The 21st Century COE Program
Center of Excellence for Research and Education on Complex Functional Mechanical Systems

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Hiroshi Kamioka 博士 & Jenneke Klein-Nulend 教授 特別講演会

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Osteocyte Biology Using Fluorescent Probes

Dr. Hiroshi Kamioka (Okayama University)

Objectives: In order to understand the physiological function of bone, bioengineering analyses use the computational solution with anatomically detailed geometric models that also reflect biological function. Therefore, it is of significance to collect configurational and conditional data of bone as to cells, tissues, and organs for establishing geometric model. In this paper, we examined 3-dimensional reconstruction of bone network, cell-cell communication via bone network, responsibility of bone cells in response to mechanical stress, and the stiffness of bone cells to have the platform in virtual-bone domain. We applied fluorescent probes for the assessment of physiological response and phenotyping of the cells. Methods: Real bone-cell network was reconstructed from optically sliced images of calvaria bone cells. Cell-cell communication was examined with Fluorescence Recovery After Photobleaching (FRAP) analysis and microinjection methods. Real time analysis of calcium response in flow-treated bone cell was performed with Fluo-3 AM. The stiffness of the bone cells was measured with Anatomic Force Microscope (AFM). Results: The average total length of the processes, the average surface area, and the average volume of one osteocyte were 1070+145 um, 1509+113um2, and 394+49um3, respectively. Functional cell-cell communications via gap junctions were confirmed among the bone cells. Flow-induce intracellular calcium was seen in 32.4% of osteoblast population and 5.5% of osteocyte populations at 1.2 Pa of fluid shear stress. The stiffness of osteoblasts was higher than that of osteocytes. Conclusions: Fluorescent probe is useful tool for analyzing osteocyte physiology.

Cell Biology of Mechano-Adaptive Bone Remodeling

Prof. Jenneke Klein-Nulend (ACTA-Vrije Universiteit)

The capacity of bone tissue to adapt to changing mechanical demands is well documented, but how the cells of bone perform this task remains poorly understood. Over the last decade significant progress has been made in understanding how bone cells may sense and transduce mechanical signals derived from bone loading. These studies emphasize the role of osteocytes as the "professional" mechanosensory cells of bone, and the lacuno-canalicular network as the structure that mediates mechanosensing. The regulatory process of mechanical adaptation produces flow of interstitial fluid in the bone lacunar-canalicular network along the surface of osteocytes, which is likely the physiological signal for bone cell adaptive responses in vivo. As a result, the maintenance of a mechanically efficient architecture is likely to depend on a balance between the intensity and spatial distribution of the mechanical stimulus and the responsiveness of the bone cells. In addition, the alignment of secondary osteons along the dominant loading direction suggests that bone remodeling is guided by mechanical strain. This means that adaptation (Wolff's Law) takes place throughout life at each remodelling cycle. We propose that alignment during remodelling occurs as a result of different canalicular flow patterns around cutting cone and reversal zone during loading. The response of cultured bone cells to fluid flow includes prostaglandin synthesis and expression of inducible cyclooxygenase-2, an enzyme that mediates mechanical loading-induced bone formation in vivo. The response of osteocytes to fluid flow includes a rapid production of nitric oxide, and expression of endothelial nitric oxide synthase. Nitric oxide has been shown to mediate the mechanical effects in bone, leading to enhanced prostaglandin E2 release. Disruption of the actin-cytoskeleton abolishes the prostaglandin response to mechanical stress in osteocytes, suggesting that the cytoskeleton is involved in cellular mechanotransduction. These studies have increased our understanding of the cell biology underlying Wolff's Law. This may lead to new strategies for combating disuse-related osteoporosis, and may also be of use in understanding and predicting the long-term integration of bone-replacing implants.