Synchrotron infrared microspectroscopy as a means of studying the chemical composition of bone: Applications to osteoarthritis

Infrared microspectroscopy combines microscopy and spectroscopy for the purpose of chemical microanalysis. Light microscopy provides a way to generate and record magnified images and visibly resolve microstructural detail. Infrared spectroscopy provides a means for analyzing the chemical makeup of materials. Combining light microscopy and infrared spectroscopy permits the correlation of microstructure with chemical composition. Inherently, the long wavelengths of infrared radiation limit the spatial resolution of the technique. However, synchrotron infrared radiation significantly improves both the spectral and spatial resolution of an infrared microspectrometer, such that data can be obtained with high signal-to-noise at the diffraction limit, which is 3-5 mu m in the mid-infrared region. In the knee joint of cynomolgus monkeys, osteoarthritis is characterized morphologically by fibrillation and clefting of the articular cartilage and marked thickening of the adjacent subchondral bone. Subchondral bone changes appear to precede the articular cartilage lesions and there is a strong correlation between the severity of the articular cartilage lesions and the thickness of the subchondral bone. It is possible that the newly-deposited subchondral bone has a different chemical structure and/or composition compared to the bone that was previously present, thereby influencing the development of the articular cartilage lesions. In this study, we use infrared microspectroscopy to study the chemical composition of the subchondral bone using two mapping methods. Using the transverse method, we are able to map the subchondral bone from the edge of the articular cartilage (older bone) to the marrow space (newer bone) and compare their chemical compositions. With the osteon method, linear maps are collected from the center of a subchondral osteon (newer bone) to the periphery (older bone). A significant advantage of this technique over other chemical methods is that the bone does not need to be homogenized for testing; we are able to study cross-sectional samples of bone in situ at a resolution better than 5 mu m and compare the results with morphological findings on stained serial sections immediately adjacent to those examined by infrared microspectroscopy. The infrared absorption bands of bone proteins and mineral are sensitive to mineral content (i.e. carbonate, phosphate, acid phosphate), mineral crystallinity, and the content/nature of the organic matrix. In this study, they are integrated and compared as a function of (1) morphology of the bone, i.e. thickness of the subchondral plate, and (2) age of bone, i.e. position in the subchondral plate. Results show that the protein/mineral ratio is higher in younger bone. As bone matures, mineralization increases, as does carbonate substitution into the hydroxyapatite lattice. Finally, most of the changes in chemical composition of bone occur within 20 mu m of the site of new bone growth, e.g. the center of an osteon, demonstrating the need for the high spatial resolution achieved only with the use of a synchrotron infrared source.