Human or mouse knee articular cartilage specimens were fixed in 4% paraformaldehyde and decalcified before paraffin embedding. Each paraffin-embedded cartilage sample was sectioned into 5-μm-thick slices for subsequent histological analysis. To evaluate the proteoglycan (PG) loss, safranin O/fast green staining (0.1% Safranin O, 0.01% Fast Green solution) and Alcian blue staining (0.1% Alcian blue solution) were performed as previously described 48, after deparaffinization and hydration. OARSI Grade 52, 53 and the MANKIN Scoring System 54, 55 were used to grade the severity of cartilage degeneration by two observers blinded to group-identifying information. OA severity was recorded with the maximal score of observed cartilage.

Reconstructed imaging of the knee joint from mice was performed using a high-resolution micro-CT instrument (InspeXio SMX-225 CT FPD HR; Shimadzu Co. Ltd., Kyoto, Japan) as previously described 56. Briefly, an X-ray energy of 225 kV and an isotropic voxel size of 4 µm was performed on knee articular cartilage as well as distal femur and proximal tibia. Each knee joint was reconstructed using a data analyzer (VGStudio MAX; Volume Graphics, Heidelberg, Germany). The number of abnormally proliferating osteophytes was counted.

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