Observation of Kinetic Response of Mineral and Collagen Phases in Bone by X-ray Diffraction

A bone has a hierarchical structure as shown in Fig. 1 [1]. Macroscopic bone properties are dependent on the microstructure and properties of structural elements. The bone is a composite material that consists of hydroxyapatite particles in a matrix of collagen fiber. Borsato et al. [2] measured the strain of hydroxyapatite by X-ray diffraction and obtained clear evidence of the stress concentration on hydroxyapatite in the bone. Sasaki et al. [3] reported the stress-strain curve for tendon collagen by X-ray diffraction. However, the relationship between the mechanical behavior of hydroxyapatite and that of collagen has been unclarified. In this study, the microscopic mechanical behavior of bone as a composite material was examined by measuring the strains of hydroxyapatite and collagen by wide-angle X-ray diffraction and small-angle X-ray scattering, respectively. Twelve cortical bone specimens with an average size of 40 × 5 × 0.5 mm3 were cut out from the middiaphysis of the bovine femur using a diamond cutter. The longer edges of the specimen were parallel to the longitudinal axis of the femur. Before the experiments, the specimens were preserved in a saline solution. X-ray energy was 15.0 keV at beamline BL40XU. The experimental setup is shown in Fig. 2. The X-ray path length was about 9 cm in wide-angle diffraction for hydroxyapatite (Fig. 2(a)), and 3 m in small-angle scattering for collagen (Fig. 2(b)). The X-ray diffraction pattern was recorded with an X-ray image intensifier (Hamamatsu Photonics, V5445P) and a fast CCD camera (Hamamatsu Photonics, C4880-80). The time resolution was 30 frames/sec. The specimens were subjected to a load using tensile loading apparatus. The tensile loading was synchronized with the X-ray shutter and CCD camera. Figures 3(a) and 3(b) show the images of X-ray diffraction from hydroxyapatite and collagen in the bone, respectively. In the wide-angle diffraction from hydroxyapatite in the bone, diffractions from (211) and (002) are strong (Fig. 3 (a)). As the (002) plane of hydroxyapatite in the bone has a preferred orientation in the bone axial direction, most of the c-axis of hydroxyapatite crystals is parallel to the bone axial direction. In the small-angle scattering from collagen in the bone, diffraction from d = 22.6 nm is strong (Fig. 3 (b)). The small-angle scattering pattern also contains a diffuse scattering perpendicular to the bone axial direction. Matsushima et al. [4] reported that this anisotropic diffuse scattering can be explained by the Fig. 1. Hierarchical structure of bone. Cancellous bone