Quantitative assessment of articular cartilage morphology via EPIC-μCT

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Quantitative assessment of articular cartilage morphology via EPIC-μCT L. Xie, Ph.D., A.S.P. Lin, M.S., M.E. Levenston, Ph.D., R.E. Guldberg, Ph.D. Osteoarthritis and Cartilage Volume 17, Issue 3, Pages 313-320 (March 2009) DOI: 10.1016/j.joca.2008.07.015 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 The segmentation method for quantifying articular cartilage morphology in the rat distal femur. Femoral cross-sections (A) were transformed into sagittal sections (B) via 3-D rotation of the grayscale image file. After drawing the contour lines to eliminate any adjacent bone marrow space (C), an appropriate threshold range was selected to segment cartilage from bone tissue according to the histogram analysis of the tissues (D). The 3-D morphology of articular cartilage was then visualized (E) and quantified in terms of cartilage thickness, volume, and surface area. Osteoarthritis and Cartilage 2009 17, 313-320DOI: (10.1016/j.joca.2008.07.015) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Optimization of incubation time and Hexabrix concentration for EPIC-μCT in the rat model. (A) The volume of analysis for each femoral condyle extended from the top surface of the cartilage down 31 slices, or 372μm, which included cartilage, subchondral bone, and some trabecular bone. (B) The average attenuation of femoral articular cartilage after incubation with each concentration of Hexabrix solution indicated that 30min of incubation was sufficient for Hexabrix to reach equilibration, regardless of the concentration. Representative X-ray attenuation histograms for femoral articular cartilage and bone incubated in 20% (C), 30% (D), 40% (E) and 50% Hexabrix solutions (F). Osteoarthritis and Cartilage 2009 17, 313-320DOI: (10.1016/j.joca.2008.07.015) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Validation of EPIC-μCT thickness measurements for rat tibial articular cartilage via needle probe testing. (A) An illustration of needle probe testing on tibial articular cartilage. (B) Representative force vs displacement curve showed regions of changing slope that indicated contact with cartilage and bone surfaces. The inset displays magnified data for the cartilage contact region. (C) Strong linear relationship between measurements of cartilage thickness obtained by EPIC-μCT and needle probing (slope=0.81, r2=0.95, n=43). (D) The differences (needle probing – EPIC-μCT) vs average cartilage thickness measured by needle probing and EPIC-μCT with 95% limits of agreement. Osteoarthritis and Cartilage 2009 17, 313-320DOI: (10.1016/j.joca.2008.07.015) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Evaluation of articular cartilage morphology during growth in male Wistar rats at 4, 8, and 16-weeks of age. (A) Representative images for intact distal femora, including bone (gray) and articular cartilage (white). (B) Representative thickness maps of segmented femoral articular cartilage. (C) Representative sagittal sections of each distal femur. Average cartilage volume (D), thickness (E), and surface area (F) for each age group (n=5, error bars indicate ±SD). *P<0.01 vs 4-week-old rats, #P<0.01 vs 8-week-old rats. Osteoarthritis and Cartilage 2009 17, 313-320DOI: (10.1016/j.joca.2008.07.015) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Histologic evaluation of rat femoral articular cartilage from male Wistar rats at 4, 8, and 16 weeks of age for validation of EPIC-μCT measurements. (A) Representative images from safranin-O staining of femoral articular cartilage. (B) Average thickness of femoral articular cartilage assessed via EPIC-μCT and histology (n=10, error bars indicate ±SD). (C) Strong linear relationship between measurements of cartilage thickness obtained by EPIC-μCT and histology (slope=0.97, r2=0.99). (D) The differences (histology – EPIC-μCT) vs average cartilage thickness measured by histology and EPIC-μCT with 95% limits of agreement (n=30). *P<0.01 vs 4-week-old rats, #P<0.01 vs 8-week-old rats. Osteoarthritis and Cartilage 2009 17, 313-320DOI: (10.1016/j.joca.2008.07.015) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions