J. P. H. J. Rutges, R. A. Duit, J. A. Kummer, F. C. Oner, M. H

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Hypertrophic differentiation and calcification during intervertebral disc degeneration J.P.H.J. Rutges, R.A. Duit, J.A. Kummer, F.C. Oner, M.H. van Rijen, A.J. Verbout, R.M. Castelein, W.J.A. Dhert, L.B. Creemers Osteoarthritis and Cartilage Volume 18, Issue 11, Pages 1487-1495 (November 2010) DOI: 10.1016/j.joca.2010.08.006 Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Immunohistochemical staining of Runx2 (A–C), collagen type X (D–F) and OPG (G–I). Runx2 staining in (A); the NP of a Thompson grade IV IVD, in (B); the transition zone from NP to AF of a Thompson grade IV IVD and (C); in a Thompson grade III non-degenerative IVD. Staining for collagen type X in (D); the NP of a Thompson grade V IVD, (E); the transition zone from NP to AF of a Thompson grade V IVD and (F); in a non-degenerative Thompson grade III IVD. Staining for OPG in (G); the NP of a Thompson grade IV IVD, (H); the transition zone from NP to AF of a Thompson grade V IVD and (I); in a non-degenerative grade I IVD. Scale bars indicate 100μm in A and B, 500μm in C and D, 200μm in E and 50μm in F–I. Osteoarthritis and Cartilage 2010 18, 1487-1495DOI: (10.1016/j.joca.2010.08.006) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 OPG levels for NP and AF per degeneration grade and age group. *=P<0.05 and **=P<0.005. Error bars indicate the 95% confidence intervals. Osteoarthritis and Cartilage 2010 18, 1487-1495DOI: (10.1016/j.joca.2010.08.006) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 A: Micro-CT image of a Thompson grade I IVD. B: Micro-CT image of a Thompson grade IV IVD with calcification in both the AF (arrows) and NP (arrowheads). Osteoarthritis and Cartilage 2010 18, 1487-1495DOI: (10.1016/j.joca.2010.08.006) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 A and B: Large calcification in the AF from a Thompson grade IV IVD stained with alizarin red (A) and von Kossa (black/brown B). C: ALP activity (red staining) in the transition zone of the NP and AF of a Thompson grade IV IVD. D: Combined staining of NADH dehydrogenase (blue), von Kossa (black/brown) and eosin (pink) in Thompson grade V IVD. E: Calcification stained with alizarin red near notochordal cells (arrow) in the NP of a 3.3-year-old child. F: Calcification stained with von Kossa (black/brown) near notochordal cells (arrow) in the NP of a 3.3-year-old child. Scale bars indicate 500μm (A, B), 100μm (C, F) 50μm (D) and 200μm (E). Osteoarthritis and Cartilage 2010 18, 1487-1495DOI: (10.1016/j.joca.2010.08.006) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions