4.10. MG63 Cell Culture

MG63 human osteoblast-like cells (BCRC 60279; Hsinchu, Taiwan) were used to examine the effects of various PACT modalities on cell function of decontaminated Ti samples. The cells were suspended in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, Langley, OK, USA) and 1% penicillin (10,000 U/mL)/streptomycin (10,000 μg/mL) solution (Gibco) in 5% CO₂ at 37 °C. A cell suspension (10⁴ cells/well) in a 24-well plate was seeded on each sample, and an uncontaminated Ti alloy sample was used as a control.

To observe cell morphology on the sample surface after initial 6 h incubation, the cells were washed three times with PBS and fixed in 2% glutaraldehyde (Wako, Tokyo, Japan) at 4 °C for 2 h. After dehydration in the graded ethanol series and drying using critical point dryer device (LADD 28000; Williston, VT, USA), the cell sample was coated with a gold layer and observed by SEM.

After 1, 3, and 7 days of incubation, cell proliferation was assayed using the MTT assay, according to the previous study [5]. The results were obtained through five independent measurements and reported in terms of absorbance at 563 nm detected with a BioTek Epoch plate reader.

To examine the early cell differentiation on the 7th and 14th days, an alkaline phosphatase (ALP) activity assay was conducted using the TRACP & ALP assay kit (Takara, Shiga, Japan) [72]. Five samples were used in each group, and the average was obtained at 405 nm absorbance using a BioTek Epoch plate reader.

Alizarin Red S staining method was used to quantify calcium deposits secreted by MG63 cells. After 7 and 14 days of incubation, the cells were washed with PBS and fixed in 4% paraformaldehyde at 4 °C for 10 min, and then stained in 0.5% Alizarin Red S (Sigma–Aldrich) in PBS for 10 min [72]. After that, the calcium mineral precipitate was extracted with a 10% cetylpyridinium chloride solution for 30 min. The absorbance of extract was detected at 562 nm using a BioTek Epoch plate reader. Five samples were used to obtain the average.