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applied within the circles to cover the tissue. The slides were 5 min, and 75% ethanol for 5 min before being rinsed in
incubated at room temperature in the dark for 50 min. The tap water. For staining, the sections were immersed in
slides were then washed 3 times with PBS (pH 7.4), each Fast Green staining solution for bone tissue for 1 – 5 min,
wash lasting 5 min. After removing the PBS, DAPI stain was and then washed to remove excess stain until the cartilage
applied within the circles and incubated in the dark at room appeared colorless. Next, they were briefly immersed in 1%
temperature for 10 min. Following the DAPI incubation, hydrochloric acid alcohol for 10 s, followed by a quick rinse
the slides were washed 3 times with PBS (pH 7.4), each with tap water. The sections were then immersed in Safranin
wash lasting 5 min. After shaking off the excess liquid, the O staining solution for bone tissue for 1 – 5 s. Following
side with the cells facing down was sealed onto a glass slide staining, the sections were quickly dehydrated in four
using an anti-fading mounting medium. The samples were grades of anhydrous ethanol for 5 s, 2 s, and 10 s, with the
observed under a Nikon inverted fluorescence microscope, fourth grade used for microscopic inspection. The sections
and images were captured. (DAPI: Excitation wavelength were then cleared in clean xylene for 5 min and sealed with
330 – 380 nm, emission wavelength 420 nm, emitting blue neutral balsam. Finally, microscopic examination, image
light; FITC: Excitation wavelength 465 – 495 nm, emission capture, and analysis were performed.
wavelength 515 – 555 nm, emitting green light; CY3:
Excitation wavelength 510 – 560 nm, emission wavelength 2.6.4. Immunofluorescence staining
590 nm, emitting red light). Hydrogel tissues were fixed in formalin, embedded

2.6.2. Alcian blue-safranin staining in paraffin, and sectioned to a thickness of 6 μm. The
deparaffinized joint sections were heated at 70°C for
Hydrogel tissues were fixed in formalin, embedded in 30 min, followed by dewaxing and dehydration through
paraffin, and sectioned to a thickness of 6 μm. The paraffin- two 10-min xylene treatments and a series of ethanol
embedded joint sections were first heated at 70°C for 30 min. treatments at concentrations of 100%, 90%, 80%, and
Following this, the sections were placed in eco-friendly 75%, each for 5 min, with three PBS washes in between.
deparaffinization solution I for 20 min, followed by a second The sections were then incubated overnight at 4°C in a
deparaffinization solution II for another 20 min. The tissues humidified chamber with primary antibody solutions for
were then treated with anhydrous ethanol I for 5 min, Col II (1:600) and MMP-13 (1:500). After incubation, the
anhydrous ethanol II for 5 min, and 5% alcohol for 5 min primary antibody solution was removed, and the sections
and were finally rinsed with tap water. To stain the sections, were rinsed once with PBS. They were then incubated with
alcian blue stain was applied for 10 – 15 min, followed by a an appropriate secondary antibody solution in a humidified
wash with tap water. In the cell culture plate, 1 mL of alcian chamber, shielded from light, for 2 h, followed by three
blue stain was added to each well and incubated for 15 min,
then washed with distilled water. The sections were then PBS washes. For imaging, neutral balsam was applied,
sequentially treated with anhydrous ethanol I for 5 min, and the sections were observed and photographed using
anhydrous ethanol II for 5 min, and anhydrous ethanol III a fluorescence microscope. Stained samples were imaged
for 5 min. Clearing was performed with xylene I for 5 min, using an Olympus IX81 inverted microscope or a Nikon
followed by xylene II for 5 min, before sealing with neutral Eclipse E800 upright microscope.
resin. For the staining procedure in the cell culture plate, 2.6.5. Western blot and RT-qPCR analysis
anhydrous ethanol I was added for 5 min and discarded,
followed by treatments with anhydrous ethanol II for 5 min The gene expression of chondrocytes in the organ-on-a-chip
and anhydrous ethanol III for 5 min. Afterward, the cover model was analyzed for Col II, SOX9, MMP-13, and Adamts5
slip with cells was removed to dry, and the side with the after introducing the inflammatory factor IL-1β, following
cells facing down was sealed onto a glass slide using neutral the procedures outlined in Sections 2.5.2 and 2.5.3.
resin. Finally, the microscopic examination was performed, 2.7. Drug screening capability of the cartilage-on-
and images were captured for analysis.
chip
2.6.3. Safranin O-fast green staining Diclofenac sodium (DS) and HC-030031 were
Hydrogel tissues were fixed in formalin, embedded administered intraarticularly at doses of 10 μM and
in paraffin, and sectioned to a thickness of 6 μm. The 5.3 μM, respectively, for 3 days to treat inflammation, and
deparaffinized joint sections were first heated at 70°C for their therapeutic effects were then evaluated accordingly
30 min. These sections were then sequentially immersed (Figure 4A). Alcian blue-safranin staining, SO/FG, and
in eco-friendly deparaffinizing and clearing solution I for immunofluorescence staining were utilized to inspect the
20 min, followed by solution II for an additional 20 min. microtissue repair post-medication, and the expression
After deparaffinization, the sections were treated with levels of OA-related genes were determined using Western
anhydrous ethanol I for 5 min, anhydrous ethanol II for blot and RT-qPCR.

Volume 1 Issue 1 (2025) 8 doi: 10.36922/or.8461