Eclipsc ti u

The cytotoxicity of BG tablet was assayed by coculturing with the osteoblast (MC3T3) cell line which was purchased from cell bank of Shanghai. The cells were cultured in complete medium that contains 90% Dulcecco's modified eagle medium (DMEM) and 10% Fetal Bovine Serum (FBS) in a humidified atmosphere at 5% CO₂ and 37 °C. The BG tablets were sterilized by high temperature and high pressure before culturing with cells. MC3T3 cells were seeded in 48 well plate with density of 10⁴ cells and volume of 200 μl complete medium per well. After material and cells coculured for 24 h, the culture medium was took out from plate and the BG tablets were washed with PBS three times, finally the Die dyeing kit (Invitrogen) was used according to the instruction. After cocultivation of the kit and cells for another 30 minutes, the growth condition of cells was observed by Inversed Fluorescent Microscope (Eclipsc Ti—U, NIKON, Japan). And in order to observe the shape of cells on BG tablet surface, 2.5% glutaraldehyde was used to fix the form of cells, then different concentration gradient of ethanol was used to dehydrate cells, finally the shape of cells was observed with SEM. Also, the quantitative analysis of cell viability was tested by CCK-8 method, the specific experiment procedures were referred to the cell counting kit (Invitrogen) instruction.

The OCP samples were sterilized by Co60 gamma radiation. Cells were directly seeded onto the culture plates at a density of 2 × 10⁴ cells/cm². The culture media were refreshed with OCP-particles-containing media (1 mg/mL) after cell adhesion and this moment was set as the initial culture time. After cultured for 24 h, the cells were rinsed with PBS thrice and fixed with 4 vol% formaldehyde solution for 4 h. The fixed cells were dehydrated with a graded series of ethanol (30, 50, 60, 70, 80, 90, 95 and 100 vol%) and air-dried at room temperature for 24 h. The attachment and morphology of cells were observed by the FESEM.
Cells were cultured with OCP particles at a density of 5 × 10³ cells/cm². After cultured for 3 days, the cells were rinsed with PBS thrice and double-stained with Calcein-AM (Biotium, USA) and propidium iodide (Biotium, USA) following the instructions. The fluorescence images of cells were acquired by an inverted fluorescence microscope (Eclipsc Ti–U, Nikon, Japan) equipped with a digital camera (40FL Axioskop, Zeiss, Germany). Quantitative cell viability was measured by a Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay. After cultured with OCP particles for 1, 3, and 5 days, the cells were incubated with the CCK-8 working solution at 37 °C for 1 h. Afterward, the absorbance of the supernatants was read at 450 nm by the microplate reader.