Glucosamine reduces anabolic as well as catabolic processes in bovine chondrocytes cultured in alginate E.J. Uitterlinden, M.D., H. Jahr, Ph.D., J.L.M.

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Glucosamine reduces anabolic as well as catabolic processes in bovine chondrocytes cultured in alginate E.J. Uitterlinden, M.D., H. Jahr, Ph.D., J.L.M. Koevoet, B.Sc., S.M.A. Bierma-Zeinstra, Ph.D., J.A.N. Verhaar, M.D., Ph.D., H. Weinans, Ph.D., G.J.V.M. van Osch, Ph.D. Osteoarthritis and Cartilage Volume 15, Issue 11, Pages 1267-1274 (November 2007) DOI: 10.1016/j.joca.2007.04.004 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 ECM status (based on the data of the control conditions from experiments 1 and 2b) and time course of experimental conditions. – GlcN addition in experiment 1. – GlcN addition in experiments 2 and 4. – GlcN addition in experiment 3. – IL-1β treatment in experiment 3. Osteoarthritis and Cartilage 2007 15, 1267-1274DOI: (10.1016/j.joca.2007.04.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Total amount of GAG per bead (expressed as μg CS; panel A) and total amount of DNA per bead (panel B) on day 38 after stimulation with GlcN. Three concentrations of three different forms of GlcN were added during the last 14 days of culture. An ECM was preformed during the first 24 days and the effects of GlcN are thus tested in a situation where a balance between synthesis and degradation exists. * indicates a statistically significant difference (P<0.05), compared to the nonsupplemented control. Osteoarthritis and Cartilage 2007 15, 1267-1274DOI: (10.1016/j.joca.2007.04.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Total GAG content (panel A) and total DNA content (panel B) per bead upon GlcN treatment on day 17. Chondrocytes were stimulated in the absence of an ECM and the GlcN effects are thus tested in an anabolic situation. Three different forms of GlcN, three concentrations each, were added during the last 14 days of culture. * indicates a statistically significant difference (P<0.05), compared to the nonsupplemented control. Osteoarthritis and Cartilage 2007 15, 1267-1274DOI: (10.1016/j.joca.2007.04.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Changes in GAG per bead (panel A) and DNA per bead (panel B) upon GlcN treatment during 17 days of culture. Cultures were treated with 5mM Gluc and 5mM GlcN-HCl during the last 14 days. Beads were harvested on days 3, 6, 10 and 17. In the absence of ECM, the GlcN effects are tested in an anabolic situation. * indicates a statistically significant difference (P<0.05), compared to the nonsupplemented control. Osteoarthritis and Cartilage 2007 15, 1267-1274DOI: (10.1016/j.joca.2007.04.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 GlcN pre-treatment protects against IL-1β triggered changes in total GAG per bead. GAG content per bead during 17 days of culture is displayed for the control condition (Control), after stimulation on day 8 for 48h with 10ng/ml IL-1β (IL-1β), after treatment with 5mM GlcN-HCl on day 6 for 96h (GlcN), and after treatment with 5mM GlcN-HCl on day 6 for 96h, followed by stimulation with 10ng/ml IL-1β on day 8 for 48h (GlcN+IL-1β). Beads were harvested on days 6, 8, 10, 13 and 17. * indicates a statistically significant difference (P<0.05), compared to 5mM GlcN-HCl treatment (GlcN). # indicates a statistically significant difference (P<0.05), compared to 10ng/ml IL-1β (IL-1β). Osteoarthritis and Cartilage 2007 15, 1267-1274DOI: (10.1016/j.joca.2007.04.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Influence of medium Gluc concentration on GlcN triggered changes in GAG content per bead. In this experimental set-up, the alleged competition between GlcN and Gluc was tested on day 17 of culture; different cultures with either GlcN-HCl or GlcN-Ac were simultaneously treated with increasing concentrations of Gluc (5–25mM). * indicates a statistically significant difference (P<0.05), compared to control condition at 5mM medium Gluc concentration. Osteoarthritis and Cartilage 2007 15, 1267-1274DOI: (10.1016/j.joca.2007.04.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions