Material Preparation
Two kinds of titanium disks (diameter = 5.8 and 31 mm respectively, thickness = 3 mm) were obtained from pure commercial titanium. The Ti disks were mechanically polished and cleaned in acetone, alcohol, and deionized water in sequence and dried in air.
The as-deposited films were fabricated in the laboratory of Shanghai Institute of Ceramics using a filtered cathodic arc system [22]. The samples were processed with deposition using filtered Ti cathodic arc plasma sources for TiO2 films and Zr cathodic arc plasma sources for ZrO2 films in oxygen atmosphere. In deposition, the pulse duration of cathodic current was 2,000 μs and the frequency was 70 Hz. The direct current voltage of 50 V and a bias of −450 V were superimposed to the sample during deposition. The working pressure was 9 × 10−3 Pa and the deposition time was 60 min. After the deposition treatment, the samples were washed with deionized water and dried in air.
The surface morphology of the films was observed by scanning electron microscopy (SEM, FEI-QUANTA 200-FEG, FEI, American) and atomic force microscopy (AFM, SPI3800N, SEIKO, Japan) (The surface of the titanium disks used as the substrate of the films was too rough to meet the demand of AFM observation. So we deposited TiO2 and ZrO2 films on silicon wafers instead for AFM observation.). The phase of films was identified by thin film X-ray diffraction (TF-XRD, D/MAX-2550, Rigaku, Japan) using a Cu Ká radiation source (1.5148Å) at 40 kV and 100 mA with a glancing angle fixed at 1°. The elemental composition of the films was determined using x-ray photoelectron spectroscopy (XPS, MicroLab 310-F) with monochromatic Al Kα radiation.
In Vitro Cell Culture
Human osteosarcoma cell line MG63 was used in this work. MG-63 cells were cultured in DMEM medium (supplemented with 10 % fetal calf serum, FCS, Eurobio) at 37 °C in a moist 5 % CO2 atmosphere. The culture medium was replaced every 3 days. After reaching confluence, the cells were released by a trypsin–EDTA solution (0.5 g/L trypsin and 0.2 g/L EDTA, Gibco) and transferred into a new tissue culture flask.
Cell Morphology
Samples with 5.8 mm in diameter were placed in 96-well plates. 5 × 103 cells were seeded on each sample and cultured under standard cell culture condition for 24 h. Then samples were washed with Phosphate buffer saline (PBS) and fixed with 2.5 % glutaraldehyde buffered by PBS for 2 h at room temperature (RT). They were then successively dehydrated in graded alcohols (30, 50, 70, 90, and 100 %), critical point-dried, sputter-coated with gold and examined using SEM (SL-30, Philips, Holland).
Counting Kit-8 Assay
Cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8, Dojindo, Japan). Samples with 5.8 mm in diameter were placed in 96-well plates. 1 × 104 cells were dispensed on each sample and incubated for 1, 4, 7, 10 days. At predetermined time points, samples were washed three times with PBS to eliminate non-viable cells. The cells on the samples were incubated with 10 μl of CCK-8 solution for 3 h in the incubator. Then the optical density was measured using a microplate reader at a wavelength of 450 nm. Three samples were tested in each group for each incubation time and the resulting absorbance for each of the samples was averaged. The experiment was run in triplicate.
Alkaline Phosphatase Activity Assay
Alkaline phosphates (ALP) activity assay was carried out from the culture supernatant. Cells were cultured on ZrO2 and TiO2 thin films as well as Ti disks as mentioned in cell proliferation assay. After incubated for 1, 4, 7 and 10 days, the supernatant was collected and assayed for ALP activity immediately using a commercial kit (Jiancheng Technology, Nanjing, China) according to the manufacturer’s instructions. Three samples were tested in each group for each incubation time and the ALP activity for each of the samples was averaged. The experiment was run in triplicate.
Type I Collagen Fluorescence Immunostain
Samples with 5.8 mm in diameter were placed in 96-well plates. 5 × 103 cells were seeded on each sample and cultured under standard cell culture condition for 4 days. The cells were fixed with 4 % paraformaldehyde for 10 min, washed three times with PBS, permeabilized with 0.1 % Triton X-100 for 5 min and blocked with blocking solution (1 % bovine serum albumin in PBS) for 60 min at RT. The cells were subsequently incubated with polyclonal rabbit anti-collagen I antibody (Novus, USA) for 12 h and washed with PBS. Then the cells were labeled with FITC conjugated goat-anti-rabbit IgG antibody (Bioworld technology, USA) for 1 h. The nucleus was counterstained with DAPI (Molecular Probes, Invitrogen, USA) for 5 min. Immunostained cells were visualized using fluorescence microscope (Axiovert 40 CFL, Zeiss, German). 6 immunofluorescence images for each group were analyzed by Image-Pro Plus 6.0 software (IPP, Media Cybernetics Inc., Silver Spring, MD). The green channel (FITC stain for COLI) was measured and the measurement parameters included area and IOD (integrated optical density). COLI expression was quantified by mean density (mean staining intensity = IOD sum/area sum).
Real-time Polymerase Chain Reaction (PCR) Analysis
Samples with 31 mm in diameter were placed in 96-well plates. 2.5 × 105 cells were dispensed on each sample and cultured for 6, 24 h and 4, 7 days. The cells on each disk were lysed using Trizol Reagent (Invitrogen, USA) and lysates were collected by pipetting and centrifugation. Total cellular RNA was isolated using Trizol Reagent according to the manufacturer’s instruction and collected by ethanol precipitation. Total RNA was quantified using UV spectrophotometry (Beckman DU-600).
First-strand complementary DNA (cDNA) was generated from each total RNA sample using an Invitrogen Superscript First-strand Synthesis system in a standard 20 μl reaction, then was amplified to generate products corresponding to mRNA encoding integrin β1, extracellular related kinase 1/2 (ERK1/2), and c-fos. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. The oligonucleotide primers used in the amplification reaction were 5′-GCGCGTGCAGGTGCAATGAAG-3′ and 5′-TGTCCGCAGACGCACTCTCC-3′ for integrin β1; 5′-GGCCGAGGAGCCCTTCACCT-3′ and 5′-CACTCCGGGCTGGAAGCGTG-3′ for ERK1; 5′-AACAGGCTCTGGCCCACCCA-3′ and 5′-ATGGTGCTTCGGCGATGGGC-3′ for ERK2; 5′-CTGTGGCCCCATCGCAGACC-3′ and 5′-CGCTCGGCCTCCTGTCATGG-3′ for c-fos; 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for GAPDH. Real-time PCR was performed using Maxima SYBR Green qPCR Master Mix (Fermentas, Canada) in a real-time PCR System (Applied Biosystems 7500, Bioscience Corporation, USA). Relative mRNA abundance was determined by the 2−ΔΔct method and reported as-fold induction. GAPDH abundance was used for normalization. Experiments were performed independently in triplicate.
Statistical Analysis
The data were expressed as mean ± standard deviation for all experiments. One way ANOVA and multiple comparison tests were performed to evaluate differences among groups. A p value < 0.05 was considered statistically significant.