C. Pauli, S. P. Grogan, S. Patil, S. Otsuki, A. Hasegawa, J. Koziol, M

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Macroscopic and histopathologic analysis of human knee menisci in aging and osteoarthritis C. Pauli, S.P. Grogan, S. Patil, S. Otsuki, A. Hasegawa, J. Koziol, M.K. Lotz, D.D. D’Lima Osteoarthritis and Cartilage Volume 19, Issue 9, Pages 1132-1141 (September 2011) DOI: 10.1016/j.joca.2011.05.008 Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 (A) Macroscopic assessment: Grade 1: Normal intact menisci, attached at both ends with sharp inner borders, no tibial or femoral surface changes. (B) Grade 2: Fraying at inner borders, tibial or femoral surface fibrillation, no tears. (C) Grade 3: Partial substance tears (→), fraying, tibial or femoral side fibrillations. (D) Grade 4: Full/complete substance tears, loss of tissue (⇐), tissue maceration. ∗Calcium deposition is marked in addition to the grade. Red marks in b–d indicate the degeneration pattern. Every location (A/F) posterior, (B/E) middle, (C/D) anterior can be given a grade 1–4. Osteoarthritis and Cartilage 2011 19, 1132-1141DOI: (10.1016/j.joca.2011.05.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Histological assessment of meniscus integrity: (A, B) Normal meniscus: the meniscus surface is smooth, no fraying or surface fibrillation, score 0. (C, D) Mild changes: The meniscus surface shows slight fibrillation or undulation, score 1. (E, F) Moderate fibrillation, fraying and/or undulation, some clefts are present, score 2. (G, H) The meniscus shows severe fraying and tears, severe disruption, score 3. Safranin-O–Fast Green, 4×. Osteoarthritis and Cartilage 2011 19, 1132-1141DOI: (10.1016/j.joca.2011.05.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Histological assessment of meniscus cellularity: (A) Normal cell distribution, score 0. (B) Diffuse hypercellularity, score 1. (C) Meniscus tissue shows hypo- to a-cellular regions, score 2. (D) Hypocellular meniscus tissue, score 3. H&E, 40×. Osteoarthritis and Cartilage 2011 19, 1132-1141DOI: (10.1016/j.joca.2011.05.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Meniscus morphology and collagen fiber organization: (A) Normal appearance of extracellular matrix and collagen fiber organization, score 0. (B) Diffuse foci (●) of degenerated extracellular matrix (hyalin and/or mucoid), most of the collagen fibers are organized, score 1. (C) Bands or confluent foci of degenerated extracellular matrix substance (hyalin and/or mucoid), most of the collagen fibers appear unorganized, score 2. (D) Fibrocartilaginous separation (edema, hyaline and/or mucoid degeneration, cyst formation (♦), tears) with unorganized collagen fibers, score 3. Safranin-O–Fast Green, 4×. Osteoarthritis and Cartilage 2011 19, 1132-1141DOI: (10.1016/j.joca.2011.05.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Safranin-O–Fast Green staining intensity: (A) No stain for Safranin-O, score 0. (B) Slight staining intensity for Safranin-O, score 1. (C) Moderate staining intensity for Safranin-O, score 2. Strong staining intensity for Safranin-O, score 3. Safranin-O–Fast Green, 4×. Osteoarthritis and Cartilage 2011 19, 1132-1141DOI: (10.1016/j.joca.2011.05.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Relationship between meniscus and cartilage gross morphology. Correlation between total cartilage scores (condyles, tibia, trochlea and patella) compared to the total menisci scores. Cartilage scores were assessed according the mapping system recommended by the ICRS and by using a modified Outerbridge system. Macroscopic grades for both menisci were obtained by using the new proposed grading systems in this study for each location (anterior, middle and posterior). Osteoarthritis and Cartilage 2011 19, 1132-1141DOI: (10.1016/j.joca.2011.05.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Gross morphology: (A) Healthy meniscus with a smooth and glistening surface. The anterior horns are smaller and the posterior horns show a broad base. (B) A larger fiber bundle/connective tissue bundle penetrates through the vascular zone into the meniscus tissue (←). (C) Degenerated menisci with severe tissue disruption (medial), fraying, small tears and calcium deposition. (D) Tissue tag or scar tissue most likely due to a flap tear (∗). Osteoarthritis and Cartilage 2011 19, 1132-1141DOI: (10.1016/j.joca.2011.05.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 8 Cell arrangements: (A) Local fibro-chondrocyte proliferation or pseudo-cloning. (B) Cells appear more arranged between the fibers. (C) Ovoid to round cell shape; the so-called fibro-chondrocyte. (D) Fusiform, elongated cell shape, the so-called fibroblast-like cell. Safranin-O–Fast Green, 40× (A, B), 100× (C, D). Osteoarthritis and Cartilage 2011 19, 1132-1141DOI: (10.1016/j.joca.2011.05.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 9 Scores for Safranin-O staining intensity of both genders among age groups. In, the age groups over 30 years Safranin-O–Fast Green staining intensity is increased. Per age group we analyzed 7–17 menisci (n=7–17). The error bars represent the 95% confidence interval. Osteoarthritis and Cartilage 2011 19, 1132-1141DOI: (10.1016/j.joca.2011.05.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions