Comparative assessment of the morphology and antigenicity of human osteochondral units using formalin and coagulant fixatives

Fixation is crucial for preserving tissue integrity during processing, and the most commonly used cross-linking fixative in immunohistochemistry is neutral buffered formalin, which requires antigen retrieval as a crucial step. The successful use of newer coagulant fixatives like methacarn and EMA to preserve isotopes and eliminate the need for antigen retrieval has been reported recently, but their role in decalcified osteochondral unit samples remains unknown. Limited information on the use of coagulant fixatives as formalin substitutes makes it important to comparatively evaluate their effects on osteochondral samples and the impact of antigen retrieval on different days. Osteochondral units from a patient with osteoarthritis who underwent total knee-replacement surgery were fixed with three studied fixatives (Formalin, Methacarn, EMA) and divided into four portions, for different time periods (Day 1, 3, 7, 10). Sections were decalcified, stained with Safranin O, Alcian Blue, Toluidine Blue, PicroSirius Red, Hematoxylin and Eosin, and immunohistochemical analysis of Collagen type II and type X with and without antigen retrieval was conducted. Formalin showed better hematoxylin uptake than coagulant-based fixatives, while all fixatives preserved tissue morphology without necrosis or cellular degeneration with comparable staining quality. Methacarn and EMA-fixed tissues showed higher uptake of collagen type II compared to formalin-fixed tissues, with collagen type II uptake occurring only in the cartilage region and collagen type X uptake occurring only in the bone region. The study highlights the effectiveness of methacarn and EMA as efficient alternatives to formalin, preserving tissue morphology and antigen specificity.