Orientation of biological apatite in rat calvaria analyzed by microbeam X-ray diffractometer

A microbeam X-ray diffractometer is a powerful tool to analyze oriented biological apatite (BAp) crystallites in bones since BAp orientation is one of the dominant controlling factors for bone mechanical function. The formation of BAp orientation seems to be partly affected by the bone formation process, including membranous or intracartilaginous ossification, the direction and the rate of bone growth, the mineral apposition rate, etc. However, the detailed process and the mechanisms of the organization of BAp orientation during the bone formation process are still not understood. In this study, we focused on a calvarial bone as a flat bone to establish a procedure to analyze BAp orientation in calvarial bone and examined the variation in BAp orientation with age and position in growing rats. Microbeam X-ray diffraction analysis was performed on the extracted calvaria of 5- to 10-week-old Wister rats. The transmission optical system was selected to analyze the orientation of the BAp c-axis along the bone surface. An incident molybdenum (Mo)-Kα X-ray, which was collimated into a 300-μm diameter, was vertically radiated on the calvaria surface, and the diffraction pattern was registered on an imaging plate. Diffraction peak intensities from the (002) and (310) planes of the hexagonal BAp were detected, and then an intensity ratio of (002)/(310) was calculated to evaluate the degree of BAp orientation. BAp orientation in a calvarial bone was successfully analyzed, and the two-dimensional distribution of the BAp c-axis along the calvarial bone surface was identified. A parietal bone, which is a part of the calvarial bone, showed a unique two-dimensional distribution of the BAp c-axis. The distribution remarkably changed depending on the position on a parietal bone and age. The anisotropy in the preferred BAp orientation was very significant at a region that showed high growth rate. Even though the bone formation process seems to affect BAp orientation in the parietal bone, further investigation is needed to understand the mechanism for the development of BAp texture, which is closely related to bone mechanical function.