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of pharmaceuticals. These models are widely used in the This may be because the cartilage-on-chip culture
early stages of drug development due to their simplicity environment not only provides a physical and biochemical
and cost efficiency. However, 2D models cannot replicate environment similar to native cartilage tissue, facilitating
the 3D microenvironment of cells in vivo and lack the cell interaction with the surrounding matrix and
complex interactions between cells and the ECM, which promoting intercellular signal transmission to support
can lead to inaccuracies in predicting the clinical effects the synthesis activities unique to cartilage but also allows
of drugs. To overcome the limitations of 2D models, cells to grow in a 3D space, better simulating real tissue
researchers have introduced microfluidic technology. structure and enhancing the interactions between cells
Microfluid allows for precise control and manipulation of closer to their natural state. 22,23 Compared to traditional
fluids within minuscule channels, simulating the blood 2D culture, the 2D + microfluid culture also demonstrated
flow and physical interactions between cells in the body’s improved results, likely due to the ability of microfluidics to
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internal environment. Nevertheless, they still fail to provide a uniform supply of nutrients and oxygen through
accurately mimic the 3D structure of cells. The technology continuous flow, thereby simulating the nourishment of
of an organ-on-a-chip, which integrates 3D cell culture cells by blood flow. In summary, while the 2D + microfluid
with microfluidic systems, offers a more comprehensive group displayed notable improvements over traditional
emulation of the complex physiological conditions of 2D culture in terms of simulating tissue structure and the
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human organs. This integration provides more accurate in vivo environment, it still falls short in fully replicating
drug testing results compared to 2D or simple microfluidic the complexity of cell-cell and cell-matrix interactions
systems. Herein, we developed a cartilage-on-chip by present in a cartilage-on-chip environment. Owing to its
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culturing primary chondrocytes in three dimensions ability to more comprehensively and accurately simulate
within a microfluidic system. To validate the superiority of the microenvironment of human organs or tissues,
our designed chip over the other two models, we conducted the cartilage-on-chip holds higher research value and
an in-depth study of the impact of 2D, 2D + microfluid potential. Thus, we chose to use the cartilage-on-chip for
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culture, and 3D + microfluid culture (cartilage-on-chip subsequent studies.
culture) on the phenotype of primary chondrocytes. The
chondrocyte phenotype refers to a specific set of gene 3.3. The construction of OA-like cartilage-on-chip
expression patterns and biochemical characteristics that are Cartilage degeneration is the central pathological change
essential for maintaining the functionality and structural in OA, characterized by dysfunction of chondrocytes,
integrity of cartilage. Chondrocytes are the primary cell degradation of the ECM, and thinning of the cartilage
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type in cartilage tissue, responsible for synthesizing and layer. This process is not confined to mechanical injury, as
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maintaining the ECM, including key components such as biochemical and inflammatory processes also play a role in
collagen and proteoglycans, which contribute to cartilage the development of OA. A large body of research indicates
structure and function. The cartilage matrix primarily that inflammation is a primary driving factor for cartilage
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consists of water, collagen (especially Col II), proteoglycans degeneration in OA. The inflammatory response triggers
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(such as ACAN), and other non-collagenous proteins. the activation and accumulation of various immune cells
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Therefore, we chose Col II and ACAN as indicators to study and mediators, which intensify chondrocyte stress and
the effects of different culture conditions on chondrocytes. accelerate ECM breakdown, ultimately contributing to
Confocal imaging was employed to observe the cartilage deterioration. Cytokines, such as IL-1β, TNF-α,
morphology of articular chondrocytes under various and IL-6, play a crucial role in the progression of OA, as they
culture conditions (Figure 3A). These results indicate exacerbate inflammation and drive degenerative changes
that while early-stage protein expression levels (day 3) within the joint. They directly promote ECM degradation
were comparable across groups, the cartilage-on-chip by regulating the metabolic balance of the cartilage matrix,
system provided a more favorable microenvironment for thereby accelerating cartilage degeneration, making them
chondrocyte differentiation and ECM production over time. important therapeutic targets for OA. Notably, IL-1β is a
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By day 10, both protein and gene expression levels of Col II key contributor to the pathological progression of OA and
and ACAN were significantly elevated in the cartilage-on- is frequently utilized in experimental research to replicate
chip group, suggesting enhanced cartilage-specific matrix the inflammatory conditions associated with the disease.
synthesis and improved chondrocyte functionality under Compared to other inflammatory factors, IL-1β provides
dynamic perfusion conditions (Figure 3B). Flow cytometry a more direct and controllable experimental model, better
results further validated this finding (Figure 3C and D, replicating the pathological characteristics of OA. 26,28
Figure A1 and A2). From these data, we can conclude that Therefore, IL-1β is an ideal choice for constructing a
under cartilage-on-chip culture conditions, chondrocytes cartilage chip model and for subsequent drug screening in
can more effectively maintain their native cell phenotype. this study.

Volume 1 Issue 1 (2025) 11 doi: 10.36922/or.8461