Macroscopic and histopathologic analysis of human knee menisci in aging and osteoarthritis

Abstract

Objective: Meniscus lesions following trauma or associated with osteoarthritis (OA) have been described, yet meniscus aging has not been systematically analyzed. The objectives of this study were to (1) establish standardized protocols for representative macroscopic and microscopic analysis, (2) improve existing scoring systems, and (3) apply these techniques to a large number of human menisci.

Design: Medial and lateral menisci from 107 human knees were obtained and cut in two different planes (triangle/cross section and transverse/horizontal section as well) in three separate locations (middle portion, anterior and posterior horns). All sections included vascular and avascular regions and were graded for (1) surface integrity, (2) cellularity, (3) matrix/fiber organization and collagen alignment, and (4) Safranin-O staining intensity. The cartilage in all knee compartments was also scored.

Results: The new macroscopic and microscopic grading systems showed high inter-reader and intra-reader intraclass correlation coefficients. The major age-related changes in menisci in joints with no or minimal OA included increased Safranin-O staining intensity, decreased cell density, the appearance of acellular zones, and evidence of mucoid degeneration with some loss of collagen fiber organization. The earliest meniscus changes occurred predominantly along the inner rim. Menisci from OA joints showed severe fibrocartilaginous separation of the matrix, extensive fraying, tears and calcification. Abnormal cell arrangements included decreased cellularity, diffuse hypercellularity along with cellular hypertrophy and abnormal cell clusters. In general, the anterior horns of both medial and lateral menisci were less affected by age and OA.

Conclusions: New standardized protocols and new validated grading systems allowed us to conduct a more systematic evaluation of changes in aging and OA menisci at a macroscopic and microscopic level. Several meniscus abnormalities appear to be specific to aging in the absence of significant OA. With aging the meniscal surface can be intact but abnormal matrix organization and cellularity were observed within the meniscal substance. The increased Safranin-O staining appears to represent a shift from fibroblastic to chondrocytic phenotype during aging and early degeneration.

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.

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, 4x.

Fig. 3

Histological assessment of meniscus cellularity: (A) Normal cell distribution, score 0. (B) Diffuse hypercellularity, score 1. (C) Meniscus tissue shows hypo- to acellular regions, score 2 (D) Hypocellular meniscus tissue, score 3. Hematoxylin & Eosin, 40x.

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, 4x.

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, 4x.

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 International Cartilage Repair Society (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).

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 (*).

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, 40x (A, B), 100x (C, D).

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.