Zonal Characteristics of Collagen Ultrastructure and Responses to Mechanical Loading in Articular Cartilage.

The biomechanical properties of articular cartilage arise from a complex bioenvironment comprising hierarchically organised collagen networks within the extracellular matrix (ECM) that interact with the proteoglycan-rich interstitial fluid. This network features a depth-dependent fibril organisation across different zones. Understanding how collagen fibrils respond to external loading is key to elucidating the mechanisms behind lesion formation and managing degenerative conditions like osteoarthritis. This study employs polarisation-resolved second harmonic generation (pSHG) microscopy to quantify the ultrastructural organisation of collagen fibrils and their spatial gradient along the depth of bone-cartilage explants under a close-to-in vivo condition. By combining with in-situ loading, we examined the responses of collagen fibrils by quantifying changes in their principal orientation and degree of alignment. The spatial gradient and heterogeneity of collagen organisation were captured at high resolution (1 μm) along the longitudinal plane of explants (0.5 mm by 2 mm). Zone-specific ultrastructural characteristics were quantified to aid in defining zonal borders, revealing consistent zonal proportions with varying overall thicknesses. Under compression, the transitional zone exhibited the most significant re-organisation of collagen fibrils. It initially allowed large deformation through the re-orientation of fibrils, which then tightened fibril alignment to prevent excessive deformation, indicating a dynamic adaptation mechanism in response to increasing strain levels. Our results provide comprehensive, zone-specific baselines of cartilage ultrastructure and micromechanics, crucial for investigating the onset and progression of degenerative conditions, setting therapeutic intervention targets, and guiding cartilage repair and regeneration efforts. STATEMENT OF SIGNIFICANCE: Achieved unprecedented quantification of the spatial gradient and heterogeneity of collagen ultrastructural organisation at a high resolution (1 μm) along the full depth of the longitudinal plane of osteochondral explants (0.5 mm by 2 mm) under close-to-in vivo condition. Suggested new anatomical landmarks based on ultrastructural features for determining zonal borders and found consistent zonal proportions in explants with different overall thicknesses. Demonstrated that collagen fibrils initially respond by reorienting themselves at low strain levels, playing a significant role in cartilage deformation, particularly within the transitional zone. At higher strain levels, more collagen fibrils re-aligned, indicating a dynamic shift in the response mechanism at varying strain levels.