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Osteoclasts play critical roles not only in normal bone homeostasis ('remodeling') , but also in the pathogenesis of bone destructive disorders such as osteoporosis, rheumatoid arthritis, and bone metastasis. However, it has not been known how osteoclast precursor monocytes migrate into the bone surface and what controls their migratory behaviors. To reveal these systems, we have recently established a new system for visualizing intact bone tissues and bone marrow cavities in live animals by using an advanced imaging technique with intravital two-photon microscopy. By means of the system we have revealed that sphingosine-1-phosphate (S1P) , a lipid mediator, dynamically regulates migration and localization of osteoclasts and their precursors in vivo . Here we show the latest data and the detailed methodology of intravital imaging of bone tissues, and also discuss its further application.

Sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. Through intravital multiphoton imaging of bone tissues, we reveal that the bidirectional function of S1P temporospatially regulates the migration of osteoclast precursors within intact bone tissues. Imaging technologies have enabled in situ visualization of the behaviors of several players in intact tissues. In addition, intravital microscopy has the potential to be more widely applied to functional analysis and intervention.

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint synovial inflammation and progressive cartilage/bone destruction. Recently it has been reported that Th17, a CD4+ T cell subset expressing RANKL on its cell surface, increases in the synovium of RA patients and enhances bone destruction associated with osteoclasts. However, the mechanism of Th17-mediated control of mature osteoclast function in vivo has remained unclear. Objectives This study aimed to investigate how the bone-resorptive functions of osteoclasts are regulated in situ and how Th17 cells control osteoclastic bone resorption in vivo. Methods To visualize fluorescently-labeled mature osteoclasts, we utilized the mice in which GFP is expressed as a fusion protein with a vacuolar type H+-ATPase “a3 subunit”, preferentially and abundantly expressed in mature osteoclasts (a3-GFP mice) [1]. Polyclonal Th17 cells were differentiated in vitro using anti-IL-4, anti-IFNγ, IL-6, TGFβ and IL-23, labeled with fluorescent dye and then adoptively transferred into a3-GFP mice before imaging. Calvaria bone tissues of a3-GFP mice were observed by using an advanced imaging system for visualizing live bone tissues with intravital multiphoton microscopy that we have originally established [2,3]. Results We have succeeded in visualization of live mature osteoclasts on the bone surface in situ. Because GFP is expressed as a fusion protein with a3 subunit, GFP fluorescence does not only serve as a marker for mature osteoclasts, but it provides information on the subcellular distribution of V-ATPase in osteoclasts. We identified different populations of live mature osteoclasts, i.e., “static – bone resorptive (R)” and “moving – non resorptive (N)”. Treatment with recombinant RANKL or bisphosphonate (risedronate) changed the composition of these populations as well as total number of mature osteoclasts. We also found that rapid RANKL injection converted the moving (N) osteoclasts to static (R) ones without any changes in total number of osteoclasts, suggesting a novel action of RANKL in controlling mature osteoclast function. Furthermore, we showed that Th17 cells could induce rapid N to R conversion of mature osteoclasts by RANKL expressed on their cell surface. Conclusions By visualizing in vivo behaviors of mature osteoclasts, we for the first time identified different populations of live osteoclasts on the bone surface, from “static – bone resorptive” to “moving – non resorptive”. Furthermore, RANKL turned out not only to promote the differentiation of osteoclasts but also to regulate the bone-resorptive function of fully differentiated mature osteoclasts. RANKL-bearing Th17 cells were shown to control bone resorption of mature osteoclasts, demonstrating novel actions of Th17 that may be a novel therapeutic target in RA. References Sun-Wada et al., J. Cell Sci. 122: 2504, 2009. Ishii et al., Nature, 458: 524, 2009. Ishii et al., J. Exp. Med.,207: 2793, 2010. Disclosure of Interest None Declared

Shearing tests on powders in a two-dimensional model were carried out for the purpose of determining the translational, rotational, rolling, and sliding movements of individual particles in a piled bed. These microscopic measurements were made by direct observation of the front surface of a two-dimensional bed and were used to calculate the overall stress-strain (τ/σ-d) relation. These calculated results were in reasonable agreement with the experimental (macroscopic) stress-strain curves.