2.10. Live/Dead Assay

After 7 days of culture, the cell-containing scaffolds were removed and placed in a new 24-well plate. The scaffolds were washed gently with PBS 3 times for 5 min each time. A live/dead staining kit (Thermo Fisher Scientific, Pittsburgh, PA) was removed from the refrigerator in advance and brought to room temperature. The reagent was then diluted with PBS to a working concentration (2 μg/mL calcitonin and 4 μg/mL bromoethorphine dimer). Each scaffold was added to 0.5 mL of the working reagent and soaked for 40 min at room temperature. PBS was used to wash the scaffolds three times, and 0.5 mL 4% paraformaldehyde solution was used to fix the cells for 1 h. Then, PBS was used to wash the cells again three times. Finally, aseptic filter paper was used to dry the scaffolds, and the sheet was sealed. The cells were then observed under a laser confocal microscope.