Dp72 charge coupled device camera

The knee joints of mice were fixed in 10% formaldehyde at 4 °C for >24 h, decalcified in 0.5 M ethylenediaminetetraacetic acid in PBS (pH 7.4) for 2 weeks, embedded in paraffin, sliced into 5-μm sections, and stained with hematoxylin and eosin, 0.1% Safranin-O (s8884; Sigma-Aldrich, St Louis, MO, USA), and 0.05% Fast green FCF (f7258; Sigma-Aldrich, St Louis, MO, USA). Sclerosis and articular cartilage destruction were identified by Safranin-O staining and measured with OsteoMeasureXP (OsteoMetrics, Inc., Atlanta, GA, USA), Image-pro plus (v4.5, Media Cybernetics, Inc., Rockville, USA), Adobe Photoshop (v9.0, San Jose, CA, USA), and an Olympus DP72 charge-coupled device camera (v2.1, Olympus Corporation, Tokyo, Japan). Articular cartilage destruction was scored by using OARSI grades (0–6), which is a standard OA-grading system^{18 (link),62 (link)} (Supplementary Data 4). The OARSI grades of the medial tibia were assessed by averaging the scores of three experienced investigators. The ratio of hyaline cartilage to calcified cartilage, osteophyte maturity, and synovitis^{65 (link)} were also determined (Supplementary Data 5). Moreover, subchondral bone sclerosis was determined by measuring the SBP thickness.

Human knee joint sections were obtained as described above and bone marrow-derived MSCs cultured in the chondrogenic medium were stained with Alcian Blue (B8438; Sigma Aldrich) and photographed with Olympus DP72 charge-coupled device camera (v2.1, Olympus Corporation, Tokyo, Japan). The Alcian-Blue activity in the dishes was also quantified by measuring the absorbance at 630 nm.

The knee joints were fixed in 10% formaldehyde at 4 °C for >48 h, decalcified in 0.5 M EDTA (pH 7.4) for 14 days, embedded in paraffin, cut into 5-μm sections, and stained with safranin-O with fast green counterstaining. Articular cartilage destruction was scored using OARSI. The tartrate-resistant acid phosphatase (TRAP)-staining kit was from Fujifilm Wako Pure Chemical Corporation (Japan). For immunohistochemistry, knee joint sections were incubated overnight at 4 °C with antibodies against MMP3 (Cat# Ab53015, 1:50), MMP13 (Cat# Ab39012, 1:50), Aggrecan (Cat# Ab1031, 1:100), pSmad3 (Cat# Ab52903, 1:100), Osterix (Cat# Ab22552, 1:400) (all from Abcam), or COL2A1 (Cat# MAB8887, 1:100; Sigma-Aldrich). Immunoactivity was detected with a DAB peroxidase-substrate detection kit (Vector Laboratories, USA). Nuclei were counterstained with hematoxylin. Samples were measured using OsteoMeasureXP (OsteoMetrics, Inc., Atlanta, GA, USA), Adobe photoshop (v19.1.3), and an Olympus DP72 charge-coupled device camera (v2.1, Olympus Corporation, Tokyo, Japan).