|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Monolayer formation of human osteoblastic cells on vertically aligned multiwalled carbon nanotube scaffolds
Anderson O Lobo*†1, Erica F Antunes*†, Mariana BS Palma*, Cristina Pacheco‑Soares‡, Vladimir J Trava‑Airoldi*† and Evaldo J Corat*†
*Laboratrio Associado de Sensores e Materiais, Instituto Nacional de Pesquisas Espaciais, CP 515, So Jos dos CamposSP, CEP 12.245970, Brazil, †Instituto Tecnolgico de AeronuticaCTA, So Jos dos CamposSP, CEP 12228900, Brazil, and ‡Laboratrio de Dinmica de Compartimentos Celulares, Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraba, So Jos dos CamposSP, CEP 12227010, Brazil
Monolayer formation of SaOS-2 (human osteoblast-like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non-toxicity and the flat spreading with monolayer formation of the SaOs-2 on VACNT scaffolds surface indicate that they can be used for biomedical applications.
Key words: cell adhesion, cytotoxicity, lactate dehydrogenase assay, SaOs-2, vertically aligned multiwalled carbon nanotubes (VACNT)
Abbreviations: CNT, carbon nanotubes, FBS, fetal bovine serum, HMDS, hexamethyldisilazane, LDH, lactate dehydrogenase, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, MWCVD, microwave plasma chemical vapour deposition, OD, optical density, SaOS-2, human osteoblast-like cells, SEM, scanning electron microscopy, SWCNT, single-walled carbon nanotube, TEM, transmission electron microscopy, VACNT, vertically aligned multiwalled carbon nanotubes
1To whom correspondence should be addressed (email email@example.com).
The most important goal in bone bioengineering is developing nanostructured scaffolds, which have the capacity to sustain bone cell growth and proliferation. CNT (carbon nanotubes) are of particular interest in bone regeneration due to their biomimmetic properties and similarity to extracellular matrix components (Bryning et al., 2007; Harrison and Atala et al., 2007; Shi et al., 2007).
In vitro cytotoxicity and cell adhesion tests with osteoblastic cells are the first steps to evaluate the biocompatibility of CNT, related to bone formation. Several studies have been performed on CNT scaffolds in recent years, showing positive and negative results to cytotoxicity. Owing to the controversial results (Casey et al., 2007; Giannona et al., 2007; Lobo et al., 2008a and 2008b; Zanello et al., 2006; Zhang et al., 2007), it is still too early to establish a general toxicological profile for this material and more systematic in vitro and in vivo investigations using biologically compatible CNT are necessary.
We herein investigated the cell monolayer formation on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds with SaOS-2 (human osteoblast-like cells) by SEM (scanning electron microscopy). The cell viability was also evaluated by LDH (lactate dehydrogenase) enzyme release assay. Cell morphology was evaluated up to 7 days. High viability and cell proliferation were observed.
2. Materials and methods
2.1. Synthesis of carbon nanotubes
The VACNT were produced as a thin film, using a MWCVD (microwave plasma chemical vapour deposition). The substrates were 10 mm Ti squares, covered by a thin Ni or Fe layer (10 nm), both deposited by an e-beam evaporator. The Ni and Fe layers were pretreated to promote nanocluster formation. The pretreatment was carried out for 5 min in plasma of N2/H2 (10/90 sccm), at a substrate temperature of around 760°C. After pretreatment, CH4 (14 sccm) was inserted into the chamber at a substrate temperature of 800°C for 2 min. The reactor was kept at a pressure of 30 torr during the whole process (Antunes et al., 2006). SEM and TEM (transmission electron microscopy) were used to observe the structure of the MWCNT.
2.2. Cell culture
SaOS-2 were provided by Cell Line Bank of Rio de Janeiro/Brazil (CR019). The cells were maintained as subconfluent monolayer’s in McCoy’s 5a (Sigma) medium (modified) with 1.5 mM l-glutamine adjusted to contain 2.2 g/l sodium bicarbonate 85%, FBS (fetal bovine serum) 15% (Gibco, BRL), 100 units/ml penicillin-streptomycin (Sigma) and 25 μg/ml l-ascorbic acid (Sigma). The incubation occurred within a CO2 (5%) atmosphere at 37°C.
2.3. LDH release assays
The enzyme LDH, located in the cytoplasm, is a marker for membrane integrity of cells, and therefore, it can be correlated to cell viability and proliferation. It is released when the cytoplasmatic membrane is damaged (Legrand et al., 1992; Decker and Lohmann-Matthes, 1988). The LDH release assays were evaluated according to ISO 10993-5 “Biological evaluation of medical devices – test for cytotoxicity: in vitro methods” (or EN 30993-5), using direct contact. All the samples were sterilized for 24 h under UV irradiation and placed in individual wells of 24-well culture plates. The cells were seeded in each well at a concentration of 5×105 cells/ml, supplemented with 10% FBS. The incubation was performed under a CO2 (5%) atmosphere, at 37°C for different times (24, 48 and 72 h). Latex fragments of proven toxic nature were used as a positive control. The cell cultures were used as negative control. The dimensions of these fragments were the same as the substrates with MWCNT. After the incubation, the substrates with MWCNT and the positive and negative controls were removed from the respective wells. The supernatants (dead cells) were analysed in accordance with the standard LDH release protocol (SIGMA cytotoxicity in vitro LDH tests). The OD (optical density) of the supernatant was measured at 490 nm wavelength with a 96-well microplate reader on a spectrophotometer Spectra Count (Packard). Aliquots were removed for testing and background subtraction (50 μl). The relative dead cell number was expressed as OD and normalized by the cell cultures
Data were collected from five different experiments (n = 5) and expressed as the average±S.D. The statistical differences were analysed by two-way ANOVA (analysis of variance; Graph Pad Prism 5®). P-values less than 0.001 were considered to indicate statistical differences.
2.4. SEM for cell morphology analysis
The capacity for cellular adhesion of the SaOS-2 cells on the VACNT films was evaluated for incubation periods of 6 h, 24 h, 48 h and 7 days. After the incubation, the attached cells on the substrate were fixed with a 3% glutaraldehyde/0.1-M sodium cacodylate buffer for 1 h and dehydrated in a graded ethanol solution series (30%, 50%, 70%, 95%, 100%) for 10 min each. The drying stage used a 1:1 solution of ethanol with HMDS (hexamethyldisilazane), and the samples were dried with pure HMDS at room temperature (23°C). After deposition of a thin gold layer, the specimens were examined by SEM. Images were recorded at magnifications of ×500 and ×2000.
In Figures 1(a) and 1(b), the SEM images of Fe (Figure 1a) and Ni (Figure 1b) nanoclusters with diameters between 30 and 70 nm are shown. Figures 1(c) and 1(d) show SEM images of the high density of the VACNT film grown on Fe and Ni, respectively. Both cases presented a length of 6–8 μm. The VACNT film produced on Fe nanoclusters are denser than those produced on Ni. The higher efficiency in the production of VACNT using Fe as catalyst is very clear from a comparison of these images. Figures 1(e) and 1(f) show TEM images of typical internal bamboo-like structures of the VACNT obtained using Fe and Ni, respectively. No contaminants from either metallic particles or amorphous carbon were observed outside the tubes. Virtually all metallic particles are enclosed by the CNT produced because the MWCVD uses all the catalyst nanoclusters formed. All of these characteristics indicate that the VACNT obtained are already quite pure, without purification or functionalization processes.
Figure 2 shows the results of LDH released assays, expressed in OD (Figure 2, left-hand panel) and normalized by the cell culture (Figure 2, right-hand panel). In Figure 2(left-hand panel), the values OD of the positive control (latex) is extremely significant compared with all other samples (P<0.001). Figure 2(right-hand panel) shows that the OD of the positive control normalized by the cell culture increased rapidly (∼60%) after 72 h, which characterizes cell death. The normalized OD values for the MWCNT samples are very close to 100%, and hence, they are considered totally non-cytotoxic. The difference in the density of VACNT produced by Fe and Ni catalyst did not affect the cell viability.
SEM images of SaOS-2 cells morphology on VACNT are shown in Figure 3, at ×500 (a) and ×2000 (b), at 6 h (1), 48 h (2) and 7 days (3). The SEM images clearly show cells with a higher number of membrane projections on VACNT. It appears that cell spread out up to contact with the neighbour cells, but they did not effectively attach among themselves, preferring to attach to the VACNT film. Figure 3 shows over time that single cells multiply (Figures 3a1 and 3b1) and evolve to tissue formation (Figures 3a3 and 3b3), spreading over the surface of VACNT substrates.
The structure and properties of CNT are highly sensitive to the production method and synthesis parameters (Cýnar et al., 2006). The majority of processes include further purification steps with oxidative acidic treatments and high-temperature annealing (Huang et al., 2003; Haddon et al., 2004). Kalbacova et al. (2007) have shown a 15% decrease in cell metabolic activity associated with the impurities of CNT synthesis and also stress of actin fibers, though with a higher number of focal adhesion points (attachment of cells in all directions).
In this work, the technique used (MWCVD) produces VACNT films with high degree of graphitization, without amorphous carbon particles, and catalyst totally encapsulated by the graphitic layers (Figures 1e–1f). In addition, the VACNT are deposited on Ti, a substrate with proven biocompatibility and largely used as implant material (Long and Rack, 1998). Hence, neither substrate nor catalyst interferes in the cytotoxicity assays. The differences in tube density (Figures 1c and 1d) using Ni or Fe nanoparticles (Figures 1a and 1b) were not sufficient to change the results of the cytotoxicity tests (Figure 2) or cell attachment features (Figure 3).
Zhang et al. (2007) investigated the effects of CNT dispersed in primary osteoblast culture using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assays. Their cytotoxicity results showed that CNT generated a time- and dose-dependent decrease in cell viability of around 60% at a CNT concentration of 0.0100 μg/ml, compared to negative control (Zhang et al., 2007). Kalbacova et al. (2007) showed a decrease of about 15% in osteoblastic metabolic activity by MTS assay for SWCNT (single-walled carbon nanotube) dispersed in culture, compared with the control, but inferior to the Ti6Al4V (20%). Some authors have reported the possibility of false positives to cytotoxicity in colorimetric assays, where a high concentration of CNT dispersed in the cell culture was used (Worle-Knirsch et al., 2006; Casey et al., 2007). The MTT assay produced a false positive to toxicity, while WST1 and LDH provided high viability. The problem with the MTT assay is that SWCNTs bind to the MTT formazan crystal and stabilize their chemical structure and, as a consequence, these crystals cannot be solubilized (Worle-Knirsch et al., 2006). Hence, to avoid doubts, as described in the Materials and methods section (section 2.3), the MWCNT samples were removed from the wells, similar to the work of Lobo et al. (2008a, 2008b). With this methodology, their scaffolds presented high biocompatibility and exceptional cell spreading and proliferation, independent of colorimetric assay or cell line used.
Zanello et al. have demonstrated that CNT can sustain osteoblast proliferation and bone-formation functions (Zanello et al., 2006). Gianonna et al. have shown the preferential cell attachment and spreading on patterned surfaces of VACNT (Giannona et al., 2007).
Some studies have shown that cells tend to migrate and adhere preferentially on CNT structures (Bryning et al., 2007; Giannona et al., 2007; Lobo et al., 2008a, 2008b). Experiments on cell attachment with scaffold made of CNT were also carried out by Giannona et al. (2007), with an osteosarcoma cell line (CAL-72 cells). The site density was controlled using polystyrene masks for deposition of catalytic Ni dots with intertube spacing of 1 μm, where the cells attach only in nanotube tips. Correa-Duarte et al. also reported the growth of a mouse fibroblast cell line (L-929) on a 3D (three-dimensional) network based on an array of interconnected MWCNT functionalized by acid solutions. The regular pattern of cavities, adjustable to the application and the different shapes and sizes of cells used, considerably contributed to the adhesion and growth of these cells (Correa-Duarte et al., 2004). Abarrategi et al. evaluated the use of MWCNT/chitosan scaffolds with a myoblastic mouse cell line (C2C12), which is a multipotent cell line able to differentiate between different phenotypes under the action of some chemical or biological factors. A confluent layer of cells that partially covers the scaffold surface was observed after 4 days (Abarrategi et al., 2008).
In Figure 3, the SaOS-2 cells showed a high proliferation on the surfaces of VACNT after 7 days. These characteristics are apparently related to the filopodium adhesion to VACNT. Despite the tubular structure of VACNT, the cell spreading has revealed a 2D character, with formation of a very flat and homogeneous cell layer, covering the whole sample area. This monolayer totally blocks the contact of cells with the biomaterial, which is desirable for further application in implants.
In summary, non-cytotoxic VACNT scaffolds were successfully obtained by MWCVD, without any purification or functionalization. The present study showed a very high capacity of fixation of human osteoblast on VACNT, which induces the formation of a very flat and homogeneous monolayer of SAOS-2 cells.
Anderson Lobo performed the CNT growth, cytotoxicity tests, and a part of this research was presented in his thesis in order to obtain the Masters Degree in Materials Science and Engineering at Instituto Tecnológico de Aeronáutica, Brazil. Erica Antunes developed the parameters for the growth of vertically aligned CNT growth using a microwave CVD process. Mariana Palma is an undergraduate in Biomedical Engineering, and was responsible for the control of cellular growth and maintenance of cell culture during the time of incubation of the CNT scaffolds. Cristina Pacheco-Soares is the co-ordinator and is responsible for the Laboratory of Cell Culture at Universidade do Vale do Paraiba, Brazil. Vladimir Trava-Airoldi is the co-ordinator of Diamantes e Materiais Relacionados (DIMARE) research group in which the CNT growth is developed. Evaldo Corat is the advisor of the three first authors and is responsible for the group of CNT growth and applications.
This work was supported by Fundação de Amparo à Pesquisa do Estado de Săo Paulo [grant numbers 08/11642-5, 07/00013-4]; scholarships from Conselho Nacional de Pesquisa e Desenvolvimento; and the Laboratório Nacional de Luz Sincrotron (high-resolution SEM images).
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Received 8 August 2008/19 May 2009; accepted 30 October 2009
Published as Cell Biology International Immediate Publication 30 October 2009, doi:10.1042/CBI20090131
© The Author(s) Journal compilation © 2010 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 1 (A, B) SEM images of Fe and Ni nanoclusters, formed after pretreatment; (C, D) SEM images of the vertically aligned MWCNT films obtained by Fe and Ni catalyst; (E, F) TEM image of bamboo-like structure of an MWCNT obtained with Fe and Ni nanoparticles
Figure 2 LDH release assays, expressed in OD (left-hand panel) and normalized by the SaOS-2 cell culture (right-hand panel)