J Mater Sci: Mater Med (2015) 26:113 DOI 10.1007/s10856-015-5450-2
BIOMATERIALS SYNTHESIS AND CHARACTERIZATION
Bioactivity behaviour of nano-hydroxyapatite/freestanding aligned carbon nanotube oxide composite Idalia A. W. B. Siqueira • Ciliana A. G. S. Oliveira • Hudson Zanin • Marco A. V. M. Grinet • Alessandro E. C. Granato • Marimelia A. Porcionatto Fernanda R. Marciano • Anderson O. Lobo
•
Received: 19 August 2014 / Accepted: 29 November 2014 Ó Springer Science+Business Media New York 2015
Abstract Bioactive and low cytotoxic three dimensional nano-hydroxyapatite (nHAp) and aligned carbon nanotube oxide (a-CNTO) composite has been investigated. First, freestanding aligned carbon nanotubes porous scaffold was prepared by large-scale thermal chemical vapour deposition and functionalized by oxygen plasma treatment, forming a-CNTO. The a-CNTO was covered with platelike nHAp crystals prepared by in situ electrodeposition techniques, forming nHAp/a-CNTO composite. After that nHAp/a-CNTO composite was immersed in simulated body fluid for composite consolidation. This novel nanobiomaterial promotes mesenchymal stem cell adhesion with the active formation of membrane projections, cell monolayer formation and high cell viability. 1 Introduction Electrodeposited nano-hydroxyapatite (nHAp) is similar to the mineral component of natural bone in terms of I. A. W. B. Siqueira C. A. G. S. Oliveira M. A. V. M. Grinet F. R. Marciano A. O. Lobo (&) Laboratory of Biomedical Nanotechnology, Institute of Research and Development (IP&D), University of Vale do Paraiba, Av. Shishima Hifumi 2911, Sao Jose dos Campos, Sa˜o Paulo CEP/ 12224-000, Brazil e-mail: [email protected]; [email protected] H. Zanin (&) Laboratory of Energy Storage & Supply (ES&S), Institute of Research and Development (IP&D), University of Vale do Paraiba, Av. Shishima Hifumi 2911, Sao Jose dos Campos, Sa˜o Paulo CEP/12224-000, Brazil e-mail: [email protected] A. E. C. Granato M. A. Porcionatto Laboratory of Neurobiology, Universidade Federal de Sao Paulo, Rua Pedro de Toledo, 669, Sa˜o Paulo CEP/04049-032, Brazil
microstructure and dimensions, and shows excellent biocompatibility, bioactivity and osteoconductivity [1–3]. Due to these properties, nHAp has long been evaluated for bone tissue reconstruction [4]. However, its poor mechanical properties (e.g. low fracture toughness and tensile strength) have limited its use in load-bearing applications and/or large bone defects [5]. Several approaches have been examined to impart mechanical integrity to the nHAp without diminishing its bioactivity [5]. Various forms of carbon seem attractive candidates in this regard [1, 6], with their unprecedented mechanical properties (high strength and toughness) and physicochemical properties (high surface area, electrical and thermal conductivity, and low mass). Combined with carbon nanotubes (CNT), nHAp forms a biocompatible, bioactive and bioabsorbable composite. Some investigations have shown synthesis of HAp on CNT using various methods such as crystal growth from simulated body fluid (SBF) [7], composite coating deposition obtained by electrophoresis [8], aerosol application [9] or sol–gel matrix formation [10]. All these methods consist of dispersing CNT in a HAp solution. The electrodeposition is an advantageous method for producing a thin, crystalline, homogeneous and adherent film and is a rapid, reproducible, efficient and low cost process [11]. We have shown that free-standing aligned carbon nanotubes (a-CNT) films grown efficiently form plate-like nHAp crystals directly on them [12]. In vitro calcification of the extracellular matrix of human osteoblast cells and in vivo bone regeneration with the lamellar bone formation after 9 weeks for these composite also were evidenced [13]. Here, we focuses on the bioactivity and cytotoxicity evaluation of freestanding nHAp/a-CNTO biocomposite. Firstly, a simple acid treatment promotes to a-CNT scaffolds hydrophilicity and 3D accessibility for biological applications. The acid treatment attaches oxygen functional
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groups to CNT surface, forming a-CNTO. The attachment of oxygen functional groups was found to be crucial for nHAp nucleation during electrodeposition. Then a-CNTO was coated by electrodeposited nHAp, forming nHAp/aCNTO biocomposite. The biocomposite was immersed in SBF and showed fast biomineralization. Also, we demonstrated low cytotoxicity, cell proliferation and cell adhesion of mesenchymal stem cells (MSC) cultivated on the nHAp/ a-CNTO and showed.
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2.4 Preparation of nHAp/a-CNTO composite The electrodeposition processes were carried out using a-CNT electrodes as anode and a platinum electrode as cathode. DC Power supply provides voltage of -4.8 V between the platinum and the work electrode. The electrolytic solution was prepared using 2.5 mM of Ca(NO3)24H2O and 1.5 mM of NH4H2PO4 in 100 ml of deionized water. During the electrodeposition, the electrolyte was magnetically stirred at the constant temperature of 90 °C.
2 Experimental 2.5 Characterization of the nHAp/a-CNTO composite 2.1 Reagents and materials Calcium-nitrate tetrahydrate (Ca(NO3)2)4H2O, diammonium hydrogen phosphate (NH4)2HPO4, and ammonium hydroxide (NH4OH) aqueous solutions were the starting compounds for nHAp precipitation. Camphor (C10H16O), ferrocene (Fe(C5H5))2 and hydrochloric acid (HCl) were used for a-CNT synthesis and purification. All chemicals used in this work were analytical grade products (greater than 99 % pure) purchased from Sigma AldrichÒ. 2.2 Synthesis of a-CNT The a-CNT was prepared using a mixture of camphor (85 wt%) and ferrocene (15 wt%) in a thermal chemical vapour deposition (CVD) furnace [14]. Camphor and ferrocene were carbon and iron catalytic source for CNT growth. The mixture was vapourised at 220 °C in an ante-chamber, and then, the vapour was carried by an argon gas flow to the chamber of the CVD furnace set at 850 °C and atmospheric pressure. The vapours were quickly inserted into the chamber, which has an apparatus to confine them into the reactive region. The large amount of precursors and the optimum conditions are perfect to convert vapours into a-CNT electrodes. The time elapsed during the process used to produce the CNT was only a few minutes. 2.3 Treatment and functionalization of CNT The removal of the catalytic particles from the CNT was performed by acid etching. The samples were immersed for 5 h in 10 M of HCl, and then, they were washed extensively in water, and dried [15]. Functionalization of the a-CNT by incorporating oxygen-containing groups was performed in a plasma enhanced CVD reactor for 10 min (85 mTorr, oxygen flow rate of 1 sccm, -400 V and 20 kHz) [16]. That oxygen treated samples we shall henceforth refer to as a-CNTO.
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The materials were characterized by scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), Raman spectroscopy and attenuated reflection infrared spectroscopy (ATR-FTIR). The surface area measurements and porosity were carried out with Quantachrome Nova Win model 1000 for multi-point Brunauer–Emmett–Teller (BET) theory using the classical helium void volume method. 2 g of the sample was dried and measured. Goniometry (Kruss DSA 100S) measured the contact angle for wettability analyses using water. The micrographs were carried out using field emission SEM (JEOL6330, FEI Inspect F50, XL30 FEG and EVO Ma10—Zeiss) to evaluate and monitor morphology. For SEM acquisition, a thin layer of gold (10 nm) was added onto the surface of the composite, due to the low electrical conductivity of nHAp. The Inca Penta FET x3 Oxford Instruments was used for EDX measurements. The samples were characterised by TEM (JEOL 3010) adjusted to 300 kV and 120 lA, using a LaB6 filament. The samples were dispersed in water and dripped on a copper mesh coated with carbon film. Raman scattering spectroscopy (Renishaw 2000 system with Ar? ion laser excitation, k = 514.5 nm) was employed to analyse the composition and structure of the samples. The curve fitting and data analysis software FitykÒ was used to assign the peak locations. The phase and crystallinity of the resulting powders were characterized by X-ray diffraction (XRD: Lab X (XRD-6000), Shimadzu X-ray Diffractometer with monochromatic CuKa radiation. The spectra were taken from 20° to 80° with a step of 0.02°. The narrow range between 24° and 36° was used to calculate the crystallinity, growth preference and crystallite size. 2.6 Cell culture Bone marrow-derived stem cells were obtained from BALB/c mice leg bones (Ethics Committee Approval 0397/11). Briefly, mice were euthanized using xylazine/
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ketamine; tibias and femurs were dissected, and the epiphyses were cut. Bone marrow was extracted from the bones under sterile conditions using a syringe needle 26G (13 mm/24, 5 mm, BD Biosciences, NJ, USA) to inject 2 ml of DMEM (Dubelcco’s modified Eagle’s medium, Invitrogen, San Francisco, USA) at one end of the bone and the marrow collected at the other end. The cell suspension was centrifuged at 4009g for 10 min at room temperature. Cells were suspended in DMEM with 10 % fetal bovine serum (FBS; Cultilab, Campinas, Brazil), 1 % L-glutamine (Invitrogen) and 1 % penicillin/1 % streptomycin (GibcoBRL, Grand Island, NY, USA). The number of cells was quantified in a Neubauer chamber, and diluted with the same medium to achieve 5 9 106 cells/ml. Cells were plated on P60 dishes and kept in a humidified incubator, at 37 °C, 5 % CO2. Change of culture medium was performed every 72 h for removal of non-adherent hematopoietic cells. When the cultures reached 90 % confluence, subcultures were performed using trypsin (Cultilab) diluted in 10 mM phosphate buffered saline (PBS). After the second subculture adherent cells were considered MSC, according to international standard [17]. 2.6.1 Cell viability assays MSC were cultured in 300 ll of DMEM low glucose in 24-well plates (Corning Incorporated, New York, NY, USA) at a density of 105 cells/well in the absence or presence of nHAp/a-CNTO during 48 and 72 h. 2.6.1.1 Live cell counting After the respective periods of incubation, the culture medium was removed and 300 ll of trypsin (Cultilab) in 10 mM PBS were added. After 10 min cells were suspended, transferred to a 2 ml tube and centrifuged at 4009g for 6 min at room temperature. After centrifugation the supernatant was discarded, cells were suspended in 10 ll of trypan blue (TB) (Life Technologies, Oregon, USA) and added to the CountessÒ Automated Cell Counter reading plates (Invitrogen). The experiment was performed in quadruplicate. 2.6.1.2 MTT assay After incubation cell viability was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. To perform the assay, culture medium was removed and 250 ll of MTT was added to each well (control or nHAp/a-CNTO). After 3 h of incubation in a humidified incubator at 37 °C, 5 % CO2, the liquid content was aspirated and 350 ll of dimethyl sulfoxide (DMSO, Sigma Aldrich) were added to each well. After 30 min, absorbance was measured at 540 nm using an ELISA reader (VersaMax ELISA Microplate Reader, CA, USA).
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2.6.2 Analysis of cell adhesion by SEM MSC adhesion on nHAp/a-CNTO composites was evaluated 96 h after plating. Cells attached to 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, we examined the specimens using an XL30 FEG scanning electron microscope (SEM).
3 Results and discussion We prepared freestanding a-CNT flakes in a way that they could be easily cleaved or cut to shape if required as shown by optical image inserted in Fig. 1a. The a-CNT has a geometric area of *100 cm2 with a thickness of 100–300 lm. Figure 1a, b presents SEM micrographs of a cross-section of porous a-CNT flake with thickness of *180 lm. Depending on the thickness of the samples, we observed that thinner samples were flexible while thicker samples are rigid. The SEM micrographs evidences high surface area scaffold and the real surface area and specific density were measured by BET and helium pycnometry to be *40.8 m2 g-1 and *2.3 g cm-3, respectively. We obtained a total interparticle porosity and intraparticle porosity around of *64.2676 and *75.7838 %, respectively. We measured contact angle *1° for all the samples due to high porosity. Figure 1c shows a high-resolution TEM micrograph from multi-walled a-CNT, revealing in˚ and deterplanar spacing of radial tubes walls of *3.4 A tails of the inner and outer diameter of *10 and 25 nm, respectively. From several TEM images, we only observed iron catalyst nanoparticle encapsulated by nanotube. This indicates that the hydrochloric acid treatment promotes iron chloride formation, removing only the outer catalyst nanoparticles. From this first analysis, we infer that this sample is a porous, lightweight material, consisting mostly of a-CNTO. Micrographs of nHAp electrodeposited on a-CNTO are presented in Fig. 2a, b. Figure 2a shows top view nHAp/aCNTO composite. Figure 2b is a higher magnification of Fig. 2a, revealing that the nHAp crystals presented a platelike faceting. Figure 2c, d show nHAp interplanar space, which ranges from 0.2 to 1 nm depending on the crystal orientation. This polycrystalline HAp with a hexagonal structure has [001] direction as preferred for crystal growth, along which the crystal planes are most densely populated with atoms, as can be observed in the inset box
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Fig. 1 Cross-section micrograph of a-CNT flake (a & b). c TEM micrograph revealing details of the multiwalled CNT. Inset (a) optical image of the a-CNT electrode before electrodeposition
Fig. 2 Micrographs of nHAp/a-CNTO (a & b). Transmission electron micrographs of nHAp crystals (c & d)
Fig. 2c. HRTEM confirms the presence of the plate-like crystals of the nHAp with hexagonal crystallographic structure. These analyses evidenced that the nHAp crystals ˚ typically exhibited the interplanar distance of *2.7 A (Fig. 2d). Raman and infrared spectroscopy are powerful physicochemical vibrational spectroscopic techniques that can
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be used for CNT and nHAp analysis. Raman spectra of nHAp/a-CNTO show three well-defined peaks (Fig. 3a). The small band around 961 cm-1, which is related to nHAp [12, 13]; and D and G bands related to CNT. The first order of the CNT Raman spectrum has two pronounced peaks (Lorentzian), centred at around 1,357 cm-1 (known as a D band) and 1,585 cm-1 (known as G band),
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Fig. 3 a Raman spectrum of nHAp/a-CNTO scaffolds. Inset Raman spectrum of precipitated HAp powder. b FTIR-ATR spectrum of nHAp/a-CNTO scaffolds
which both are related to carbon sp2 hybridization [18, 19]. The D band is related to defects of the CNT and disordered carbon. The G band (E2g) is relative to the well crystallized graphite. The second order of Raman spectrum of a-CNT (no data shown) revealed that the G’ peak is very pronounced, also confirming the good crystallinity observed by HRSEM image (Fig. 1). For appropriated deconvolution fitting, two Gaussian peaks centred at around 1,250 and 1,480 cm-1 were added necessarily. The shoulder has its origin in the double resonance process, because its Raman shift (*1200 cm-1) is a point on phonon dispersion curves. The band at 1,250 cm-1 can be attributed to transverse and longitudinal acoustic modes or longitudinal optic modes very close to the K-point at the Brillouin zone, or a convolution of them [20]. The origin of 1,480 cm-1 band is correlated with the polar groups grafting onto CNT surfaces [21]. The D’ peak (1,622 cm-1) is also observed. The D’ band shoulder appears in the Raman spectra and is also correlated to disorder [20]. Raman spectrum was also taken from nHAp powder (inset in Fig. 3a). Lattice vibrations of anti-symmetrical deformation mode of PO43can be observed at around 429 and 590 cm-1 [12, 13]. The symmetrical and the anti-symmetrical stretching mode of PO43-are observed at around 960 and 1,044 cm-1, respectively [17]. Figure 3b shows the FTIR ATR analysis of nHAp/a-CNTO scaffolds showing three modes referred to the t4 and t2 CO32- [22]. The P–O stretching IR mode, which appears at *962 cm-1 in the spectra of all the samples, is attributed to the phosphates. The PO4 region appears as a very strong band at *1,029 cm-1, a strong shoulder at *1,060 cm-1, and a third clearly distinguished band at *1,092 cm-1. All these peaks and bands presented, evidencing the successful behaviour of this method to produce nHAp composite [23, 24]. Figure 4a shows the XRD pattern of the nHAp grown on a-CNTO scaffold. The principal diffraction peaks of nHAp appear as 2h values of 25.9° for reflection (002), at 30°–34° (triplet) for reflections (211), (122), (300) and (202) (JCPDS 024-0033) [11, 12]. The broadening of a
diffraction peak can be related to the mean crystallite size via the Scherrer equation (t = 0.89k/B coshB) [25], where t is the mean crystallite size, B is the peak line-width at half maximum (in radian), hB is the Bragg diffraction angle, and k is the X-ray wavelength (Cu Ka radiation in our case). A preference calculated from the XRD data was further explored as a structure indicator [12]. The relative intensity (RI) of the (002) peak in comparison to the intensities of the three strongest peaks for HAp powder-sample standard (JCPDS 024-0033) is defined as: RI ¼ Ið002Þ =Ið211Þ þ Ið112Þ þ Ið300Þ ;
ð1Þ
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 nHAp 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 equation: P ¼ RI RIs:
ð2Þ
A similar value was obtained by Hu et al. [26]. The refined structural cell parameters for plate-like nHAp are a = 0.9430 and c = 0.6891 nm (interplanar space group P63/m (C26h)). The O–H groups are ordered on the c-axis or in the (002) plane The O–H groups are ordered on the c-axis or in the (002) plane. Figure 4b shows the typical EDX spectrum of nHAp/aCNTO composite. The distribution of the chemical elements onto the nHAp/a-CNTO composite is presented in Fig. 4c. One can observe the nHAp forms a uniform coating on freestanding a-CNTO electrode surface. Direct plate-like nHAp crystals were electrodeposited onto freestanding a-CNT electrodes, without any thermal treatment. This can be interpreted with respect to super-saturation (due to a higher current density and electrical field) and crystal growth on –COO– groups attached to the a-CNT electrodes, as we discussed in details in our previous works [11–13].
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Fig. 4 a X-ray diffraction pattern from the nHAp/a-CNTO composite. nHAp planes compared to JCPDS card 00-024-0033. b EDX spectrum from the nHAp/a-CNTO composite and c distribution of
chemical elements in the nHAp/a-CNTO composite from left to right: Carbon, Iron, Oxygen, Calcium and Potassium
Table 1 XRD data of the prepared samples Sample
Cristallinity (%)a
Average crystallite size (nm)
Preference growth plane [002]
Score specificity (%)b
Ca/P molar ratioc
nHAp/aCNTO
65
66
0.23
63
1.67
a
Expressed by percentage
b
Compared to JCPDS card: 024-0033 (Hydroxyapatite)
c
Obtained by EDX
Table 1 shows the crystallinity, average crystallite size, preference growth plane, score specificity, and Ca/P molar ratio. This data was calculated from scaffold samples. We identified preferential growth along the (002) plane, and secondly, along the (112) plane. The thickness of the platelike structure is very close to the crystalline size obtained using the Scherrer formula from the XRD (Table 1). This indicates that each platelet is a single plate-like nHAp crystal. Therefore, the (002) crystalline planes, whose axes are along in the [001] direction, would grow preferentially [12]. The higher intensity of the (002) plane shows a standard nHAp growth pattern on a-CNT. The high frequency of these planes may be related to a plate-like morphology, as evidenced in Fig. 2. The expression of previous plate-like morphology of nHAp indicates some selectivity in surface binding. The bioactivity of the nHAp/a-CNTO composites was evaluated after the samples had been soaked in SBF 1.59
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solution. Figure 5 shows the formation of nanocrystals of apatite after the samples had been immersed in SBF 1.5 9 solution for (c & d) 7 days and (e & f) 14 days. Figure 5a, b are the negative control. A growth of uniform layer on the surface of the biomineralised samples was observed, in both times, with heterogeneous distribution of crystal agglomerations on the surface. The grain sizes of apatite are bigger after longer time of incubation. The longer the incubation time of the nHAp/a-CNTO composites in SBF 1.59 solution, the thicker and denser are the layers formed by globular nanoapatites, which elucidates that the biomineralization process can be successfully induced, confirming the composite bioactivity. Energy dispersive spectra were taken to semi-quantify CA/P ratios. The values vary according to those found in literature around 1.55, which is consistent with Calcium poor HAp (CHAp). Samples immersed in SBF 1.59 for the respective biomineralisation showed a CA/P ratio lower
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Fig. 5 Typical micrograph of nHAp/a-CNTO before (a & b) and after soaking in SBF 1.59 for 7 days (c & d) and 15 days (e & f)
than stoichiometric HAp obtained by nHAp/a-CNTO flakes, which was 1.67 (Table 1), and this decrease was accentuated by increasing the interval time of incubation as is shown by EDX (Table 2). The reduction in Ca/P ratio is Table 2 Atomic % of chemical elements of nHAp/a-CNTO biomineralised in SBF 1.59 Time
Ca (%)
P (%)
Ca/P (%)
7 days
18.37
11.07
1.65
15 days
16.93
10.73
1.57
explained by globular like formation of CHAp. This globular-like structure is formed electrostatic of positive calcium ions in the SBF and negatively surface of electrodeposited HAp, forming the Ca-rich CHAp. The electrodeposited film (eHAp) exposes hydroxyl and a phosphate unit in its crystal structure due to its isoelectric point is about 5–7, which is lower than the pH of the SBF *7.4. That cause negatively charged surface on eHAp, accumulating Ca2? of the solution, which makes the Ca-rich CHAp formation. That Ca-rich CHAp formation changes progressively the charge on the surface, inducing PO43-
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Fig. 6 MSC viability assessed by counting living cells using CountessÒ Automated Cell Counter 48 and 72 h post culture on nHAp/a-CNTO surface. Results are shown as average ± standard error (*P \ 0.05, **P \ 0.01, ***P \ 0.001, One way ANOVA and Tukey’s post test). Control: MSC plated on plastic surfaces
and CO32- competitive adsorption. The consequence is the formation of the Ca-poor CHAp, which has been documented to take place in the process of apatite formation on various bioactive ceramics [27, 28]. Commonly, associations between two or more colorimetric assays were used to biomaterials evaluation. TB is a widely used assay for staining dead cells. The TB method is a very common assay for evaluating cytotoxicity in experimental investigations [29] where dead cells absorb TB into the cytoplasm because of loss of membrane selectivity, whereas live cells remain unstained. However, TB staining cannot be used to distinguish between the healthy cells and the cells that are alive but losing cell functions. Comparatively, MTT assay is a colorimetric assay for assessing cell viability by mitochondrial energy metabolism. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the viable cells presented [30]. For this way, we analyzed nHAp/a-CNTO using these two colorimetric assays. The nHAp/a-CNTO composite surface presented low cytotoxic for MSC cells and results are illustrated in Fig. 6. Cells cultured for 3 days on nHAp/a-CNTO showed an increase in the number of cells, suggesting that the surface is not only biocompatible in terms of cell survival, but may also stimulate cell proliferation (Fig. 6). Cell survival, measured by MTT assay is in agreement with cell counting analysis, since both results show that nHAp/a-CNTO does not reduce cell viability (Fig. 7). A-CNT showed adhesion, biocompatibility and induced the cartilage formation using chondrocytes [31]. In another study of our group the nHAp/ a-CNTO composite showed to be a good scaffold for human osteoblast [32] and in vivo bone regeneration [13].
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Fig. 7 MSC viability assessed by MTT assay. MTT assay was performed in cells cultured for 48 and 72 days on nHAp/a-CNTO surface. Results are shown as average ± standard error (*P \ 0.05 One way ANOVA and Tukey’s post test). Control: MSC plated on plastic surfaces
It is known that the molecular mechanisms governing the interaction of MSC and the anchoring nanomaterial mostly involve mechanotransduction due to physical stimuli converted into biochemical signals and subsequently integrated into the cellular responses [33]. However, this is highly complex and not completely understood. Figure 8 shows the micrographs of murine MSC adhered to nHAp/a-CNTO composite surfaces after 96 h in culture. The MSCs used in this study were obtained from adult animals and have the ability to differentiate into a variety of cell types, such as osteoblasts and osteocytes [34, 35]. The cells spread with no preferential direction, acquiring a flat roughly circular form over the surface. A healthy cell pattern is observed on the nHAp/a-CNTO composites demonstrating that the cells have an active formation of membrane projections all over the cell surface. On hydrophilic surfaces, cells generally showed good spreading, proliferation and differentiation [36]. Our results show that hydrophilicty and porosity (pycnometer analysis) are important for better cell attachment and spreading. Also, we suggested that the nHAp/CNTO bioscaffolds are bioactive and favorable to apatite formation.
4 Conclusion We have shown a fast method to produce nHAp/a-CNTO bioscaffolds. We have prepared freestanding a-CNTO scaffolds without using binding materials, which often bring impurities into the structure. The a-CNTO scaffolds have high porosity and are superhydrophilic, and the lack of a substrate means the liquid phase has three-dimensional access to it. We electrodeposited homogeneous plate-like nHAp crystal films onto these a-CNTO electrodes without any thermal treatment. In addition nHAp/a-CNTO has shown in vitro bioactivity after immersing tests in SBF,
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Fig. 8 Micrographs of the murine MSC adhesion on nHAp/a-CNTO after 96 h
forming globular nHAp crystals. nHAp/a-CNTO has shown in vitro biocompatibility by two different assays with no reduction of cell viability, actually it promoted cell proliferation. The new nanobiomaterial has suitable surface for MSC adhesion with active formation of membrane projections and cell monolayer formation. Acknowledgments The authors thank to the Sa˜o Paulo Research Foundation (FAPESP Grant/2011/17877-7), (Grant/2013/07696-0), (Grant/2011/20345-7) and (Grant/2014/02163-7) and CNPq (Grant/ 474090/2013-2) (Grant/202439/2012-7) (Grant/404646/2012-3) for financial support. Special thanks to Priscila Leite for scanning
electron microscopy analyses, Laboratorio Nacional de Luz Sincroton for HR-TEM, Laboratorio de Combusta˜o e Propulsao (LCP) of Instituto Nacional de Pesquisas Espaciais, (INPE/Cachoeira Paulista).
References 1. LeGeros RZ. Properties of osteoconductive biomaterials: calcium phosphates. Clin Orthop Relat Res. 2002;395:81–98. 2. Oyane A, Onuma K, Ito A, Kim HM, Kokubo T, Nakamura T. Formation and growth of clusters in conventional and new kinds of simulated body fluids. J Biomed Mater Res A. 2003;64A:339–48.
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BIOMATERIALS SYNTHESIS AND CHARACTERIZATION
Bioactivity behaviour of nano-hydroxyapatite/freestanding aligned carbon nanotube oxide composite Idalia A. W. B. Siqueira • Ciliana A. G. S. Oliveira • Hudson Zanin • Marco A. V. M. Grinet • Alessandro E. C. Granato • Marimelia A. Porcionatto Fernanda R. Marciano • Anderson O. Lobo
•
Received: 19 August 2014 / Accepted: 29 November 2014 Ó Springer Science+Business Media New York 2015
Abstract Bioactive and low cytotoxic three dimensional nano-hydroxyapatite (nHAp) and aligned carbon nanotube oxide (a-CNTO) composite has been investigated. First, freestanding aligned carbon nanotubes porous scaffold was prepared by large-scale thermal chemical vapour deposition and functionalized by oxygen plasma treatment, forming a-CNTO. The a-CNTO was covered with platelike nHAp crystals prepared by in situ electrodeposition techniques, forming nHAp/a-CNTO composite. After that nHAp/a-CNTO composite was immersed in simulated body fluid for composite consolidation. This novel nanobiomaterial promotes mesenchymal stem cell adhesion with the active formation of membrane projections, cell monolayer formation and high cell viability. 1 Introduction Electrodeposited nano-hydroxyapatite (nHAp) is similar to the mineral component of natural bone in terms of I. A. W. B. Siqueira C. A. G. S. Oliveira M. A. V. M. Grinet F. R. Marciano A. O. Lobo (&) Laboratory of Biomedical Nanotechnology, Institute of Research and Development (IP&D), University of Vale do Paraiba, Av. Shishima Hifumi 2911, Sao Jose dos Campos, Sa˜o Paulo CEP/ 12224-000, Brazil e-mail: [email protected]; [email protected] H. Zanin (&) Laboratory of Energy Storage & Supply (ES&S), Institute of Research and Development (IP&D), University of Vale do Paraiba, Av. Shishima Hifumi 2911, Sao Jose dos Campos, Sa˜o Paulo CEP/12224-000, Brazil e-mail: [email protected] A. E. C. Granato M. A. Porcionatto Laboratory of Neurobiology, Universidade Federal de Sao Paulo, Rua Pedro de Toledo, 669, Sa˜o Paulo CEP/04049-032, Brazil
microstructure and dimensions, and shows excellent biocompatibility, bioactivity and osteoconductivity [1–3]. Due to these properties, nHAp has long been evaluated for bone tissue reconstruction [4]. However, its poor mechanical properties (e.g. low fracture toughness and tensile strength) have limited its use in load-bearing applications and/or large bone defects [5]. Several approaches have been examined to impart mechanical integrity to the nHAp without diminishing its bioactivity [5]. Various forms of carbon seem attractive candidates in this regard [1, 6], with their unprecedented mechanical properties (high strength and toughness) and physicochemical properties (high surface area, electrical and thermal conductivity, and low mass). Combined with carbon nanotubes (CNT), nHAp forms a biocompatible, bioactive and bioabsorbable composite. Some investigations have shown synthesis of HAp on CNT using various methods such as crystal growth from simulated body fluid (SBF) [7], composite coating deposition obtained by electrophoresis [8], aerosol application [9] or sol–gel matrix formation [10]. All these methods consist of dispersing CNT in a HAp solution. The electrodeposition is an advantageous method for producing a thin, crystalline, homogeneous and adherent film and is a rapid, reproducible, efficient and low cost process [11]. We have shown that free-standing aligned carbon nanotubes (a-CNT) films grown efficiently form plate-like nHAp crystals directly on them [12]. In vitro calcification of the extracellular matrix of human osteoblast cells and in vivo bone regeneration with the lamellar bone formation after 9 weeks for these composite also were evidenced [13]. Here, we focuses on the bioactivity and cytotoxicity evaluation of freestanding nHAp/a-CNTO biocomposite. Firstly, a simple acid treatment promotes to a-CNT scaffolds hydrophilicity and 3D accessibility for biological applications. The acid treatment attaches oxygen functional
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groups to CNT surface, forming a-CNTO. The attachment of oxygen functional groups was found to be crucial for nHAp nucleation during electrodeposition. Then a-CNTO was coated by electrodeposited nHAp, forming nHAp/aCNTO biocomposite. The biocomposite was immersed in SBF and showed fast biomineralization. Also, we demonstrated low cytotoxicity, cell proliferation and cell adhesion of mesenchymal stem cells (MSC) cultivated on the nHAp/ a-CNTO and showed.
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2.4 Preparation of nHAp/a-CNTO composite The electrodeposition processes were carried out using a-CNT electrodes as anode and a platinum electrode as cathode. DC Power supply provides voltage of -4.8 V between the platinum and the work electrode. The electrolytic solution was prepared using 2.5 mM of Ca(NO3)24H2O and 1.5 mM of NH4H2PO4 in 100 ml of deionized water. During the electrodeposition, the electrolyte was magnetically stirred at the constant temperature of 90 °C.
2 Experimental 2.5 Characterization of the nHAp/a-CNTO composite 2.1 Reagents and materials Calcium-nitrate tetrahydrate (Ca(NO3)2)4H2O, diammonium hydrogen phosphate (NH4)2HPO4, and ammonium hydroxide (NH4OH) aqueous solutions were the starting compounds for nHAp precipitation. Camphor (C10H16O), ferrocene (Fe(C5H5))2 and hydrochloric acid (HCl) were used for a-CNT synthesis and purification. All chemicals used in this work were analytical grade products (greater than 99 % pure) purchased from Sigma AldrichÒ. 2.2 Synthesis of a-CNT The a-CNT was prepared using a mixture of camphor (85 wt%) and ferrocene (15 wt%) in a thermal chemical vapour deposition (CVD) furnace [14]. Camphor and ferrocene were carbon and iron catalytic source for CNT growth. The mixture was vapourised at 220 °C in an ante-chamber, and then, the vapour was carried by an argon gas flow to the chamber of the CVD furnace set at 850 °C and atmospheric pressure. The vapours were quickly inserted into the chamber, which has an apparatus to confine them into the reactive region. The large amount of precursors and the optimum conditions are perfect to convert vapours into a-CNT electrodes. The time elapsed during the process used to produce the CNT was only a few minutes. 2.3 Treatment and functionalization of CNT The removal of the catalytic particles from the CNT was performed by acid etching. The samples were immersed for 5 h in 10 M of HCl, and then, they were washed extensively in water, and dried [15]. Functionalization of the a-CNT by incorporating oxygen-containing groups was performed in a plasma enhanced CVD reactor for 10 min (85 mTorr, oxygen flow rate of 1 sccm, -400 V and 20 kHz) [16]. That oxygen treated samples we shall henceforth refer to as a-CNTO.
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The materials were characterized by scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), Raman spectroscopy and attenuated reflection infrared spectroscopy (ATR-FTIR). The surface area measurements and porosity were carried out with Quantachrome Nova Win model 1000 for multi-point Brunauer–Emmett–Teller (BET) theory using the classical helium void volume method. 2 g of the sample was dried and measured. Goniometry (Kruss DSA 100S) measured the contact angle for wettability analyses using water. The micrographs were carried out using field emission SEM (JEOL6330, FEI Inspect F50, XL30 FEG and EVO Ma10—Zeiss) to evaluate and monitor morphology. For SEM acquisition, a thin layer of gold (10 nm) was added onto the surface of the composite, due to the low electrical conductivity of nHAp. The Inca Penta FET x3 Oxford Instruments was used for EDX measurements. The samples were characterised by TEM (JEOL 3010) adjusted to 300 kV and 120 lA, using a LaB6 filament. The samples were dispersed in water and dripped on a copper mesh coated with carbon film. Raman scattering spectroscopy (Renishaw 2000 system with Ar? ion laser excitation, k = 514.5 nm) was employed to analyse the composition and structure of the samples. The curve fitting and data analysis software FitykÒ was used to assign the peak locations. The phase and crystallinity of the resulting powders were characterized by X-ray diffraction (XRD: Lab X (XRD-6000), Shimadzu X-ray Diffractometer with monochromatic CuKa radiation. The spectra were taken from 20° to 80° with a step of 0.02°. The narrow range between 24° and 36° was used to calculate the crystallinity, growth preference and crystallite size. 2.6 Cell culture Bone marrow-derived stem cells were obtained from BALB/c mice leg bones (Ethics Committee Approval 0397/11). Briefly, mice were euthanized using xylazine/
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ketamine; tibias and femurs were dissected, and the epiphyses were cut. Bone marrow was extracted from the bones under sterile conditions using a syringe needle 26G (13 mm/24, 5 mm, BD Biosciences, NJ, USA) to inject 2 ml of DMEM (Dubelcco’s modified Eagle’s medium, Invitrogen, San Francisco, USA) at one end of the bone and the marrow collected at the other end. The cell suspension was centrifuged at 4009g for 10 min at room temperature. Cells were suspended in DMEM with 10 % fetal bovine serum (FBS; Cultilab, Campinas, Brazil), 1 % L-glutamine (Invitrogen) and 1 % penicillin/1 % streptomycin (GibcoBRL, Grand Island, NY, USA). The number of cells was quantified in a Neubauer chamber, and diluted with the same medium to achieve 5 9 106 cells/ml. Cells were plated on P60 dishes and kept in a humidified incubator, at 37 °C, 5 % CO2. Change of culture medium was performed every 72 h for removal of non-adherent hematopoietic cells. When the cultures reached 90 % confluence, subcultures were performed using trypsin (Cultilab) diluted in 10 mM phosphate buffered saline (PBS). After the second subculture adherent cells were considered MSC, according to international standard [17]. 2.6.1 Cell viability assays MSC were cultured in 300 ll of DMEM low glucose in 24-well plates (Corning Incorporated, New York, NY, USA) at a density of 105 cells/well in the absence or presence of nHAp/a-CNTO during 48 and 72 h. 2.6.1.1 Live cell counting After the respective periods of incubation, the culture medium was removed and 300 ll of trypsin (Cultilab) in 10 mM PBS were added. After 10 min cells were suspended, transferred to a 2 ml tube and centrifuged at 4009g for 6 min at room temperature. After centrifugation the supernatant was discarded, cells were suspended in 10 ll of trypan blue (TB) (Life Technologies, Oregon, USA) and added to the CountessÒ Automated Cell Counter reading plates (Invitrogen). The experiment was performed in quadruplicate. 2.6.1.2 MTT assay After incubation cell viability was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. To perform the assay, culture medium was removed and 250 ll of MTT was added to each well (control or nHAp/a-CNTO). After 3 h of incubation in a humidified incubator at 37 °C, 5 % CO2, the liquid content was aspirated and 350 ll of dimethyl sulfoxide (DMSO, Sigma Aldrich) were added to each well. After 30 min, absorbance was measured at 540 nm using an ELISA reader (VersaMax ELISA Microplate Reader, CA, USA).
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2.6.2 Analysis of cell adhesion by SEM MSC adhesion on nHAp/a-CNTO composites was evaluated 96 h after plating. Cells attached to 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, we examined the specimens using an XL30 FEG scanning electron microscope (SEM).
3 Results and discussion We prepared freestanding a-CNT flakes in a way that they could be easily cleaved or cut to shape if required as shown by optical image inserted in Fig. 1a. The a-CNT has a geometric area of *100 cm2 with a thickness of 100–300 lm. Figure 1a, b presents SEM micrographs of a cross-section of porous a-CNT flake with thickness of *180 lm. Depending on the thickness of the samples, we observed that thinner samples were flexible while thicker samples are rigid. The SEM micrographs evidences high surface area scaffold and the real surface area and specific density were measured by BET and helium pycnometry to be *40.8 m2 g-1 and *2.3 g cm-3, respectively. We obtained a total interparticle porosity and intraparticle porosity around of *64.2676 and *75.7838 %, respectively. We measured contact angle *1° for all the samples due to high porosity. Figure 1c shows a high-resolution TEM micrograph from multi-walled a-CNT, revealing in˚ and deterplanar spacing of radial tubes walls of *3.4 A tails of the inner and outer diameter of *10 and 25 nm, respectively. From several TEM images, we only observed iron catalyst nanoparticle encapsulated by nanotube. This indicates that the hydrochloric acid treatment promotes iron chloride formation, removing only the outer catalyst nanoparticles. From this first analysis, we infer that this sample is a porous, lightweight material, consisting mostly of a-CNTO. Micrographs of nHAp electrodeposited on a-CNTO are presented in Fig. 2a, b. Figure 2a shows top view nHAp/aCNTO composite. Figure 2b is a higher magnification of Fig. 2a, revealing that the nHAp crystals presented a platelike faceting. Figure 2c, d show nHAp interplanar space, which ranges from 0.2 to 1 nm depending on the crystal orientation. This polycrystalline HAp with a hexagonal structure has [001] direction as preferred for crystal growth, along which the crystal planes are most densely populated with atoms, as can be observed in the inset box
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Fig. 1 Cross-section micrograph of a-CNT flake (a & b). c TEM micrograph revealing details of the multiwalled CNT. Inset (a) optical image of the a-CNT electrode before electrodeposition
Fig. 2 Micrographs of nHAp/a-CNTO (a & b). Transmission electron micrographs of nHAp crystals (c & d)
Fig. 2c. HRTEM confirms the presence of the plate-like crystals of the nHAp with hexagonal crystallographic structure. These analyses evidenced that the nHAp crystals ˚ typically exhibited the interplanar distance of *2.7 A (Fig. 2d). Raman and infrared spectroscopy are powerful physicochemical vibrational spectroscopic techniques that can
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be used for CNT and nHAp analysis. Raman spectra of nHAp/a-CNTO show three well-defined peaks (Fig. 3a). The small band around 961 cm-1, which is related to nHAp [12, 13]; and D and G bands related to CNT. The first order of the CNT Raman spectrum has two pronounced peaks (Lorentzian), centred at around 1,357 cm-1 (known as a D band) and 1,585 cm-1 (known as G band),
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Fig. 3 a Raman spectrum of nHAp/a-CNTO scaffolds. Inset Raman spectrum of precipitated HAp powder. b FTIR-ATR spectrum of nHAp/a-CNTO scaffolds
which both are related to carbon sp2 hybridization [18, 19]. The D band is related to defects of the CNT and disordered carbon. The G band (E2g) is relative to the well crystallized graphite. The second order of Raman spectrum of a-CNT (no data shown) revealed that the G’ peak is very pronounced, also confirming the good crystallinity observed by HRSEM image (Fig. 1). For appropriated deconvolution fitting, two Gaussian peaks centred at around 1,250 and 1,480 cm-1 were added necessarily. The shoulder has its origin in the double resonance process, because its Raman shift (*1200 cm-1) is a point on phonon dispersion curves. The band at 1,250 cm-1 can be attributed to transverse and longitudinal acoustic modes or longitudinal optic modes very close to the K-point at the Brillouin zone, or a convolution of them [20]. The origin of 1,480 cm-1 band is correlated with the polar groups grafting onto CNT surfaces [21]. The D’ peak (1,622 cm-1) is also observed. The D’ band shoulder appears in the Raman spectra and is also correlated to disorder [20]. Raman spectrum was also taken from nHAp powder (inset in Fig. 3a). Lattice vibrations of anti-symmetrical deformation mode of PO43can be observed at around 429 and 590 cm-1 [12, 13]. The symmetrical and the anti-symmetrical stretching mode of PO43-are observed at around 960 and 1,044 cm-1, respectively [17]. Figure 3b shows the FTIR ATR analysis of nHAp/a-CNTO scaffolds showing three modes referred to the t4 and t2 CO32- [22]. The P–O stretching IR mode, which appears at *962 cm-1 in the spectra of all the samples, is attributed to the phosphates. The PO4 region appears as a very strong band at *1,029 cm-1, a strong shoulder at *1,060 cm-1, and a third clearly distinguished band at *1,092 cm-1. All these peaks and bands presented, evidencing the successful behaviour of this method to produce nHAp composite [23, 24]. Figure 4a shows the XRD pattern of the nHAp grown on a-CNTO scaffold. The principal diffraction peaks of nHAp appear as 2h values of 25.9° for reflection (002), at 30°–34° (triplet) for reflections (211), (122), (300) and (202) (JCPDS 024-0033) [11, 12]. The broadening of a
diffraction peak can be related to the mean crystallite size via the Scherrer equation (t = 0.89k/B coshB) [25], where t is the mean crystallite size, B is the peak line-width at half maximum (in radian), hB is the Bragg diffraction angle, and k is the X-ray wavelength (Cu Ka radiation in our case). A preference calculated from the XRD data was further explored as a structure indicator [12]. The relative intensity (RI) of the (002) peak in comparison to the intensities of the three strongest peaks for HAp powder-sample standard (JCPDS 024-0033) is defined as: RI ¼ Ið002Þ =Ið211Þ þ Ið112Þ þ Ið300Þ ;
ð1Þ
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 nHAp 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 equation: P ¼ RI RIs:
ð2Þ
A similar value was obtained by Hu et al. [26]. The refined structural cell parameters for plate-like nHAp are a = 0.9430 and c = 0.6891 nm (interplanar space group P63/m (C26h)). The O–H groups are ordered on the c-axis or in the (002) plane The O–H groups are ordered on the c-axis or in the (002) plane. Figure 4b shows the typical EDX spectrum of nHAp/aCNTO composite. The distribution of the chemical elements onto the nHAp/a-CNTO composite is presented in Fig. 4c. One can observe the nHAp forms a uniform coating on freestanding a-CNTO electrode surface. Direct plate-like nHAp crystals were electrodeposited onto freestanding a-CNT electrodes, without any thermal treatment. This can be interpreted with respect to super-saturation (due to a higher current density and electrical field) and crystal growth on –COO– groups attached to the a-CNT electrodes, as we discussed in details in our previous works [11–13].
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Fig. 4 a X-ray diffraction pattern from the nHAp/a-CNTO composite. nHAp planes compared to JCPDS card 00-024-0033. b EDX spectrum from the nHAp/a-CNTO composite and c distribution of
chemical elements in the nHAp/a-CNTO composite from left to right: Carbon, Iron, Oxygen, Calcium and Potassium
Table 1 XRD data of the prepared samples Sample
Cristallinity (%)a
Average crystallite size (nm)
Preference growth plane [002]
Score specificity (%)b
Ca/P molar ratioc
nHAp/aCNTO
65
66
0.23
63
1.67
a
Expressed by percentage
b
Compared to JCPDS card: 024-0033 (Hydroxyapatite)
c
Obtained by EDX
Table 1 shows the crystallinity, average crystallite size, preference growth plane, score specificity, and Ca/P molar ratio. This data was calculated from scaffold samples. We identified preferential growth along the (002) plane, and secondly, along the (112) plane. The thickness of the platelike structure is very close to the crystalline size obtained using the Scherrer formula from the XRD (Table 1). This indicates that each platelet is a single plate-like nHAp crystal. Therefore, the (002) crystalline planes, whose axes are along in the [001] direction, would grow preferentially [12]. The higher intensity of the (002) plane shows a standard nHAp growth pattern on a-CNT. The high frequency of these planes may be related to a plate-like morphology, as evidenced in Fig. 2. The expression of previous plate-like morphology of nHAp indicates some selectivity in surface binding. The bioactivity of the nHAp/a-CNTO composites was evaluated after the samples had been soaked in SBF 1.59
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solution. Figure 5 shows the formation of nanocrystals of apatite after the samples had been immersed in SBF 1.5 9 solution for (c & d) 7 days and (e & f) 14 days. Figure 5a, b are the negative control. A growth of uniform layer on the surface of the biomineralised samples was observed, in both times, with heterogeneous distribution of crystal agglomerations on the surface. The grain sizes of apatite are bigger after longer time of incubation. The longer the incubation time of the nHAp/a-CNTO composites in SBF 1.59 solution, the thicker and denser are the layers formed by globular nanoapatites, which elucidates that the biomineralization process can be successfully induced, confirming the composite bioactivity. Energy dispersive spectra were taken to semi-quantify CA/P ratios. The values vary according to those found in literature around 1.55, which is consistent with Calcium poor HAp (CHAp). Samples immersed in SBF 1.59 for the respective biomineralisation showed a CA/P ratio lower
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Fig. 5 Typical micrograph of nHAp/a-CNTO before (a & b) and after soaking in SBF 1.59 for 7 days (c & d) and 15 days (e & f)
than stoichiometric HAp obtained by nHAp/a-CNTO flakes, which was 1.67 (Table 1), and this decrease was accentuated by increasing the interval time of incubation as is shown by EDX (Table 2). The reduction in Ca/P ratio is Table 2 Atomic % of chemical elements of nHAp/a-CNTO biomineralised in SBF 1.59 Time
Ca (%)
P (%)
Ca/P (%)
7 days
18.37
11.07
1.65
15 days
16.93
10.73
1.57
explained by globular like formation of CHAp. This globular-like structure is formed electrostatic of positive calcium ions in the SBF and negatively surface of electrodeposited HAp, forming the Ca-rich CHAp. The electrodeposited film (eHAp) exposes hydroxyl and a phosphate unit in its crystal structure due to its isoelectric point is about 5–7, which is lower than the pH of the SBF *7.4. That cause negatively charged surface on eHAp, accumulating Ca2? of the solution, which makes the Ca-rich CHAp formation. That Ca-rich CHAp formation changes progressively the charge on the surface, inducing PO43-
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Fig. 6 MSC viability assessed by counting living cells using CountessÒ Automated Cell Counter 48 and 72 h post culture on nHAp/a-CNTO surface. Results are shown as average ± standard error (*P \ 0.05, **P \ 0.01, ***P \ 0.001, One way ANOVA and Tukey’s post test). Control: MSC plated on plastic surfaces
and CO32- competitive adsorption. The consequence is the formation of the Ca-poor CHAp, which has been documented to take place in the process of apatite formation on various bioactive ceramics [27, 28]. Commonly, associations between two or more colorimetric assays were used to biomaterials evaluation. TB is a widely used assay for staining dead cells. The TB method is a very common assay for evaluating cytotoxicity in experimental investigations [29] where dead cells absorb TB into the cytoplasm because of loss of membrane selectivity, whereas live cells remain unstained. However, TB staining cannot be used to distinguish between the healthy cells and the cells that are alive but losing cell functions. Comparatively, MTT assay is a colorimetric assay for assessing cell viability by mitochondrial energy metabolism. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the viable cells presented [30]. For this way, we analyzed nHAp/a-CNTO using these two colorimetric assays. The nHAp/a-CNTO composite surface presented low cytotoxic for MSC cells and results are illustrated in Fig. 6. Cells cultured for 3 days on nHAp/a-CNTO showed an increase in the number of cells, suggesting that the surface is not only biocompatible in terms of cell survival, but may also stimulate cell proliferation (Fig. 6). Cell survival, measured by MTT assay is in agreement with cell counting analysis, since both results show that nHAp/a-CNTO does not reduce cell viability (Fig. 7). A-CNT showed adhesion, biocompatibility and induced the cartilage formation using chondrocytes [31]. In another study of our group the nHAp/ a-CNTO composite showed to be a good scaffold for human osteoblast [32] and in vivo bone regeneration [13].
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Fig. 7 MSC viability assessed by MTT assay. MTT assay was performed in cells cultured for 48 and 72 days on nHAp/a-CNTO surface. Results are shown as average ± standard error (*P \ 0.05 One way ANOVA and Tukey’s post test). Control: MSC plated on plastic surfaces
It is known that the molecular mechanisms governing the interaction of MSC and the anchoring nanomaterial mostly involve mechanotransduction due to physical stimuli converted into biochemical signals and subsequently integrated into the cellular responses [33]. However, this is highly complex and not completely understood. Figure 8 shows the micrographs of murine MSC adhered to nHAp/a-CNTO composite surfaces after 96 h in culture. The MSCs used in this study were obtained from adult animals and have the ability to differentiate into a variety of cell types, such as osteoblasts and osteocytes [34, 35]. The cells spread with no preferential direction, acquiring a flat roughly circular form over the surface. A healthy cell pattern is observed on the nHAp/a-CNTO composites demonstrating that the cells have an active formation of membrane projections all over the cell surface. On hydrophilic surfaces, cells generally showed good spreading, proliferation and differentiation [36]. Our results show that hydrophilicty and porosity (pycnometer analysis) are important for better cell attachment and spreading. Also, we suggested that the nHAp/CNTO bioscaffolds are bioactive and favorable to apatite formation.
4 Conclusion We have shown a fast method to produce nHAp/a-CNTO bioscaffolds. We have prepared freestanding a-CNTO scaffolds without using binding materials, which often bring impurities into the structure. The a-CNTO scaffolds have high porosity and are superhydrophilic, and the lack of a substrate means the liquid phase has three-dimensional access to it. We electrodeposited homogeneous plate-like nHAp crystal films onto these a-CNTO electrodes without any thermal treatment. In addition nHAp/a-CNTO has shown in vitro bioactivity after immersing tests in SBF,
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Fig. 8 Micrographs of the murine MSC adhesion on nHAp/a-CNTO after 96 h
forming globular nHAp crystals. nHAp/a-CNTO has shown in vitro biocompatibility by two different assays with no reduction of cell viability, actually it promoted cell proliferation. The new nanobiomaterial has suitable surface for MSC adhesion with active formation of membrane projections and cell monolayer formation. Acknowledgments The authors thank to the Sa˜o Paulo Research Foundation (FAPESP Grant/2011/17877-7), (Grant/2013/07696-0), (Grant/2011/20345-7) and (Grant/2014/02163-7) and CNPq (Grant/ 474090/2013-2) (Grant/202439/2012-7) (Grant/404646/2012-3) for financial support. Special thanks to Priscila Leite for scanning
electron microscopy analyses, Laboratorio Nacional de Luz Sincroton for HR-TEM, Laboratorio de Combusta˜o e Propulsao (LCP) of Instituto Nacional de Pesquisas Espaciais, (INPE/Cachoeira Paulista).
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