Computed tomography detects changes in contrast agent diffusion after collagen cross- linking typical to natural aging of articular cartilage  H.T. Kokkonen, J. Mäkelä, K.A.M. Kulmala, L. Rieppo, J.S. Jurvelin, V. Tiitu, H.M. Karjalainen, R.K. Korhonen, V. Kovanen, J. Töyräs  Osteoarthritis and Cartilage  Volume 19, Issue 10, Pages 1190-1198 (October 2011) DOI: 10.1016/j.joca.2011.07.008 Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 (A) An osteochondral disc (Ø=25.4 mm) was drilled from a bovine patella and trimmed to leave ∼1mm of bone under the cartilage. The disc was then cut into four similar pieces and smaller osteochondral plugs (Ø=6.0mm) were punched from each piece. (B) A CT-image of one representative sample after 2h of immersion in the contrast agent bath. The analyzed cartilage area is delineated with a solid line. The width of the analysis area was 100pixels (i.e., 3,010μm), and the height was matched with the full cartilage thickness. (C) A magnified image of the analyzed area. (D) A profile curve corresponding to the depth-wise contrast agent distribution within cartilage tissue. The pixel rows in subfigure C were horizontally averaged to give one profile for the depth-dependent X-ray absorption. Finally, the acquired profiles were normalized with the average bath concentration above the cartilage surface. Osteoarthritis and Cartilage 2011 19, 1190-1198DOI: (10.1016/j.joca.2011.07.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 (A) Average contrast agent distribution profiles in reference (n=7) and threose treated (n=7) samples at different time points. 0 denotes articular surface and 1 deep cartilage. The partition is defined as the contrast agent concentration in the cartilage divided by that in the bath. (B) The difference in contrast agent partition between the threose treated and reference samples. The difference is especially great at the equilibrium in the deep cartilage. (C) Average contrast agent content in full thickness cartilage as a function of time. After 5h, the contrast agents show significantly greater partition in the reference samples than in the threose treated samples. (D) The diffusion flux in the threose treated and reference samples. The contrast agent flux is significantly lower in the threose treated samples at 2h. In the subfigures B and D, the 95% confidence interval (CI) is expressed with error bars. Osteoarthritis and Cartilage 2011 19, 1190-1198DOI: (10.1016/j.joca.2011.07.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 (A) Representative stress–relaxation curves recorded for paired threose treated and reference samples during the non-destructive testing. The threose treated sample shows higher equilibrium modulus. (B) The average (n=7) stress–strain curves recorded during the destructive testing. Since no significant abrupt decrease in stress during the destructive test was consistently detected, the yield/failure points could not be determined for all samples. The 95% CI is expressed with error bars. Osteoarthritis and Cartilage 2011 19, 1190-1198DOI: (10.1016/j.joca.2011.07.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 The spatial collagen (A) and PG (B) distribution of the threose treated samples and the reference samples were virtually identical (a.u. refers to absorption unit). No statistically significant cross-linking induced changes in the orientation (C) or anisotropy (D) of the collagen fibrils were observed. (E) As revealed by OD measurements of Safranin-O stained sections, the fixed charge distribution was altered by the threose treatment. Osteoarthritis and Cartilage 2011 19, 1190-1198DOI: (10.1016/j.joca.2011.07.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions