Calcein am

To detect the cell viability and attachment of the RBMSCs seeded on the pure scaffold, RAOECs–ECM scaffold or RBMSCs–ECM scaffold. RBMSCs (4 ×10⁵ cells/scaffold) were seeded onto the scaffolds and incubated at 37°C. After 1, 3 and 7 days, the medium was removed, and the scaffolds were washed with PBS three times and then treated with Calcein AM (Bestbio, Shanghai, China). The live cells on the scaffolds were observed under a fluorescence microscope. To observe the attachment and morphology of cells grown on the scaffolds, the cell–scaffold composites were collected at Day 3, washed twice with PBS and chemically fixed using 2.5% glutaraldehyde solution. The specimens were subsequently dehydrated twice with a series of graded ethanol (30%, 50%, 70%, 80%, 90%, and 100%). After dehydration, the scaffolds were immersed in hexamethyl–disilazane for 2 min and vacuum–dried overnight. Finally, the scaffolds were sputter–coated for 60 s at 10 mA with gold, and the gold–coated scaffolds were observed via SEM.

To study the specific elimination of senescent chondrocytes by the NPs, DOX-induced senescent ATDC5 cells were seeded on a 48-well cell culture plate (5 × 10⁴ cells per well). CuS and B2M-CuS NPs (50 µg mL^− 1) were added to the plate and cultured with cells for 24 h, and cell viability was assayed with CCK-8. Live/dead staining was also performed. The above-treated cells were gently washed with PBS and stained with 200 µL of Calcein AM and propidium iodide (PI; BestBio, China) for 30 min at 37 ℃ in the dark. Cells were imaged with a fluorescence microscope.