercial reagent of stoichiometric hydroxyapatite (Aldrich, synthetic Ca10(PO4)6(OH)2, grade 99.999%, lot 10818HA) as reference. The measurements were collected using the fundamental parameters of characteristic X-ray emission of the elements Ca and P. The elements O and H were used as chemical balance. The reference was also used as a point in the intensity curve calibration. The energy calibration was performed using internal standards for light elements.23 2.4. Cellular Adhesion on Biomineralized Superhydrophilic VACNT Films. The samples of biomineralized superhydrophilic VACNT after 21 days soaked in SBF were sterilized for 24 h under UV irradiation and were placed in individual wells of 24-well culture

plates. The cells were maintained as subconfluent monolayers in minimum essential medium (MEM) with 1.5 mL 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 mg/mL L-ascorbic acid (Sigma). The incubation occurred within a CO2 (5%) atmosphere at 37 °C.22 The cells were seeded in each well at a concentration of 5 × 105 cells/mL, supplemented with 10% FBS. The incubation was performed under a CO2 (5%) atmosphere, at 37 °C. The adhesion of the human osteoblast cells on the biomineralized VACNTs films was monitored up to 7 days. 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%, and 100%) for 10 min each. The drying stage at room temperature used a 1:1 solution of ethanol with hexamethyldisilazane. The samples were coated by a thin gold film to improve the visualization. SEM imaging was performed for morphological analysis of human osteoblast cell on biomineralized VACNT films. 4415

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Figure 3. SEM images of superhydrophilic VACNTs films soaked in SBF after (a) 7, (b) 14, and 21 days of incubation. The left picture shows the surface view, the central picture is a higher magnification of the surface view, and the right picture is taken at a 45° to see details of the cross section. The culture of human osteoblast cells on biomineralized superhydrophilic VACNTs films were also analyzed by fluorescence microscopy. After 7 days, it was fixed using a solution containing 4% paraformaldehyde plus 4% sucrose, for 15 min. The samples were rinsed three times in PBS buffer 0.1 M for 5 min each. They were blocked with 3% milk plus 0.6% Triton for 1 h. Then, they were rinsed again and incubated overnight with primary antibody anti-Phaloidin (1:25) (Santa Cruz Biotechnology) or unspecific rabbit IgG rabbit (1:50) diluted in 1% milk. Next, they were rinsed for 2 h in PBS and incubated with secondary antibody Alexa Flour 568 antigoat and Alexa Fluor 488 antirabbit, respectively, diluted 1:200 in 1% milk. Before observation, the slides were kept on DAPI 1:100 solution for 10 min, and then, they were rinsed and observed on a fluorescence microscope using red (560 nm) and blue (460 nm) filters.11

3. RESULTS AND DISCUSSION Figure 1 shows the morphological and structural VACNT analyses before and after exposure to the oxygen plasma. Figure 1a shows a SEM image of the high density of the VACNT film as-grown on Fe catalyst. VACNTs presented a length of 6−8 μm. Figure 1b shows a TEM image of typical internal bamboolike structures of the VACNT. Figure 1c shows a SEM image after the oxygen plasma etching and Figure 1d shows structural changes by TEM. Only the morphological VACNT tips changes could be observed on conversion to superhydrophilic VACNT films. No contaminants from either metallic particles or amorphous carbon were observed outside the tubes (Figure 1d). Virtually all metallic particles are enclosed by the VACNT produced, because the MWCVD uses all catalyst nanoclusters. 4416

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Figure 4. FEG-SEM images shown the bioactivity of superhydrophilic VACNTs films after 21 days soaked in SBF. (a) Detail of the biological apatites deposited and (b) cross section showing the densification of superhydrophilic VACNT films.

To assume two different oxygen species is further supported in our case by the large full widths at half maxima (fwhm) for the experimental O1s peaks. For as-grown VACNT (Figure 2e), these four components are found at 531.2 (CO), 532.5 (C− OH), 533.4 (COOH−), and 534.5 eV (C−O) assigned as carboxylate,27−30 oxygen doubly bonded to carbon in carboxylic acids and esters (35−35), and possibly −C−O (534.5 eV) or absorbed H2O. Exposure to the oxygen plasma (Figure 2f) promoted a progressive increase in all oxygen groups, mainly in the fwhm's, which increased (more description in ref 15). In the quantitative analysis, the oxygen content on VACNT surface increased from 2.8% (Figure 2e) to 18.9%, after the oxygen plasma treatment (Figure 2f). The use of oxygen plasma treatment on VACNT surfaces has already been reported in the literature.29,30 However, all reported works showed only a partial change on the wettability of the CNT surface due to the low concentration of carboxyl (carboxylic acid)/carboxylate groups attached to CNT tips (values up to 14%).29 Chirila et al.31 demonstrated that the wettability increased up to 68% after the treatment with microwave plasma and 20% with radiofrequency plasma as compared to the untreated CNT. Chirila et al. also showed that the contact angle with water decreased from 88° to 58° after plasma treatment.31 The plasma conditions (oxygen flow rate of 1 sccm, at a pressure of 85 mTorr, −700 V, and total time 120 s) used in the present work show a higher efficiency compared to the previous reports due to higher concentration of carboxylic groups directly attached to the VACNT tips. The superhydrophilicity of the VACNT films obtained after the oxygen plasma treatment was a requirement for obtaining the bioactivity. As previously described, this is a new method to obtain biomineralized superhydrophilic VACNTs composites that achieve excellent bioactivity and citocompatibility characteristics. Figure 3 shows the SEM images of the superhydrophilic VACNT films after different times of SBF incubation. These incubation times were (a) 7, (b) 14, and (c) 21 days. The left picture is a top view, the central picture is the top view at a higher magnification, and the right picture is a tilted view to show the cross section details. SEM evidenced mineralized nodules on all samples. Mineralized nodules had a rounded shape (calcospherites) and were made of elementary tablets or plates of polycrystalline biological apatite packed together. VACNT were completely recovered with the calcospherites and

The high atomic hydrogen concentration in the gas mixture efficiently removes residues from the amorphous carbon. Figure 2 shows the efficiency of the oxygen plasma treatment on converting VACNT surfaces from superhydrophobic to superhydrophilic behavior. The comparison of as-grown VACNT before and after the plasma treatment was studied using CA (a,b) and XPS (c−f) techniques. Systematic studies using polar and dispersive components using different liquids are shown in other publication.21 From the pulsed−direct current oxygen plasma treatment used in this work, a significant change of the contact angle from ∼154° (Figure 2a) to ∼0° (Figure 2b) was achieved. Hence, the VACNT surface switched from superhydrophobic to superhydrophilic, showing the high efficiency of this treatment. To assess specific carboxyl (carboxylic acid)/carboxylate groups attached on surfaces, the C1s and O1s spectra were deconvoluted (Figure 2c−f). Comparison between as-grown VACNT (Figure 2c and e) and after plasma functionalization (Figure 2d and f) are shown by the respective deconvolution of XPS spectra. All binding energies were referenced to C1s at 284.1 eV. The spectra were deconvoluted by assuming a Lorentzian−Gaussian sum of functions (20% Lorentzian maximum contribution).21 The spectra were analyzed using Spectrum software Fityk 0.9.8. 33 The C1s band was decomposed into five Gaussian components, referring to the bonds: CC (∼284.1 eV), C−O (∼286.2 eV), CO (286.8 eV), −COO− (288.1 eV), and the last one at 290.1 eV assigned to the shakeup peak (σ−σ* transitions).24−27 The intensity of the CO peak, especially the −COO− peak, increased after the plasma etching. From these fits, the widths at half maxima (fwhm) showed an increase and suitable shifts for all bands after oxygen plasma treatment (data shown in Figure 2d). This implies strong C and O bond formation,24−26 mainly carboxyl (carboxylic acid)/carboxylate groups situated at the ends of the tubes. Datsyuka et al.24 also showed that a suitable shift occurs for all bands attributed to carboxyl (carboxylic acid)/carboxylate groups after oxidation using HCl solution. Figure 2e and f shows deconvolution of the O1s spectra. In the literature, the oxygen peak for carbon materials is frequently decomposed into two components: one in the range 531.2− 532.6 eV, attributed to oxygen doubly bound to carbon, and the second in the range 532.8−533.1 eV, attributed to oxygen singly bounded to carbon.27 Figure 2e and f shows four peaks. 4417

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apatite layer.34 Tanahashi et al.35 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 lead to the most satisfying apatite growth rate. Boskey et al.36 further found that the presence of Ca-phospholipid-PO4 complexes could also cause apatite formation in vivo. Xiao et al.37 established that a prephosphorylation of CNTs was necessary to improve the nonhomogeneous apatite crystals formation after soaked by 24 h with SBF. Biomineralization is a quite complicated but powerful approach for the synthesis of advanced materials.38 Niu et al.39 showed that the biomineralization process of CNT films occurs after 35 days with homogeneous apatite layer along the entire length of the CNTs. In Figure 4a, we show better characteristics and in only 21 days. Nucleation, in general, represents an activation energy barrier to the spontaneous formation of a solid phase from a supersaturated solution. This kinetic constraint may be sufficient to offset the thermodynamic driving force for precipitation, resulting in metastable solutions, which do not undergo phase transformations over a long period of time. The activation energy for nucleation (ΔGN) is related to γ and ΔGB by the equation41

progressively became entrapped within them. In general, superhydrophilic VACNT films are bioactive. Commonly, globular apatites, including biological ones, shows up when SBF is used in this biomimetic approach.32 The SEM images of Figure 3 show the massive growth of the apatite crystallites clusters with a globular-like shape on the superhydrophilic VACNTs films (Figure 3). After 7 days of SBF incubation (Figure 3a) notice that considerable differences are observed compared to the superhydrophilic VACNTs topography (Figure 1). The central picture shows porous apatites on superhydrophilic VACNT films. However, it does not form a complete apatite layer deposit yet (right picture of Figure 3a). In this initial stage, only the biological apatites are precipitate on superhydrophilic VACNT tips due to the carboxylic groups attached of them (Figure 2). After 14 days, many globular apatite layers are clearly observed (Figure 3b). At this time the biological apatites formed all over the superhydrophilic VACNT films. Notice that a compact apatite layer formed. This figure is highly illustrative and also shows the strong bioactivity of superhydrophilic VACNT films after the oxygen plasma treatment. Bioactivity is really proven after 21 days of incubation time. Figure 3c shows many cracks in the structure formed by the globular apatites grown on the superhydrophilic VACNT films. These cracks are directly attributed to a higher density of apatites formed. The cross section image shows an increase of the apatite layer deposited all over the superhydrophilic VACNT films. Details of the densification of superhydrophilic VACNTs films after 21 days incubation in SBF are shown in Figure 4. The FEG-SEM in Figure 4a shows details of biological apatites formed. The needle-like apatites formed are the typical morphology of apatites deposited on implant surfaces.32 Figure 4b shows in detail the exceptional bioactivity of superhydrophilic VACNTs films. This cross section view shows the biological apatites formed on and between the superhydrophilic VACNT films. It is very illustrative to define a complete densification among the VACNT. From these findings, clearly we have shown the exceptional bioactivity of superhydrophilic VACNTs films. We have shown an increase of the apatite layer deposited (thickness between 1.5 and 2.0 μm). Biomineralization is a quite complicated but powerful approach for the synthesis of advanced materials.1−3 Many researchers pay great attention to the mechanism of biomineralization on the different material surfaces, especially based on the CNTs. 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.34−40 Akasaka et al.40 concluded that the CNTs in the biomineralization process may act as an effective nucleation surface to induce apatite formation. According to Liao et al.,32 there was 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 carbonate ions was necessary to obtain mineralization after 1 day. However, the specific apatite nucleation site and the influence of the biomineralization ambient conditions were ignored in their research. In fact, many factors, especially the surface chemistry, can greatly influence the growth rate of the

ΔG N = 16πγ33(ΔGB)2

(1)

and ΔGB = kT ln S υ

(2)

where k is the Boltzmann constant, T is the nucleation temperature, S is the relative supersaturation of the solution, γ is the solid/liquid interfacial energy, υ is the molecular volume, and ΔGB is the energy released in the formation of bonds in the bulk of the aggregate. The −COOH groups formed on superhydrophilic VACNT films after the oxygen plasma treatment (Figure 2c−f) constructed ordered “recognized sites″ with high polarity and charged density, which could draw the biological apatite formation on them (Figures 3 and 4). This top surface is heavily attacked by oxygen plasma, which is responsible for the grafting of the oxygen groups onto CNTs and a further roughening of the VACNT surfaces. Clearly, the natural biological apatite formation on superhydrophilic VACNT films is highly influenced by the COOH− groups identified by XPS (Figure 2). The enlargement of the COO− band (288.1 eV) is due to a higher concentration of oxygen after the oxygen plasma treatment (around 18.93%). Both characteristics seem to be important for promoting biological apatite grown: some authors have already shown better apatite growth on rough surfaces,42−45 and the mechanism of HA deposition depends on the liberation of OH− ions, which may be enhanced by the presence of oxygen groups on surface.46 Besides this, carboxylic groups can attract Ca2+ cations and initiate calcium−phosphate precipitation and reproduce the effect of biological molecules such as BSP.47 The natural apatite formation also can be explained due to the higher activation energy of the defects on the VACNT tips after the oxygen plasma that corroborate with nucleation of the apatites in extension of the tubes. Besides, the higher SBF ion concentration brings more Ca2+ and PO43‑ in solution, thus implying higher ion collision probability, in other words, higher ion activation energy. Thereafter, the chance of apatite nucleation and growth increased significantly. 4418

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Figure 5. Structural analysis of biological apatites formed on superhydrophilic VACNT films after biomineralization at different times. (a) Raman and (b) IR spectra of biological apatites obtained on the superhydrophilic VACNT films and (c) X-ray diffractograms identify the biomineralization process. * Diffractogram of as-grown VACNT for comparison (data shown in ref 22).

phenomena indicate that a kind of interaction existed between formed biological apatites and the −COOH groups formed after the exposure of VACNT at the oxygen plasma. The reason could be explained from the structures and ion properties of superhydrophilic VACNTs. There are many interior unsaturated bonds such as CC in the superhydrophilic VACNT, especially at the defect tip position. These bonds (infrared and Raman spectra) are usually considered to be electron deficient. Therefore, the direct bindings between Ca2+ and superhydrophilic VACNT defect tips are almost impossible. On the other hand, the oxygen atoms of the −C−O−C− and −COO− in the superhydrophilic VACNT tips possess many lone pairs of electrons and thus could easily create the ionic interaction with Ca2+. Moreover, others authors also show that there may be a kind of hydrogen bond interaction between the −COO in functionalized nanocomposites and −OH in HA, which could also enhance the growth of HA.50 Therefore, we could reach the conclusion that the biological apatites are initially deposited on the VACNT tips, and after this occurred the complete recovery of the extension of the tubes. Figure 5c illustrates the X-ray diffraction patterns of biomineralized superhydrophilic VACNTs immersed in SBF after three different times (7, 14, and 21 days). The diffraction pattern of the as-grown superhydrophilic VACNT is also shown for comparison. The diffraction peaks were indexed to the Joint Committee on Powder Diffraction Standards using X’pert HighScore software (www.panalytical.com). For this comparison, the diffracted peaks were indexed with their respective crystallographic planes: 29.47° (104), 36.39° (110), 39.45° (113), 43.05° (202), 46.1° (211), 47.82° (018), and 48.77° (116). In addition, they were in close agreement with the values reported in ICDD (PDF 00−005−0586) to Calcite, had rhombohedral structure,51 and remained stable during the whole biomineralization period. The XRD data of the samples depicted in Figure 5c were in good agreement with those previously reported by Liao et al.,32 Barrere et al.,33 and Tasis.54 The XRD results were confirmed by FT-IR; according to Tasis et al. the simultaneous occurrence of peaks at 711, 870, and 1420 cm‑1 indicate the presence of calcite.53 The crystallite sizes for different soaking times of the VACNT films in SBF were 32.4 nm (7 days), 26.1 nm (14 days), and 20.9 nm (21 days),

Raman and infrared spectroscopies are powerful physicochemical vibrational spectroscopic techniques that can be used for HA structural analysis. Typical Raman and IR bands of calcium phosphates grown on superhydrophilic VACNT films after soaking in SBF for different times are shown in Figure 5a and b, respectively. In vitro, biomimetism consists of initiating HA growth on biomaterial surfaces. Figure 5a shows the sharp Raman band at 961 cm−1, characteristic of crystalline phosphate groups. Bands of lower intensities were observed at ∼420, 580, and 780 cm−1 and 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 n-HA.43 It is remarkable to observe it in the biomineralization on superhydrophilic VACNTs films after soaking in SBF. The Raman band recorded at 1040−1045 cm−1 from an ex vivo human bone is assigned to P−O stretching.43 Clearly, a relation between the higher intensity of these peaks with the time of soaking in SBF is observed. Figure 5a also shows the VACNT Raman bands (D and G).48 These bands are not observed after 21 days of superhydrophilic VACNT film soaking in SBF. These analyses are very conclusive and corroborate with SEM images shown in Figure 4a and b. Due to the biomineralization process, the superhydrophilic VACNT films were completely covered by biological apatites. The D and G band peaks decrease accompanied by the biomineralization time due to the density of apatite layer formation that completely recovered the superhydrophilic VACNT films. Figure 5b shows the FT-IR ATR analysis. The multiplets located around 1000 cm−1 are attributed to phosphate modes. The split bands, mainly at 1030 and 1090 cm−1, seem to correlate with the formation of a well-crystallized apatite. A more detailed analysis indicates the presence of a carbonated component, where carbonate ions are substituting in A and B sites of the apatite structure50 (corresponding to phosphate and hydroxyl ions, respectively). Carbonate bands have been detected at 879, 1415, and 1455 cm−1. Molecular and adsorbed water bands are also seen at 1640 cm−1 (Figure 5b). These results are very illustrative and conclusive to determine the bioactivity of superhydrophilic VACNT films. All these 4419

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Figure 6. Micro X-ray fluorescence spectrometry by energy-dispersive mapping of biological apatites formed on superhydrophilic VACNT films after (a) 7, (b) 14, and (c) 21 days soaked in simulated body fluid. (1) show the mapping analyses and (2) quantitative values (mass %) versus line length media.

calcium carbonate hexahydrate, calcium carbonate monohydrate, vaterite, aragonite, and calcite), calcite is the most thermodynamically stable at room temperature which favors its formation, even slightly above these conditions. Other studies involving immersion of carbon nanotubes with some type of functionalization in SBF also found calcite on the surface of the nanotubes after a certain immersion time.52,53 X-ray diffraction patterns of superhydrophilic VACNT after 14 days in SBF (Figure 5c) shown another peak which can be assigned to 31.63° (211), 44.00° (400), and 45.43° (203) of crystalline apatite; this was also related for Yan and co-workers.54 In 2θ = 56.46° only one intense peak appears after 14 days, and disappears unexpectedly after 21 days in SBF. A similar type of event has been reported in XRD pattern for nanotube samples immersed in SBF by Aryal et al.;52 probably in this stage apatitic nanoseeds were grown in matrix which later developed into macrocrystallites, which were diffused in SBF during the

calculated from the most intense calcite peak, at 29.4° (2θ). This reduction was expected, since the calcification process tends to increase with increasing deposition time. The crystal becomes more compact and crystalline until the process stabilizes. Note that the intensity of the peaks does not vary linearly for the most intense peak of calcite (29.4°). From 14 days, SBF intensity starts to reduce. The reduction phase of calcite and apatite is also reduced and tends to become poorly crystalline as can be seen by the shape of the peaks of the apatite phase after 21 days of incubation. These results also show that the method favors control over crystal growth, probably due to the slow initiation of seed formation caused by SBF. The temperature control at 37 °C of samples immersed in SBF can produce sharper peaks than those under ambient conditions,53,54 and the strong intensity of the peaks indicated a very crystalline calcite. According to Tasis et al.,53 of the six phases of calcium carbonate (amorphous calcium carbonate, 4420

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biomineralization process. The biomineralization in vitro process ceased at this time due to the saturation of nanocomposites. We have analyzed in other times and observed that the biological apatite layer is released in the medium (no data show). From this paper, we have shown the limit time of the calcification in vitro process in SBF solution. Finally, note that we did not find characteristics of the hydroxyapatite process cited in the literature.52,53 Our results showed that VACNT was capable of nucleating calcite crystals from SBF within 7 days, and formatted crystals of the calcite are sensitive to synthesis conditions. During the natural biological apatite formation, the carboxyl (carboxylic acid) groups may lose their hydrogen atoms and obtain negative electrical charges. The negative charge is evenly distributed on the two oxygen atoms. This negative charge is prone to attracting positively charged calcium ions (Figure 5b). Then, by exposing this reactive assemblage to phosphate and hydroxide ions, calcium phosphate is expected to form on the superhydrophilic VACNT surface through the carboxyl functionalized site (after plasma treatment). This mechanism is believed to be responsible for initiation of the biomineralization process on the superhydrophilic VACNT surface.52,53 Figure 6 shows that the elemental composition of the coating was investigated by μEDX mapping analysis of biological apatites grown on the superhydrophilic VACNTs after different SBF incubation times of (a) 7, (b) 14, and (c) 21 days. The pictures on the left are a μEDX line mapping analysis,1 and the pictures on the right show the quantitative values (mass %) versus line length media analyses in these different SBF soaking times.2 The Ca and P content profiles (% wt) as a function of depth (μm) for each group are shown. The Ca/P ratios determined from the analyses were as follows: 1.28 (7 days), 1.48 (14 days), and 1.55 (21 days). These data were collected from the data media of 3 points, area: 0.8 mm × 0.6 mm. All the data show values near the biological apatites present in bone tissue.33 It is very illustrative and corroborates with the SEM, Raman, FTIR, and X-ray analyses to prove the bioactivity of the superhydrophilic VACNT films. Figure 7 illustrates the establishment of ionic bonding between carboxylate groups and calcium ions, after superhydrophilic VACNT soaking in SBF. It has been suggested that the carboxylate groups facilitate the initial deposition of calcium ions, and the attraction of calcium ions is an important initial step in calcium phosphate formation.55 With the increasing density of carboxylate groups, the abundant supply of coordination sites available for complexation with calcium ions led to a very large number of nuclei for the natural apatite deposition. The large amount of nuclei facilitates the precipitation and formation of apatite on the superhydrophilic VACNT films. With this sequence of reactions, it is possible to revise the Saffar et al.56 mechanism, and a revised one can be proposed as presented in Figure 7. Cui et al.57 proved that electrostatic attraction could be further facilitated by strong −COO−Ca2+ or (−COO−)2Ca2+ interactions and the weaker −O−Ca2+ interaction during the induction periods for apatite nucleation on polymer nanofibers using SBF solutions. After this, the positive charge of amino groups could form ionic bonds with carboxylate groups, which could initiate phosphate ions on the RCa+ to form HA. The proposed model presented here is the same, due to the higher network of carboxyl or carboxylate groups attached on superhydrophilic VACNT tips.

Figure 7. Schematic illustration of biological apatite formation and dissolution during soaking of the superhydrophilic VACNT surfaces in SBF based on the model of Saffar et al.56

Saffar et al.57 proposed a model of carboxylate/carboxyl (carboxylic acid) functionalized CNTs (CNT−COOH) to chemically synthesize HA, as an effective template. The authors showed that the calcium phosphate-like material induces ionic interaction with the ACOO headgroup as a result of oppositely charged ions at the contact sites. Biomimetic bone tissue engineering materials could be used to restore and improve damaged bone. The objective of the study was the generation of superhydrophilic VACNT films to induce in vitro calcification. Nanotubes mimic collagen due to their microfibrillar structure, as revealed by electron microscopy. Thus, the nanotube scaffolds represent suitable 3-D arrangements similar to the randomly distributed collagen fibrils in woven bone.5,6 Carbon nanotubes appear to be privileged candidates for inducing mineralization of biological apatites. In this way, for the first time we have shown the cellular adhesion on the biomineralized superhydrophilic VACNTs. For this, human osteoblast cell adhesion on the biomineralized superhydrophilic VACNT films was examined by the SEM (Figure 8a) and immunostaining of phaloidin (Figure 8b) at 7 days. Highly attached cells were observed on biomineralized superhydrophilic VACNTs. A specific antiphaloidin was used to observe the adhesion (Figure 8b). DAPI was used to mark nucleolus (Figure 8b). A high degree of biomineralization seemed to facilitate the cell adhesion, raising the number of adhered cells (Figure 8b). In addition, a considerable increase of cell cytoplasmic projections was observed on biomineralized superhydrophilic VACNTs, with all the actin filaments of the cells completely spreading and strained (highlighted by red in Figure 8b). Detail of this actin filament is shown in Figure 8c. Cells spread horizontally across the surface on biomineralized superhydrophilic VACNTs (Figure 8a), showing a very confluent layer. Fluorescence microscopy results (Figure 8b) were consistent with the enhanced spreading in human osteoblast cell morphology and filopodia extension on 4421

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Figure 8. Cellular adhesion of human osteoblast cells on biomineralized superhydophilic VACNT nanocomposites after soaking in SBF at 21 days. (a) Scanning electron microscopy of human osteoblast cell after 7 days of incubation. (b) Fluorescence microscopy images of human osteoblast cells marked by the immunostaining reaction of actin filaments (red) and DAPI (blue) at 7 days. (c) Details of (b).

the in vitro biomineralization with biological apatite deposition. All changes promoted by the plasma etching can only assert that the treatment was beneficial for biomineralization, and consequently cell adhesion and growth. This may indicate that not only the superhydrophilicity, but also the chemical modifications on CNT surface may positively influence the cell adhesion and spreading. Also, the cell preference by a high number of attachment sites provided by biomineralization of superhydrophilic VACNT facilitates cell growth and spreading, as well as encourages monolayer formation. In general, considering the use of SWCNTs, studies have shown an altered organization and decreased amount of those adhesive proteins inside the cells grown with CNTs. The actin stress fiber was thinner and diffused when the SaOS-2 cells were grown with SWCNTs. Furthermore, vinculin formed small patches all around the cells and actin filaments did not convincingly end in these spots, which is different from what happened when those cells were cultured on glass or titanium alloy.43 On the other hand, human cervical carcinoma HeLa cells showed altered morphology when grown on SWCNTs, but their morphology was similar to that of the cells on glass when they were grown on an MWCNT slide. The FAK distribution inside the cells was also different on SWCNTs (peripheral) and MWCNTs (homogenously distributed in whole cell body).59 This suggests a variation in the cell behavior according to the type of CNTs grown on. Our results on biomineralization, cell spreading, and adhesion of super-

biomineralized superhydrophilic VACNT films found above. This suggests that biomineralization also induced distinct intracellular processes that may modify the behavior of the cells. Simple observation of the extension of the cell corroborates this. Consequently, more surface contact by the cell to superhydrophilic VACNT films results in an increased number of actin filaments (red). Although the expressions of adhesion proteins were not measured, the images might indicate greater expression of adhesion proteins. Those yields are already being built by our group. This dot expression (white arrows) can be related to two different phenomena: higher adhesion of both cell-to-cell and cell-to-biomineralized VACNT substrates. Surfaces play an important role in a biological system for most biological reactions occurring at surfaces and interfaces. Cell attachment and proliferation strongly depend on the chemical and physical properties of the surface. In culturing cells on biomaterial surfaces, surface free energy is an important parameter that guides the first events occurring at the biomaterial/biological interface, such as interaction of water and proteins with biomaterial, guiding further responses.58,59 The results presented here indicate that biomineralization of superhydrophilic VACNT scaffolds may become of great interest for biomaterial manufacture for future use in biomedicine. A simple oxygen plasma etching modified the surface characteristics (variation of CA ∼160°) and incorporated carboxylic and other oxygen polar groups onto VACNTs. The superhydrophilic VACNT films after oxygen plasma treatment presented a bioactivity characteristic that induces 4422

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hydrophilic VACNT films support the concept that wettability of VACNT scaffolds affects cell behavior.

4. CONCLUSION In conclusion, we have demonstrated the biomimetic mineralization of superhydrophilic VACNT films. The biomineralization VACNT template offers a new perspective for mineralization and carbon based nanocomposites under mild processing conditions. The detailed structural analysis revealed that the polycrystalline biological apatite formation is due to−COOH end groups that were attached to VACNT tips after oxygen plasma etching. Our approach not only provides a novel route for nanostructured materials, but also suggests that −COOH termination sites can play a significant role in biomimetic mineralization. The new nanocomposites presented here are very promising as nanobiomaterials due to the excellent biomineralization process and human osteoblast adhesion.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by FAPESP (Proc: 2011/17877-7). Special thanks to Priscila Leite from Universidade do Vale do Paraiba by scanning electron microscopy images.

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REFERENCES

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