Molecular differentiation between osteophytic and articular cartilage – clues for a transient and permanent chondrocyte phenotype K. Gelse, A.B. Ekici,

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Molecular differentiation between osteophytic and articular cartilage – clues for a transient and permanent chondrocyte phenotype K. Gelse, A.B. Ekici, F. Cipa, B. Swoboda, H.D. Carl, A. Olk, F.F. Hennig, P. Klinger Osteoarthritis and Cartilage Volume 20, Issue 2, Pages 162-171 (February 2012) DOI: 10.1016/j.joca.2011.12.004 Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Gene expression profile by cDNA array-analysis. Heat map visualizing hierarchical clustering analysis based on the expression levels of genes that are differentially expressed in osteophytic cartilage compared to articular cartilage. RNA preparations from N = 15 different donors were pooled to yield N = 3 paired sets of articular and osteophytic cartilage. The genes with a differential expression greater than 20-fold are shown. P-value < 0.005. (Red = higher expressed genes; blue = lower expressed genes). Osteoarthritis and Cartilage 2012 20, 162-171DOI: (10.1016/j.joca.2011.12.004) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Genes involved in chondrogenesis and endochondral ossification that are characterized by striking differential expression in osteophytic chondrocytes (OC) and articular chondrocytes (AC). The values represent the mean and 95% CI (error bars) of the fold-change in mRNA expression between osteophytic and articular chondrocytes of N = 15 different donors detected by quantitative RT-PCR. Osteoarthritis and Cartilage 2012 20, 162-171DOI: (10.1016/j.joca.2011.12.004) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Immunohistochemical analysis of osteophytic and articular cartilage of the border zone of an osteoarthritic joint. (A): Overviews of the transition area of osteophyte and articular cartilage. Insert with high magnification depicts the non-hypertrophic phenotype of articular chondrocytes in deep zones. (B): Higher magnifications of osteophyte cartilage demonstrate a superficial Col1-positive fibrocartilaginous layer, a deeper Col2-positive hyaline cartilage layer, and underlying osseous tissue. (C): High magnifications (Toluidine blue staining) of the lower half of osteophyte cartilage depict hypertrophic chondrocytes only in the deepest cell layer but not in the overlying middle zone. The deep zone of articular cartilage is characterized by a clear tide mark without cellular hypertrophy within the overlying non-calcified cartilage. (D): GREM1 immunohistochemistry shows positive staining for endothelial structures (∗) but negative staining for subchondral bone. Higher magnifications demonstrate negative staining for osteophytic chondrocytes (open arrowhead) but strong intracellular staining for articular chondrocytes (arrow). (E): BGLAP immunohistochemistry shows strong positive staining for subchondral bone. Osteophytic chondrocytes are characterized by intracellular staining for osteocalcin, whereas articular chondrocytes are completely negative. (F): No non-specific staining was evident in negative controls incubated with isotype normal mouse IgG. Bar = 1 mm in A, B; Bars = 50 μm in C, D, E, F. Safranin-O–Fast green staining; Toluidine blue staining. Osteoarthritis and Cartilage 2012 20, 162-171DOI: (10.1016/j.joca.2011.12.004) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions