Histological Analysis

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.