8 μm filters

Transwell assays were performed using 24-well plates with 8-μm filters (Corning, USA). 12 hours after transfection of the corresponding plasmids, C33A cells were treated with serum starvation. 1×10⁵ cells in 200 μl of serum-free medium were placed in the upper chamber and 500 μl 18% FBS medium was injected in the lower chamber. After 48 hours of incubation, the migrant cells attached to the lower surface were fixed, photographed and quantitatively processed, and each experiment was repeated three times.

According to previous studies, co-cultured cells were mixed at a ratio of 5:1 (HUVECs: hBMSCs) in the study^{16 (link), 35 (link), 36 (link)}. Growth factor-reduced Matrigel (50 μL, BD, USA) was plated in 96-transwell plates with 8 μm filters (Corning, USA), and seeded with HUVECs (3.6 × 10⁴), with a mix of cells suspension consisting of 6 × 10³ cells/well hBMSCs labelled with CellTracker^TM CM-Dil (Invitrogen, USA) and 3 × 10⁴ cells/well HUVECs transfected with lentivirus to express green fluorescent protein (GFP-HUVECs), and with GFP-HUVECs (3.6 × 10⁴), respectively. hBMSCs were labelled with CM-Dil according to the manufacturer’s instructions, while the lentiviral vector for transducing GFP was constructed according to published methods⁴⁹ . To study real-time stimulation of angiogenesis, β-TCP or 5%CS/β-TCP scaffolds were added to transwell inserts. After incubation at 37 °C for 4 h, HUVECs groups were stained with Calcein AM Fluorescent Dye (BD, USA), then tubes were imaged on an inverted fluorescence microscope. The number of formed tubes was calculated using Image Pro Plus 6.0.