Comparative Assessment of Primary Osteoarthritis Progression Using Conventional Histopathology, Polarized Light Microscopy, and Immunohistochemistry

Abstract

Objective: Evaluation of collagen orientation and arrangement in articular cartilage can improve our understanding of primary osteoarthritis (OA) progression and targeted therapies. Our goal was to determine if polarized light microscopy (PLM) for collagen organization is useful in identifying early primary OA features in comparison to current standard histopathological methods.

Design: Osteochondral specimens from 90 total knee arthroplasty patients with relatively preserved lateral femoral condyle were scored using (1) histological-histochemical grading system (HHGS); (2) Osteoarthritis Research Society International (OARSI); (3) PLM-Changoor system for repair cartilage, scores ranging between 0 (totally disorganized cartilage) and 5 (healthy adult cartilage); and (4) new PLM system for primary OA cartilage with superficial zone PLM (PLM-SZ) and deep zone PLM (PLM-DZ) scores, each ranging between 0 (healthy adult SZ and DZ collagen organization) and 4 (total loss of collagen organization). Serial sections were stained for collagen I and II antibodies. Spearman correlation coefficients (r_s) were determined.

Results: The associations between: (1) PLM-Changoor and HHGS or OARSI were weak (r_s = -0.36) or moderate (r_s = -0.56); (2) PLM-SZ and HHGS or OARSI were moderate (r_s = 0.46 or r_s = 0.53); and (3) PLM-DZ and HHGS or OARSI were poor (r_s = 0.31 or r_s = 0.21), respectively. Specimens exhibiting early and mild OA (HHGS < 5 and OARSI < 8.6) had PLM-SZ and PLM-DZ scores between 0 and 4 and between 0 and 3, respectively, and indicated new histopathological features not currently considered by HHGS/OARSI.

Conclusions: PLM was effective at identifying early SZ and DZ collagen alterations that were not evident in the traditional scoring systems. Incorporating PLM scores and/or additional HHGS/OARSI features can help improve characterization of early primary OA cartilage.

Keywords: cartilage; histopathology; osteoarthritis; polarized light microscopy.

Figure 1.

Lateral femoral condyles (LFC) were obtained from varus knee total knee arthroplasty patients for assessment in the study. (A) A schematic representation of the LFC is shown. Orientation of each sample was noted (A, anterior; P, posterior; L, lateral; M, medial). For each LFC, the 3 central arches were subdivided into multiple specimens and the cartilage specimen marked with the cross was collected for histology. (B) Human LFC is systematically subdivided into multiple specimens ~4 mm × 4 mm in the operating room under sterile conditions using a custom-built miter box and stainless-steel hand saw (0.25 mm kerf). The specimen marked with a cross is used for histology. (C) Representative image of a cartilage specimen (~4 mm × 4 mm) used for histology.

Figure 2.

Joint plots showing distribution of traditional histopathological scores (HHGS, OARSI) and polarized light microscopy (PLM) score for repair cartilage (PLM-Changoor) for 90 osteochondral specimens obtained from lateral femoral condyle. (A) HHGS vs OARSI average scores show a strong Spearman correlation coefficient and the histogram shows data distribution with median HHGS score = 5 and median OARSI score = 4.9. (B) PLM-Changoor versus HHGS average scores indicated a poor Spearman correlation coefficient and the histogram shows data distribution with median PLM-Changoor score = 3. (C) PLM-Changoor versus OARSI average scores indicated a moderate Spearman correlation coefficient. HHGS, histological-histochemical grading system; OARSI, Osteoarthritis Research Society International.

Figure 3.

Joint plots showing distribution of traditional histopathological scores (HHGS, OARSI) and polarized light microscopy (PLM) score for primary osteoarthritis cartilage (PLM-SZ and PLM-DZ) for 90 osteochondral specimens obtained from lateral femoral condyle. (A) HHGS versus PLM-SZ average scores show a moderate spearman correlation coefficient and the histogram shows data distribution with median HHGS score = 5 and median PLM-SZ score = 3. (B) OARSI versus PLM-SZ average scores show a moderate spearman correlation coefficient and the histogram shows data distribution with median OARSI score = 4.9. (C) HHGS versus PLM-DZ average scores show a poor Spearman correlation coefficient and the histogram shows data distribution with median PLM-DZ score = 2. (D) OARSI versus PLM-DZ average scores show a poor Spearman correlation coefficient. HHGS, histological-histochemical grading system; OARSI, Osteoarthritis Research Society International; SZ, superficial zone; DZ, deep zone.

Figure 4.

Representative human cartilage specimens obtained from lateral femoral condyle displaying traditional human osteoarthritis (OA) progression features (OA degradation progressing from surface down) as seen in hematoxylin and eosin (HE) (column 1: A1-E1), safranin-O/fast green (SafO-FG) (column 2: A2-E2), unstained polarized light microscopy (UNS-PLM) (column 3: A3-E3), bright-field images for collagen II (Col II-BF) (column 4: A4-E4) and bright-field images for collagen I (Col I-BF) immunohistochemical staining (column 5: A5-E5) at 5× magnification. Scale bar represents 1 mm. (A) Best-quality cartilage (HHGS total score: 2; structure score = 0, cell score = 1, safranin-O staining score = 0, tidemark integrity score = 1). (B) Cartilage with surface irregularities (HHGS total score: 3; structure score = 1, cell score = 0, safranin-O staining score = 1, tidemark integrity score = 1). (C) Cartilage with clefts to transitional zone (HHGS total score: 6; structure score = 3, cell score = 1, safranin-O staining score = 1, tidemark integrity score = 1). (D) Cartilage with multiple clefts through transitional zone (HHGS total score: 7; structure score = 3, cell score = 2, safranin-O staining score = 2, tidemark integrity score = 0). (E) Cartilage with multiple clefts through radial zone (HHGS total score: 8; structure score = 4, cell score = 2, safranin-O staining score = 2, tidemark integrity score = 0). HHGS, histological-histochemical grading system.

Figure 5.

Higher magnification (10×) of the boxed regions shown in Figure 4 to illustrate the osteoarthritic features in detail. Box regions near the superficial zone (top region) for unstained polarized light microscopy, collagen II, and collagen I cartilage sections are shown in columns 1, 2, and 3. Box regions near the deep zone (bottom region) for unstained polarized light microscopy, collagen II, and collagen I cartilage sections are shown in columns 4, 5, and 6. Scale bar represents 200 µm. Note: # indicates Col I-stained regions near the crevice.

Figure 6.

Representative human cartilage specimens displaying normal cartilage structure but exhibiting varying patterns of slight safranin-O stain loss as seen under hematoxylin and eosin (HE) (column 1: A1-D1), safranin-O/fast green (SafO-FG) (column 2: A2-D2), unstained polarized light microscopy (UNS-PLM) (column 3: A3-D3), bright-field images for collagen II (Col II-BF) (column 4: A4-D4), and bright-field images for collagen I (Col I-BF) immunohistochemical staining (column 5: A5-D5) at 5× magnification. Scale bar represents 1 mm. (A) Cartilage with interterritorial matrix loss and territorial matrix loss (HHGS total score: 4; structure score = 1, cell score = 1, safranin-O staining score = 1, tidemark integrity score = 1). (B) Cartilage with interterritorial matrix loss near tidemark (HHGS total score: 4; structure score = 1, cell score = 1, safranin-O staining score = 1, tidemark integrity score = 1). (C) Cartilage with necrotic matrix in the radial zone (HHGS total score: 5; structure score = 1, cell score = 3, safranin-O staining score = 1, tidemark integrity score = 0). (D) Cartilage with territorial matrix loss (HHGS total score: 5; structure score = 1, cell score = 2, safranin-O staining score = 1, tidemark integrity score = 1). HHGS, histological-histochemical grading system.

Figure 7.

Higher magnification (10×) of the boxed regions shown in Figure 6 to illustrate the osteoarthritic features in detail. Box regions of interest from the cartilage presented in Figure 6 (SafO-FG staining features), as seen under safranin-O/fast green (column 1 SafO-FG), unstained polarized light microscopy (column 2 UNS-PLM), collagen II immunohistochemical staining (column 3 Col II-BF), and collagen I immunohistochemical staining (column 4 Col I-BF). Scale bar represents 200 µm. Note that since the staining was performed on different sections of cartilage, sometimes overlapping of same regions on different sections was not possible. Asterix indicates inter-territorial matrix loss; star indicates territorial matrix loss; arrow indicates matrix necrosis; # indicates some of the Col I stained regions).

Figure 8.

Representative human cartilage specimens displaying normal cartilage structure but exhibiting varying patterns of modifications near tidemark as seen under hematoxylin and eosin (HE) (column 1: A1-D1), safranin-O/fast green (SafO-FG) (column 2: A2-D2), unstained polarized light microscopy (UNS-PLM) (column 3: A3-D3), bright-field images for collagen II (Col II-BF) (column 4: A4-D4), and bright-field images for collagen I (Col I-BF) immunohistochemical staining (column 5: A5-D5) at 5× magnification. Scale bar represents 1 mm. (A) Cartilage with multiple tidemarks (HHGS total score:4; structure score = 1, cell score = 2, safranin-O staining score = 1, tidemark integrity score = 1). (B) Cartilage with unidentified matrix deposition near tidemark (HHGS total score: 6; structure score = 3, cell score = 1, safranin-O staining score = 1, tidemark integrity score = 1). (C) Cartilage with multiple tidemarks breached by blood vessels (HHGS total score: 6; structure score = 1, cell score = 1, safranin-O staining score = 3, tidemark integrity score = 1). (D) Cartilage with cartilage-bone-cartilage interface (HHGS total score: 5; structure score = 1, cell score = 1, safranin-O staining score = 2, tidemark integrity score = 1). HHGS, histological-histochemical grading system.

Figure 9.

Higher magnification (10×) of the boxed regions shown in Figure 8 to illustrate the osteoarthritic features in detail. Box regions of interest from the cartilage presented in Figure 8 (tidemark features), as seen under hematoxylin and eosin (column 1 HE), unstained polarized light microscopy (column 2 UNS-PLM), collagen II immunohistochemical staining (column 3 Col II-BF), and collagen I immunohistochemical staining (column 4 Col I-BF). Scale bar represents 200 µm. Note: Hash indicates some of the Col I stained regions.