5 g/L sodium bicarbonate, 0 1 mM non-essential amino acids, and 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 selleck chemical adhesion assay and 1 × 104 cells/cm2 for 1 week for cell proliferation assay, respectively. Cell adhesion 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 NVP-BSK805 cell line fluorescent microscope to examine the cell adhesion morphology and selleck chemicals 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). Cell proliferation 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 during 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 Figure 1a,b,c,d shows the SEM micrographs of as-anodized TiO2 nanotubes with the diameters of 10, 25, 50, and 100 nm produced by electrochemical anodization at the applied voltages of 5, 10, 20, and 40 V, respectively.

This entry was posted in Antibody. Bookmark the permalink.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>