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2.9. Statistical analysis permitting researchers to observe and quantify the effects
of pharmaceuticals, chemical substances, or alternative
Each experiment included at least three biological replicates. therapeutic strategies on the behavior of chondrocytes. This
Data are expressed as the mean ± standard deviation unless
stated otherwise. Specific details regarding sample size, approach facilitates disease modeling and drug screening
pre-processing steps, and statistical methods are provided research without the need for animal experiments or
in the figure legends. Statistical analyses were conducted clinical trials, offering an efficient and ethical avenue for the
using Prism 9 (GraphPad, USA). A significance threshold discovery of new medications and treatment modalities.
of 0.05 was applied, with statistical significance indicated as The osmotic function of the chip chambers simulates
follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. the osmosis process within human organs. By adjusting the
composition and concentration of the culture fluid inside
3. Results the chip, it is possible to mimic the internal physiological

3.1. Construction of the cartilage-on-chip environment of human organs for biological processes such
as intercellular substance exchange and nutrient supply.
The knee joint is covered by cartilage, which plays a In this study, we visualized the osmotic action between
crucial role in shock absorption and reducing friction microfluidics and the hydrogel chambers through the
between bones during movement, thereby lowering the transmission of dye, ensuring cell-matrix interactions. Over
risk of injury. Articular cartilage consists of chondrocytes time, the dye’s osmotic action became stronger, indicating
that are embedded in an extracellular matrix (ECM), increased interaction between cells and the matrix. The
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and these chondrocytes rely on the surrounding synovial concentration difference between the upper and lower
fluid for the exchange of nutrients. The 3D structure of chambers was greatest at 0 h. After continuous perfusion for
cartilage is essential for its function, making 3D culturing 72 h, the concentrations in the upper and lower chambers
a key approach to emulate this complex structure in a reached equilibrium. At 96 h, there was no concentration
laboratory setting. Recent studies have highlighted the use difference compared to 72 h, indicating that the environment
of 10% GelMA as a scaffold material in the development had reached a state of balance after 3 days. This indicates
of cartilage-on-a-chip models. These studies demonstrate that growth factors can permeate into the hydrogel within
that GelMA offers sufficient mechanical stability and 3 days, providing an environment similar to the natural
favorable cell viscosity while also effectively supporting the ECM and ensuring cell-matrix interactions (Figure 2B).
maintenance of chondrocyte expression. 20-22 Consequently, Therefore, we set the culture cycle of the environment in the
this study adopted 10% GelMA to create a supportive 3D later model construction to 3 days. Primary chondrocytes
matrix that facilitates chondrocyte growth and phenotype were embedded within the hydrogel and cultured in the
preservation. Furthermore, the microfluidic chip system cartilage-on-chip system with continuous perfusion at a
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introduces a microfluidic environment that, through flow rate of 0.5 mL/min. Samples were taken on days 3, 6,
microfluidic flow, provides a continuous supply of nutrients and 10, observing almost no dead cells in all groups, whereas
and waste removal, thereby simulating the natural living cells displayed good viability. Notably, green staining
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environment of cartilage. This dynamic culturing method increased as the culture time extended, indicating a rise in
is vital for maintaining the viability and functionality of cell density. This suggests that the hydrogel scaffold is non-
chondrocytes. Ultimately, the cartilage-on-chip combines toxic to cells and can be used to construct the cartilage-on-
3D cell culture techniques with microfluidics technology chip. Although the number of dead cells increased over time,
to create a platform that closely mimics the natural the number of living cells remained significantly higher,
environment of joint cartilage (Figure 2A). indicating that primary chondrocytes maintained viability

The main body of the chip consists of 24 cell inoculation under a flow rate of 0.5 mL/min for 10 days, demonstrating
chambers and four bottom flow channels. The inserts are good biocompatibility (Figure 2C).
designed to accommodate cell-laden hydrogels, forming These results collectively indicated that the construction
modular microtissues with a height of 3 mm and an inner of the cartilage-on-chip was successful, enabling cells to
diameter of 6 mm. The chip’s flow channel has a height grow and perform their biological functions normally. This
of 1 mm and extends 180 mm in length, whereas the ensured the accuracy and reproducibility of experimental
tube diameter measures 6.3 mm. The perfusion channels outcomes, thereby better preparing the groundwork for
connect the bottom flow channels of the chip’s main body subsequent drug screening processes.
to a centrifuge tube, forming a closed loop. The flow rate
control and liquid movement are regulated by a peristaltic 3.2. Cartilage-on-chip maintains the chondrocyte
pump and perfusion channels, maintaining a flow rate of phenotype
0.5 mL/min. Within such a system, each chamber of the Traditional 2D models for drug screening utilize monolayer
bioreactor serves as a microenvironment akin to knee joint, cell cultures on flat plates to test the activity and toxicity

Volume 1 Issue 1 (2025) 10 doi: 10.36922/or.8461