24 well plate

2 × 10⁴/well cells were seeded into a 24-well plate (Cellvis, USA) coated with Col I, CSM/Col I and BMP2-CSM/Col I hydrogels in the 2D model, and Col I, CSM/Col I and BMP2-CSM/Col I hydrogels with 1 × 10⁶/ml cells were added into a 24-well plate (Cellvis, USA), respectively, in the 3D model. All samples were stained with the Calcein-AM/PI Double Staining Kit (Dojindo Molecular Technologies, Japan) according to the operation manual at Days 1 and 4, and then visualized under a laser scanning confocal microscope (LSCM; Nikon, Japan).

2 × 10⁴/well cells were seeded into a 24-well plate (Cellvis, USA) coated as above-mentioned in the 2D model, and different hydrogels embedded with 1 × 10⁶/ml cells were added into a 24-well plate (Cellvis, USA) in the 3D model. Fibrous actin (F-actin) cytoskeletons of all samples were stained with 100 nM TRITC Phalloidin solution (No. 40734ES75, Yeason, China), and nuclei were further visualized using DAPI solution (No. C1005, Beyotime Biotech, China) at Days 1 and 4.
Similarly, circular glass culture plates were coated with different hydrogels in 24-well plates, and then 2 × 10⁴/well cells were seeded. At Days 1 and 4, the samples were harvested and fixed by 4% paraformaldehyde, dehydrated through a series of alcohol concentrations (10%, 20%, 30%, 50%, 70%, 85%, 90% and 100% twice), dried with CO₂ critical-point drying, and then spray-coated with gold. Samples were observed under SEM.