Intermittent mechanical loading of articular cartilage explants modulates chondroitin sulfate fine structure  K. Sauerland, Ph.D., J. Steinmeyer, Ph.D.  Osteoarthritis and Cartilage  Volume 15, Issue 12, Pages 1403-1409 (December 2007) DOI: 10.1016/j.joca.2007.05.004 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Schematic illustration of the loading protocols. (A) Explants were cyclically loaded for 10s with a peak stress of 0.5MPa followed by a period of unloading lasting 10, 100 or 1000s. This intermittent loading cycle was repeated for the total duration of experiments lasting 3 days. (B) Explants were cyclically loaded for 10s with a peak stress of 0.1, 0.5 or 1.0MPa followed by a period of unloading lasting 1000s. This intermittent loading cycle was repeated for the total duration of experiments lasting 3 days. (C) Explants were cyclically loaded for 10s with a peak stress of 0.5MPa followed by a period of unloading lasting 1000s. This intermittent loading cycle was repeated for the total duration of experiments lasting 1, 3 or 6 days. Osteoarthritis and Cartilage 2007 15, 1403-1409DOI: (10.1016/j.joca.2007.05.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Percentage of newly synthesized internal disaccharides of unloaded articular cartilage explants cultured for up to 6 days in serum-free and supplemented Ham's F-12 media. [3H]-glcNH2-labeled CS produced during the final 18h of the 1-, 3- or 6-day culture period was digested with chondroitin lyases, and the products were separated by high-performance anion-exchange chromatography and subsequently quantified by liquid scintillation counting. Data represent the mean values±S.D. (N=6). Osteoarthritis and Cartilage 2007 15, 1403-1409DOI: (10.1016/j.joca.2007.05.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effect of the duration of intermittence on the percentage of the internal disaccharides ΔDi4S and ΔDi6S as well as on the abundance of galNAc4,6S termini on CS chains that were newly synthesized by loaded cartilage explants; data are expressed as percentage of unloaded controls (=100%). Using a 0.5Hz frequency and a sinusoidal waveform, a pressure of 0.5MPa was cyclically applied on cartilage discs for 10s followed by a period of unloading ranging from 10 to 1000s. Explants were intermittently loaded for 3 days [see also Fig. 1(A)]. Data shown are mean values±S.D. (N=6). Statistically significant differences from unloaded control values are given as follows: *0.01<P≤0.05; **0.001<P≤0.01; ***P≤0.001. Osteoarthritis and Cartilage 2007 15, 1403-1409DOI: (10.1016/j.joca.2007.05.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effect of different stress levels on the percentage of the internal disaccharides ΔDi4S and ΔDi6S as well as on the abundance of galNAc4,6S termini on CS chains that were newly synthesized by loaded cartilage explants; data are expressed as percentage of unloaded controls (=100%). Using a 0.5Hz frequency and a sinusoidal waveform, a maximum pressure ranging from 0.1 to 1.0MPa was cyclically applied on cartilage discs for 10s followed by a period of unloading lasting 1000s. Explants were intermittently loaded for 3 days [see also Fig. 1(B)]. Data shown are mean values±S.D. (N=6). Statistically significant differences from unloaded control values are given as follows: *0.01<P≤0.05. Osteoarthritis and Cartilage 2007 15, 1403-1409DOI: (10.1016/j.joca.2007.05.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effect of the duration of loading on the percentage of the internal disaccharides ΔDi4S and ΔDi6S as well as on the abundance of galNAc4,6S termini on CS chains that were newly synthesized by loaded cartilage explants; data are expressed as percentage of unloaded controls (=100%). Using a 0.5Hz frequency and a sinusoidal waveform, a pressure of 0.5MPa was cyclically applied on cartilage discs for 10s followed by a period of unloading lasting 1000s. Explants were intermittently loaded for 1–6 days [see also Fig. 1(C)]. Data shown are mean values±S.D. (N=6). Statistically significant differences from unloaded control values are given as follows: *0.01<P≤0.05. Osteoarthritis and Cartilage 2007 15, 1403-1409DOI: (10.1016/j.joca.2007.05.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions