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treatment resulted in a 25.1% higher amount of IL-1β released in the OA model after exposure to

the LPS concentration of 0.5 μg/mL for 48 h, compared to that from the saline-treated group.

Similarly, the TNF-α expression was also markedly increased by 29.9% at 48 h following LPS

treatment. It appears that the secretion of inflammatory cytokines increased over time, which may

be attributed to the reaction between osteochondral cells and LPS to further release of cytokines.

These inflammatory markers are implicated in cartilage inflammation and chondrocyte apoptosis

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during the development and progression of OA .

To further investigate the performance of the OA model, we used Dic, Dex and Cur as the

LPS-induced OA treatment model. Dic is a NSAID, Dex is a glucocorticoid, and Cur is a natural

polyphenolic compound. All three are drugs for the treatment of OA, and their therapeutic

mechanisms are different. 43-45 . Firstly, CCK-8 assay was used to detect the effect of different Dic,

Dex and Cur concentrations on cell viability for 48 h. Cellular uptake experiments of Cur-treated

microtissues were performed, and the results showed that Cur had completely entered the cell

interior at 4 h (Figure S7). Dic, Dex and Cur treatment significantly reduced the expression levels

of TNF-α, IL-β, IL-6 in the LPS-induced model, bringing them close to those in the untreated

control group, and then the expression levels of IL-10 promote close to untreated control group

which significantly higher than LPS-induced model (Figure 9B-D). These three drugs regulate

osteoarthritis inflammation through a dual mechanism: they reduce pro-inflammatory factors

(TNF-α, IL-1β and IL-6), while simultaneously promoting the elevation of the anti-inflammatory

factor IL-10 via improvement of the inflammatory microenvironment and immunomodulation.

Collectively, these actions ultimately restore the balance of the pro-inflammatory-anti-

inflammatory cytokine network, approaching the physiological state observed in the untreated

group, thereby demonstrating the effective regulatory effect of the drugs on osteoarthritis

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