Quantitative pre-clinical screening of therapeutics for joint diseases using contrast enhanced micro-computed tomography  N.J. Willett, T. Thote, M. Hart, S. Moran, R.E. Guldberg, R.V. Kamath  Osteoarthritis and Cartilage  Volume 24, Issue 9, Pages 1604-1612 (September 2016) DOI: 10.1016/j.joca.2016.04.021 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Representative CT images indicating articular cartilage volume of interest (VOI) and cartilage degeneration. A: Rat tibial articular cartilage overlay on bone indicating VOI. B: Representative image illustrating erosions (cartilage defect < 50% of thickness) and lesion (cartilage defect > 50% of thickness). C–F: Representative images showing coronal section of rat medial tibial joint illustrating no osteophyte (OP) in sham joint (C), outline of osteophyte in MMT joint (D), total osteophyte (indicated in white) including mineralized and cartilaginous matrix in MMT joint (E) and mineralized matrix (indicated in white) in MMT joint (F). Osteoarthritis and Cartilage 2016 24, 1604-1612DOI: (10.1016/j.joca.2016.04.021) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Representative histology and EPIC-μCT images depicting OA progression over 3 weeks. A–D: Toluidine blue stained MMT joints showing no damage in sham (A), cartilage surface erosion at week 1 in MMT joint (B), increased cartilage surface erosion at week 2 in MMT joint (C) and development of osteophyte (red arrow) and severe cartilage erosion at week 3 in MMT joint (D). E–H: Corresponding EPIC-uCT images for the samples as sectioned via histology indicating progression of cartilage degradation. No damage observed in sham (E) or week 1 MMT joint (B). Increased areas of X-ray attenuation in cartilage loaded with contrast agent observed in MMT joints at week 2 (G) and 3 (H) as indicated by black arrow. Red arrow indicates area of mineralization within marginal osteophyte at week 2 and week 3 in MMT joints. Red indicates higher X-ray attenuation corresponding to lower proteoglycan content. Osteoarthritis and Cartilage 2016 24, 1604-1612DOI: (10.1016/j.joca.2016.04.021) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Quantification of cartilage degeneration and osteophyte development in MMT joints over 3 weeks. A: Histopathological three zone sum score is significantly increased in MMT joints at weeks 1, 2 and 3 compared to shams. B: X-ray attenuation in cartilage loaded with contrast agent (referred to as cartilage attenuation) is significantly increased in MMT joints at weeks 2 & 3 compared to shams. C: Cartilage lesion volume is significantly increased in MMT joints at week 3 compared to shams, and MMT joints at week 1 & 2. D: Osteophyte widths as calculated via histo-pathology scoring show an increase in MMT joints compared to shams at week 2 and week 3. E–G: Osteophyte average 3D thickness (E), osteophyte total volume (F) and osteophyte mineral volume (G) as calculated via EPIC-μCT show an increase in MMT joints compared to shams at week 2 and 3. n = 5–6/group for CT data; n = 3 for sham and n = 5–6 for MMT for histology data. * = P < 0.05, sham significantly different from MMT within time point; ** = P < 0.05, sham significantly different from MMT at week 2. Osteoarthritis and Cartilage 2016 24, 1604-1612DOI: (10.1016/j.joca.2016.04.021) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Representative histology and EPIC-μCT images depicting MMT progression over 3 weeks. A–B: Toluidine blue stained MMT joints showing reduced osteophyte formation in MMPi treated joint (B) compared to vehicle treated joint (A). C–D: Corresponding EPIC-μCT images for the samples as sectioned via histology indicating reduced osteophyte formation in MMPi treated joint (D) compared to vehicle treated joint (C). Red indicates higher X-ray attenuation corresponding to lower proteoglycan content. Osteoarthritis and Cartilage 2016 24, 1604-1612DOI: (10.1016/j.joca.2016.04.021) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 MMP inhibitor (MMPi) treatment reduces cartilage damage and osteophyte development compared to vehicle treated groups. A: MMPi treatment significantly reduces three zone sum score compared to vehicle treated group. B: X-ray attenuation in cartilage loaded with contrast agent (referred to as cartilage attenuation) is significantly lower in sham group compared to both vehicle treated and MMPi treated groups. C: Lesion volume is significantly reduced in MMPi treated group compared to vehicle treated group. D: Osteophyte widths as calculated via histo-pathology scoring are significantly lower in MMPi treated group compared to vehicle treated group. E–G: Osteophyte average 3D thickness (E), osteophyte total volume (F) and osteophyte mineral volume (G) as calculated via EPIC-uCT are significantly lower in MMPi treated group compared to vehicle treated group. n = 15–21/group, * = P < 0.05, sham significantly different from vehicle & MMPi; $ = P < 0.05, vehicle significantly different from MMPi; # = P < 0.05, vehicle significantly different from sham. Osteoarthritis and Cartilage 2016 24, 1604-1612DOI: (10.1016/j.joca.2016.04.021) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions