Subchondral plate porosity colocalizes with the point of mechanical load during ambulation in a rat knee model of post-traumatic osteoarthritis H. Iijima,

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Subchondral plate porosity colocalizes with the point of mechanical load during ambulation in a rat knee model of post-traumatic osteoarthritis H. Iijima, T. Aoyama, J. Tajino, A. Ito, M. Nagai, S. Yamaguchi, X. Zhang, W. Kiyan, H. Kuroki Osteoarthritis and Cartilage Volume 24, Issue 2, Pages 354-363 (February 2016) DOI: 10.1016/j.joca.2015.09.001 Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Trajectories of knee joint excursions during rat ambulation recorded using a 3-dimensional motion capture apparatus at 4 weeks after surgery. (A) Sham-operated knee. (B) DMM-operated knee. Zero percent step cycle means paw contact, and 100% is the next paw contact of the same limb. Knee flexion angle = 0° means that the trochanter major (hip), knee joint cavity (knee), and lateral malleolus (ankle) are located in alignment. Lines denote the mean ± 95% confidence intervals (n = 4). Vertical line: transition from stance phase to swing phase during ambulation in sham- and DMM-operated knees. Osteoarthritis and Cartilage 2016 24, 354-363DOI: (10.1016/j.joca.2015.09.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Representative 3-dimensional micro-computed tomography (μ-CT) images of subchondral bone. (A) Three-dimensional reconstruction of the femoral condyle and tibia, with subchondral bone perforations (red arrow) (a), and with subchondral bone perforations of all samples (n = 8 for each time point) superimposed (red circle) on the same 3-dimensional reconstruction images (b). Asterisk denotes the medial side. (B) Three-dimensional μ-CT images of the sagittal plane with various knee flexion angles at 4 weeks after surgery. Red box presents a magnification of the figure on the below. Red arrow indicates SBCs without trabecular structure, as confirmed by perforations from surface view (as shown [A]). Osteoarthritis and Cartilage 2016 24, 354-363DOI: (10.1016/j.joca.2015.09.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Histological analysis of cartilage and subchondral bone. (A) Representative photographs of toluidine blue-stained sections in the medial compartment. Asterisk denotes severe subchondral bone damage, with replacement by fibrous tissue. Magnification: ×100. Scale bars = 200 μm. (B) OARSI score of medial the femur and tibia. Boxplots denote median values and interquartile ranges. Vertical bars denote ranges. Significantly different values (P < 0.05) are displayed in bold (n = 8; Mann–Whitney U test). (C) Calcified cartilage and subchondral bone (SB) damage score of the medial femur and tibia. Boxplots denote median values and interquartile ranges. Vertical bars denote ranges. Significantly different values (P < 0.05) are displayed in bold (n = 8; Mann–Whitney U test). Osteoarthritis and Cartilage 2016 24, 354-363DOI: (10.1016/j.joca.2015.09.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Immunohistochemistry findings. (A) Representative photographs of type II collagen and Col2-3/4c. Magnification: ×100. Scale bars = 200 μm. (B) Mean staining intensity of type II collagen (0–255: 0 indicates no staining and 255 indicates maximum staining) in the cartilage. Bars denote the mean ± 95% confidence intervals. Significantly different values (P < 0.05) are displayed in bold (n = 8; paired t-test). Osteoarthritis and Cartilage 2016 24, 354-363DOI: (10.1016/j.joca.2015.09.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Ultrastructure of the cartilage surface of the medial tibial plateau using scanning electron microscopy (SEM). (A) Comparison of subchondral bone perforations (red circle) confirmed by 3-dimensional micro-computed tomography (μ-CT) images (a) as shown Fig. 2, and cartilage degeneration observed by SEM (b). Black box delineates the region of interest for SEM observation. Scale bars = 200 μm. (B) Magnification of the SEM image in the medial tibial plateau in sham (a, c) and DMM (b, d) cartilage surfaces. White boxes (a, b) present a magnification of the corresponding figure (c and d, respectively). Scale bars = 100 μm (a, b); 10 μm (c, d). Osteoarthritis and Cartilage 2016 24, 354-363DOI: (10.1016/j.joca.2015.09.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Biomechanical properties of the medial femoral posterior condyle and medial tibial plateau. (A) Dynamic stiffness (N/mm) calculated by deformation (mm) until the test load achieved to 0.1 N. Bars show the mean ± 95% confidence intervals (CIs). Significantly different values (P < 0.05) are displayed in bold (n = 8; paired t-test). (B) Creep response (μm), which is the equilibrium deformation between the initially applied test load and after 300 s maintaining a 0.1 N test load. Bars show the mean ± 95% CIs. Significantly different values (P < 0.05) are displayed in bold (n = 8; paired t-test). Osteoarthritis and Cartilage 2016 24, 354-363DOI: (10.1016/j.joca.2015.09.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Supplementary Fig. 1 Gait parameters obtained from 3-dimensional motion analysis at 4 weeks after surgery: maximum knee extension angle, flexion angle, and total knee range of motion. (A) Stance phase. (B) Swing phase. Positive value of maximum extension angle indicates that the knee joint is flexed (e.g., maximum extension angle = 60° means that the knee is flexed at a 60° angle). No significant differences between sham- and destabilized medial meniscus-operated knees are seen in the 3 gait parameters. Bars show the mean ± 95% confidence intervals (n = 4; pared t-test). Osteoarthritis and Cartilage 2016 24, 354-363DOI: (10.1016/j.joca.2015.09.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions