Diffraction Enhanced X-ray Imaging of the Articular Cartilage of Experimental Animals

Introduction: The clinical techniques utilized in the assessment of the health status of articular cartilage of synovial joints include x-ray radiography, magnetic resonance imaging and ultrasound (1). Conventional radiography provides the highest spatial resolution (2) and is the most commonly used technique. However, because cartilage is not visible with standard x-rays, the radiographic evaluation of joint disease is based on joint space narrowing and the associated pathological bone changes, thus rendering radiography of diagnostic value only in advanced disease. We show, for the first time, that a novel x-ray technology, Diffraction Enhanced Imaging (DEI,) allows visualization of articular cartilage in both disarticulated and intact knee joints of experimental animals. DEI is an x-ray radiographic technique in its very early stages of development and biological application. It provides dramatic gains in contrast over conventional radiography by utilizing x-ray refraction and scatter rejection in addition to the absorption of conventional radiography. Here we show that both normal and damaged articular cartilage of animal knee joints can be imaged through DEI and that the contrast heterogeneities observed in the resultant images can be verified as cartilage damage through both gross visual and histological examination. Materials and Methods: The adolescent New Zealand white rabbits used for this study were obtained with IACUC approval. The rabbits had a control knee in which no damage was induced, or an experimental knee in which injury was carried out by intra-articular injection of a sterile solution of 3.0 mg of the chymopapain activated by sodium L-cysteinate hydrochloride in 0.3 ml sterile saline (3). The animals were sacrificed one week post injection, the intact knee joints with all surrounding soft tissues were x-rayed, DE imaged and then opened for gross examination and further imaging. The DEI technique (4,5) was carried out on the knee joint specimens at the X15 beam line at the National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY. Briefly, an imaging beam is prepared by diffracting the polychromatic beam from the synchrotron with two matching crystals to create a nearly monochromatic, highly collimated beam of a single x-ray energy. This beam is then passed through the subject as in conventional radiography. In DEI, however, a third crystal (analyzer crystal) is placed between the subject and the image detector or radiographic plate. X-ray diffraction from a perfect crystal, with its narrow reflection angular width provides the tools necessary to prepare and analyze x-ray beams traversing an object on the microradian scale (Zachariasen, 1945). The condition for x-ray diffraction from a crystal is met only when the incident beam makes the correct angle to the atomic lattice planes in the crystal for a given x-ray photon energy or wavelength. If the analyzer crystal is rotated a few microradians in the horizontal direction through the Bragg condition for diffraction, an image profile will be obtained as a result of scatter rejection at the microradian level. The character of the images obtained changes according to the setting of the analyzer crystal. In this study, both 18 keV and 30 keV energy levels were used for a range of between 2 and 6 seconds exposure time. The actual resultant x-ray dosage ranged from 300 to 750 mR All images were recorded on CD58 maxi speed x-ray film. For comparison, all specimens were also radiographed in the standard x-ray mode without the DEI technique. After imaging, the specimens were fixed in paraformaldehyde and prepared for histology. The DE images were subsequently compared to the gross visual and histological appearance of the specimens. Results: We have applied DEI to both disarticulated and intact rabbit knee joints with the outcome of visualization of the articular cartilage. Imaging in the standard radiography mode in the absence of of the DEI technique resulted in no visualization of articular cartilage. The figure to the right is a DE image of a femoral condyle from a rabbit that had previously been injected with chymopapain to induce cartilage damage. The articular cartilage of both condyles is visible (between the white arrows) and it is of interest that even the cartilage of the condyle that is superimposed on the other is visible. The cartilage of the higher condyle has an intact surface and homogeneous appearance except at a focal point of contrast heterogeneity (arrowhead). Upon histological examination, it was shown that this focal contrast heterogeneity corresponded to a site of cartilage degeneration as a result of chymopapain injection. In all cases, the DE images were representative of the gross visual and histological appearance of the specimens in terms of surface integrity and correlation of degeneration to image contrast heterogeneity. DE images of intact knee joints also allowed visualization of the articular cartilage even with the superimposition of surrounding soft tissues such as tendons, ligaments, skin and hair.