Papers by Keyword: Microbeam X-Ray Diffraction (XRD)

Abstract: The diagnosis of hard tissues is generally carried out by bone mineral density (BMD) measurement as a bone quantity parameter. BMD, however, does not necessarily explain bone fracture risks in some clinical cases. Recently, various parameters relating to bone strength have been investigated. These additional parameters, so-called bone quality, reflect intrinsic bone conditions. We have been studying the preferential alignment of the biological apatite (BAp) c-axis among various bone quality parameters. BAp, a dominant component of hard tissue, is an ionic crystal that crystallizes in a hexagonal lattice accompanied with the anisotropic property. In this article, we investigated the osteoclast role in the recovery process of BAp orientation during bone regeneration using osteopetrotic (op/op) mice in which the number of osteoclasts decreases. A surgically drilled, 500-μm diameter hole on each tibia of both control and op/op 8-week-old mice was introduced from the medial surface into the medullary cavity located at a 30% length from the proximal tibia end. After surgery, tibiae injuries were regularly observed by in situ micro-CT, and then the mice were sacrificed four to eight weeks after surgery. BAp orientation was analyzed in and near the regenerated portion by the microbeam X-ray diffraction system. As a result, we found the insufficient recovery of BAp orientation in spite of the apparent repair of bone appearance and quantity from CT images, even eight weeks after surgery in both cases of control and op/op mice. We conclude that this defective animal model can be used to evaluate bone quantity and quality at the cortical portion during bone regeneration in gene-defect mice in which the expression of bone cells is controlled, for example.

Abstract: 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)-K X-ray, which was collimated into a 300-m 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.

Abstract: Bone microstructure and its functions are maintained by the activity of bone cells such as osteoclast for bone resorption and osteoblast for bone formation. In this study, we examined the role of osteoclast on the formation of the preferential orientation of biological apatite (BAp) as a bone quality parameter using OPG-KO and op/op mouse models in which the expression of osteoclast increases for osteoporosis and decreases for osteopetrosis. The orientation degree of the BAp c-axis was analyzed by a microbeam X-ray diffraction system. We found more decrease in the preferential alignment of the BAp c-axis along the longitudinal direction of bone in the femoral bones of both OPG-KO and op/op mice at 12 weeks compared with normal control mice. We concluded that changes in the amount and activity of osteoclast affect BAp alignment, resulting in the degradation of bone microstructure in osteoporosis and osteopetrosis.

Abstract: Bone mechanical function is given as a result of the material and structural parameters of bone tissue. We previously reported that the material parameter of regenerated bone can be evaluated dominantly using two indices of the density and the preferred orientation degree of biological apatite (BAp). In addition, bone morphology remarkably changes during bone regeneration, which may lead to a dynamic change in the mechanical function of whole bone. In this study, therefore, material and structural parameters of regenerated bone are analyzed separately. A 5-mm-long defect was introduced in rabbit ulna and spontaneously regenerated, and then a three-point bending test was conducted at the regenerated portion. The important parameter which dominantly controls the whole bone mechanical function shifts from a structural to material parameter during bone regeneration. Moreover, it was statistically demonstrated that the increase in the material parameter is strongly determined by recovery of the orientation degree of the BAp c-axis.

Abstract: Since preferential orientation of c-axis of biological apatite (BAp) crystallites depends strongly on the shape of hard tissue, closely relating to the in vivo stress distribution, it is a useful parameter to judge the bone quality. In this study, preferential alignment of BAp crystallites in original and regenerated hard tissues were analyzed by the micro-beam X-ray diffractometer (μ-XRD) with a beam spot of 50 or 100 μm in diameter. Regenerating processes of bone defects introduced artificially in the rabbit ulna or skull were healed by inserting a biodegradable gelatin hydrogel incorporating basic fibroblast growth factor-2 (FGF-2). Recovery of BAp orientation alignment depends strongly on the regenerated portion and period, which is insufficient to recover the original level, while bone mineral density (BMD) is almost improved to the original level. This means that BMD recovers prior to improvement of the BAp orientation and the related mechanical function in the regenerated tissues. Thus, reloading on the regenerated portion caused by BMD restoration is suggested to accelerate to produce the appropriate BAp preferential alignment due to the remodeling process. The BAp orientation was finally concluded to be one of the most important indices to check the regenerative degree and process in the regenerated bone under the tissue engineering technique.

Abstract: A dominant inorganic substance in hard tissue is known to be a biological apatite (BAp)^nano-crystal which basically crystallizes in an anisotropic hcp lattice, and the BAp c-axis is parallel to extended collagen fibrils. We applied the microbeam X-ray diffractometer system with an incident beam spot 100µm or 50µm in diameter to the original, regenerated and pathological hard tissues in order to analyze the preferential alignment of the BAp c-axis as a parameter of bone quality closely relating to the mechanical function. We conclude that the BAp orientational distribution in the hard tissues is a new measure to evaluate stress distribution in vivo, nano-scale microstructure and the related mechanical function, healing process of the regenerated bone and progress of the bone diseases.