Thickness and diameter of the CP-2 titanium (Ti) specimens (Seoul Titanium Inc., Korea) were 0.3 mm and 13 mm. The specimens were solvent-cleaned by a serial ultrasonic treatment in acetone, methyl alcohol and de-ionized water, and then dried with oxygen blow.
Treatment of microwave-induced argon plasma
Self-designed microwave-induced argon plasma was used in this study as previously described . Briefly, this system consisted of 2.45 GHz magnetron power supply (1 kW), an applicator including a tuning section and the nozzle made of quartz. The microwave was introduced through a WR-284 copper waveguide with internal cross section dimensions of 72 mm × 24 mm. The plasma generated at the end of a nozzle was formed by an interaction between the high electrical fields, which is generated by the microwave power supply, the waveguide aperture and the gas nozzle. The electric field intensity around the nozzle was calculated by a high frequency structure simulator (HFSS) code simulation as previously described . Argon was used as a working gas for this plasma system, which was chosen for its inertness, and the gas flow rate is approximately 100 min−1 at 8 kgf/cm2. For evaluating the plasma efficiency on Ti disks, Ti disks were placed in front of a nozzle in the plasma system and exposed to plasma for 1 s, 5 s at ‘short treatment (3 cm)’ and 10 s, 30 s, 90 s at ‘long treatment (7 cm)’, respectively.
Measurement of emission spectra
The emission spectra from the plasma according to the gas type were acquired through the optical fiber in the range of 200-1200 nm wavelength. The optical emission spectrometer (HR4000, Ocean Optics Inc, USA) was used to analyze the emission spectra and the electronically excited species generated by the plasma device. The measurement point has been set to be just region of plasma plume. The optical emission spectrum was obtained from the plasma device.
Contact angle measurement
Contact angle was measured using the sessile drop method . to evaluate hydrophilicity of prepared specimens. De-ionized water was utilized as probing liquid. Surface energy was also measured by same method. Three samples were used to collect the contact angle data in diiodomethane, formamide and glycerol for each plasma treated titanium. Lewis acids and bases were used to calculate the surface energy .
MC3T3-E1 mouse pre-osteoblastic cell line (Riken, Japan) was maintained in αMEM medium modified with ascorbic acid supplemented with 10 % fetal bovine serum and 1 % antibiotic antimycotic solution (WelGENE, Korea). Cells were cultured at 37 °C in a humidified 5 % CO2 atmosphere incubator.
Cell attachment and proliferation test
Microwave-induced argon plasma treated Ti disks were placed to 24 well plate and the titanium disks were sterilized in 70 % ethanol for 30 min and washed three times in distilled water. After sterilization, MC3T3-E1 cells were seeded on titanium disks at a density of 6 × 104 cells/disk and incubated for 2 h for cell attachment test. For cell proliferation test, cells were seeded at a density of 3 × 104 cells/disk and cultured for 1, 3, 5 days in a CO2 incubator. The viability of the cells was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, MTT was added to each well at 500 μg/mL and further incubated for 4 h at 37 °C. After incubation, dimethyl sulfoxide and glycine buffer was added to each well for the solubilization of the formed formazan salts. The absorbance of each well was measured spectrophotometrically at 570 nm by a microplate reader.
Cell morphometric analysis
For cell morphometric analysis, the cells were seeded at the initial seeding density of 1.0 × 104 cells/disk and after 2 h of incubation they were fixed with ice cold 70 % ethanol. Cell plasma membrane was visualized by staining with Texas Red C2-maleimide (Texas Red, 30 ng/ml in PBS, Invitrogen) . Cell nuclei were counterstained with Hoechst 33258 (1 g/ml in PBS, Sigma). Ten random pictures were taken by IX-70 microscope, equipped with a DP-71 digital camera (Olympus, Japan). Cell attachment area, perimeter and Feret’s diameter of 30 cells for each group were measured by Image J software (NIH, USA) .
All results were statistically analyzed by Student t-test. Data are expressed as mean ± standard error of mean. P values <0.05 were considered statistically significant.