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scaffolds were stained using the BCIP/NBT ALP chromogenic kit (Beyotime, Shanghai, China);

on day 21, calcium deposition on the scaffolds was stained with ARS.

For cellular tracking, C-MTs and EO-MTs were labeled with Qtracker®655 and

Qtracker®525, respectively. The labeled MTs were then mixed with GelMA hydrogel and printed

under light-protected conditions, followed by blue light-induced photocrosslinking. The constructs

were transferred to a cell culture incubator for subsequent culture, and all subsequent procedures

were performed under dark conditions. On days 7 and 14, the interface region between the osseous

layer and cartilaginous layer was observed using CLSM.

2.6. Osteoarthritis Model Induced by LPS

Medium containing LPS at different concentrations (0, 0.01, 0.5, 1, and 10 μg/mL) was

added to cultured chondrocytes for 48 h, and the cell viability was investigated by CCK-8 assay.

After the biomimetic osteochondral tissue model was constructed, LPS (0.5 μg/mL) was added in

the culture medium for 6, 24, and 48 h to induce OA. At various time points, the supernatants were

collected, and the levels of TNF-α and IL-1β were then detected by respective ELISA assay kits

(LunChangShuoBiotech, Xiamen, China). In addition, Dic, Dex and Cur was used as the drug

model to treat OA. Different concentrations of Dic (75, 100, and 125 μM), Dex (1.5, 3, and 4.5

μM), and Cur (30, 40, and 50 μM) were added to the culture medium. After 48 hours of incubation,

cell viability was assessed using the CCK-8 assay. Cur was labeled with FITC, added to the cells

and incubated for a certain period of time, then the cells were fixed with 4% paraformaldehyde,

nuclear stained with DAPI, and observed by CLSM imaging. The designed biomimetic joint tissue

was treated with LPS for 48 h, and was then added Dic, Dex and Cur solution for incubation within

48 h. The contents of inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-10 in the supernatant

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