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After stabilizing the microenvironment of the chip, channel. 30,31 Extensive animal studies and laboratory
IL-1β was introduced into the growth medium on research indicate that it reduces cellular activation
day 4 to stimulate the chondrocytes. After 3 days, the and inflammatory responses by inhibiting the TRPA1
tissue phenotype was evaluated using histological and channel and alleviates pain through neural pathways,
immunostaining analyses to assess its response to the demonstrating the potential for OA treatment. Given that
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inflammatory factor (Figure 4A). The TUNEL assay existing models cannot fully simulate the OA environment
demonstrated that in the control group, cell nuclei stained in humans, the development and screening of drugs face
with blue DAPI exhibited little to no green fluorescence, various challenges. To address this, we have developed an
indicating minimal or absent DNA fragmentation. In the OA-like cartilage-on-chip, which has been preliminarily
IL-1β-treated group, along with the blue DAPI-stained tested using DS to confirm its drug-screening functionality.
nuclei, some cells exhibited green fluorescence (marked Subsequent research investigated the potential therapeutic
by red arrows), indicating that IL-1β treatment led to effects of HC-030031 for OA.
apoptosis to some extent. Immunohistochemical staining Drugs were continuously injected into the inflamed
further demonstrated that, compared to the control chip at a flow rate of 0.5 mL/min for 3 days. To evaluate
group, the IL-1β-treated group displayed lighter and their therapeutic effect, gene expression levels were
more dispersed staining, indicating collagen depletion analyzed on day 10 (Figure 5A). RT-qPCR results revealed
and glycosaminoglycan degradation, which reflected that the treatment with DS and HC-030031 effectively
cartilage damage and structural deterioration. In addition, downregulated the expression levels of MMP13 and
protein expression levels of Col II and MMP13 were Adamts5, alleviating the inflammation induced by IL-1β
evaluated using immunofluorescence imaging and Western and further validating the anti-inflammatory action of the
blot analysis. Both techniques consistently indicated a drugs. At the same time, cartilage matrix markers Col II
reduction in Col II expression and an increase in MMP13 and SOX9 were upregulated, protecting chondrocytes and
expression after IL-1β treatment, further supporting the promoting cartilage repair to some extent (Figure 5D).
evidence of cartilage degeneration (Figure 4B and C). In This was further confirmed by immunofluorescence
addition, we further analyzed four biomarkers extensively staining and Western blot. Moreover, SO/FG staining
studied in joint disease research using RT-qPCR: Col II, displayed more GAG retention in tissues treated with the
SOX9, MMP13, and Adamts5. In the IL-1β-treated model, drugs, indicating the drugs’ efficacy in inhibiting cartilage
the expression of Col II and SOX9 decreased, whereas degeneration (Figure 5B and C). This is consistent with the
Adamts5 and MMP13 exhibited a significant upregulation RT-qPCR findings mentioned earlier.
(Figure 4D), indicating inflammation and cartilage matrix
degradation. In summary, chondrocytes in the cartilage-on-chip,
treated as described, regained their ECM formation
These findings demonstrated that inflammation capabilities, not only enhancing the clinical relevance of the
induction through IL-1β was successful, and the cartilage- model and demonstrating its potential for drug screening
on-chip can effectively serve as a novel model for simulating but also further supporting DS and HC-030031 as the
OA and facilitating subsequent research. effective chemical agents for protecting cartilage cells from
inflammation-induced degeneration.
3.4. OA-like cartilage-on-chip for chemical drug
screening 3.5. OA-like cartilage-on-chip for biological drug

OA significantly impacts daily activities, with treatment screening
strategies typically including pharmacotherapy and joint Given the limited variety and effects of current drugs
replacement surgery. Pharmacotherapy is an essential available for treating cartilage degeneration, researchers
component of OA treatment, with most patients requiring are exploring new biologics and regenerative medicine
either short- or long-term medication to manage symptoms strategies, such as the use of stem cells and biomaterials
effectively throughout the course of their treatment. For for cartilage regeneration. Notably, recent studies
instance, DS, a widely used non-steroidal anti-inflammatory indicate that gut microbiome dysbiosis is linked to the
drug, is often selected to relieve OA-related symptoms development of OA. By modulating the gut microbiome,
because of its strong anti-inflammatory and pain-relieving probiotics may help reduce joint inflammation and slow
properties. 28,29 However, these chemical drugs primarily the progression of OA. For example, LGG, a widely studied
focus on pain management and inflammation control, with and used probiotic, has been reported to indirectly affect
limited effects on cartilage repair, thus highlighting the inflammation and immune processes by regulating the gut
importance of developing new drugs that can both manage microbiome, potentially improving the inflammatory
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symptoms and promote cartilage regeneration. HC-030031 environment of OA, thereby alleviating symptoms and
is an experimental drug that specifically blocks the TRPA1 enhancing joint function. Although direct research on OA

Volume 1 Issue 1 (2025) 12 doi: 10.36922/or.8461