Df7518

Tail veins of AIA rats were administered with free DiD or DiD-NPs. Ankle joints were collected 24 h after the last administration to prepare sections. The prepared sections of 10 μm thick slices were stained with CD44 antibody (Affinity Biosciences, DF6392, 1:500), CD68 antibody (Affinity Biosciences, DF7518, 1:500), and FOLR2 antibody (Affinity Biosciences, DF9518, 1:300). Nuclei was stained by DAPI. A laser scanning confocal microscope (LSM 800, Zeiss, Germany) was taken to record the fluorescent distributions in synovial joints.

The fluorescently labeled dye with an excitation wavelength of 550 nm (EFL-DYE-UF-ENE-R) was dissolved in LAP at 5 mg/ml to prepare fluorescent hydrogels. All samples were divided into SilMA, MSN/SilMA, Ag@MSN/SilMA and Ag@MSN-BMP-2/SilMA groups. The hydrogels were made into cylinders with a diameter of 1 cm and a height of 5 mm using a mold. Mice were anesthetized, then the backs of the mice were shaved and sterilized. The samples were implanted into the backs of the mice (n = 40, 10 mice per group). Photographs were taken immediately after the operation (day 0) and at weeks 1, 4 and 8 using a subcutaneously small-animal live imaging system (Bruker) to observe and record hydrogel degradation in different groups.
All samples in SilMA, MSN/SilMA, Ag@MSN/SilMA and Ag@MSN-BMP-2/SilMA groups were crosslinked to assess the biocompatibility of the composite hydrogels by UV light in vivo, and subcutaneously implanted into the back of rats (n = 40, 10 rats per group). The rats were anesthetized at 1, 4 and 8 weeks after implantation, and each implanted hydrogel scaffold was removed and weighed to plot the degradation curve. Skin tissue fixed, embedded in paraffin, and sectioned. Hematoxylin–eosin (H&E) staining and CD68 (DF7518, Affinity) immunofluorescence were subsequently performed to assess the in vivo biocompatibility of the hydrogels.

IHC staining was performed according to the commercial kits (PV-6001 and PV-6002, ZSGB-Bio, Beijing, China). Antigen retrieval of the paraffin sections was performed using the same protocol described for IF staining. Subsequently, sections were incubated with 0.3% H₂O₂ for 10 min to inactivate endogenous peroxidase activity and then washed with PBS. After being blocked with 5% goat serum for 15 min, slices were incubated overnight at 4 °C with the primary antibodies as follows: rabbit anti-NLRP3 antibody (ab214185, 1:200, Abcam), rabbit anti-cleaved-caspase 1 p20 (1:100, AF4005, Affinity), IL-1β (1:100, GTX74034, Gentex), rabbit anti-GSDMD (1:800, ab219800, Abcam), mouse anti-IBA-1 (1:300, GB12105, Servicebio), rabbit anti-Ly6G (1:500, GB11229, Servicebio), rabbit anti-CD68 (1:100, DF7518, Affinity). The sections were incubated with enzyme-conjugated goat anti-mouse IgG or goat anti-rabbit IgG polymer for 30 min. Finally, immunoreactivity was visualized using 3,3-diaminobenzidine (DAB, ZLI-9017, ZSGB-Bio) followed by restaining with hematoxylin. Images were captured by a light microscope (Leica, DM2500, Germany).