NANO LETTERS
Fabrication and Biocompatibility of Carbon Nanotube-Based 3D Networks as Scaffolds for Cell Seeding and Growth
2004 Vol. 4, No. 11 2233-2236
Miguel A. Correa-Duarte,† Nicholas Wagner,‡,§ Jose´ Rojas-Chapana,† Christian Morsczeck,‡ Michael Thie,‡ and Michael Giersig*,† Center of AdVanced European Studies and Research (Caesar) Received September 1, 2004; Revised Manuscript Received October 1, 2004
ABSTRACT Thin film networks of multiwalled carbon nanotubes (MWCNTs) were prepared by exerting chemically induced capillary forces upon the nanotubes. During this process MWCNTs undergo a transformation from being a vertically aligned structure to an interlocking resistive network of interconnected nanotubes, whose main feature is a regular three-dimensional (3D) sieve architecture. Due to their structural characteristics at the nanoscale level, 3D-MWCNT-based networks are in principle ideal candidates for scaffolds/matrices in tissue engineering. Their potential application in this field was confirmed by extensive growth, spreading, and adhesion of the common mouse fibroblast cell line L929.
Carbon structures have been a subject of extensive research since the discovery of fullerenes1 and carbon nanotubes2 (CNTs) because of their unique structure-dependent electrical and mechanical properties. In recent years, considerable efforts have been made to fabricate different carbon morphologies and explore new applications in different fields including, among others, composites,2-4 sensors,5 electrochemical,6 field emission,7-12 and nanoscale electronic devices.13-17 Furthermore, there has been a tremendous interest in applying the properties of CNTs for promising biological applications.18 Several studies have been done of interactions between carbon nanotubes and living mammalian cells. Webster et al.19 describe the cellular response of neuron and osteoblast cells to composites made up of carbon nanofibers as “fillers in polycarbonate urethane substrates”. According to the authors, the cell response to the composite may lead to successful integration of neural and bone tissue implants. Khan20 performed a study to evaluate the feasibility of CNTbased composites for cartilage regeneration and in vitro cell proliferation of chondrocytes. Supronowicz et al.21 reported the application of nanocomposites consisting of blends of polylactic acid and CNTs that can be used to expose cells to electrical stimulation, and Mattson22 showed how the conductivity of CNTs coated with bioactive molecules can help neuron growth on nanotubes. Also, the effect of charge * Corresponding author. † Nanoparticle Technology Division. ‡ Dental Cell Biology Division. § Fachhochschule Bonn-Rhein-Sieg, Rheinbach/St. Augustin. 10.1021/nl048574f CCC: $27.50 Published on Web 10/13/2004
© 2004 American Chemical Society
manipulation on functionalized CNTs vs neuronal growth was reported.23 Many interesting applications for cell growth and tissue modeling can be envisioned if carbon nanotubes are structured in two (2D) and three (3D) architectures of interconnected cavities. Although the above-mentioned studies19-23 have investigated the cellular response to carbon nanofibers/ nanotubes including dose-dependent effect24 cell proliferation on aligned carbon nanofibers,25 intracellular transport26 and cytotoxicity,27 2D and 3D scaffolds of CNTs with periodic and biomimetic internal architecture to support biocompatible cell growth have not been widely considered to date. The MWCNTs are not only mechanically strong and electrically conductive, but they are also capable of being shaped into 3D architectures. They could be ideal for cell seeding and in vitro cell modeling, leading to the design of promising new tissue engineered products in biological applications. This study reports growth of mouse fibroblast cell line L929 on a 3D network based on an array of interconnected MWCNTs. The regular pattern of cavities, adjustable to the application and the different shapes and sizes of cells used, considerably contributes to the adhesion and growth of these cells. The general procedure of the experimental design is outlined in Figure 1. The process starts from perpendicularly aligned MWCNTs, which were provided by Nanolab Inc. and were grown by chemical vapor deposition (PCVD)28 on a silicon substrate using Ni as a catalyst. The substrate with the aligned MWCNTs was functionalized in an acid solution (12 h in a
Figure 1. Representation of the general method used to fabricate 3D MWCNT-based network and its application as a scaffold for cell growth. (A) MWCNTs perpendicularly aligned to the substrate. (B) The latter were chemically treated to obtain a cross-linked 3D structure. (C) The 3D network, like a scaffold, favors the cell growth.
Figure 3. Examination by SEM of the MWCNT-based walls. (A) 3D networks of 50 µm length carbon nanotubes. (B) High magnification image of intercrossed carbon nanotubes in the walls of the cavities.
Figure 2. Examination by SEM of the different MWCNT-based structures. (A) Perpendicular aligned carbon nanotubes. (B) The latter after a physicochemical treatment forming pyramid-like structure with basal planes of ca. 3 µm. (C) Network of crosslinked carbon nanotube walls forming cavities.
nitric/sulfuric (1:3) acid solution) by means of an oxidation process, which generates carboxylic groups at the ends and in the defects of the sidewalls of the MWCNTs. At the same time, parallel to the functionalization, the acid solution creates capillary and tensile forces, which are involved in creating the 3D assembly, as was previously reported by Liu et al.29 Thus, the results show that by treating the MWCNT substrate with the acid solution, this will lead to capillary and tensile forces between the aligned tubes, which spread the solution over the whole silicon substrate in such a way that all the interspaces among the aligned tubes become soaked. 2234
The latter process generates a hydrostatic dilation stress to the solution, which is larger at higher densities, leading to the flattening of the nanotubes. When nanotubes collapsing from opposite directions meet between two regions of lower density, a 3D structure is formed, composed of honeycomblike polygons with walls perpendicular to the substrate. Subsequently, a cell seeding onto the scaffold was performed, depositing fibroblasts onto the surface of the 3D structure. Figure 2 shows SEM images of aligned carbon nanotubes 50 µm in length (Figure 2A) bonded to a silicon substrate which form a 3D scaffold after functionalization treatment. Depending on the length of the aligned MWCNTs, it is possible to modulate their spatial distribution. Thus, this technique can be used to create pyramid-like structures (Figure 2B) or interconnected cavities with a volume large enough to harbor a specific cell type under investigation (Figure 2C). Cavities varying between ∼5 and 60 µm and ∼5-15 µm diameter were obtained by modeling MWCNTs of 50 and 35 µm in length, respectively. High-resolution SEM image of the walls of the CNTs network (Figure 3A) shows the large surface area available for cell attachment (Figure 3B). In addition, the cross-linking of MWCNTs should improve the mechanical properties of the network. To investigate the response of, e.g., connective tissue cells on a 3D network of MWCNTs as introduced above, the Nano Lett., Vol. 4, No. 11, 2004
Figure 4. Scanning electron micrographs of L929 mouse fibroblasts growing on MWCNT-based network. (A,B) After 1 day. (C,D) After 7 days.
common mouse fibroblast cell line L929 (ATCC Cat. No.: CCL-1; Rockville, MD) was used. For experiments, the scaffolds were first sterilized with UV light for 30 min. Subsequently, a MWCNT scaffold (∼0.5 cm2) was placed into a six-well plate (Corning, Netherlands), and 1 × 105 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin, in a humidified atmosphere containing 5% CO2 for 1 day or 7 days, respectively. (DMEM and all supplements were obtained by Biochrom, Germany.) Cell growth onto the MWCNT-based networks and cell morphology were determined using SEM. Samples were washed with Earle’s buffered saline solution (Biochrom, Germany) and fixed in 2.5% glutardialdehyde (Roth, Germany) in 0.1 mol/L cacodylate buffer, pH 7.4, for 2 h at 4 °C. After repeated washing in buffer, samples were dehydrated in ethanol and dried using hexamethyldisilazane (Polysciences, Germany) as intermedium. A 7 nm gold layer (0.35 nm/s) was sputtered onto the samples prior to analysis in the LEO Supra 55 SEM (Zeiss, Germany). Figure 4 shows the specific morphology of L929 cells on the MWCNT-based network. After 1 day, most of the L929 cells had adhered to the MWCNT surface (Figure 4A,B). Isolated large stretched cells were found with elongated cytoplasm projections attaching to the walls of the cavities (Figure 4B). Additionally, cells appeared to attach to the bottom of the cavities by shorter projections arising from their lower surface (arrows, magnified inset Figure 4B). After Nano Lett., Vol. 4, No. 11, 2004
7 days of growth, fibroblasts had formed a confluent layer covering the surface of the network (Figure 4C,D), i.e., most of the surface of the MWCNT-based network was hidden beneath the cell layer (Figure 4C). The vitality of L929 cells when cultured in the presence of MWCNTs was essentially the same when compared to controls using [3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazoliumbromide] (MTT) reduction assay.30,31 Furthermore, the MWCNTs did not exhibit cytotoxic activity against the L 929 cells used (data not shown). Though the MWCNTs possess strong bonding properties that are ideal for forming biocompatible composites, studies were aimed at testing the cell compatibility of MWCNTs in 3D architectures without any additional modification. Collectively, these results support our hypothesis that MWCNTbased networks with regular 3D structures are highly biocompatible and provide a good basis to stimulate robust tissue formation. Moreover, these results show that 3D structuring via cross-linking of MWCNTs is a key factor to successfully using MWCNT-based scaffolds for tissue engineering. The objective of this study was to characterize the MWCNT-based networks in terms of architecture, surface nanotexture, and chemistry as supportive 3D matrices for cell seeding and growth. It was shown that 3D MWCNTbased networks can be fabricated with a defined number, size, spacing, and degree of periodicity of cavities, which together with cell compatibility are important design criteria for tissue engineering scaffolds. Finally, the architecture 2235
designs can be optimized such that 3D scaffold meets functional, mechanical, and mass transport requirements. Kotov et al.32 recently pointed out the importance of synthetic 3D scaffolds having regular geometric interconnected cavities. Scaffolds seeded with cells should not only match tissue morphology but also allow systematic studies of the cell interaction and motility during tissue generation.32,33 Although not bioresorbable, MWCNT-based 3D networks were shown to support the cell attachment and growth of mouse fibroblasts. Unlike synthetic polymers,33 MWCNTs as nonresorbable materials do not present toxic byproducts which raise concerns that the scaffold microenvironment may not be ideal for tissue growth. Furthermore, MWCNT-based 3D networks possess structural integrity and stability to retain shape in vivo, with strong mechanical strength to support developing tissue and withstand in vivo forces. As described in previous works,19-23,34 they could serve as biocompatible matrices in situations where the MWCNTs can be used to restore, maintain, or reinforce damaged or weakened tissues, or where MWCNTs can act as drug delivery devices, as well as in situations where the bioresorption process itself causes damage or injury. Acknowledgment. We thank Nanolab. Inc (www.nanolab.com) for kindly supplying the MWCNTs.
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Nano Lett., Vol. 4, No. 11, 2004
