Synchrotron phase-contrast microtomography reveals the impact of degeneration on the 3D structure of human articular cartilage

OBJECTIVE: To evaluate quantitative and qualitative cell and tissue properties of human articular cartilage using phase-contrast synchrotron microtomography to determine what biological information can be obtained in comparison to traditional histopathology. DESIGN: Human knee cartilage from 49 deceased donors without known osteoarthritis (ages 18-85 years, 25 females and 24 males) and 4 patients undergoing total knee replacement due to advanced osteoarthritis (ages 61-75 years, all female) were imaged at 2.75 µm voxel size using synchrotron-based x-ray phase-contrast microtomography. Chondrocytes were segmented using an adaptive thresholding algorithm and cell properties were analyzed in relation to their depth within the tissue. Segmentations were validated by acquiring higher-resolution (0.65 µm voxel size) tomographs of two donors. Properties derived from microtomography were compared to the OARSI grades obtained by Safranin-O histological staining. RESULTS: Tomographic images show both chondrocytes and degenerative features such as cell clustering and fibrillation. Consistent chondrocyte segmentation was achievable, although separation of clustered cells was limited by resolution. Cell density, cell angle and cell shape show consistent profiles along the cartilage depth and depth-dependent associations with OARSI grade. CONCLUSIONS: Synchrotron based phase-contrast microtomography provides fully three-dimensional (3D) data and allows for clear visualization of articular cartilage degeneration, e.g. fibrillation and cell clustering. Furthermore, when combined with automatic image processing this approach provides whole-volume quantitative information about 3D chondrocyte properties and their relation to degeneration. Additionally, the technique is suitable for combination with other imaging techniques and in-situ experiments as it does not require invasive sample preparation.