Af1785

The different groups of chondrocyte sheets were fixed in 4% paraformaldehyde and embedded in paraffin. Cell sheets were incubated with Type II Collagen primary antibodies (rabbit anti human, Servicebio, GB11021, 1:100 dilution), MMP16 (goat anti human, R&D, AF1785, 1:40 dilution) and a secondary antibody (donkey anti rabbit, Servicebio, GB22403, 1:200 dilution and donkey anti goat, Servicebio, GB21404, 1:300 dilution). The cell nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI, Servicebio, G1012). The samples were then observed and photographed observed under a high-quality fluorescence microscope.

The protein expression of COL2A1 and MMP16 were examined by western blot analysis. The chondrocytes were treated with miR-193b-3p mimics, mimics-NC, MMP16 expression or DMEM. The specimens were washed twice using PBS and lysed using a RIPA lysis reagent (Thermo Fisher Scientific, Rockford, Illinois, USA). The protein concentrations were determined by bicinchoninic acid (BCA) assay (Pierce Biotechnology, Waltham, Massachusetts, USA). The samples were incubated overnight with primary antibodies including rabbit anti human COL2A1 (Abcam, ab188570, 1:1000 dilution) and goat anti human MMP16 (R&D, AF1785, 1:10000 dilution) at 4°C. The membrane for each antibody was then visualized with horseradish peroxidase (HRP) conjugated secondary antibodies (goat anti-rabbit and donkey anti-goat, Daixuan Bio, 1:500 dilution). The intensities of the protein bands, representative of protein levels, were determined using Quantity One Image software. β-actin was used to normalize target proteins. All experiments were performed in triplicate.