Second Harmonic Generation Imaging Links Collagen Structure and Biochemical Composition in Human Osteoarthritic...

Articular cartilage is a highly hydrated, collagen-rich tissue that plays a critical role in joint function. Early degenerative changes in osteoarthritis (OA) are associated with alterations in collagen organization that precede gross structural damage; however, current imaging modalities, including radiography, computed tomography, and magnetic resonance imaging, often lack sufficient sensitivity to detect these microstructural changes. Second harmonic generation (SHG) imaging is a label-free optical technique sensitive to collagen microstructure. Previously, we developed an image-based parameter, the volumetric fraction of organized collagen (Φ_col), which strongly correlates with cartilage mechanical properties. In this study, we investigated whether Φ_col reflects collagen-related biochemical composition in human OA cartilage. Osteochondral samples (n = 29 blocks, 81 sections) were obtained from tibial plateaus of 11 patients undergoing total knee arthroplasty. SHG imaging was performed on superficial cryosectioned cartilage (~100-150 µm from the articular surface) to quantify Φ_col, followed by biochemical hydroxyproline assays to estimate collagen content normalized to wet tissue weight. We observed a robust positive correlation between Φ_col and hydroxyproline content (Spearman's ρ = 0.95, p < 0.05), with both measures decreasing progressively with QP-derived tissue degeneration grade. Tissue wet weight exhibited a moderate negative correlation with hydroxyproline content (ρ =  - 0.49, p < 0.05). These findings indicate that SHG-derived Φ_col is strongly associated with collagen-related structural and compositional features within the superficial cartilage layer in this standardized ex vivo imaging workflow. While the study is limited to ex vivo tissue sections from osteoarthritic joints and does not establish clinical feasibility, the results provide quantitative validation of SHG-derived metrics that may support the future development of non-destructive imaging approaches for cartilage assessment, which is currently not available.