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Osteoblast cell response to surface-modified carbon nanotubes
Materials Science and Engineering C 32 (2012) 1057–1061
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
Osteoblast cell response to surface-modified carbon nanotubes Faming Zhang a,⁎, Arne Weidmann b, J. Barbara Nebe b, Eberhard Burkel a a b
Institute of Physics, University of Rostock, 18055 Rostock, Germany Biomedical Research Center, Department of Cell Biology, University of Rostock, 18057 Rostock, Germany
a r t i c l e
i n f o
Article history: Received 26 October 2009 Received in revised form 22 June 2010 Accepted 9 July 2010 Available online 14 July 2010 Keywords: Carbon nanotubes Surface modification Cell response Biomedical applications
a b s t r a c t In order to investigate the interaction of cells with modified multi-walled carbon nanotubes (MWCNTs) for their potential biomedical applications, the MWCNTs were chemically modified with carboxylic acid groups (–COOH), polyvinyl alcohol (PVA) polymer and biomimetic apatite on their surfaces. Additionally, human osteoblast MG-63 cells were cultured in the presence of the surface-modified MWCNTs. The metabolic activities of osteoblastic cells, cell proliferation properties, as well as cell morphology were studied. The surface modification of MWCNTs with biomimetic apatite exhibited a significant increase in the cell viability of osteoblasts, up to 67.23%. In the proliferation phases, there were many more cells in the biomimetic apatite-modified MWCNT samples than in the MWCNTs–COOH. There were no obvious changes in cell morphology in osteoblastic MG-63 cells cultured in the presence of these chemically-modified MWCNTs. The surface modification of MWCNTs with apatite achieves an effective enhancement of their biocompatibility. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Carbon nanotubes (CNTs) have attracted broad attention because of their unique structure and novel properties. In recent years, many research efforts have focused on the exploration of the application of both single-wall (SWCNT) and multi-wall (MWCNT) carbon nanotubes in the biological and biomedical field as nerve cell stimuli, diabetes sensors, cancer therapy, drug delivery carrier and bone scaffold materials etc. [1,2]. Compared with other bone scaffold materials such as polymers and peptide fibers, the high tensile strength, excellent flexibility, and low density of CNTs make them ideal for the production of bone [3]. However, the CNTs have yet to cross many technological hurdles. The lack of solubility of CNTs in aqueous media and the bioinert surfaces have been major technical barriers [2,4]. The recent expansion in methods to chemically modify and functionalize CNTs with water-soluble polymers has made it possible to solubilize and disperse CNTs in water, thus opening the way for their facile manipulation and processing in physiological environments [5,6]. These functional groups, modified and functionalized on the surface of CNTs, could activate their bio-inert surface to attract calcium cations and thereby nucleate and initiate the crystallization of apatite. Many water-soluble polymers have been modified on the surface of CNTs [7–10]. Poly (vinyl alcohol) (PVA) is a type of water-soluble and biocompatible polymer. The PVA/CNT composites and coatings have been studied by a number of researchers [11–13]. Apatite has been functionalized on the modified CNTs by using simulated body fluid soaking [14], G/P solutions [15] or chemical
⁎ Corresponding author. Tel.: + 49 381 4986864; fax: + 49 381 4986862. E-mail addresses: [email protected], [email protected] (F. Zhang). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.07.007
mineralization methods [2]. Now the surface modification and functionalization with water-soluble polymer and apatite is no longer a big scientific problem. Up to the present, however, there have been few publications reporting the cell response to chemically-modified carbon nanotubes. Understanding the interaction of cells with the modified MWCNTs is very important for the potential medical application of CNTs. In this study, based on the surface modification of MWCNTs with PVA and apatite as a potential substrate for bone scaffold applications, the osteoblast cells' response to the surface-modified MWCNTs were studied. The MWCNTs were functionalized using a direct reaction of polyvinyl alcohol (PVA) polymer with carboxylic acid groups on the CNTs by the “graft to” method, based on the procedures of Lin et al. [16]. After this, biomimetic nanostructured apatite was modified on the PVA-modified MWCNTs by simulated body fluid soaking based on the research of Aryal et al. [14]. MG-63 osteoblast cells were cultured in the presence of the MWCNT–COOH, MWCNT–PVA and MWCNT– Apatite samples. The metabolic activity, cell proliferation, and cell morphology of MG-63 osteoblasts in response to the modified MWCNTs were studied.
2. Experimental 2.1. Materials The MWCNTs were obtained from Shenzhen Nanotech Port, Ltd., China. They were produced by catalytic chemical vapor deposition (CCVD) in which CH4 or C2H2 was converted into CNTs at 1000 °C in the presence of Ni and La catalysts. The purity of the MWCNTs as received was higher than 95%.
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2.2. Surface modification The schematic illustration of the surface modification and chemical structure of the MWCNTs is shown in Fig. 1. The starting MWCNTs (Fig. 1a) were refluxed in nitric acid (HNO3) at 120 °C for 24 h to functionalize the carbon nanotubes with carboxylic acid groups (–COOH) (Fig. 1b). After vacuum filtration to remove the liquid phase, the remaining solids were washed repeatedly with deionized water until a neutral pH was achieved, and then dried under vacuum. The resulting carboxylated CNTs were then mixed with a PVA solution, followed by ultrasonic shaking for 60 min and stirring at a temperature of about 100 °C for tens of hours to modify with a polyvinyl alcohol (PVA) polymer (Fig.1c). To remove the liquid phase via vacuum filtration, the remaining PVA-modified black solids were washed repeatedly with acetone and deionized water and dried under a vacuum. The PVA is a biocompatible, water-soluble synthetic polymer with the chemical function of (CH2CHOH)n. Finally, the MWCNTs–PVA samples were soaked in the SBF solution for 14 days to modify the apatite nanostructures (Fig. 1d). The apatite formed in the SBF is a biomimetic apatite which is biocompatible [14,17]. The modified MWCNTs were soaked in a simulated body fluid (SBF) with an ion composition similar to human blood plasma. The SBF solution was also prepared according to the procedure described by Kokubo [17]. The MWCNTs were dispersed in polystyrene bottles containing SBF solution at 37.0 °C in a shaking water bath. The ratio of weight (mg) to solution volume (ml) was 1/10. The soaking solution was not refreshed during soaking of up to 14 days. At that time point, the sample was taken out, the MWCNTs were filtrated and rinsed with deionized water and dried in a vacuum oven at 150 °C for 48 h. Then the modified MWCNTs were subjected to Fourier transform infrared spectroscopy (FT-IR) analysis on an FT-IR Nexus Thermo Nicolet instrument. The modified MWCNTs were also subjected to transmission electronic microscopy (TEM) analysis. The TEM observation was undertaken with a JEOL JEM-2010; the microscopes were operated at 120 keV.
Invitrogen) with 10% fetal calf serum (FCS, PAA), 4500 mg/l glucose (high glucose), GlutaMAX, pyruvate, 1% gentamicin (Ratiopharm) and 0.02% Plasmocin (Invivogen) at 37 °C and in a 5% CO2 atmosphere. 2.4. MTS-assay The inhibitory influence of surface-modified MWCNTs was studied by measuring the osteoblast MG-63's cell metabolism. The CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega) was used. The principle of the test is an enzymatic cleavage of the methyltetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)) (MTS) by active cells into a violet Formosan product. The amount of Formosan product is directly proportional to the number of living cells. Cells were incubated in 500 μl DMEM for 24 h with modified MWCNTs. 100 μl of the MTS solution were added to each well and incubated for 2 h at 37 °C and in a 5% CO2 atmosphere. The spectrophotometric absorption was analyzed in a 96-well plate by an ELISA reader at 490 nm. 2.5. Cell proliferation The MG-63 cells were cultured in 6-well plates (Greiner Bio-One) at 37 °C and in a 5% CO2 atmosphere in the 24 h presence of nanotubes which were placed on culture insert (Thin Cert, pore size 0.4 μm, Greiner Bio-One) in these 6-well plates. For flow cytometry, cells were suspended using 0.05% trypsin/0.02% EDTA solution (5 min at 37 °C). Then cells were washed in phosphate buffered saline (PBS), fixed with 70% ethanol overnight at − 20 °C, and intensively washed again. After treatment with 1% RNase (Sigma) for 20 min at 37 °C, the DNA of the cells was labelled with propidium iodide (50 μg/ml, Sigma) overnight at 4 °C. Cells were measured in a FACSCalibur™ flow cytometer (BD Biosciences) equipped with a 488 nm argon-ion laser and a Macintosh Power PC (G4). In general, 25,000 events were acquired using CellQuest Pro 4.0.1. Cell cycle phases were then calculated in percent using ModFIT version 3.0 (BD Biosciences). 2.6. SEM observation
2.3. Cell culture Human osteoblastic cells of the cell line MG-63 (ATCC-CRL-1427) were cultivated in Dulbecco's modified Eagle medium (DMEM,
The MG-63 cells were cultured on cover glass (Ø 12 mm, Karl Hecht KG) in 6-well plates (Greiner Bio-One) at 37 °C and in a 5% CO2 atmosphere in the presence of nanotubes which were placed on culture
Fig. 1. Schematic illustration of the surface modification and chemical structure of the MWCNTs.
F. Zhang et al. / Materials Science and Engineering C 32 (2012) 1057–1061
inserts (Thin Cert, pore size 0.4 μm, Greiner Bio-One) in these 6-well plates. After 24 h, cells were fixed with 4% glutaraldehyde and dehydrated through a grade series of acetone. After critical point drying (Emitech K850, Emitech) and sputter-coating with gold (SCD 004, BALTEC), the samples were examined using a scanning electron microscope DSM 960A (Carl Zeiss). 2.7. Statistics Statistical analysis was performed with SPSS 14.0 for Windows. The differences between the concentrations were evaluated using Student's t-test for independent samples because variables present normal distribution (Kolmogorov–Smirnov test). Data were expressed as mean and standard error of the mean. A probability value of p b 0.05 was considered significant.
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bands at 3430 cm− 1 and 1635 cm− 1 on the three surfaces are corresponding to the O–H stretching vibration. These bands are also possibly associated with water (O–H from the water). But the bands from the water absorption at 3430 cm− 1 and 1635 cm− 1 are generally broad and weak in the dry samples. In this study, these bands at 3430 cm− 1 and 1635 cm− 1 are sharp and strong, especially for the MWCNT–Apatite sample. This indicates that the O–H stretching for the MWCNT–COOH, –PVA, and MWCNTs–Apatite were mainly from the carboxylic acid or hydroxyapatite (Ca5(PO4)3(OH)) besides the possible water absorption. Fig. 3 shows the TEM micrographs of the MWCNT–Apatite sample. Apatite grown on the CNTs surfaces are found in the sample of MWCNT– Apatite (Fig. 3a). The selected area diffraction (SAED) pattern corresponded to a low-crystalline hydroxyapatite (Fig. 3b). The FT-IR and TEM results confirmed that the apatite was successfully grafted onto the surface of MWCNTs.
3. Results and discussion
3.2. Cytotoxicity of the modified MWCNTs
3.1. Surface modification of the MWCNTs
Fig. 4 shows the metabolic activity of osteoblast cells after a 24 h influence of the surface-modified MWCNTs by MTS assay. The activity
The FT-IR spectra of the MWCNT–COOH, MWCNT–PVA and MWCNT–Apatite samples are shown in Fig. 2. In the spectrum of MWCNT–COOH, the absorption band at 3430 cm− 1 and 1635 cm− 1 correspond to the O–H stretching vibration of carboxylic acid (COOH). The band at 2922 cm− 1 belongs to the C–H stretching and the band at 1390 cm− 1 corresponds to the H bonds. The FR-IR spectrum of the MWCNT–PVA sample shows absorption bands centered at 800, 1151, 1560 cm− 1, which are due to the C–C bonding, C–O–C stretching and C–H bending [18]. The C–O band at 1058 cm− 1 in MWCNT–COOH shifted to 1076 cm− 1 in MWCNTs–PVA. Bond length is shortened resulting in the shift of the peak to a higher wave number after esterification [19]. The intensity of this C–O band in the MWCNT–PVA exhibits more strongly than that of the MWCNT–COOH. The peak for the O–H stretching at 1390 cm− 1 in MWCNT–PVA from the carboxyl acid was significantly reduced in comparison to the MWCNT–COOH. Additionally, a new absorbance band formed at 1022 cm− 1 in the MWCNT–PVA sample, which is attributed to the interfacial covalent reaction between the MWCNT–COOH and PVA [20]. These indicate that the esterification reaction happened in the MWCNT–PVA. The FT-IR result of MWCNT–Apatite shows strong adsorption bands at 3430 and 1635 cm− 1 corresponding to the O–H stretching from the hydroxyapatite. The band at 2922 cm− 1 belongs to the C–H stretching. The adsorption band at 1087 cm− 1 is due to the PO3− 4 group. The adsorption
Fig. 2. FT-IR spectra of the MWCNT–COOH, MWCNT–PVA and MWCNTs–Apatite samples exhibiting the loading of–COOH groups, PVA and apatite onto the MWCNTs.
Fig. 3. TEM micrographs of the MWCNT–Apatite sample (a) with inserted SAED pattern of apatite (b) showing that apatite was modified onto the MWCNTs.
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Fig. 4. Metabolic activity of MG-63 osteoblasts in the presence of modified MWCNTs (n = 10, mean ± SEM, # TCPS significant to modifications; * sample significant to other modifications) exhibiting higher cell viability in the MWCNT–Apatite sample.
of the cells' metabolism correlates with their viability and the cytotoxicity of the sample. The activity of the cells cultured on the tissue culture polystyrene (TCPS) without MWCNTs as control was normalized to 100%. The osteoblasts in the presence of MWCNTs show a significant decrease in their metabolic activity, exhibiting a decrease in cell viability compared to the TCPS control. The MWCNT–COOH and the MWCNT–PVA sample reduced the cell viability to 20.69 ± 4.04% and to 44.55 ± 11.69%, respectively. The MWCNT–Apatite shows 67.23 ± 23.09% activity of cells. After modification in the biomimetic SBF solution with apatite, the MWCNTs exhibit a relatively higher cell viability and lower cytotoxicity in comparison to the other modified MWCNTs. The formation of the apatite in SBF is a bio-mineralization process [21]. The low-crystalline bone-like apatite can provide a suitable environment for osteoblast cell growth [21]. Thus, the MWCNT–Apatite sample shows higher cell viability than that of the MWCNT–COOH and MWCNT–PVA. For bio-applications, the cytotoxicity of the MWCNTs is very important. A cytotoxicity in the range of 20–30% is acceptable for biomedical applications [22]. The surface modification with apatite effectively reduces the cytotoxicity of the CNTs.
3.3. Cell proliferation on the modified MWCNTs Fig. 5 shows the proliferation of osteoblast cells in the presence of the surface-modified MWCNTs using flow cytometric cell cycle analysis. In addition to the MTS-test (Fig. 4), the cell cycle analysis shows that a higher percentage of MG-63 osteoblast cells with surface-modified MWCNTs in the non-proliferating static cell cycle
phase (G0/G1) compared to the TCPS control. There were more cells on MWCNT–COOH (68.79 ± 1.09%) in the static cell cycle phase. The cell proliferation was decreased in general with surface-modified MWCNTs. In the proliferation phase, where cells synthesize DNA (S and G2/M), the percentage of cells influenced by the modified MWCNTs decreased in contrast to the TCPS control (56.83 ± 4.54%). Comparing the surface-modified MWCNTs, there were significantly more proliferating cells in the case of MWCNT–PVA (42.98 ± 0.26%) and MWCNT–Apatite (35.08 ± 2.62%) than with MWCNT–COOH (31.21 ± 1.09%). Generally, the cells show a decrease in cell proliferation and their metabolic activity in response to the modified MWCNTs in comparison to the control. In the proliferation phases (S + G2/M), there were more cells on the MWNT–PVA and MWNT– Apatite. The MWCNT–PVA shows higher osteoblast proliferation but lower viability than those of MWCNT–Apatite. Further studies using in-depth cell experiments are necessary to compare and confirm the cytocompatibility of the MWCNT–PVA and MWCNT–Apatite samples. Generally speaking, the surface modification of MWCNTs with apatite represents an effective enhancement of the cell proliferation properties. 3.4. Cell morphology observation Fig. 6 shows the SEM micrographs of the cell morphology of osteoblasts cultured in the presence of the modified MWCNTs for 24 h. In this, the unmodified MWCNTs were used as a control. The MG-63 osteoblast cells under the influence of the modified MWCNTs show similar sizes and shapes to the control. The modified MWCNTs have no obvious influence on the size and shape of the osteoblast cells since they were not changed. But from the SEM images it can be seen that there were generally more cells in the modified CNTs. Cell adhesion was enhanced, especially on the MWCNT–Apatite. The biocompatible nanoapatite may provide an anchor for portions of the cells, thus improving cell adhesion on the modified CNTs. The increment of cell adhesion on the apatite-modified CNTs will be verified and proved by protein adsorption in the next step research. Zanello et al. [23] reported a dramatic change in cell shapes in ROS 17/2.8 osteoblasts cultured on chemically-modified MWCNTs with poly (m-aminobenzene sulfonic acid) homopolymer (PABS) and poly(ethylene glycol) (PEG). However, in this study the MG-63 osteoblasts were cultured in the presence of COOH, PVA and Apatite-modified MWCNTs in the medium. We investigated the influence of the modified MWCNTs on growing osteoblasts. Here they show similar sizes and shapes with the control grown on unmodified MWCNTs. This may be due to the differences in the modified chemicals (PABS, PEG, PVA and Apatite) and types of osteblast cell lines (Ros 17/2.8 and MG 63). In general, the MWNT–Apatite sustained osteoblast growth, showing an enhanced biocompatibility with MG-63 osteoblast cells. The surface modification of MWCNTs with apatite is an effective enhancement of its biocompatibility. Carbon nanotubes have unique mechanical, electrical and optical properties etc. MWNT–Apatite may have the potential to be used in the field of bone repair as a new tissue engineering scaffold, a reinforcement of biopolymers and bio-cements, and even as a new bone graft material. 4. Conclusions
Fig. 5. Cell proliferation of MG-63 osteoblasts in the presence of modified MWCNTs (n = 3, mean ± SD, # TCPS significant to modifications; * sample significant to other modifications) exhibiting higher proliferation of cells in the MWCNT–PVA and MWCNT–Apatite samples.
MWCNTs were surface-modified chemically with –COOH groups, PVA polymer, and biomimetic apatite by soaking in the SBF. Human osteoblast MG-63 cells were cultured in the presence of these surfacemodified MWCNTs. The surface modification of MWCNTs with biomimetic apatite exhibited significantly increased cell viability, up to 67.23%. In the proliferation phases (S+ G2/M), there were more cells under the influence of MWCNT–Apatite than that of MWCNT–COOH. There were no obvious changes in the cell morphology of osteoblastic MG-63 cells after cultivation with these modified MWCNTs. The surface
F. Zhang et al. / Materials Science and Engineering C 32 (2012) 1057–1061
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Fig. 6. SEM micrographs of MG-63 osteoblasts in the presence of modified MWCNTs (a) unmodified MWCNTs, (b) MWCNT–COOH, (c) MWCNT–PVA and (d) MWCNT–Apatite exhibiting no obvious changes in cell morphology.
modification of MWCNTs with apatite is an effective enhancement of its biocompatibility. References [1] A.V. Liopo, M.P. Stewart, J. Hudson, J.M. Tour, T.C. Pappas, Journal of Nanoscience and Nanotechnology 6 (5) (2006) 1365. [2] W. Yang, P. Thordarson, J.J. Gooding, S.P. Ringer, F. Braet, Nanotechnology 18 (2007) 412001. [3] B. Zhao, H. Hu, S.K. Mandal, R.C. Haddon, Chem. Mater. 17 (12) (2005) 3235. [4] K.A.S. Fernando, Y. Lin, Y.P. Sun, Langmuir 20 (2004) 4777. [5] Z. Huang, J. Li, Q. Chen, H. Wang, Materials Chemistry and Physics 114 (1) (2009) 33. [6] V. Goran, M. Aleksandar, O. Maja, R. Velimir, Č. Miodrag, A. Radoslav, S.U. Petar, Applied Surface Science 255 (18) (2009) 8067. [7] L. Vaisman, G. Marom, H.D. Wagner, Advanced Functional Materials 16 (3) (2005) 357. [8] X. Pei, L. Hu, W. Liu, J. Hao, European Polymer Journal 44 (8) (2008) 2458. [9] H. Kong, P. Luo, C. Gao, D. Yan, Polymer 46 (8) (2005) 2472. [10] L. Zhao, T. Nakayama, H. Tomimoto, Y. Shingaya, Q. Huang, Nanotechnology 20 (2009) 325501.
[11] C. Bartholome, A. Derre, O. Roubeau, C. Zakri, P. Poulin, Nanotechnology 19 (2008) 325501. [12] P. Xue, K.H. Park, X.M. Tao, W. Chen, X.Y. Cheng, Composite Structures 78 (2) (2007) 271. [13] H. Vedala, J. Huang, X.Y. Zhou, G. Kim, S. Roy, W.B. Choi, Applied Surface Science 252 (2006) 7987. [14] S. Aryal, K.C.R. Bahadura, N. Dharmaraj, K.W. Kim, H.Y. Kim, Scripta Materialia 54 (2) (2006) 131. [15] Q. Tan, K. Zhang, S. Gua, J. Ren, Applied Surface Science 255 (15) (2009) 7036. [16] Y. Lin, B. Zhou, K.A.S. Fernando, P. Liu, L.F. Allard, Y.P. Sun, Macromolecules 36 (2003) 7199. [17] T. Kokubo, H. Takadama, Biomaterials 15 (2006) 2907. [18] S. Krimm, C.Y. Liang, G.B.B.M. Sutherland, Journal of Polymer Science 22 (1956) 227. [19] K. Pal, A.K. Banthia, D.K. Majumdar, AAPS PharmSciTech 8 (1) (2007) E1. [20] F. Zeng, Z. Li, Journal of Functional Materials 35 (2004) 2670 (In Chinese). [21] F. Zhang, J. Chang, J. Lu, C. Ning, Materials Science and Engineering: C 8 (2008) 1330. [22] Draft International Standard. Biological evaluation of medical devices—Part 5: Tests for in vitro cytotoxicity (ISO/DIS 10993-5:2007). [23] L.P. Zanello, B. Zhao, H. Hu, R.C. Haddon, Nano Letters 6 (3) (2006) 562.
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
Osteoblast cell response to surface-modified carbon nanotubes Faming Zhang a,⁎, Arne Weidmann b, J. Barbara Nebe b, Eberhard Burkel a a b
Institute of Physics, University of Rostock, 18055 Rostock, Germany Biomedical Research Center, Department of Cell Biology, University of Rostock, 18057 Rostock, Germany
a r t i c l e
i n f o
Article history: Received 26 October 2009 Received in revised form 22 June 2010 Accepted 9 July 2010 Available online 14 July 2010 Keywords: Carbon nanotubes Surface modification Cell response Biomedical applications
a b s t r a c t In order to investigate the interaction of cells with modified multi-walled carbon nanotubes (MWCNTs) for their potential biomedical applications, the MWCNTs were chemically modified with carboxylic acid groups (–COOH), polyvinyl alcohol (PVA) polymer and biomimetic apatite on their surfaces. Additionally, human osteoblast MG-63 cells were cultured in the presence of the surface-modified MWCNTs. The metabolic activities of osteoblastic cells, cell proliferation properties, as well as cell morphology were studied. The surface modification of MWCNTs with biomimetic apatite exhibited a significant increase in the cell viability of osteoblasts, up to 67.23%. In the proliferation phases, there were many more cells in the biomimetic apatite-modified MWCNT samples than in the MWCNTs–COOH. There were no obvious changes in cell morphology in osteoblastic MG-63 cells cultured in the presence of these chemically-modified MWCNTs. The surface modification of MWCNTs with apatite achieves an effective enhancement of their biocompatibility. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Carbon nanotubes (CNTs) have attracted broad attention because of their unique structure and novel properties. In recent years, many research efforts have focused on the exploration of the application of both single-wall (SWCNT) and multi-wall (MWCNT) carbon nanotubes in the biological and biomedical field as nerve cell stimuli, diabetes sensors, cancer therapy, drug delivery carrier and bone scaffold materials etc. [1,2]. Compared with other bone scaffold materials such as polymers and peptide fibers, the high tensile strength, excellent flexibility, and low density of CNTs make them ideal for the production of bone [3]. However, the CNTs have yet to cross many technological hurdles. The lack of solubility of CNTs in aqueous media and the bioinert surfaces have been major technical barriers [2,4]. The recent expansion in methods to chemically modify and functionalize CNTs with water-soluble polymers has made it possible to solubilize and disperse CNTs in water, thus opening the way for their facile manipulation and processing in physiological environments [5,6]. These functional groups, modified and functionalized on the surface of CNTs, could activate their bio-inert surface to attract calcium cations and thereby nucleate and initiate the crystallization of apatite. Many water-soluble polymers have been modified on the surface of CNTs [7–10]. Poly (vinyl alcohol) (PVA) is a type of water-soluble and biocompatible polymer. The PVA/CNT composites and coatings have been studied by a number of researchers [11–13]. Apatite has been functionalized on the modified CNTs by using simulated body fluid soaking [14], G/P solutions [15] or chemical
⁎ Corresponding author. Tel.: + 49 381 4986864; fax: + 49 381 4986862. E-mail addresses: [email protected], [email protected] (F. Zhang). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.07.007
mineralization methods [2]. Now the surface modification and functionalization with water-soluble polymer and apatite is no longer a big scientific problem. Up to the present, however, there have been few publications reporting the cell response to chemically-modified carbon nanotubes. Understanding the interaction of cells with the modified MWCNTs is very important for the potential medical application of CNTs. In this study, based on the surface modification of MWCNTs with PVA and apatite as a potential substrate for bone scaffold applications, the osteoblast cells' response to the surface-modified MWCNTs were studied. The MWCNTs were functionalized using a direct reaction of polyvinyl alcohol (PVA) polymer with carboxylic acid groups on the CNTs by the “graft to” method, based on the procedures of Lin et al. [16]. After this, biomimetic nanostructured apatite was modified on the PVA-modified MWCNTs by simulated body fluid soaking based on the research of Aryal et al. [14]. MG-63 osteoblast cells were cultured in the presence of the MWCNT–COOH, MWCNT–PVA and MWCNT– Apatite samples. The metabolic activity, cell proliferation, and cell morphology of MG-63 osteoblasts in response to the modified MWCNTs were studied.
2. Experimental 2.1. Materials The MWCNTs were obtained from Shenzhen Nanotech Port, Ltd., China. They were produced by catalytic chemical vapor deposition (CCVD) in which CH4 or C2H2 was converted into CNTs at 1000 °C in the presence of Ni and La catalysts. The purity of the MWCNTs as received was higher than 95%.
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2.2. Surface modification The schematic illustration of the surface modification and chemical structure of the MWCNTs is shown in Fig. 1. The starting MWCNTs (Fig. 1a) were refluxed in nitric acid (HNO3) at 120 °C for 24 h to functionalize the carbon nanotubes with carboxylic acid groups (–COOH) (Fig. 1b). After vacuum filtration to remove the liquid phase, the remaining solids were washed repeatedly with deionized water until a neutral pH was achieved, and then dried under vacuum. The resulting carboxylated CNTs were then mixed with a PVA solution, followed by ultrasonic shaking for 60 min and stirring at a temperature of about 100 °C for tens of hours to modify with a polyvinyl alcohol (PVA) polymer (Fig.1c). To remove the liquid phase via vacuum filtration, the remaining PVA-modified black solids were washed repeatedly with acetone and deionized water and dried under a vacuum. The PVA is a biocompatible, water-soluble synthetic polymer with the chemical function of (CH2CHOH)n. Finally, the MWCNTs–PVA samples were soaked in the SBF solution for 14 days to modify the apatite nanostructures (Fig. 1d). The apatite formed in the SBF is a biomimetic apatite which is biocompatible [14,17]. The modified MWCNTs were soaked in a simulated body fluid (SBF) with an ion composition similar to human blood plasma. The SBF solution was also prepared according to the procedure described by Kokubo [17]. The MWCNTs were dispersed in polystyrene bottles containing SBF solution at 37.0 °C in a shaking water bath. The ratio of weight (mg) to solution volume (ml) was 1/10. The soaking solution was not refreshed during soaking of up to 14 days. At that time point, the sample was taken out, the MWCNTs were filtrated and rinsed with deionized water and dried in a vacuum oven at 150 °C for 48 h. Then the modified MWCNTs were subjected to Fourier transform infrared spectroscopy (FT-IR) analysis on an FT-IR Nexus Thermo Nicolet instrument. The modified MWCNTs were also subjected to transmission electronic microscopy (TEM) analysis. The TEM observation was undertaken with a JEOL JEM-2010; the microscopes were operated at 120 keV.
Invitrogen) with 10% fetal calf serum (FCS, PAA), 4500 mg/l glucose (high glucose), GlutaMAX, pyruvate, 1% gentamicin (Ratiopharm) and 0.02% Plasmocin (Invivogen) at 37 °C and in a 5% CO2 atmosphere. 2.4. MTS-assay The inhibitory influence of surface-modified MWCNTs was studied by measuring the osteoblast MG-63's cell metabolism. The CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega) was used. The principle of the test is an enzymatic cleavage of the methyltetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)) (MTS) by active cells into a violet Formosan product. The amount of Formosan product is directly proportional to the number of living cells. Cells were incubated in 500 μl DMEM for 24 h with modified MWCNTs. 100 μl of the MTS solution were added to each well and incubated for 2 h at 37 °C and in a 5% CO2 atmosphere. The spectrophotometric absorption was analyzed in a 96-well plate by an ELISA reader at 490 nm. 2.5. Cell proliferation The MG-63 cells were cultured in 6-well plates (Greiner Bio-One) at 37 °C and in a 5% CO2 atmosphere in the 24 h presence of nanotubes which were placed on culture insert (Thin Cert, pore size 0.4 μm, Greiner Bio-One) in these 6-well plates. For flow cytometry, cells were suspended using 0.05% trypsin/0.02% EDTA solution (5 min at 37 °C). Then cells were washed in phosphate buffered saline (PBS), fixed with 70% ethanol overnight at − 20 °C, and intensively washed again. After treatment with 1% RNase (Sigma) for 20 min at 37 °C, the DNA of the cells was labelled with propidium iodide (50 μg/ml, Sigma) overnight at 4 °C. Cells were measured in a FACSCalibur™ flow cytometer (BD Biosciences) equipped with a 488 nm argon-ion laser and a Macintosh Power PC (G4). In general, 25,000 events were acquired using CellQuest Pro 4.0.1. Cell cycle phases were then calculated in percent using ModFIT version 3.0 (BD Biosciences). 2.6. SEM observation
2.3. Cell culture Human osteoblastic cells of the cell line MG-63 (ATCC-CRL-1427) were cultivated in Dulbecco's modified Eagle medium (DMEM,
The MG-63 cells were cultured on cover glass (Ø 12 mm, Karl Hecht KG) in 6-well plates (Greiner Bio-One) at 37 °C and in a 5% CO2 atmosphere in the presence of nanotubes which were placed on culture
Fig. 1. Schematic illustration of the surface modification and chemical structure of the MWCNTs.
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inserts (Thin Cert, pore size 0.4 μm, Greiner Bio-One) in these 6-well plates. After 24 h, cells were fixed with 4% glutaraldehyde and dehydrated through a grade series of acetone. After critical point drying (Emitech K850, Emitech) and sputter-coating with gold (SCD 004, BALTEC), the samples were examined using a scanning electron microscope DSM 960A (Carl Zeiss). 2.7. Statistics Statistical analysis was performed with SPSS 14.0 for Windows. The differences between the concentrations were evaluated using Student's t-test for independent samples because variables present normal distribution (Kolmogorov–Smirnov test). Data were expressed as mean and standard error of the mean. A probability value of p b 0.05 was considered significant.
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bands at 3430 cm− 1 and 1635 cm− 1 on the three surfaces are corresponding to the O–H stretching vibration. These bands are also possibly associated with water (O–H from the water). But the bands from the water absorption at 3430 cm− 1 and 1635 cm− 1 are generally broad and weak in the dry samples. In this study, these bands at 3430 cm− 1 and 1635 cm− 1 are sharp and strong, especially for the MWCNT–Apatite sample. This indicates that the O–H stretching for the MWCNT–COOH, –PVA, and MWCNTs–Apatite were mainly from the carboxylic acid or hydroxyapatite (Ca5(PO4)3(OH)) besides the possible water absorption. Fig. 3 shows the TEM micrographs of the MWCNT–Apatite sample. Apatite grown on the CNTs surfaces are found in the sample of MWCNT– Apatite (Fig. 3a). The selected area diffraction (SAED) pattern corresponded to a low-crystalline hydroxyapatite (Fig. 3b). The FT-IR and TEM results confirmed that the apatite was successfully grafted onto the surface of MWCNTs.
3. Results and discussion
3.2. Cytotoxicity of the modified MWCNTs
3.1. Surface modification of the MWCNTs
Fig. 4 shows the metabolic activity of osteoblast cells after a 24 h influence of the surface-modified MWCNTs by MTS assay. The activity
The FT-IR spectra of the MWCNT–COOH, MWCNT–PVA and MWCNT–Apatite samples are shown in Fig. 2. In the spectrum of MWCNT–COOH, the absorption band at 3430 cm− 1 and 1635 cm− 1 correspond to the O–H stretching vibration of carboxylic acid (COOH). The band at 2922 cm− 1 belongs to the C–H stretching and the band at 1390 cm− 1 corresponds to the H bonds. The FR-IR spectrum of the MWCNT–PVA sample shows absorption bands centered at 800, 1151, 1560 cm− 1, which are due to the C–C bonding, C–O–C stretching and C–H bending [18]. The C–O band at 1058 cm− 1 in MWCNT–COOH shifted to 1076 cm− 1 in MWCNTs–PVA. Bond length is shortened resulting in the shift of the peak to a higher wave number after esterification [19]. The intensity of this C–O band in the MWCNT–PVA exhibits more strongly than that of the MWCNT–COOH. The peak for the O–H stretching at 1390 cm− 1 in MWCNT–PVA from the carboxyl acid was significantly reduced in comparison to the MWCNT–COOH. Additionally, a new absorbance band formed at 1022 cm− 1 in the MWCNT–PVA sample, which is attributed to the interfacial covalent reaction between the MWCNT–COOH and PVA [20]. These indicate that the esterification reaction happened in the MWCNT–PVA. The FT-IR result of MWCNT–Apatite shows strong adsorption bands at 3430 and 1635 cm− 1 corresponding to the O–H stretching from the hydroxyapatite. The band at 2922 cm− 1 belongs to the C–H stretching. The adsorption band at 1087 cm− 1 is due to the PO3− 4 group. The adsorption
Fig. 2. FT-IR spectra of the MWCNT–COOH, MWCNT–PVA and MWCNTs–Apatite samples exhibiting the loading of–COOH groups, PVA and apatite onto the MWCNTs.
Fig. 3. TEM micrographs of the MWCNT–Apatite sample (a) with inserted SAED pattern of apatite (b) showing that apatite was modified onto the MWCNTs.
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Fig. 4. Metabolic activity of MG-63 osteoblasts in the presence of modified MWCNTs (n = 10, mean ± SEM, # TCPS significant to modifications; * sample significant to other modifications) exhibiting higher cell viability in the MWCNT–Apatite sample.
of the cells' metabolism correlates with their viability and the cytotoxicity of the sample. The activity of the cells cultured on the tissue culture polystyrene (TCPS) without MWCNTs as control was normalized to 100%. The osteoblasts in the presence of MWCNTs show a significant decrease in their metabolic activity, exhibiting a decrease in cell viability compared to the TCPS control. The MWCNT–COOH and the MWCNT–PVA sample reduced the cell viability to 20.69 ± 4.04% and to 44.55 ± 11.69%, respectively. The MWCNT–Apatite shows 67.23 ± 23.09% activity of cells. After modification in the biomimetic SBF solution with apatite, the MWCNTs exhibit a relatively higher cell viability and lower cytotoxicity in comparison to the other modified MWCNTs. The formation of the apatite in SBF is a bio-mineralization process [21]. The low-crystalline bone-like apatite can provide a suitable environment for osteoblast cell growth [21]. Thus, the MWCNT–Apatite sample shows higher cell viability than that of the MWCNT–COOH and MWCNT–PVA. For bio-applications, the cytotoxicity of the MWCNTs is very important. A cytotoxicity in the range of 20–30% is acceptable for biomedical applications [22]. The surface modification with apatite effectively reduces the cytotoxicity of the CNTs.
3.3. Cell proliferation on the modified MWCNTs Fig. 5 shows the proliferation of osteoblast cells in the presence of the surface-modified MWCNTs using flow cytometric cell cycle analysis. In addition to the MTS-test (Fig. 4), the cell cycle analysis shows that a higher percentage of MG-63 osteoblast cells with surface-modified MWCNTs in the non-proliferating static cell cycle
phase (G0/G1) compared to the TCPS control. There were more cells on MWCNT–COOH (68.79 ± 1.09%) in the static cell cycle phase. The cell proliferation was decreased in general with surface-modified MWCNTs. In the proliferation phase, where cells synthesize DNA (S and G2/M), the percentage of cells influenced by the modified MWCNTs decreased in contrast to the TCPS control (56.83 ± 4.54%). Comparing the surface-modified MWCNTs, there were significantly more proliferating cells in the case of MWCNT–PVA (42.98 ± 0.26%) and MWCNT–Apatite (35.08 ± 2.62%) than with MWCNT–COOH (31.21 ± 1.09%). Generally, the cells show a decrease in cell proliferation and their metabolic activity in response to the modified MWCNTs in comparison to the control. In the proliferation phases (S + G2/M), there were more cells on the MWNT–PVA and MWNT– Apatite. The MWCNT–PVA shows higher osteoblast proliferation but lower viability than those of MWCNT–Apatite. Further studies using in-depth cell experiments are necessary to compare and confirm the cytocompatibility of the MWCNT–PVA and MWCNT–Apatite samples. Generally speaking, the surface modification of MWCNTs with apatite represents an effective enhancement of the cell proliferation properties. 3.4. Cell morphology observation Fig. 6 shows the SEM micrographs of the cell morphology of osteoblasts cultured in the presence of the modified MWCNTs for 24 h. In this, the unmodified MWCNTs were used as a control. The MG-63 osteoblast cells under the influence of the modified MWCNTs show similar sizes and shapes to the control. The modified MWCNTs have no obvious influence on the size and shape of the osteoblast cells since they were not changed. But from the SEM images it can be seen that there were generally more cells in the modified CNTs. Cell adhesion was enhanced, especially on the MWCNT–Apatite. The biocompatible nanoapatite may provide an anchor for portions of the cells, thus improving cell adhesion on the modified CNTs. The increment of cell adhesion on the apatite-modified CNTs will be verified and proved by protein adsorption in the next step research. Zanello et al. [23] reported a dramatic change in cell shapes in ROS 17/2.8 osteoblasts cultured on chemically-modified MWCNTs with poly (m-aminobenzene sulfonic acid) homopolymer (PABS) and poly(ethylene glycol) (PEG). However, in this study the MG-63 osteoblasts were cultured in the presence of COOH, PVA and Apatite-modified MWCNTs in the medium. We investigated the influence of the modified MWCNTs on growing osteoblasts. Here they show similar sizes and shapes with the control grown on unmodified MWCNTs. This may be due to the differences in the modified chemicals (PABS, PEG, PVA and Apatite) and types of osteblast cell lines (Ros 17/2.8 and MG 63). In general, the MWNT–Apatite sustained osteoblast growth, showing an enhanced biocompatibility with MG-63 osteoblast cells. The surface modification of MWCNTs with apatite is an effective enhancement of its biocompatibility. Carbon nanotubes have unique mechanical, electrical and optical properties etc. MWNT–Apatite may have the potential to be used in the field of bone repair as a new tissue engineering scaffold, a reinforcement of biopolymers and bio-cements, and even as a new bone graft material. 4. Conclusions
Fig. 5. Cell proliferation of MG-63 osteoblasts in the presence of modified MWCNTs (n = 3, mean ± SD, # TCPS significant to modifications; * sample significant to other modifications) exhibiting higher proliferation of cells in the MWCNT–PVA and MWCNT–Apatite samples.
MWCNTs were surface-modified chemically with –COOH groups, PVA polymer, and biomimetic apatite by soaking in the SBF. Human osteoblast MG-63 cells were cultured in the presence of these surfacemodified MWCNTs. The surface modification of MWCNTs with biomimetic apatite exhibited significantly increased cell viability, up to 67.23%. In the proliferation phases (S+ G2/M), there were more cells under the influence of MWCNT–Apatite than that of MWCNT–COOH. There were no obvious changes in the cell morphology of osteoblastic MG-63 cells after cultivation with these modified MWCNTs. The surface
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Fig. 6. SEM micrographs of MG-63 osteoblasts in the presence of modified MWCNTs (a) unmodified MWCNTs, (b) MWCNT–COOH, (c) MWCNT–PVA and (d) MWCNT–Apatite exhibiting no obvious changes in cell morphology.
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