Diameter-sensitive biocompatibility of anodic TiO2 nanotubes treated with supercritical CO2 fluid
© Lan et al.; licensee Springer. 2013
Received: 13 February 2013
Accepted: 21 March 2013
Published: 2 April 2013
This work reports on the diameter-sensitive biocompatibility of anodic TiO2 nanotubes with different nanotube diameters grown by a self-ordering process and subsequently treated with supercritical CO2 (ScCO2) fluid. We find that highly hydrophilic as-grown TiO2 nanotubes become hydrophobic after the ScCO2 treatment but can effectively recover their surface wettability under UV light irradiation as a result of photo-oxidation of C-H functional groups formed on the nanotube surface. It is demonstrated that human fibroblast cells show more obvious diameter-specific behavior on the ScCO2-treated TiO2 nanotubes than on the as-grown ones in the range of diameters of 15 to 100 nm. This result can be attributed to the removal of disordered Ti(OH)4 precipitates from the nanotube surface by the ScCO2 fluid, thus resulting in purer nanotube topography and stronger diameter dependence of cell activity. Furthermore, for the smallest diameter of 15 nm, ScCO2-treated TiO2 nanotubes reveal higher biocompatibility than the as-grown sample.
Titanium (Ti) and its alloys have been widely used for dental and orthopedic implants because of their favorable mechanical properties, superior corrosion resistance, and good biocompatibility [1–3]. When exposed to the atmosphere, the Ti metal spontaneously forms a thin, dense, and protective oxide layer (mainly TiO2, approximately 10 nm thick) on its surface, which acts like a ceramic with superior biocompatibility. When the Ti implant is inserted into the human body, the surrounding tissues directly contact the TiO2 layer on the implant surface. Therefore, the surface characteristics of the TiO2 layer determine the biocompatibility of Ti-based implants. Earlier studies primarily investigated the influence of surface topography of implants on cell behaviors at the micrometer scale [4–6]. Recently, the interaction of nanometric scale surface topography, especially in the sub-100-nm region, with cells has been recognized as an increasingly important factor for tissue acceptance and cell survival [7–9]. Various nanotopography modifications have been proposed to enhance the cell responses to the Ti-based implants. For example, TiO2 nanowire scaffolds fabricated by hydrothermal reaction of alkali with the Ti metal, mimicking the natural extracellular matrix in structure, can promote the adhesion and proliferation of mesenchymal stem cells (MSCs) on Ti implants . Chiang et al. also proposed that a TiO2 multilayer nanonetwork causes better MSC adhesion and spreading, as well as faster cell proliferation and initial differentiation .
In the recent years, self-organized TiO2 nanotubes fabricated by electrochemical anodization of pure Ti foils have attracted considerable interest owing to their broad applications in photocatalysis , dye-sensitized solar cells , and biomedical field [14, 15]. A major advantage of anodic oxidation is the feasibility to well control the diameter and shape of the nanotubular arrays to the desired length scale, meeting the demands of a specific application by precisely controlling the anodization parameters. In a number of studies on the cell response to TiO2 nanotubes, nanosize effects have been demonstrated for a variety of cells [16–18]. Park et al. reported that vitality, proliferation, migration, and differentiation of MSCs and hematopoietic stem cells, as well as the behavior of osteoblasts and osteoclasts, are strongly influenced by the nanoscale TiO2 surface topography with a specific response to nanotube diameters between 15 and 100 nm . Furthermore, even if the surface chemistry of the nanotubes is completely modified with a dense alloy coating onto the original nanotube layers, the nanosize effects still prevail . In other words, the cell vitality has an extremely close relationship with the geometric factors of nanotube openings.
On the other hand, using supercritical CO2 (ScCO2) as a solvent has shown many advantages when chemically cleaning or modifying the surface of materials. The high diffusivity and low surface tension of ScCO2 enable reagents to access the interparticle regions of powders, buried interfaces, or even nanoporous structures that cannot be reached using conventional solution or gaseous treatment methods [21, 22]. Recent studies have shown that ScCO2 is an effective alternative for terminal sterilization of medical devices . It was also reported that the contact of ScCO2 with P25 TiO2 (70% anatase and 30% rutile), which contains both anatase and rutile phases, leads to the formation of a variety of functional groups and substantially modifies the surface chemistry . Moreover, the ScCO2 drying technique has been proven to effectively reduce intertube contacts and to produce bundle-free and crack-free TiO2 nanotube films . The aim of this study is to gain an understanding of the influence of ScCO2 on surface topography and chemistry of anodic TiO2 nanotubes and also to study the diameter-specific biocompatibility of these ScCO2-treated TiO2 nanotubes with human fibroblast cells. The human fibroblast cell behavior, including cell adhesion, proliferation, and survival, in response to the diameter of TiO2 nanotubes is investigated.
Preparation of ScCO2-treated TiO2 nanotubes
Self-organized TiO2 nanotubes were prepared by electrochemical anodization of Ti foils (thickness of 0.127 mm, 99.7% purity, ECHO Chemical Co. Ltd., Miaoli, Taiwan). A two-electrode electrochemical cell with Ti anode and Pt as counter electrode was used. All anodization experiments were carried out in ethylene glycol electrolytes containing 0.5 wt.% NH4F at 20°C for 90 min. All electrolytes were prepared from reagent-grade chemicals and deionized water. Anodization voltages applied were between 10 and 40 V, and resulted in nanotube diameters ranging from 15 up to 100 nm. The TiO2 nanotubes with the diameter of 100 nm annealed at 400°C for 2 h were also prepared as the reference sample. After the electrochemical process, the nanotube samples were cleaned ultrasonically with deionized water for 1 h to remove the residual by-products on the surface. Subsequently, ScCO2 fluid (99.9% purity) was utilized to treat the nanotubes at the temperature of 53°C and in the pressure of 100 bar for 2 h. For the in vitro experiments, low-intensity UV light irradiation (<2 mW/cm2) was performed on all nanotube samples using fluorescent black-light bulbs for 8 h.
Field emission scanning electron microscopy (FE-SEM; FEI Quanta 200 F, FEI, Hillsboro, OR, USA) was employed for the morphological characterization of the TiO2 nanotube samples. X-ray diffraction (XRD) was utilized to determine the phase of the TiO2 nanotubes. The surface wettability of materials was evaluated by measuring the contact angle between the TiO2 nanotubes and water droplets in the dark. Contact angle measurements were performed at room temperature by the extension method, using a horizontal microscope with a protractor eyepiece. In addition, in order to investigate the functional groups possibly formed during the ScCO2 process, X-ray photoelectron spectroscopy (XPS) was employed to analyze the carbon spectra (in terms of C 1s) on the nanotube surfaces.
MRC-5 human fibroblasts were received from the Bioresource Collection and Research Center, Taiwan. Cells were plated in a 10-cm tissue culture plate and cultured with Eagle's minimum essential medium (Gibco, Life Technologies Corporation, Grand Island, NY, USA) containing 10% fetal bovine serum, 2 mM l-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2. Cells were then seeded onto the autoclaved titanium samples placed in a 12-well culture plate (Falcon, BD Biosciences, San Jose, CA, USA) at a density of 5 × 103 cells/cm2 for 3 days for cell adhesion assay and 1 × 104 cells/cm2 for 1 week for cell proliferation assay, respectively.
For cell adhesion experiments, 3 days after cell plating, non-adherent cells were washed with phosphate-buffered saline (PBS). The adherent cells were fixed in 4% paraformaldehyde (USB Corp., Cleveland, OH, USA) for 1 h at room temperature and washed with PBS. After fixation, the cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich Corporation, St. Louis, MO, USA) in PBS for 15 min at 4°C. Cells were then washed with PBS and incubated with rhodamine phalloidin (Life Technologies Corporation, Grand Island, NY, USA) for 15 min for actin filament stain and with diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific Inc., Waltham, MA, USA) for 5 min for nuclei stain. The images of the stained fibroblasts were taken using a fluorescent microscope to examine the cell adhesion morphology and cytoskeletal arrangement. For SEM observation, cells were fixed with 2.5% glutaraldehyde solution (Merck & Co., Inc., Whitehouse Station, NJ, USA) for 1 h at room temperature. Samples were rinsed in PBS solution twice, dehydrated in a series of ethanol (40%, 50%, 60%, 70%, 80%, 90%, and 100%) and critical point dried with a critical point dryer (CPD 030, Leica Microsystems, Wetzlar, Germany).
Additional cell proliferation was quantified 1 week after cell plating at a density of 1 × 104 cells/cm2 using cell proliferation reagent WST-1 (Roche, Woerden, Netherlands) according to the manufacturer's instructions. On the 7th day, cells on the nanotubes were washed with PBS twice. The cells were incubated with a medium containing 10% WST-1 cell proliferation reagent at 37°C in a humidified atmosphere of 5% CO2 for 2 h. The solution was then retrieved from each well to a 96-well plate, and optical densities were measured using a spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland) at 450 nm. All experiments were carried out in triplicate, and at least three independent experiments were performed. Data were presented as mean ± standard deviation and analyzed by analysis of variances using SPSS 12.0 software (SPSS Inc., Chicago, IL, USA). A p value of <0.05 was considered statistically significant.
Results and discussion
In conclusion, this study investigates the diameter-sensitive biocompatibility of ScCO2-treated TiO2 nanotubes of different diameters prepared by electrochemical anodization. We find that ScCO2-treated TiO2 nanotubes can effectively recover their surface wettability under UV light irradiation as a result of photo-oxidation of C-H functional groups formed on the surface. It is demonstrated that human fibroblast cells show more obvious diameter-specific behavior on the ScCO2-treated nanotubes than on the as-grown ones, which can be attributed to the removal of disordered Ti(OH)4 precipitates from the nanotube surface by the ScCO2 fluid. This results in purer nanotube topography, stronger diameter dependence of cell activity, and thus higher biocompatibility for the 15-nm-diameter ScCO2-treated TiO2 nanotubes than the as-grown sample. This study demonstrates that the use of ScCO2 fluid can be an effective, appropriate, and promising approach for surface treatments or modifications of bio-implants.
MYL is currently a visiting staff of the Department of Otolaryngology at Taipei Veterans General Hospital and also a Ph.D. candidate of National Yang-Ming University (Taiwan). CPL is currently a Master's degree student of National Central University (Taiwan). HHH is a professor of the Department of Dentistry at National Yang-Ming University (Taiwan). JKC is an assistant professor of the Institute of Materials Science and Engineering at National Central University (Taiwan). SWL is an associate professor of the Institute of Materials Science and Engineering at National Central University (Taiwan).
The research is supported by the Veterans General Hospitals University System of Taiwan Joint Research Program under contract nos. VGHUST101-G4-3-1 and VGHUST101-G4-3-2 and by the National Science Council of Taiwan under contract no. NSC-100-2221-E-008-016-MY3. The authors also thank the Center for Nano Science and Technology at National Central University and Clinical Research Core Laboratory at Taipei Veterans General Hospital for the facility support.
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