Contrast-enhanced x-ray microscopy of bovine articular cartilage

Osteoarthritis (OA) is a common chronic disease of joints typically characterized by degenerative changes of articular cartilage, which is comprised of chondrocytes embedded in a composite of water-imbibing proteoglycans restrained by fibrillar collagen network. Early diagnosis of OA requires sensitive imaging, ideally at the cellular-molecular level. Whereas cartilage histopathology is destructive, time-consuming and limited to 2D views, contrast-enhanced x-ray microscopy (XRM) brings the possibility to non-destructively image the cartilage collagen network in 3D at high resolution. This study establishes a correlation between contrast-enhanced XRM and the gold-standard histology for the evaluation of the cartilage collagen network. Cartilage with subchondral bone was excised in 3 x 3 mm 2 cross-sectional area from healthy bovine knees and stained in phosphotungstic acid (PTA) for 0, 4, 8, 12, 16, 20, 24, 28 and 32 hours. XRM imaging was performed after each staining time, analyzed and determined an optimal staining time of 16 hrs and a saturated staining time of 24 hrs for this sample. Polarized light microscopy and second harmonic generation dual-photon microscopy of a histology section from the same sample were analyzed and compared with the matching XRM slice. Cartilage collagen network from PTA-enhanced XRM was well correlated with histology. We validated the PTAenhanced XRM for the evaluation of the cartilage collagen network non-destructively. The 3D cartilage volume from this technique will provide a non-destructive approach to investigate OA pathology.