Multi-laboratory Validation of Join of the Five Dyes Revealing Collagenous Tissue (JFRL) Staining for Bone Histology Across Animal Species and Bone Implant Materials

Histological staining is essential for understanding bone structure and pathology; however, variations in decalcification agents can compromise reproducibility. We have previously developed a novel osteochondral staining method, Join of the Five dyes Revealing coLlagenous tissue (JFRL) staining, that is independent of the decalcification method. To promote its widespread adoption, this study confirms the robustness of JFRL staining through intra- and inter-laboratory validation. JFRL staining demonstrated consistent patterns across different manufacturers and facilities, with proper dehydration steps being crucial for optimal results. We applied JFRL staining to diverse vertebrate species prepared under various fixation and decalcification conditions to effectively visualize species-specific bone structures, including distinct osteoid and mineralized bone features from fish to large mammals. Furthermore, JFRL staining proved useful in evaluating bone biomaterials within defect models and clearly depicts the complex architecture of bone-healing processes and material integration. The staining qualitatively distinguished osteoid, mineralized bone, hyaline cartilage, and bone cells of different colors across all applications. These findings establish JFRL staining as a robust and versatile method for bone histology. Future studies focusing on quantitative assessment and pathological applications will prove that JFRL staining presents a reliable tool for both basic research and clinical diagnostics of bone disorders.