Optimizing osteochondral decalcification and histological processing in ovine joint models

Decalcification is a critical step in bone and cartilage histology that directly influences tissue preservation and staining quality. This study aimed to establish an efficient decalcification protocol for ovine stifle joint tissues while optimizing their morphological preservation and staining performance. Samples from the distal femoral condyles of four sheep were allocated to three experimental groups according to the decalcification protocol: 2% ethylenediaminetetraacetic acid (EDTA), 0.1% nitric acid, and a combination of both reagents. Samples were evaluated at different time points for mineral removal, preservation of cellular and tissue architecture, and staining quality using hematoxylin and eosin and Safranin O/Fast Green techniques. Histological preservation was assessed using the O'Driscoll scoring criteria. The 0.1% nitric acid protocol demonstrated the best balance between decalcification efficiency and tissue preservation, achieving satisfactory mineral removal within 48 h while maintaining cartilage and subchondral bone morphology. In contrast, EDTA requires longer decalcification periods (5-7 days), resulting in slower mineral removal and mild, progressive loss of cellular detail over time. The combined EDTA-nitric acid protocol promoted rapid decalcification but caused substantial tissue degradation, reducing histological quality. Histological evaluation confirmed that the preservation of hyaline cartilage architecture, chondrocyte morphology, extracellular matrix staining, and osteochondral organization was superior in the nitric acid group. These findings indicate that 0.1% nitric acid is a reliable and time-efficient protocol for decalcifying ovine osteochondral tissues, enabling adequate histological evaluation while preserving tissue integrity. This study provides a practical methodological reference for the histological processing of ovine joint tissues in experimental and translational research settings.