α mem medium

The sterilized Ti-B12 and Ti6Al4V materials were placed in sterile Petri dishes, and the growth medium containing α-MEM medium (Cellmax), 10% FBS (MRC), and 1% penicpstreptomycin mixture (Solarbio) was added and placed in the cell incubator for 72 h to prepare the extract. MC3T3-E1 cells in the logarithmic growth phase were collected and seeded in 96-well cell culture plates at 1×10⁵ mL^-1 (100 μL per well). They were divided into three groups: A, B, and C, with five wells set at each time point in each group. The culture medium was changed after 24 h in the cell incubator. Groups A and B were added with 200 μL of Ti-B12 and Ti6Al4V material extract, respectively. Group C was added with an ordinary growth medium, which was changed every 3 days. At 1, 3, and 7 days, cell growth status was observed by microscope and photographed, and then cell proliferation rate was detected by the Cell Counting Kit 8 (CCK8) kit (Biyuntian, Shanghai, China).

A mouse calvarial preosteoblast cell line (MC3T3-E1 Subclone 4, ATCC-LGC, standards, MeisenCTCC, Zhejiang, China) was cultured in α-MEM medium (CellMax, Beijing, China) supplemented with 10% fetal bovine serum (CellMax) and 1% penicillin–streptomycin solution (Solarbio) in a humidified 37°C incubator with 5% CO₂. The culture medium was changed every 2–3 days. Cells between passages 5 and 9 were used for all experiments.