Frozen cartilage specimens without subchondral bone were embedded in Tissue-Tek O.C.T. (Sakura, Alphen aan den Rijn, The Netherlands) for cryosectioning. Serial transverse frozen sections (12-μm thickness) were collected starting from the cartilage surface; 20 adjacent sections were collected at different distances from the surface to represent the superficial (0 to 240 μm), middle (480 to 720 μm), and deep (960 to 1200 μm) layers. Twenty sections between each layer were skipped to avoid cross-contamination. In all, a total of 54 samples were acquired, representing each joint site, disease state, and cartilage depth.

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