Analysis of Demineralized Mammalian Bone Tissue by Fourier Transform Infrared Microscopy

Abstract

The amounts and properties of cartilage matrix components such as collagen and proteoglycans change in joint and bone related diseases, and spectroscopic studies of these components are important for developing new diagnostic and therapeutic methods. Infrared spectroscopy provides information on chromophores and vibrations related to characteristic molecules present in cells and tissues. In addition, infrared spectroscopy is a non-invasive and useful spectroscopy based analytical technique that does not use antibody labeling. In this study, we used decalcifi ed unstained specimens of canine mandibular bone and rat and rabbit tibia. The specimens were subjected to microscopic infrared spectroscopy, and analyzed by determining the infrared spectrum, with a focus on fi ber proteins (collagen) and carbohydrates (proteoglycans) present in the specimens. Normally, a large signal originating from the PO stretching mode of phosphate (PO 4 3-) is observed during measurements of the infrared absorption spectrum of bone sections; however, the above mentioned signal was not observed in our study as the samples used in our study were decalcifi ed. Instead, we observed signals from proteoglycans that are usually masked by signals originating from PO stretching and are diffi cult to observe. Measurements were performed on the same types of bone sections, and the results showed that the infrared spectrum varied greatly depending on the portion subjected to the measurements. However, in contrast, the results of the measurements conducted on the cartilage regions of rat and rabbit tibia coincided in terms of the amide I, amide II, and amide III bands, as well as the signal intensity ratio of the proteoglycan signal. Our fi ndings indicated that infrared spectrum patterns allowed the identifi cation of cartilaginous tissues in bone sections. By using this characteristic signal as a marker and carrying out two dimensional mapping on the cartilage part, for example, it is suggested that it can be applied to analysis of collagen distribution in demineralized fossil samples.