International Cartilage Repair Society (ICRS) Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials

Abstract

Cartilage repair strategies aim to resurface a lesion with osteochondral tissue resembling native cartilage, but a variety of repair tissues are usually observed. Histology is an important structural outcome that could serve as an interim measure of efficacy in randomized controlled clinical studies. The purpose of this article is to propose guidelines for standardized histoprocessing and unbiased evaluation of animal tissues and human biopsies. Methods were compiled from a literature review, and illustrative data were added. In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points. In human clinical therapy, treatments are applied to developed lesions, and biopsies are obtained, usually from a subset of patients, at a specific time point. In striving to standardize evaluation of structural endpoints in cartilage repair studies, 5 variables should be controlled: 1) location of biopsy/sample section, 2) timing of biopsy/sample recovery, 3) histoprocessing, 4) staining, and 5) blinded evaluation with a proper control group. Histological scores, quantitative histomorphometry of repair tissue thickness, percentage of tissue staining for collagens and glycosaminoglycan, polarized light microscopy for collagen fibril organization, and subchondral bone integration/structure are all relevant outcome measures that can be collected and used to assess the efficacy of novel therapeutics. Standardized histology methods could improve statistical analyses, help interpret and validate noninvasive imaging outcomes, and permit cross-comparison between studies. Currently, there are no suitable substitutes for histology in evaluating repair tissue quality and cartilaginous character.

Keywords: animal models; articular cartilage; biopsy; cartilage repair; collagen type I; collagen type II; fibrocartilage; glycosaminoglycan; histology; polarized light microscopy; subchondral bone; tidemark.

Figure 1.

Different features of the osteochondral junction in normal and repair cartilage are revealed by hematoxylin and eosin (H & E) (A, C, E, G) and Safranin O/fast green/iron hematoxylin (SafO) (B, D, F, H). In normal human cartilage (A and B, adult hip surgical waste, femoral neck fracture), H&E clearly stains the tidemark (A, white arrows), while SafO readily discriminates cartilage from fast green–stained bone (below the black arrows, B). For heterogeneous human repair cartilage (C and D, biopsy taken 1 year postmicrofracture^,), H&E is better for determining the cartilage-bone boundary (black arrows, C) and abnormal mineralization (dashed circle), while SafO discriminates fibrocartilage from fast green–stained fibrous repair and bone (D). In hyaline cartilage repair elicited in a sheep model (E-H, 6 months posttreatment), the tidemark is beginning to form (white arrows, 10x magnification for E and F, 40x magnification for G and H). White arrows = tidemark; black arrows = cartilage-bone interface; AC = articular cartilage; cc = calcified cartilage; FC = fibrocartilage; HC = hyaline cartilage; b = bone.

Figure 2.

Appearance of human 2-mm-diameter biopsy obtained with a Jamshidi 11-gauge needle (A, C) and corresponding decalcified Safranin O–stained paraffin section (B, D). Samples were obtained ex vivo with an ethics-approved protocol from the same lateral condyle (nonlesional area) obtained after total knee arthroplasty (74-year-old female). (A and B) A biopsy cored perpendicular to the surface and (C and D) a biopsy cored deliberately at an oblique angle to the surface are shown. Both biopsies were initially 6 mm long, but the subchondral bone was missing from the oblique biopsy prior to histoprocessing (C). Part of the subchondral bone in the perpendicular biopsy (B) was lost during histoprocessing.

Figure 3.

Histoprocessing and histomorphometry of large animal defects. The example is taken from a sheep cartilage repair model (6 months repair). In this unilateral cartilage repair model, the repaired defect (top panels) was decalcified, trimmed at 2 levels in the defect (midproximal and middistal), and stained with Safranin O/Fast Green. Repair tissue above the projected tidemark was cropped using histomorphometric software, and total area (TA) and total stained repair tissue area (TS) were used to determine percentage of Safranin O–stained repair. The contralateral intact condyle was decalcified, trimmed through the middle, and cropped with matching defect width, and total area was used to determine percentage fill of the defect with repair tissue.

Figure 4.

Example of standardized histoprocessing to evaluate a human biopsy (A-D, from cadaveric knee medial femoral condyle) or sheep hyaline repair cartilage 6-month repair after treatment with microfracture and chitosan-GP/blood implant (E-H). Sections were stained for Safranin O, immunostained for collagen type II and collagen type I, and observed by polarized light microscopy (PLM). SZ = superficial zone; DZ = deep zone; AC = articular cartilage. Note the abnormal vascular invasion and mineralization (*) in this particular human biopsy above the tidemark (horizontal arrow, A-D), which is frequently observed in osteoarthritis.

Figure 5.

Histomorphometry of chondral versus subchondral soft repair tissues. The example is from a 2-month repair of a trochlear full-thickness rabbit knee defect with two 0.9-mm microdrill holes. (A) Safranin O–stained trochlear repair tissue, with the “projected tidemark” drawn through the defect area. (B) The chondral repair is cropped separately from the subchondral soft tissue repair for further histomorphometric analysis.