Figure 2.

Histological anatomo‐pathological analysis of hematoxylin and eosin stained AKU cartilage. (A,a) Healthy cartilage from the knee of a healthy young male subject who underwent surgery for a traumatic accident (Control 12, Table I). (b) AKU cartilage in non‐fibrillated regions, distant from the damaged cartilage. Hypocellularity and empty lacunae (arrow) are showed. Specimen was from Patient 1 (Table I). (c) Image from the most damaged region of AKU cartilage. Different grading of ochronotic pigmentation was clearly visible, being more pronounced in the subchondral zone. Specimen was from Patient 2 (Table I). (d) Chondrocyte aggregates or “clones” were found in great matrix lacunae. Clustered chondrocytes in the pigmented areas organized in clones are shown (arrows). Specimen was from Patient 3. Scale bar = 100 μm. (B) Photomicrograph showing the different patterns of ochronotic pigmentation in AKU cartilage. Ochronotic pigmentation was intense at the deep and middle cartilage areas. Scale bar = 100 μm. (a) Superficial zone showing the articular surface with matrix scarcely pigmented but presenting a defined area more ochronotic where dismorphic pigmented chondrocytes were random positioned; (b) Middle hypertrophic zone with dense ochronotic pigment in the extracellular matrix. Chondrocytes showed intracellular deposits and appeared phenotipically normal; (c) Deep zone showing intense pigment deposition in the extracellular matrix. Chondrocytes showed intracellular pigmentation, and some of them were chondroptotic. Their dimension was larger in volume than chondrocytes present in the hypertrophic zone. Resorption of the subchondral plate was also appreciable. (C) 4‐HNE detection in AKU, OA, and normal cartilage. (a) Healthy cartilage, as a negative control, from Control 12 (Table I); (b–d) AKU cartilage from Patient 1 (Table I); (e–g) AKU cartilage from Patient 2 (Table I); (h) OA cartilage, as a positive and reference control. Immuno‐fluorescence to 4‐HNE was relevant in all AKU cartilage specimens (b–g). Immuno‐specificity of the staining was assessed by the absence of fluorescence in the superficial zones of AKU cartilage (e–g) evidenced only by DAPI fluorescence (blue). The deep zone of cartilage surface, the pericellular matrix, the intracellular matrix, and the matrix around the cells, all showed positive staining which suggested the presence of strong lipid peroxidation in these areas. Scale bar = 100 μm.