48 well plates

The membranes were cropped into round samples 1 cm in diameter, and samples from the different groups were plated on 48-well plates (Jet Biofil, China). The cells were then seeded at 5 × 10³ cells per membrane. After culturing for 3 days, FITC phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) staining was performed to stain the nuclei and F-actin of the cells, which were observed under a confocal laser scanning microscope (CLSM, Leica TCS SP8 X, Germany). After removing the culture media, the samples were washed with phosphate-buffered solution (PBS) before fixing the cells with a 4% paraformaldehyde (PFA) solution. Triton X-100 solution (0.5%) was used to permeabilize cells for 10 min, and the samples were washed again with PBS. FITC phalloidin (Yeasen, China) was diluted with 1% bovine serum albumin (BSA) solution at a ratio of 1:200. The solution was then added to the cells and incubated at room temperature for 1 h. After three rounds of washing for 5 min, 100× diluted DAPI (Yeasen, China) solution was added to the samples for nuclear staining and incubated for 10 min. After cleaning the residual dye with PBS, the membranes were transferred to confocal dishes for observation.

Murine MC3T3-E1 osteoblastic cells were obtained from the American Type Culture Collection (CRL-2593). Cells were cultured in complete Alpha-MEM (Alpha-MEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS and 1% penicillin and streptomycin (PenStrep, Thermo Fisher Scientific, Waltham, MA, USA).
Upon confluence, cells were trypsinized and seeded at a density of 10,000 cells/disc on titanium discs positioned in 48-well plates (JET BIOFIL, Guangzhou, China) and pre-conditioned as reported in Section 2.3.1.
To avoid the possibility that the presence of 10% FBS in the culturing medium masked the effects of surface conditioning during the experiments, cells were cultured in complete Alpha-MEM additioned with 0.5% FBS and 1% PenStrep.