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Cytocompatibility studies of vertically-aligned multi-walled carbon nanotubes: Raw material and functionalized by oxygen plasma
Materials Science and Engineering C 32 (2012) 648–652
Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Cytocompatibility studies of vertically-aligned multi-walled carbon nanotubes: Raw material and functionalized by oxygen plasma A.O. Lobo a,b,c,⁎, M.A.F. Corat d, E.F. Antunes a,b, S.C. Ramos b, C. Pacheco-Soares e, E.J. Corat a,b a
Laboratório Associado de Sensores e Materiais, INPE, São José dos Campos/SP, Brazil Instituto Tecnológico de Aeronáutica, ITA, São José dos Campos/SP, Brazil Laboratório de Nanotecnologia Biomédica, Universidade do Vale do Paraíba, São José dos Campos/SP, Brazil d Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório, CEMIB, UNICAMP, Campinas/SP, Brazil e Laboratório de Dinâmica de Compartimentos Celulares, UNIVAP, São José dos Campos/SP, Brazil b c
a r t i c l e
i n f o
Article history: Received 9 February 2010 Received in revised form 25 May 2010 Accepted 11 August 2010 Available online 20 August 2010 Keywords: Cytocompatibility Multi-walled carbon nanotubes Functionalization Oxygen plasma Superhydrophilic
a b s t r a c t It was presented a strong difference on cell adhesion and proliferation of functionalized vertically-aligned multi-walled carbon nanotube (VACNT) scaffolds compared to raw-VACNT. Biocompatibility in vitro tests were performed on raw-VACNT after superficial modification by oxygen plasma, which changes its superhydrophobic character to superhydrophilic. Two cytocompatibility tests were applied: 1) total lactate dehydrogenase colorimetric assay for the study of proliferating cells; and 2) cellular adhesion by scanning electron microscopy. Results showed that superhydrophilic VACNT scaffolds stimulate cell growth with proliferation up to 70% higher than normal growth of cell culture. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Controlling surface energy of biomaterials is of significant interest in biomedical applications involving cell-biomaterial interactions. Surface wetting phenomena significantly affect various biological events at the sub-cellular and cellular level, as in protein adsorption, or cell attachment and spreading [1,2]. Numerous studies have implicated a role for surface nano-topography for nano-biomaterials development affecting the cell response, with both increased [3] and decreased [4] adhesions were reported. For binding interactions between cells and biomaterial surfaces, it has become increasingly evident that cells are influenced by spatial domains, structural composition and mechanical forces at the micro and nanoscale [5]. Among other nano-biomaterials, carbon nanotubes (CNT) promise a great role for the study of tissues regeneration [6–10]. The electronic structure, the surface morphology, and exceptional mechanical properties of CNT are typical of graphite-like structures, but they can be distinguished by their tubular construction with nanometric diameters and high aspect ratio, i.e., they are considered a fibrous
⁎ Corresponding author. Present address: Laboratório de Nanotecnologia Biomédica, Instituto de Pesquisa e Desenvolvimento, UNIVAP, Av. Shishima Hifumi, 2911, Urbanova, São Jose dos Campos, São Paulo, Brazil. CEP: 12.244-000. Tel.: +55 1239471166; fax: +55 1239471149. E-mail address: [email protected] (A.O. Lobo). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.08.010
material [11,12]. CNT, hence, present physical dimensions similar to extracellular matrix (ECM) components and are appropriated to mimic their features [13]. It has been recognized that hydrophilic surfaces are generally favorable to the adhesion, spreading and proliferation of various cell types [1–4,13]. Some studies have shown that raw vertically-aligned multi-walled carbon nanotubes (VACNT) are superhydrophobic [14–18], which may be a limitation for their application as biomaterial [19,20]. Some studies have already shown that VACNT favors cell growth and adhesion [21,22] despite its superhydrophobic character. Somehow the cells appear to overcome the surface superhydrophobicity and interact directly with the VACNT by its adhesion, spreading and proliferation. It seems that changes on surface morphology by tube bending may be responsible for a change on VACNT wettability during the wetting process. However, the literature do not report any study about cellular interaction with true superhydrophilic VACNT. The wettability of carbon nanotube (CNT) may be controlled by several chemical and physical treatments for CNT functionalization. Chemistry effect is employed by changing wettability nanotubes. As know, oxygen-containing functional groups are formed on the CNT surfaces by treatment with oxidation [23,24], or treatment acid [25]. The wettability for polar liquids such as water can be enhanced significantly in this way, leading to more reactive VACNT surfaces [26,27]. Compared to these methods, the exposure of VACNT to oxygen plasma is the most efficient way to introduce simultaneously polar functional groups (COH, OH, C O, COOH) and roughness [28,29] to the CNTs.
A.O. Lobo et al. / Materials Science and Engineering C 32 (2012) 648–652
This work reports comparative cytocompatibility studies of mouse fibroblasts (L-929) on raw-VACNT films, which are superhydrophobic, and VACNT functionalized by pulsed-direct current oxygen plasma, which are superhydrophilic. Comparisons of cell growth on superhydrophobic and superhydrophilic VACNT films were performed for up to 7 days of incubation. Cell proliferation was evaluated by the lactate dehydrogenase (LDH) colorimetric assay and cellular adhesion was evaluated by scanning electron microscopy. True superhydrophilic VACNT films considerably enhanced cell proliferation to be up to 70% higher than normal growth of cell culture. 2. Materials and methods 2.1. VACNT synthesis The VACNT films were produced using a microwave-plasma chamber at 2.45 GHz [30,31]. The Ti substrates (100 mm2) were covered by Fe or Ni layers of 10 nm deposited by e-beam evaporation. The Fe or Ni layers were pre-treated to promote nanocluster formation, which served as the catalyst for VACNT growth. The pretreatment was carried out during 5 min in plasma of N2/H2 (10/ 90 sccm), at a substrate temperature of approximately 760 °C. After the pre-treatment, 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 entire process. 2.2. VACNT functionalized by polar groups Functionalization of the nanotube tips by the incorporation of oxygen-containing groups was performed in a pulsed-direct current plasma reactor with an oxygen flow rate of 1 sccm, at a pressure of 85 mTorr, −700 V and with a frequency of 20 kHz [32]. The contact angle of deionised water drops (2 μl) with the VACNT films was measured by using the sessile drop method with a Kruss EasyDrop instrument (DSA 100). Each CA measurement was taken in five different values. It was performed immediately after the drop on surface in order to avoid the evaporation process. A chemical surface modification was calculated using liquids with different surface tensions and polarities e it was calculated elsewhere [32]. The incorporation of the polar groups were monitored by X-Ray Photoelectron Spectroscopy (XPS), using an equipment from VG Microtech (XR 705), operating at 486.5 eV (AlKα). Scanning and transmission electron microscopy were used to observe the morphology and structure of the VACNTs, respectively. 2.3. Cell cultures Mouse fibroblast cells were provided by Cell Line Bank from Rio de Janeiro/Brazil (CR019). The cells were maintained as sub-confluent monolayer's in a minimum essential medium with 1.5 mM Lglutamine adjusted to contain 2.2 g/l sodium bicarbonate 85%; 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. All 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% fetal bovine serum. The incubation was performed under a CO2 (5%) atmosphere at 37 °C for different periods (6 h, 48 h, 72 h and 7 days). Fragments of non-harmful filter paper were used as a negative control, whereas latex fragments were applied as a positive control. The dimensions of these fragments were the same as the substrates containing VACNTs. After the incubations, the substrates with VACNTs, and the positive and negative controls were removed from the respective wells.
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2.4. LDH total assay for cell proliferation analysis The lactate dehidrogenase assay (LDH) 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. The proliferation on both scaffolds, the ones of raw-VACNT and the ones functionalized by oxygen plasma, were evaluated by LDH. The enzyme LDH, located in the cytoplasm, was used as a marker for membrane integrity of cells and therefore it might be correlated to cell viability and proliferation. This enzyme is released when a damage of the cytoplasm membrane occurs [33]. After incubation time of 48 and 72 h, respectively, 50 μl (1/10 vol.) of a LDH assay lysis solution was added to each well, and the plates were returned to the incubator for 45 min. Aliquots of the medium (50 μl) were removed for testing and background subtraction. The LDH assay mixture from SIGMA® TOX 7 Kit was added in an amount equal to twice the volume of medium removed from each well, i.e. 100 μl. After this stage, aliquots of 150 μl were transferred to a clean flat-bottom plate with 96 wells, and incubated at room temperature for 20–30 min with an opaque material to protect them from light. The reaction was terminated with the addition of a 1/10 volume of 1 M HCl (50 μl) to each well. The optical density was measured at a wavelength of 490 nm with a 96-well microplate reader using a spectrophotometer Spectra Count (Packard). The total LDH was expressed as [ODsample − ODblank], where OD refers to optical density. The cell proliferation on raw-VACNT and on VACNT treated by O2 plasma, in comparison to the negative control, were expressed as h
i ODsample ODblank = ODnegative control ODblank T100:
2.5. Cellular adhesion test Scanning electron microscopy (JEOL JSM 5610 VPI) was used to evaluate the potential of cellular adhesion of the L-929 cells on the superhydrophobic and superhydrophilic VACNT scaffolds, for incubation periods of 6 h and 7 days. After culturing for a period of time, the attached cells on the substrate were fixed with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h. After fixation they were dehydrated in a graded ethanol solution series (30, 50, 70, 95 and 100%) for 10 min each. To dehydrate completely, a solution of ethanol (50%) with hexamethyldisilazane (HMDS) and HMDS (100%) were used. HMDS drying preserves excellent surface detail in the cell, due reduced surface tension and also cross-links proteins, therefore adding strength to the sample during air-drying [34]. Subsequently, the samples were dried at room temperature for 30 min. Finally, a thin layer of gold was deposited on the samples in order to observe them by scanning electron microscopy. 2.6. Statistical analysis Data were collected from five different experiments and expressed as the average±the standard deviation. The statistical differences were analyzed by 2-way Anova (Graph Pad Prism 5®). The populations from the multi-walled carbon nanotube films were obtained with normal distribution and independent to each experiment. The P-values of less than 0.05 were considered to indicate statistical differences. 3. Results and discussion Fig. 1 shows typical scanning (a) and transmission electron microscopy images after the oxygen plasma etching (b) compared to as-grown (c) VACNT. Visually, no significant morphological and structural changes could be observed on superhidrophilic VACNT films (c). Details of the TEM and SEM analysis of the as-grown VACNT films obtained in this work can be observed in elsewhere [35].
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A.O. Lobo et al. / Materials Science and Engineering C 32 (2012) 648–652
Fig. 1. (a) Scanning electron microscopy image of VACNT films and transmission electron microscopy image of internal structures, before (c) and after (b) the oxygen plasma etching.
The effect of oxygen plasma on the wettability of VACNT surfaces due the increase of the number of polar groups is shown in Fig. 2. The upper part shows the contact angle measurements while the lower part shows the XPS analyses of C1s peak of the raw-VACNT (left) and of the functionalized-VACNT (right). The contact angle of a liquid droplet in thermal equilibrium on a horizontal surface is the most common way to judge the wettability of a solid surface. If the liquid used is water, the CA can define the wettability degree, thus classifying the surface as: hydrophobic (contact angle N90°), hydrophilic (b90°), superhydrophobic (N150°) and superhydrophilic (b5°). From the pulsed-direct current oxygen plasma treatment used in this work, a significant change on the contact angle from ~154° (Fig. 2a) to ~0° (Fig. 2b) was achieved. Hence, the VACNT surface switched from superhydrophobic to superhydrophilic, showing the high efficiency of this treatment. The use of oxygen plasma treatment on CNT surfaces has already been reported in literature
[31,32]. All reported works showed only a partial change on the wettability of the CNT surface. Chirila et al. demonstrated that the wettability increased up to 68% after the treatment with microwaveplasma and 20% with radio frequency-plasma as compared to the untreated CNT [36]. Brandl and Marginean showed that the contact angle with water decreased from 88° to 58° after plasma treatment [37]. The plasma conditions used in the present work shows a higher efficiency as compared to previous reports. Fig. 2 shows the efficiency of the oxygen plasma treatment on VACNT surfaces from superhydrophobic to superhydrophilic behavior. The comparisons between as-grown VACNT and after the plasma treatment were studied using CA (a–b) and XPS (c–d) techniques. To asses specific carboxylic groups attached on surface, the spectra were deconvoluted at the C1s. A deconvolution comparison between as-grown VACNT (c) and after plasma functionalization (d), were showed. All binding energies were referenced to C1s at 284.5 eV. The spectra were deconvoluted by assuming a Lorentzian–Gaussian sum of functions (20% Lorentzian maximum contribution) [38]. The spectra were analyzed using Spectrum software XPS peak41 [39]. The C1s peak was decomposed into four Gaussian components, referring to the bounds: C C (~284 eV), C–O (~286.2 eV), C O (286.8 eV), and –COO (287.9 eV) [40–43]. The intensity of the C O and mainly the –COO peak increased after the oxidation. From these fit the full width at half maximum (FWHM) show an enlargement for all bands (data not shown) after oxygen plasma treatment (Fig. 2d). In the quantitative analysis was observed that the oxygen content increased from 2.8% to 18.9%, after the oxygen plasma treatment. Fig. 3 shows the influence of superhydrophobic and superhydrophilic VACNT surfaces on the cell proliferation, as obtained by LDH total assay. It is a consensus in the literature that the total LDH is an efficient colorimetric assay to identify cell proliferation. In total LDH assay all cells of each well were quantified. The behavior of the positive control significantly shows cell death. It presented highly significant differences (p b 0.0001) in the interval of 48–72 h, confirming cytotoxicity. It is also clear that VACNT does not affect cell growth, as compared to the negative control. The values increase with the incubation time for all the VACNT films, i.e. raw and functionalized, independent of the catalyst, iron or nickel. Worle-Knirsch et al. also observed no reduction in cell viability for the LDH assay with several cell lines (A549, ECV304) and singlewalled carbon nanotubes for 24 h and longer periods at different concentrations [44]. On the other hand, these results presented here are an association between cell proliferation and viability up to 72 h, different of the results presented by Worle-Knirsch et al. A 20% increase in the number of cells in contact with raw-VACNT scaffolds was observed. The statistical results show a highly significant (pb 0.0001) increase in the number of cells in contact with the VACNTs between 48 and 72 h of incubation as compared to negative control. An increase in LDH yield for the superhydrophilic VACNT sample compared to raw-VACNT scaffolds, indicated further cell proliferation. In general, it is shown that cell proliferation on superhydrophilic VACNT is 70% higher than on the negative control. Cell attachment observed after 6 h and up to 7 days of culturing time was observed by scanning electron microscopy either on superhydrophobic or superhydrophilic VACNT, which typical micrographies are shown in Fig. 4. The high efficiency of cell growth on superhydrophilic surfaces by formation of concise cell monolayer is highlighted in Fig. 4c and d. The images in Fig. 4 are for 6 h (left side) and for 7 days (right) of incubation. Upper images are for raw-VACNT (a and b) and lower images are for functionalized-VACNT. For raw-VACNT the images have shown larger spaces between the cells, indicating a smaller proliferation and spreading as compared with the functionalized-VACNT, despite that a very healthy cell behavior have been observed in both substrates. Cellular adhesion is generally dependent on time, adhesive forces at the cell/material interface and surface topography. Several authors have shown that an initial period of time (e.g. 6 to 12 h) is essential for
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Fig. 2. Effect of oxygen plasma functionalization on the VACNTs. Optical microscopy images of the contact angle between deionised water and VACNT before (a) and after (b) the oxygen plasma treatment (magnification 200×). C1s XPS peak analysis before (c) and after (d) the oxygen plasma treatment.
the cell adhesion due to migration and proliferation of the cells on biomaterial surface [45,46]. The scanning electron microscopy images of initial phase (6 h) of cellular adhesion clearly show a higher number of cells with membrane projections totally flat and starting the monolayer formation on superhydrophilic VACNT (Fig. 4b) scaffolds compared to raw-VACNT (Fig. 4a). Lobo et al. have shown a high level of L-929 cell biocompatibility with raw-VACNT films [21,22,35], with a monolayer formation after 7 days. Their results are very similar to the ones observed in the present work for the raw-VACNT. However, the behavior of the L-929
cells on functionalized-VACNT is much better. Clearly, there is an increase in the number of cells on the superhydrophilic VACNT scaffolds compared to the superhydrophobic VACNT scaffolds for both time intervals. The high degree of wettability seemed to accelerate the cell adhesion and the proliferation process (Fig. 4b and d). This might apparently facilitate filopodium adhesion on superhydrophilic VACNT scaffold. Despite the tubular structure of superhydrophilic VACNT (Fig. 4d), the cell spreads with the formation of a flat and homogenous cell monolayer after 7 days, covering the whole substrate area. Finally, high cellular affinity to superhydrophilic VACNT scaffolds expands the field of applications and/or production of biomaterials. 4. Conclusions Superhydrophilic VACNT films were successfully produced by incorporation of carboxylic groups using pulsed-direct current oxygen plasma. Wettability of this material might increase and facilitate the contact from substrate to the cells. Superhydrophilic VACNT surfaces efficiently obtained by oxygen plasma functionalization accelerate the adhesion and increase proliferation of L-929 cells, which is a great feature and desirable for biomaterial industry. Acknowledgements
Fig. 3. Proliferation of L-929 cells measured by total LDH in the interaction with rawand functionalized-VACNT by O2 plasma, normalized and compared to cellular growth. Results are the average ± the standard deviation for n = 5.
We gratefully acknowledge funding by Fundação de Amparo à Pesquisa do Estado de São Paulo under the grants 2008/11642-5 and 07/00013-4, scholarships from Conselho Nacional de Pesquisa e Desenvolvimento, and high resolution scanning electron microscopy images from Laboratório Nacional de Luz Sincrotron. The contribution of Marcelo Henrique Maia Costa with XPS analysis is also gratefully acknowledged.
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Fig. 4. Scanning electron microscopy images of L-929 after 6 h (a and c) and 7 days (b and d) of incubation on superhydrophobic VACNT (a and b) and superhydrophilic VACNT (c and d).
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Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Cytocompatibility studies of vertically-aligned multi-walled carbon nanotubes: Raw material and functionalized by oxygen plasma A.O. Lobo a,b,c,⁎, M.A.F. Corat d, E.F. Antunes a,b, S.C. Ramos b, C. Pacheco-Soares e, E.J. Corat a,b a
Laboratório Associado de Sensores e Materiais, INPE, São José dos Campos/SP, Brazil Instituto Tecnológico de Aeronáutica, ITA, São José dos Campos/SP, Brazil Laboratório de Nanotecnologia Biomédica, Universidade do Vale do Paraíba, São José dos Campos/SP, Brazil d Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório, CEMIB, UNICAMP, Campinas/SP, Brazil e Laboratório de Dinâmica de Compartimentos Celulares, UNIVAP, São José dos Campos/SP, Brazil b c
a r t i c l e
i n f o
Article history: Received 9 February 2010 Received in revised form 25 May 2010 Accepted 11 August 2010 Available online 20 August 2010 Keywords: Cytocompatibility Multi-walled carbon nanotubes Functionalization Oxygen plasma Superhydrophilic
a b s t r a c t It was presented a strong difference on cell adhesion and proliferation of functionalized vertically-aligned multi-walled carbon nanotube (VACNT) scaffolds compared to raw-VACNT. Biocompatibility in vitro tests were performed on raw-VACNT after superficial modification by oxygen plasma, which changes its superhydrophobic character to superhydrophilic. Two cytocompatibility tests were applied: 1) total lactate dehydrogenase colorimetric assay for the study of proliferating cells; and 2) cellular adhesion by scanning electron microscopy. Results showed that superhydrophilic VACNT scaffolds stimulate cell growth with proliferation up to 70% higher than normal growth of cell culture. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Controlling surface energy of biomaterials is of significant interest in biomedical applications involving cell-biomaterial interactions. Surface wetting phenomena significantly affect various biological events at the sub-cellular and cellular level, as in protein adsorption, or cell attachment and spreading [1,2]. Numerous studies have implicated a role for surface nano-topography for nano-biomaterials development affecting the cell response, with both increased [3] and decreased [4] adhesions were reported. For binding interactions between cells and biomaterial surfaces, it has become increasingly evident that cells are influenced by spatial domains, structural composition and mechanical forces at the micro and nanoscale [5]. Among other nano-biomaterials, carbon nanotubes (CNT) promise a great role for the study of tissues regeneration [6–10]. The electronic structure, the surface morphology, and exceptional mechanical properties of CNT are typical of graphite-like structures, but they can be distinguished by their tubular construction with nanometric diameters and high aspect ratio, i.e., they are considered a fibrous
⁎ Corresponding author. Present address: Laboratório de Nanotecnologia Biomédica, Instituto de Pesquisa e Desenvolvimento, UNIVAP, Av. Shishima Hifumi, 2911, Urbanova, São Jose dos Campos, São Paulo, Brazil. CEP: 12.244-000. Tel.: +55 1239471166; fax: +55 1239471149. E-mail address: [email protected] (A.O. Lobo). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.08.010
material [11,12]. CNT, hence, present physical dimensions similar to extracellular matrix (ECM) components and are appropriated to mimic their features [13]. It has been recognized that hydrophilic surfaces are generally favorable to the adhesion, spreading and proliferation of various cell types [1–4,13]. Some studies have shown that raw vertically-aligned multi-walled carbon nanotubes (VACNT) are superhydrophobic [14–18], which may be a limitation for their application as biomaterial [19,20]. Some studies have already shown that VACNT favors cell growth and adhesion [21,22] despite its superhydrophobic character. Somehow the cells appear to overcome the surface superhydrophobicity and interact directly with the VACNT by its adhesion, spreading and proliferation. It seems that changes on surface morphology by tube bending may be responsible for a change on VACNT wettability during the wetting process. However, the literature do not report any study about cellular interaction with true superhydrophilic VACNT. The wettability of carbon nanotube (CNT) may be controlled by several chemical and physical treatments for CNT functionalization. Chemistry effect is employed by changing wettability nanotubes. As know, oxygen-containing functional groups are formed on the CNT surfaces by treatment with oxidation [23,24], or treatment acid [25]. The wettability for polar liquids such as water can be enhanced significantly in this way, leading to more reactive VACNT surfaces [26,27]. Compared to these methods, the exposure of VACNT to oxygen plasma is the most efficient way to introduce simultaneously polar functional groups (COH, OH, C O, COOH) and roughness [28,29] to the CNTs.
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This work reports comparative cytocompatibility studies of mouse fibroblasts (L-929) on raw-VACNT films, which are superhydrophobic, and VACNT functionalized by pulsed-direct current oxygen plasma, which are superhydrophilic. Comparisons of cell growth on superhydrophobic and superhydrophilic VACNT films were performed for up to 7 days of incubation. Cell proliferation was evaluated by the lactate dehydrogenase (LDH) colorimetric assay and cellular adhesion was evaluated by scanning electron microscopy. True superhydrophilic VACNT films considerably enhanced cell proliferation to be up to 70% higher than normal growth of cell culture. 2. Materials and methods 2.1. VACNT synthesis The VACNT films were produced using a microwave-plasma chamber at 2.45 GHz [30,31]. The Ti substrates (100 mm2) were covered by Fe or Ni layers of 10 nm deposited by e-beam evaporation. The Fe or Ni layers were pre-treated to promote nanocluster formation, which served as the catalyst for VACNT growth. The pretreatment was carried out during 5 min in plasma of N2/H2 (10/ 90 sccm), at a substrate temperature of approximately 760 °C. After the pre-treatment, 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 entire process. 2.2. VACNT functionalized by polar groups Functionalization of the nanotube tips by the incorporation of oxygen-containing groups was performed in a pulsed-direct current plasma reactor with an oxygen flow rate of 1 sccm, at a pressure of 85 mTorr, −700 V and with a frequency of 20 kHz [32]. The contact angle of deionised water drops (2 μl) with the VACNT films was measured by using the sessile drop method with a Kruss EasyDrop instrument (DSA 100). Each CA measurement was taken in five different values. It was performed immediately after the drop on surface in order to avoid the evaporation process. A chemical surface modification was calculated using liquids with different surface tensions and polarities e it was calculated elsewhere [32]. The incorporation of the polar groups were monitored by X-Ray Photoelectron Spectroscopy (XPS), using an equipment from VG Microtech (XR 705), operating at 486.5 eV (AlKα). Scanning and transmission electron microscopy were used to observe the morphology and structure of the VACNTs, respectively. 2.3. Cell cultures Mouse fibroblast cells were provided by Cell Line Bank from Rio de Janeiro/Brazil (CR019). The cells were maintained as sub-confluent monolayer's in a minimum essential medium with 1.5 mM Lglutamine adjusted to contain 2.2 g/l sodium bicarbonate 85%; 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. All 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% fetal bovine serum. The incubation was performed under a CO2 (5%) atmosphere at 37 °C for different periods (6 h, 48 h, 72 h and 7 days). Fragments of non-harmful filter paper were used as a negative control, whereas latex fragments were applied as a positive control. The dimensions of these fragments were the same as the substrates containing VACNTs. After the incubations, the substrates with VACNTs, and the positive and negative controls were removed from the respective wells.
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2.4. LDH total assay for cell proliferation analysis The lactate dehidrogenase assay (LDH) 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. The proliferation on both scaffolds, the ones of raw-VACNT and the ones functionalized by oxygen plasma, were evaluated by LDH. The enzyme LDH, located in the cytoplasm, was used as a marker for membrane integrity of cells and therefore it might be correlated to cell viability and proliferation. This enzyme is released when a damage of the cytoplasm membrane occurs [33]. After incubation time of 48 and 72 h, respectively, 50 μl (1/10 vol.) of a LDH assay lysis solution was added to each well, and the plates were returned to the incubator for 45 min. Aliquots of the medium (50 μl) were removed for testing and background subtraction. The LDH assay mixture from SIGMA® TOX 7 Kit was added in an amount equal to twice the volume of medium removed from each well, i.e. 100 μl. After this stage, aliquots of 150 μl were transferred to a clean flat-bottom plate with 96 wells, and incubated at room temperature for 20–30 min with an opaque material to protect them from light. The reaction was terminated with the addition of a 1/10 volume of 1 M HCl (50 μl) to each well. The optical density was measured at a wavelength of 490 nm with a 96-well microplate reader using a spectrophotometer Spectra Count (Packard). The total LDH was expressed as [ODsample − ODblank], where OD refers to optical density. The cell proliferation on raw-VACNT and on VACNT treated by O2 plasma, in comparison to the negative control, were expressed as h
i ODsample ODblank = ODnegative control ODblank T100:
2.5. Cellular adhesion test Scanning electron microscopy (JEOL JSM 5610 VPI) was used to evaluate the potential of cellular adhesion of the L-929 cells on the superhydrophobic and superhydrophilic VACNT scaffolds, for incubation periods of 6 h and 7 days. After culturing for a period of time, the attached cells on the substrate were fixed with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h. After fixation they were dehydrated in a graded ethanol solution series (30, 50, 70, 95 and 100%) for 10 min each. To dehydrate completely, a solution of ethanol (50%) with hexamethyldisilazane (HMDS) and HMDS (100%) were used. HMDS drying preserves excellent surface detail in the cell, due reduced surface tension and also cross-links proteins, therefore adding strength to the sample during air-drying [34]. Subsequently, the samples were dried at room temperature for 30 min. Finally, a thin layer of gold was deposited on the samples in order to observe them by scanning electron microscopy. 2.6. Statistical analysis Data were collected from five different experiments and expressed as the average±the standard deviation. The statistical differences were analyzed by 2-way Anova (Graph Pad Prism 5®). The populations from the multi-walled carbon nanotube films were obtained with normal distribution and independent to each experiment. The P-values of less than 0.05 were considered to indicate statistical differences. 3. Results and discussion Fig. 1 shows typical scanning (a) and transmission electron microscopy images after the oxygen plasma etching (b) compared to as-grown (c) VACNT. Visually, no significant morphological and structural changes could be observed on superhidrophilic VACNT films (c). Details of the TEM and SEM analysis of the as-grown VACNT films obtained in this work can be observed in elsewhere [35].
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Fig. 1. (a) Scanning electron microscopy image of VACNT films and transmission electron microscopy image of internal structures, before (c) and after (b) the oxygen plasma etching.
The effect of oxygen plasma on the wettability of VACNT surfaces due the increase of the number of polar groups is shown in Fig. 2. The upper part shows the contact angle measurements while the lower part shows the XPS analyses of C1s peak of the raw-VACNT (left) and of the functionalized-VACNT (right). The contact angle of a liquid droplet in thermal equilibrium on a horizontal surface is the most common way to judge the wettability of a solid surface. If the liquid used is water, the CA can define the wettability degree, thus classifying the surface as: hydrophobic (contact angle N90°), hydrophilic (b90°), superhydrophobic (N150°) and superhydrophilic (b5°). From the pulsed-direct current oxygen plasma treatment used in this work, a significant change on the contact angle from ~154° (Fig. 2a) to ~0° (Fig. 2b) was achieved. Hence, the VACNT surface switched from superhydrophobic to superhydrophilic, showing the high efficiency of this treatment. The use of oxygen plasma treatment on CNT surfaces has already been reported in literature
[31,32]. All reported works showed only a partial change on the wettability of the CNT surface. Chirila et al. demonstrated that the wettability increased up to 68% after the treatment with microwaveplasma and 20% with radio frequency-plasma as compared to the untreated CNT [36]. Brandl and Marginean showed that the contact angle with water decreased from 88° to 58° after plasma treatment [37]. The plasma conditions used in the present work shows a higher efficiency as compared to previous reports. Fig. 2 shows the efficiency of the oxygen plasma treatment on VACNT surfaces from superhydrophobic to superhydrophilic behavior. The comparisons between as-grown VACNT and after the plasma treatment were studied using CA (a–b) and XPS (c–d) techniques. To asses specific carboxylic groups attached on surface, the spectra were deconvoluted at the C1s. A deconvolution comparison between as-grown VACNT (c) and after plasma functionalization (d), were showed. All binding energies were referenced to C1s at 284.5 eV. The spectra were deconvoluted by assuming a Lorentzian–Gaussian sum of functions (20% Lorentzian maximum contribution) [38]. The spectra were analyzed using Spectrum software XPS peak41 [39]. The C1s peak was decomposed into four Gaussian components, referring to the bounds: C C (~284 eV), C–O (~286.2 eV), C O (286.8 eV), and –COO (287.9 eV) [40–43]. The intensity of the C O and mainly the –COO peak increased after the oxidation. From these fit the full width at half maximum (FWHM) show an enlargement for all bands (data not shown) after oxygen plasma treatment (Fig. 2d). In the quantitative analysis was observed that the oxygen content increased from 2.8% to 18.9%, after the oxygen plasma treatment. Fig. 3 shows the influence of superhydrophobic and superhydrophilic VACNT surfaces on the cell proliferation, as obtained by LDH total assay. It is a consensus in the literature that the total LDH is an efficient colorimetric assay to identify cell proliferation. In total LDH assay all cells of each well were quantified. The behavior of the positive control significantly shows cell death. It presented highly significant differences (p b 0.0001) in the interval of 48–72 h, confirming cytotoxicity. It is also clear that VACNT does not affect cell growth, as compared to the negative control. The values increase with the incubation time for all the VACNT films, i.e. raw and functionalized, independent of the catalyst, iron or nickel. Worle-Knirsch et al. also observed no reduction in cell viability for the LDH assay with several cell lines (A549, ECV304) and singlewalled carbon nanotubes for 24 h and longer periods at different concentrations [44]. On the other hand, these results presented here are an association between cell proliferation and viability up to 72 h, different of the results presented by Worle-Knirsch et al. A 20% increase in the number of cells in contact with raw-VACNT scaffolds was observed. The statistical results show a highly significant (pb 0.0001) increase in the number of cells in contact with the VACNTs between 48 and 72 h of incubation as compared to negative control. An increase in LDH yield for the superhydrophilic VACNT sample compared to raw-VACNT scaffolds, indicated further cell proliferation. In general, it is shown that cell proliferation on superhydrophilic VACNT is 70% higher than on the negative control. Cell attachment observed after 6 h and up to 7 days of culturing time was observed by scanning electron microscopy either on superhydrophobic or superhydrophilic VACNT, which typical micrographies are shown in Fig. 4. The high efficiency of cell growth on superhydrophilic surfaces by formation of concise cell monolayer is highlighted in Fig. 4c and d. The images in Fig. 4 are for 6 h (left side) and for 7 days (right) of incubation. Upper images are for raw-VACNT (a and b) and lower images are for functionalized-VACNT. For raw-VACNT the images have shown larger spaces between the cells, indicating a smaller proliferation and spreading as compared with the functionalized-VACNT, despite that a very healthy cell behavior have been observed in both substrates. Cellular adhesion is generally dependent on time, adhesive forces at the cell/material interface and surface topography. Several authors have shown that an initial period of time (e.g. 6 to 12 h) is essential for
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Fig. 2. Effect of oxygen plasma functionalization on the VACNTs. Optical microscopy images of the contact angle between deionised water and VACNT before (a) and after (b) the oxygen plasma treatment (magnification 200×). C1s XPS peak analysis before (c) and after (d) the oxygen plasma treatment.
the cell adhesion due to migration and proliferation of the cells on biomaterial surface [45,46]. The scanning electron microscopy images of initial phase (6 h) of cellular adhesion clearly show a higher number of cells with membrane projections totally flat and starting the monolayer formation on superhydrophilic VACNT (Fig. 4b) scaffolds compared to raw-VACNT (Fig. 4a). Lobo et al. have shown a high level of L-929 cell biocompatibility with raw-VACNT films [21,22,35], with a monolayer formation after 7 days. Their results are very similar to the ones observed in the present work for the raw-VACNT. However, the behavior of the L-929
cells on functionalized-VACNT is much better. Clearly, there is an increase in the number of cells on the superhydrophilic VACNT scaffolds compared to the superhydrophobic VACNT scaffolds for both time intervals. The high degree of wettability seemed to accelerate the cell adhesion and the proliferation process (Fig. 4b and d). This might apparently facilitate filopodium adhesion on superhydrophilic VACNT scaffold. Despite the tubular structure of superhydrophilic VACNT (Fig. 4d), the cell spreads with the formation of a flat and homogenous cell monolayer after 7 days, covering the whole substrate area. Finally, high cellular affinity to superhydrophilic VACNT scaffolds expands the field of applications and/or production of biomaterials. 4. Conclusions Superhydrophilic VACNT films were successfully produced by incorporation of carboxylic groups using pulsed-direct current oxygen plasma. Wettability of this material might increase and facilitate the contact from substrate to the cells. Superhydrophilic VACNT surfaces efficiently obtained by oxygen plasma functionalization accelerate the adhesion and increase proliferation of L-929 cells, which is a great feature and desirable for biomaterial industry. Acknowledgements
Fig. 3. Proliferation of L-929 cells measured by total LDH in the interaction with rawand functionalized-VACNT by O2 plasma, normalized and compared to cellular growth. Results are the average ± the standard deviation for n = 5.
We gratefully acknowledge funding by Fundação de Amparo à Pesquisa do Estado de São Paulo under the grants 2008/11642-5 and 07/00013-4, scholarships from Conselho Nacional de Pesquisa e Desenvolvimento, and high resolution scanning electron microscopy images from Laboratório Nacional de Luz Sincrotron. The contribution of Marcelo Henrique Maia Costa with XPS analysis is also gratefully acknowledged.
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Fig. 4. Scanning electron microscopy images of L-929 after 6 h (a and c) and 7 days (b and d) of incubation on superhydrophobic VACNT (a and b) and superhydrophilic VACNT (c and d).
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