Live dead kit

After culturing for 1, 3, 7, and 10 days, the viability of the spheroid cells was assessed using a LIVE/DEAD kit (Sigma-Aldrich, St. Louis, MO, USA). The cells were stained with 4 µM Calcein AM and 4 µM PI for 30 min in 37 °C. Calcein-AM broken down by esterase in a viable cell results in a strong green fluorescence signal (excitation: 490 nm, emission: 515 nm), whereas dead cells are stained as red by the aqua-fluorescent reactive dye PI (excitation: 535 nm, emission: 617 nm). Cell survival was observed with a laser confocal microscope (LSM 700, Zeiss, Oberkochen, Germany), and 3D cell images were reconstructed.
Cells and spheroids were dissociated into single cells by treatment with 0.25% trypsin-EDTA for 10 min. The cell survival rate was also quantified by the CCK-8 assay (CCK-8, Dojindo, Kumamoto, Japan). The cell suspension (100 µL; 5000 cells/well) to a 96-well plate and the plate was incubated for 24 h in a humidified incubator. After adding 10 µL of the CCK-8 solution to each well and incubation for 2 h, cell proliferation was observed with a TECAN 200/200Pro multimode microplate reader (TECAN Trading AG, Männedorf, Switzerland).

Cell viability and proliferation on the scaffolds was assessed by the CCK-8 assay (Boster) and a LIVE/DEAD kit (Sigma-Aldrich). Briefly, a suspension of 1 × 10⁴ BMSCs in 100 μl GM were loaded onto the cubic PLA/HA composite scaffolds. After 1, 3, 5, and 7 days of culturing, viability and proliferation of BMSCs were visualized using LIVE/DEAD kit. Live cells per microscopic field were counted using Image-J software. Furthermore, CCK-8 kit was employed to quantitatively evaluate the cell viability. The optical density (OD) value was read by a microplate reader at 450 nm (Bio Tek Instruments, Winooski, VT, USA).