MWCNT nanocomposites

Materials Science and Engineering C 33 (2013) 4305–4312

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Effect of ultrasound irradiation on the production of nHAp/MWCNT nanocomposites Anderson O. Lobo a,⁎, Hudson Zanin b, Idalia A.W.B. Siqueira a, Nelly C.S. Leite a, Fernanda R. Marciano a, Evaldo J. Corat b a Laboratory of Biomedical Nanotechnology, Development Research Institute (IP&D), Universidade do Vale do Paraiba (Univap), Av. ShishimaHifumi, 2911, São José dos Campos, 12244-000 SP, Brazil b 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

a r t i c l e

i n f o

Article history: Received 15 January 2013 Received in revised form 23 May 2013 Accepted 19 June 2013 Available online 28 June 2013 Keywords: Nanohydroxyapatite Carbon nanotubes Wettability Nanocomposites Tissue regeneration

a b s t r a c t Large amounts of nanohydroxyapatite (nHAp)-multiwall carbon nanotube (MWCNT) nanocomposites are produced by two different aqueous precipitation methods. The ultrasonic irradiation (UI) and slow-drip addition under continuous magnetic stirring (DMS) methods were used to investigate the precipitation of nHAp acicular crystals. Calcium-nitrate, diammonium hydrogen phosphate, and ammonium hydroxide were used as precursor reagents. Superhydrophilic MWCNT were also employed. XPS analysis evidences that the functionalized MWCNTs are composed of 18 to 20 at.% of oxygen and that this property influences the nHAp formation. The high surface area of the MWCNT decreases the mean free path of ions, favoring the nHAp formation assisted by UI. The crystallinity was evaluated using the Scherrer equation. Semi-qualitative energy dispersive spectroscopy (EDS) analysis showed that the main components of HAp powders were calcium and phosphorus in the ratio Ca/P around of 1.67. Bioactivity properties of the nHAp/MWCNT-UI nanocomposites could be evaluated after 14 days soaking in simulated body fluid medium. Scanning electron microscopy, EDS, Fourier transform infrared attenuated total reflection spectroscopy, and X-ray diffraction techniques proved that the apatites formed on the surface and to points that the nHAp/MWCNT-UI have potential biological applications. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Hydroxyapatite (Ca10(PO4)6(OH)2, HAp) is used as a biomaterial to regenerate bone tissue, because of its chemical and crystallographic similarities with the main inorganic component of natural bone [1,2]. The close chemical likeness of HAp to natural bone has led to extensive research to use synthetic HAp as a bone substitute and/or replacement in biomedical applications [3,4]. However, its poor mechanical properties such as brittleness and low wear resistance have limited the use of bulk HAp coatings in implant applications [5]. To overcome this problem, HAp composites are reinforced with polymers used [6,7]. HAp has better osteoconductivity if the crystal is biological apatite likeness. For this reason, reducing the size of the HAp crystal to nanoscale is of current interest [8]. Many methods have been developed to prepare HAp powders. The techniques include sol–gel [9], homogeneous precipitation [10], hydrothermal [11], mechano-chemical [12], RF plasma spray [13], spray dry [14], combustion synthesis [15], supersonic rectangular jet impingement [16], ultrasonic spray freeze-drying [17], and sonochemical ⁎ Corresponding author. Tel.: +55 12 3947 1100; fax: +55 12 3947 1117. E-mail addresses: [email protected], [email protected] (A.O. Lobo). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.06.032

synthesis [18] methods. In this work, an aqueous precipitation process is used to prepare HAp powders because it is simple and does not require expensive equipment. Multiwalled carbon nanotubes (MWCNTs) are arousing the interest of researchers in the biomedical area due to their exceptional combination of mechanical and chemical properties with biocompatibility [19]. Hydrophilic surfaces are generally favorable to cell attachment and biomineralization of bone tissue [20]. Some investigations have been performed on the synthesis of nHAp and MWCNT using various methods to obtain nHAp/MWCNT nanocomposites [21]. However, these methods consist in nonhomogeneous dispersion of the MWCNT in nHAp solution. Recent studies have shown reactivity of chemical species in solution involved in a synthesis stimulated by ultrasonic irradiation (UI) of the reaction mixture. This type of treatment causes cavitation in an aqueous medium inducing formation, growth, and collapse of micro bubbles. This intense agitation process provokes the dissolution–precipitation of solids through which a reduction of particle size and surface activation of the product is achieved [22–24]. The efficiency of the synthesis process depends on many variables, the most important of which is the type of ultrasonic agitation device; the use of a tip ultrasonic homogenizer is more efficient than an ultrasonic bath.

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In vitro and in vivo studies showed bioactivity properties of ceramic biomaterials due to apatite layer formed on their surface [25,26]. Simulated body fluid (SBF) is used for in vitro bioactivity tests for biomaterials due to its similarity to blood plasma components [27]. In the literature, the SBF solution preparation is made through different methodologies. The SBF solution is used to demonstrate the bioactivity of the material, by immersing the samples in this solution, which induces biomimetic process promoted by the Ca–P nucleation on the surface of the biomaterial [28,29]. In this work, for the first time, nHAp/MWCNT composites produced with an aqueous precipitation process, and two different experimental conditions (assisted or non-assisted by UI) are compared for their resulting crystallinity, homogeneity, and stoichiometry. nHAP/MWCNT-UI composites showed bioactivity properties after 14 days soaking in SBF solution due to a layer of apatite formation and the carbonated calcium phosphate precipitation. These findings make these new nanocomposites very attractive for further studies in bone tissue regeneration because they can be functionalized with the biomolecules of interest such as collagen and chitosan to improve the biological response for clinical applications. 2. Experimental 2.1. Reagents Calcium-nitrate (Ca(NO3)2), diammonium hydrogen phosphate ((NH4)H2PO4), and ammonium hydroxide (NH4OH) water solution were used as the starting materials for hydroxyapatite precipitation. The camphor (C10H16O), ferrocene (Fe(C5H5)2), and hydrochloric acid (HCl) were used for MWCNT synthesis and treatment. All chemicals used in this work were analytical grade products purchased from Sigma Aldrich, Co. 2.2. Synthesis of MWCNT The MWCNTs were prepared using a mixture of camphor (85 wt.%) and ferrocene in a thermal chemical vapor deposition (CVD) furnace, as reported elsewhere [30]. The mixture vaporized at 220 °C in an ante-chamber and then, the vapor was carried by an argon gas flow at atmospheric pressure, to the chamber of the CVD furnace set at 850 °C. The time elapsed during the process used to produce the MWCNTs was only a few minutes. 2.3. Treatment and functionalization of MWCNT The removal of the catalytic particles from the MWCNTs was performed by acid etching. The samples were subjected to UI for 5 h in 10 M of HCl, and then, washed extensively in water, and dried. The incorporation of oxygen-containing groups was carried out 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 pulse frequency of 20 kHz, as in a previous work [31]. 2.4. Synthesis of nHAp/MWCNT nanocomposites The nHAp/MWCNT nanocomposites were prepared using two methods for comparison. The main difference between these methods is the activation energy that promotes the precipitation of nHAp crystals. The UI and dropwise addition under continuous magnetic stirring (DMS) methods were used for the investigation. The solutions used for both of the samples were as follows: 100 mL of 0.167 M of Ca(NO3)2·4H20 and 100 mL of 0.1 M of (NH4)H2PO4·NH4OH were added to both solutions to fix the pH ~10. These initial proportions yield theoretical calcium to phosphorous ratio (Ca/P) of 1.67. The MWCNT (1 wt.%) powder was mixed with diammonium

hydrogen phosphate water solution. Eq. (1) shows the yield calculated. 

 CaðNO3 Þ2 ðgÞþ ðNH4 ÞH2 PO4 ðgÞ −½nHAp=MWCNT nanocomposites ðgÞ

ð1Þ 2.4.1. Ultrasonic irradiation (UI) In the ultrasonic irradiation procedure, the calcium-nitrate and diammonium hydrogen phosphate/MWCNT aqueous solutions were mixed before UI. The solutions were mixed by pouring one solution into the other. Thereafter, the precipitations were subjected to ultrasound for 30 min (Ultrasonic Processor 500 W; 20 kHz; 13 mm probe; model: SO-VCX-500, SONICS), maintaining the pH above 10 every 10 min. The resulting suspensions were left to age for 120 h. After the samples were filtered and washed with water, they were left to dry at 60 °C for 4 h. 2.4.2. Dropwise magnetic stirring (DMS) For sample nHAp/MWCNT, the ammonium phosphate solution was stirred constantly and kept at 60 °C while adding the solution of Ca(NO3)2·4H2O using a slow drip (dropwise) at a rate of 1.2 mL/min. During the dropwise procedure, the solution was heated to 100 °C and the temperature was kept constant for about 15 min. From this, a milky, somewhat gelatinous precipitate was produced. This precipitate filtered was washed with deionized water, and dried in an oven for 2 days at 60 °C. Subsequently, the nanocomposites were calcinated in a kilnat 650 °C under N2 flow for 1 h. 2.5. Characterization of nHAp/MWCNT nanocomposites Morphological analyses were performed by FEI Inspect F50–High Resolution Scanning Electron Microscope (HRSEM) with coupled energy dispersive X-ray (EDS) instrument operated at 20–30 kV. Raman spectra were recorded at ambient temperature using a Renishaw microprobe system, employing an argon laser for excitation (λ = 514.5 nm) with a laser power of approximately 6 mW. The spot size used was 15 μm. The surface area measurements were carried out using Quanta chrome Nova Win model 1000 for multi-point BET using the classical helium void volume method. The phase and crystallinity of the resulting nanopowders were characterized by X-ray diffraction (XRD: Lab X (XRD-6000), Shimadzu. X-ray Diffractometer) with monochromatic CuKα radiation. The data were collected over the course of 2 h over a range of 20–80° with a scanning step of 0.02°, and data over a narrow range of 24–36° angles were collected to calculate the crystallinity and crystallite size. The HAp preferential growth plane was calculated using the equation suggested by Hu et al. [32]. The crystallinity could be evaluated by the following empirical relation (Eq. (2)) [33]:  Xc ¼ 1− V ð112Þ=ð300Þ =Ið300Þ

ð2Þ

where Xc is the crystallinity, I(300) is the intensity of the (300) reflection, and V(112)/(300) is the intensity of the hollow between (112) and (300) reflections, which completely disappear in nanocrystalline samples. The crystallite size in the [002] direction could estimate from the peak broadening of XRD reflection according to the Scherrer formula (Eq. (3)) [34]: r ¼ Kλ=B cosθ

ð3Þ

where r is the crystallite size [nm], K is a constant taken as 0.9, λ is the wavelength of monochromatic X-ray beam [nm] (λCuKα = 0.15418 nm), B is full width of the peak at half maximum (FWHM) intensity of (002) reflection [rad], and is the exact diffraction angle [°] satisfying the Bragg's law for the (002) plane.

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The relative intensity (RI) of the (002) peak in comparison to the intensities of the three strongest peaks for HAp powder-sample standard (JCPDS 9-432) is defined as (Eq. (4)): RI ¼ Ið002Þ =Ið211Þ þ I ð112Þ þ I ð300Þ

ð4Þ

where I(002), I(211), I(112), and I(300) are the XRD peak intensities of (002), (211),(112), and (300) planes, respectively. For the HAp powder standard calculated in reference to JCPDS 024-0033, RIs = 0.1818 for the (002) peak. Each peak has its own RI and RIs. In this work, the preference, P, is defined as the relative difference between RI and RIs, as presented in the Eq. (5): P ¼ RI−1

ð5Þ

Fourier transform infrared attenuated total reflection spectroscopy (FTIR-ATR: Spotlight 400–Perkin-Elmer) and FT-Raman spectroscopy (RFS100: λ = 1064 nm, Bruker) were used to analyze the surface chemical composition of the powders produced. All measurements were conducted at room temperature. 2.6. Bioactivity of nHAp/MWCNT-UI nanocomposites The SBF solution was prepared by dissolving ions in distilled water in a stir plate. The SBF compositions were proposed by Barrere et al. (Table 1) [35], whose component concentration is five times higher than the original solution proposed by Kokubo [36]. The pH of the solutions was adjusted with a pH meter (Metrohm), so that values were 7.40 (adding NaOH) in order for the pH of the fluid to be near that of blood plasma. The powder samples (nHAp and nHAp/MWCNT-UI) were bonded by biological adhesive compound of cyanoacrylate on the titanium plates (1 cm2). The nHAp and nHAp/VAMWCNT-UI nanocomposites were immersed into polyethylene containers with 15 mL of each SBF pH solution for 14 days and placed in a refrigerated bench top incubator (Cientec CT-713), shaken at 75 rpm at a temperature around 36.5 °C. The pH values of the nHAp and nHAp/MWCNT-UI nanocomposites were soaked in SBF solution and this solution was collected in different pre-determined time intervals using a pH meter. After the incubation time, the samples were washed with deionized water at 80 °C. HRSEM, EDS, FTIR-ATR, and XRD analyses were used to identify the apatite formation on the surface of the samples after immersion in SBF medium, according to characterization methodology. For XRD analyses, the carbonated HAp reference to JCPDS 00-019-0272 was used. 3. Results and discussion Fig. 1 shows the (a) C1s and (b) O1s fitted photoemission spectra recorded for superhydrophilic MWCNT. The C1s curve deconvoluted into four peaks at around 284.5, 285.8, 288.5, and 291.6 eV. The peaks correspond to aliphatic carbons (with C―C single bonds); carbon atoms with C―O, C―O―C, or C―OH single bonds; carbon atoms with C O double bonds; and with carbon atoms with the COO― carboxylate bonds, respectively. The last one at 291.6 eV is used to assign the shake-up peak (p–p* transitions). The O1s curve

Table 1 Components and concentration of SBF. Reagents

Quantity (mM)

NaCl MgCl2·6H2O CaCl2·2H2O Na2HPO4·2H2O NaHCO3

733.5 7.5 12.5 5.0 21.0

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deconvoluted into three peaks at around 535.5, 533.5, and 532.3 eV, which correspond to C―O, COOH―, and C―OH, respectively. This infers the strong C and O bond formations [37,38] of mainly oxygen content groups that are situated along the tubes. The oxygen plasma creates open-end termini in the structure, which are stabilized by ―COOH and ―OH groups, which are left bonded to the nanotubes at the end termini and/or the sidewall defect sites. The ―COOH was expected to covalently bond to MWCNT due to the strong interaction of CNT carbon atoms. These functionalized MWCNTs were present around 18–20 at.% of oxygen. This is obviously due to increased functionalization from increased surface area and/or open-end termini from the open tubes because functionalization of the sidewalls is harder to realize. The superhydrophilicity properties presented by MWCNT powders after oxygen plasma treatment were essential to produce, for the first time, the nHAp/MWCNT-UI nanocomposites. Fig. 2 shows the X-ray diffraction pattern of the (a) nHAp/ MWCNT-UI; (b) nHAp/MWCNT-DMS; (c) nHAp, and (d) MWCNT powders that we obtained. The band width of the pattern could relate to the mean crystallite size using the Scherrer equation (r = 0.89λ/ Bcosθ). Each nHAp crystal orientation is specified in the XRD pattern, where the main diffraction peaks of HA appear at 2θ = 28.93° (Fig. 2(a)). The nHAp peaks with higher intensity are identified with an asterisk (*), because of the 85% specificity compared to JCPDS: 024-0033 card. The direct precipitation of nHAp depends on the availability of 2+ PO3− , and OH− ions in the solution, where the agitation is 4 , Ca the key to dissolution of the solid substances. Nanocrystalline HAp powders can be obtained by adjusting the reaction conditions and using reduced time periods of UI (7–10 min) compared to those reported in the literature (60 min). It was reported that the crystallinity of HAp prepared by the conventional precipitation method at low temperature (15 °C) for 24 h was only 0.03 [39]. Pang et al. showed that the crystallinity of HAp increased to 53% after calcination [39]. Recently, Zou et al. associated the ultrasound and microwave irradiation to nHAp production [40]. However, these authors showed a lower crystallinity (~ 33%) for all the nHAp crystals produced. For comparison, Table 2 presents the results obtained here. In our results, we obtained 25% crystallinity in as-preparednHAp/ MWCNT-DMS, and 80% crystallinity in as-prepared nHAp/MWCNT-UI without calcination. The results of crystallinity obtained by UI were higher than the results obtained by DMS method. We observed the great influence of hydrophilic MWCNTs on the formation of nHAp. The presence of MWCNT, with its high surface area of 40 m2/g, decreases the mean free path of ions, favoring nHAp formation. Furthermore, the presence of carboxyl groups on the CNT walls, facilitates nHAp crystallization and large amounts of precipitates. The typical morphology of the nHAp/MWCNT reveals excellent interaction between the two components of the composite, which was observed previously by XRD and confirmed by Raman spectroscopy. Raman spectroscopy is a powerful tool for structural analysis of HAp and MWCNT. The evaluation of the carbon hybridization of the hydrophilic MWCNT was performed, employing laser wavelength of 1064 nm for excitation. This wavelength interacts strongly with carbon sp2 hybridization, because it is more sensitive to amorphous carbon than typical visible laser lines, employed for Raman scattering [41–43]. Furthermore, this laser can attenuate sample fluorescence from most organic molecules, which is usually important for biological tissues [44]. Fig. 3 shows the Raman spectra of (a) nHAp/MWCNT-UI and (b) nHAp/MWCNT-DMS nanocomposites. The peak at 961 cm−1 is characteristic of crystallized HAp. The sharp peak at 1030–1050 cm−1 is assigned to apatitic phosphate groups and is observed only in well-crystallized nHAp [45]. Bands of lower intensities were observed at ~ 420, 580, and 780 cm−1 and

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Fig. 1. XPS spectra of the superhydrophilic MWCNT: (a) and (b) are the deconvolution of the regions containing C and O, respectively.

attributed to other forms of apatites such as octacalcium phosphate and dicalcium phosphate dehydrates [46]. The peak at 725 cm−1 is associated to the carbonate group. The two peak clusters at about 900 cm−1 to 1150 cm−1 and 550 cm−1 to 600 cm−1 represent a typical apatite spectrum. Generally, these peaks are attributed to the B-type substitution and the lower intensity band at 872 cm−1 is assigned to the A-type carbonate substitution. The P–O stretching IR mode, which appears at ~ 962 cm−1 in the spectra of all the samples, is attributed to phosphates. The PO4 region appears as a very strong band at ~ 1029 cm−1, a strong shoulder at ~ 1060 cm−1, and a third clearly distinguished band at ~ 1092 cm−1 in all the spectra of nHAp [47–49]. The Raman band recorded at 1040–1045 cm−1 from a human bone ex vivo is assigned to P–O stretching [47–49] and is clearly more evident in nHAp/MWCNT-UI. The first order D and G bands of superhydrophilic MWCNT are shown in the Raman spectra. The ID/IG ratio is smaller in (a) nHAp/ MWCNT-UI(FWHM = 1.55) compared to (b) nHAp/MWCNT-DMS (FWHM = 2.32) nanocomposites, from which one could conclude that the formation of nHAp changes the shape of the Raman spectrum of the superhydrophilic MWCNT. That change could be explained by the direct participation of the superhydrophilic MWCNT in the synthesis of the nHAp. There are many interior unsaturated bonds such as C C in the superhydrophilic MWCNT, especially in the vicinity of the defects. These bonds (identified by infrared and Raman spectra analyses)

are usually considered to be electron deficient. Therefore, bonds between Ca2+ and superhydrophilic MWCNT defects are almost impossible. On the other hand, the oxygen atoms of the ―C―O―C― and ―COO− in the superhydrophilic MWCNT possess many lone pairs of electrons and thus could easily create the ionic interaction with Ca2+. Moreover, other authors also show that a hydrogen bond interaction between the ―COO in functionalized nanocomposites and ―OH in nHAp may exist, which could also enhance the growth of HAp [50] minimizing apparent defects in superhydrophilic MWCNTs. Furthermore, we analyzed the Raman spectra of the superhydrophilic MWCNT, which was also sonicated in deionized water. We did not observe any appreciable change in the Raman spectra, even though the ultrasonic irradiation was damaged and broke the superhydrophilic MWCNT, principally in areas with defects. From our results, we conclude that the UI per se is not what causes the change of the Raman spectra of the MWCNT. Instead, we suggest that the nHAp crystals are formed primarily onto MWCNT defects, covering those regions, blocking the access of the laser line, and consequently masking the real Raman spectra. Fig. 4 shows FTIR-ATR spectra of (a) nHAp/MWCNT-UI nanocomposites, (b) nHAp/MWCNT-DMS nanocomposites, and (c) nHAp. In a general overview, several peaks and bands are observed and identified. The bands at 564 and 601 cm−1 are from the vibrations of the O–P–O mode [51]. The 961 cm−1 band resulted from the P–O symmetric stretching vibrations [52]. The two weak peaks at 725 cm−1 and 832 cm−1 are associated with the carbonate group which reveals the presence of carbonates in our sample [53]. The carbonate ion is likely to have come from the reaction between alkaline HAp samples and atmospheric carbon dioxide as observed elsewhere [54]. The multiplets located around 1000 cm−1 are attributed to phosphate modes. The split bands, mainly at 1030 and 1090 cm−1, seem to correspond to the formation of a well-crystallized apatite [51–54]. Detailed analysis permits indication of the presence of a carbonated component, where carbonate ions substitute others in Table 2 XRD of the prepared samples. Sample

Fig. 2. X-ray diffraction patterns of the (a) nHAp/MWCNT-UI nanocomposites prepared by ultrasound irradiation method; (b) nHAp/MWCNT-DMS nanocomposites prepared by mixture addition method; (c) nHAp and (d) MWCNT powders.

¥ PreferenceGrowth ≠Score Ca/P *Cristallinity Average specificity molar crystallite Plane (%) ratio size

nHAp/ 25 MWCNT-DMS 80 nHAp/ MWCNT-US

20.3 35

0.00495 −0.721

72

1.96

42

1.66

*Expressed by percentage. ≠Compared to JCPDS card: 024-0033 (Hydroxyapatite). ¥ Obtained by EDS. Error ± 10%.

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aqueous solution were used as the starting materials. There are reactions or equilibrium reactions as follows: 5CaðNO3 Þ2 :4H2 0 þ 3NH4 H2 PO4 þ 7NH4 OH→Ca5 ðPO4 Þ3 OH þ 10NH4 NO3 þ 7H2 O þ

NH3 þ H2 O→NH4 þ OH

−

ð6Þ ð7Þ

However, only during the ultrasonic precipitation process is the CO2 quickly removed by the ultrasonic irradiation, and the pH value of the solution is increased to maintain the following reaction: 2þ

10Ca

Fig. 3. Raman spectra of (a) nHAp/MWCNT-UI nanocomposites prepared by ultrasound irradiation method and (b) nHAp/MWCNT-DMS nanocomposites prepared by dropwise addition method, under continuous magnetic stirring.

A and B sites of the apatitic structure (corresponding to phosphate and hydroxyl ions, respectively). Carbonate bands are detected at 879, 1415, and 1455 cm−1. Exposure to the oxygen plasma promoted a progressive increase in all oxygen groups [55]. The quantitative analysis showed that the oxygen content on the CNT surface was 18.9% after the oxygen plasma treatment [29,31]. The plasma conditions in the present work show a higher efficiency compared to the previous reports [29,31] due to a higher concentration of carboxyl groups directly attached to the CNT. The superhydrophilicity of the CNT powder obtained after the oxygen plasma treatment is a requirement for obtaining the nHAp/MWCNT nanocomposites. For both processes, assisted and non-assisted by the UI, the formation of nHAp is the combination of several chemical reactions, as reported elsewhere [34–39]. In this work, calcium-nitrate, diammonium hydrogen phosphate, and ammonium hydroxide

Fig. 4. FTIR spectra of (a) nHAp/MWCNT-UI nanocomposites by ultrasound irradiation method; (b) nHAp/MWCNT-DMS nanocomposites prepared by dropwise addition method, under continuous magnetic stirring; and (c) typical nHAp powders.

þ 6H2 PO4

−

Ultrasound

þ 14OH → Ca10 ðPO4 Þ6 x ðOHÞ2 þ 12H2 O ð8Þ

The presence of OH− in the solution is crucial to force the reaction to completion (9). Therefore, the continuous addition of ammonium hydroxide is important to obtain nHAp particles attached to superhydrophilic MWCNT, and the pH of the solution is adjusted close to 10, in order to produce nHAp during the whole process. Both methods are very efficient for producing nanosized crystals of HAp. In this work, we obtained a yield of around 98% with UI and 90% yield with DMS methods, respectively (measured by Eq. (1)). There are some advantages and disadvantages associated with each method. We will present a general discussion pointing out the similarities and differences, based on the improvements made and the current state of the art. Few works that address the mechanism of formation of apatitic calcium phosphates by precipitation methods are available. Heughebaert proposed a thermally activated mechanism that would proceed in several stages [56]. These stages include the formation of an initial precipitate with a Ca/P ratio close to 1.5 followed by an increase of the Ca/P ratio accompanied by a decrease in the pH of the solution. Raynaud et al. agree with this mechanism and counteract the decrease in pH of the solution by adding an NH4OH solution, which maintains a constant pH during the synthesis [34]. For the first time, we have shown the growth preference calculations of nHAp attached to superhydrophilic MWCNT produced using an aqueous synthesis method. For the nHAp–MWCNT-DMS nanocomposites, we verified that the crystals grow preferentially in another plane (no data shown). This result is coherent, because nHAp has a hexagonal space group of P63/m (C6h2) with a = 0.9430 nm and c = 0.6891 nm. The O–H groups are ordered on the c-axis or in the (002) plane. For nHAp with a hexagonal structure, the [001] direction is the usual direction for preferred growth, along which the crystal planes are most densely populated with atoms. These findings are corroborated by the Ca/P ratio and specificity presented by nHAp crystals produced by mixture method. Current literature shows that the aqueous precipitation method either assisted or not assisted by ultrasound, produces acicular or globular nHAp [39–57]. To produce globular nHAp, the authors kept the phosphate solution under UI, with the pH close to 9 and then added the calcium solution using a slow drip. However, other authors show that when the phosphate solution is dripped into the calcium solution without the effect of UI keeping the pH around 10.8 before it is subject to UI, the acicular form is observed after 15 min of UI. In this case, we produced nanocomposites of acicular nHAp with MWCNT from a simple mixture of both solutions assisted with UI for 30 min. We associated these results with the presence of the carboxyl groups formed on the MWCNT structure and to the high surface area that is available in the solution with the presence of the carbon nanotubes (surface area of 40 m2/g). This large area could explain the large amount of nHAp crystals produced in a single process. To the best of our knowledge, the literature reports nHAp production only in association with lower mass (laboratory scale)

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Fig. 5. pH changes of SBF solution versus time due to bioactivity of nHAp/MWCNT-UI nanocomposites by ultrasound irradiation method and typical nHAp powders.

[58,59]. Based on the production, the MWCNT and the precipitation method influence the crystallinity of nHAp. In fact, UI can induce primary nucleation in nominally particle-free solutions and even at much lower supersaturation levels [60]. UI not only induces nucleation, but also increases reproducibility and efficiency. Notably, the nHAp crystals produced using the sonication method have a better yield and stoichiometry compared to the DMS method. This is surprising because apparently all of the Ca and P ions in the solution formed nHAp (yield ~ 98%). Another effect of the ultrasound irradiation on nucleation is reduced induction time between the establishment of supersaturation and the onset of nucleation and crystallization [57]. All of the effects are due to the highly spatially resolved regions of extreme excitation, temperature, and pressure created by bubble collapse and concomitant release of shock waves. Other postulates suggest that (i) subsequent rapid local cooling rates, calculated at

107–1010 K/s, play a significant role in increasing supersaturation; (ii) localized pressure increases reduce the crystallization temperature; and (iii) the cavitation events allow the excitation energy barriers associated with nucleation to be surmounted, in which case it should be possible to correlate the number of cavitations and nucleation events in a quantitative way [58]. Another important factor associated with UI is the time of exposure. Research shows that short times of the UI (~5–15 min) produce nHAp crystals. The results presented here demonstrate the effect of a long exposure time (30 min) of UI on nHAp crystals production. It is known that at short times, the ultrasound wave fails to blend the solution and precipitate uniformly, so little precipitate is obtained after sonication; longer times produce apparent crystals the size of which decreases under continuous sonication [60]. The difference in crystal size distributions between the extreme cases of a short burst of ultrasound to nucleate at lower levels of supersaturations and smaller crystals via continuous ultrasound application throughout the process evidences that a tradeoff between high degrees of nucleation and crystal size exists. The literature shows that there is a decrease in crystallinity with exposure to ultrasonic radiation exceeding 15 min. Applying the equation of crystallinity and Table 2 demonstrate correlation between an increase in crystallinity of nHAp and the presence of MWCNT. After an extensive structural and chemistry analyses of the nHAp/MWCNT-UI nanocomposites produced here, we have shown, also for the first time, the bioactivity properties of this nanobiomaterial. SBF (5 ×) has a higher HCO3― concentration (21 mM), similar to human blood plasma. This concentration induces the release of CO2 from the solution, resulting in an increase in pH value, and demonstrates that the Ca–P depositions occur, by precipitations of nucleated apatite particles on the sample surface, due to the instability of the solution [27]. Fig. 5 shows the increase of pH in the first days, indicating the reactivity of nHAp/MWNCT-UI nanocomposites soaked in SBF. The pH decrease trend in the nHAp samples used as control shows bioresorbability similar to that in biological apatite of bone [29].

Fig. 6. SEM on the surface of the samples of nHAp/MWCNT-UI before (a, b) and after (c, d) soaking in SBF by 14 days.

A.O. Lobo et al. / Materials Science and Engineering C 33 (2013) 4305–4312 Table 3 Ca/P ratio — data provided by EDS (at.%).

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Table 4 Area calculation of carbonate and phosphate region in FTIR spectrum.

Samples

Ca/P

Sd

SBF

nHAp

nHAp/MWCNT-UI

nHAp nHAp/MWCNT-UI

1.57 1.70

0.01 0.04

Carbonate

12.947

17.953

The increasing pH value observed in nHAp/MWCNT-UI after three days of immersion indicated the higher reactivity in this solution. Zhou et al. biomineralized samples of PLLA/bioactive glass composite and measured pH values. They found that during two days, the pH values increase due to higher reactivity of the biomaterial, which is a behavior characteristic of bioglasses [61]. Different from these authors, we have shown for the first time that the nHAp/MWCNT-UI has short reactivity time and after seven days the pH value remained constant in SBF solution with pH values approximately that of plasma. Hashimi et al. stated that increased pH values were due to the release of Ca ions and the exchange with H+ or H3O+ ions; this phenomenon occurs in the first day of immersion in the SBF solution, which results in formation of carbonated HAp [62]. Fig. 6 shows the images obtained by the MEV on the surface of the nHAp/MWCNT-UI samples before (a,b) and after (c,d) soaking in SBF solution. Fig. 6a shows details of the nHAp crystals produced. More details of the nHAp crystals are in Fig. 5b. These images show small acicular particles agglomerated onto larger particles (~ 40 nm). The SEM results are coherent with the calculated grain size from the XRD spectrum (data shown in Table 2). Recently, other authors have found similar particle morphologies [63]. Fig. 6c and d proved that the bioactivity of the nHAp/MWCNT-UI nanocomposites is due to morphology alterations on the surface with incorporation of apatites on the surface without defined orientation. A homogeneously deposition of Ca and P on the nHAp/MWCNT-UI nanocomposite verified after the biomineralization process (14 days). Table 3 shows the results collected from EDS analyses. The Ca/P ratio of the apatite formation on nHAp/MWCNT-UI after soaked in SBF were compared to the as-prepared nHAp/MWCNT-UI and nHAp control. An increase of Ca/P ratio after the biomineralization process can be verified. In this study, the Ca/P ratio obtained with nHAp/MWCNT-UI nanocomposites soaked in SBF was calculated with three means of each sample in different zones and the results were almost similar to stoichiometric apatite [64]. The nHAp/MWCNT-UI had increased Ca/P ratio when compared to the nHAp control.

Fig. 7. FTIR spectra of nHAp/MWCNT-UI nanocomposites by ultrasound irradiation method and typical nHAp powders after immersion in SBF.

Fig. 7 shows the FTIR-ATR spectra of the nHAp and nHAp/ MWCNT-IU nanocomposites after soaking in SBF for 14 days. In general, there was an enlargement in some regions of the FTIR-ATR spectrum collected on nHAp/MWCNT-UI nanocomposites after soaking SBF. FTIR-ATR spectrum showed amorphous carbonate and phosphate. The stretching vibration mode of the phosphate group (PO4) at 960 cm−1 indicates the presence of nHAp [13–16]. The bands in the 1069 cm−1, 870 cm−1, and 1650–1300 cm−1 regions were assigned to the vibrational modes of carbonate [13–16]. The band at 1245 cm−1 corresponding to the ester group indicates the presence of cyanoacrylate in biological adhesive [65]. The area calculation corresponding to the carbonate region demonstrates increased intensity of these bands in nHAp/MWCNT-UI nanocomposite compared to the nHAp group (Table 4). The increase of the intensity of carbonate group bands is related to carbonated HAp formation due to the substitution of carbonate ion inside the structure of nHAp. Evidence of bioactivity of the nHAp/MWCNT-UI nanocomposites could be defined by the increase in the region of carbonate HAp compared to the control (nHAp). The presence of these bands indicates the enhanced incorporation of the carbonate apatite from the reaction under non-inert conditions [66]. Data shows that both biomineralized nHAp/MWCNT-UI and nHAp have carbonated HAp peaks in XRD analyses (Fig. 8). However, Table 5 shows the nHAp/MWCNT-UI area calculation was higher compared to nHAp, evidenced by larger intensity peaks. The nHAp/ MWCNT-UI crystallite size was smaller than nHAp control due to the nanosize of the formed nanoparticles. During the biomineralization in SBF solution, CaCO3 and calcium phosphate amorphous layer formed on the surface due to migration 2− of Ca2+, CO2− and PO3− PO3− ions by ion transferred 3 , CO3 4 4 channels, and all these events form new apatite crystal likeness to bone tissue [67]. The results of SEM, EDS, FTIR, and XRD analyses proved the apatites formed on the surface and indicate that these new nHAp/ MWCNT-UI nanocomposites have a potential use in biological applications.

Fig. 8. X-ray diffraction patterns of the nHAp/MWCNT-UI nanocomposites prepared by ultrasound irradiation method and nHAp powders after immersion in SBF.

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Table 5 Full width at half maximum calculation of carbonated hydroxyapatite after soaking in SBF and crystallite size obtained from XRD analyses. Groups

FWHM

Cristallite size (Å)

nHAp nHAp/MWCNT-UI

0.540 0.696

15.2 11.9

[13] [14] [15] [16] [17] [18] [19] [20]

4. Conclusion In this work, we prepared and characterized nHAp/MWCNT nanocomposites produced in relatively large amounts. Two different aqueous precipitation methods were studied and ultrasonic irradiation presents some advantages such as dispensing a need for calcination. The presence of high surface area of 40 m2/g MWCNT decreased the mean free path of ions, favoring nHAp formation. Furthermore, the presence of carboxyl groups on the CNT walls, facilitates acicular nHAp crystallization. The typical morphology of the nHAp/MWCNT-UI nanocomposites reveals strong interaction of the acicular nHAp in relation to the MWCNTs. The nHAp crystallinity was evaluated by Scherrer equation, XRD pattern, FTIR, and Raman spectra. Semi-qualitative μEDX analysis shows that the main components of HAp powders were calcium and phosphorus in the ratio Ca/P of the 1.67. nHAp/MWCNT-UI nanocomposites induce in vitro bioactivity after soaking in SBF solution for 14 days. In addition, nHAp/MWCNT-UI nanocomposites not only promote apatite formation but also increase pH values according to time of immersion. All these chemical characteristics of nHAp/MWCNT-UI nanocomposites favor the carbonate calcium deposition. These findings indicate the applicability of nHAp/MWCNT-UI nanocomposites as scaffolds for bone regenerative medicine. Other in vitro and in vivo assays using polymeric and ceramic matrices will be carried out by our group to better characterize the bioactivity, extracellular matrix calcification, and mechanical properties of the nHAp/MWCNT-UI nanocomposites for future applications.

[21] [22] [23] [24] [25] [26]

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Acknowledgment The authors would like to thank the following institutions for financial support: São Paulo Research Foundation (FAPESP) under the grants (2011/17877-7), (2011/20345-7) and CNPq (800042/2012-3) for this project.

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