Optimization of imaging methods for an enhanced structural assessment of the tissues of the osteochondral unit

Imaging plays a pivotal role in the investigation of heterogeneous tissues such as articular cartilage, whose hierarchical organization underpins its unique mechanical properties and proper joint function. This aspect is particularly relevant for understanding the pathophysiology of degenerative diseases, including osteoarthritis. Accordingly, the present PhD thesis explores advanced X-ray imaging techniques combined with mechanical testing to enhance structural characterization in preclinical context. Given the intrinsic radiotransparency of cartilage, alternative X-ray approaches were assessed to improve contrast and structural detectability. The first part of the thesis provides a comprehensive overview of advanced X-ray imaging modalities for articular cartilage, including absorption-, refraction-, and scattering-based methods. Absorption-based techniques, notably contrast-enhanced imaging, improve visualization of internal structures but may affect tissue integrity. Refraction- and scattering-based methods offer quantitative information down to mesoscopic scale without contrast agents, although they require specific instrumentation. Among the reviewed approaches, propagation-based phase-contrast imaging emerged as the most promising for rheological investigations due to its relatively simple setup and short acquisition times. The second part focuses on the iodine-based cationic contrast agent CA4+ for quantitative imaging of proteoglycan content. Indentation tests demonstrated that CA4+ temporarily alters the mechanical response of cartilage, although immersion in saline solution promoted contrast agent resorption and recovery of mechanical properties. High-resolution peripheral quantitative computed tomography further showed that CA4+-enhanced imaging can predict mechanical behaviour through correlations with proteoglycan distribution. Building on these findings, CA4+ was integrated with detector-based spectral imaging, enabling energy-resolved density mapping of cartilage and bone and improved visualization of features such as the calcified cartilage layer, a key marker of degeneration. Finally, synchrotron-based propagation phase-contrast imaging was applied to visualize cartilage under compression, revealing radiation-induced stiffening effects dependent on imaging protocol parameters. Overall, this thesis demonstrates the significant potential of advanced X-ray techniques for high-resolution, minimally invasive, preclinical assessment of articular cartilage.