Osteocytes play a pivotal role in the regulation of skeletal mass. Osteocyte processes are thought to sense the flow of interstitial fluid that is driven through the osteocyte canaliculi by mechanical stimuli placed upon bone, but how this flow elicits a cellular response is virtually unknown. Modern theoretical models assume that osteocyte canaliculi contain ultrastructural features that amplify the fluid flow-derived mechanical signal. Unfortunately the calcified bone matrix has considerably hampered studies on the osteocyte process within its canaliculus. Using one of the few ultra high voltage electron microscopes (UHVEM) available worldwide, we applied UHVEM tomography at 2 MeV to reconstruct unique three-dimensional images of osteocyte canaliculi in 1 μm sections of human bone. A realistic three-dimensional image-based model of a single canaliculus was constructed, and the fluid dynamics of a Newtonian fluid flow within the canaliculus was analyzed. We created virtual 2.2 nm thick sections through a canaliculus and found that traditional TEM techniques create a false impression that osteocyte processes are directly attached to the canalicular wall. The canalicular wall had a highly irregular surface and contained protruding axisymmetric structures similar in size and shape to collagen fibrils. We also found that the microscopic surface roughness of the canalicular wall strongly influenced the fluid flow profiles, whereby highly inhomogeneous flow patterns emerged. These inhomogeneous flow patterns may induce deformation of cytoskeletal elements in the osteocyte process, thereby amplifying mechanical signals. Based on these observations, new and realistic models can be developed that will significantly enhance our understanding of the process of mechanotransduction in bone.

Runx1 is expressed in medial edge epithelial (MEE) cells of the palatal shelf. Conditionally rescued Runx1(-/-) mice showed limited clefting in the anterior junction between the primary and the secondary palatal shelves, but not in the junction between the secondary palates. In wild type mice, the fusing epithelial surface exhibited a rounded cobblestone-like appearance, while such cellular prominence was less evident in the Runx1 mutants. We also found that Fgf18 was expressed in the mesenchyme underlying the MEE and that locally applied FGF18 induced ectopic Runx1 expression in the epithelium of the palatal explants, indicating that Runx1 was induced by mesenchymal Fgf18 signaling. On the other hand, unpaired palatal explant cultures revealed the presence of anterior-posterior (A-P) differences in the MEE fates and fusion mechanism. Interestingly, the location of anterior clefting in Runx1 mutants corresponded to the region with different MEE behavior. These data showed a novel function of Runx1 in morphological changes in the MEE cells in palatal fusion, which is, at least in part, regulated by the mesenchymal Fgf signaling via an epithelial-mesenchymal interaction.

Periodontal tissue homeostasis depends on a complex cellular network that conveys cell-cell communication. Gap junctions (GJs), one of the intercellular communication systems, are found between adjacent human periodontal ligament (hPDL) cells; however, the functional GJ coupling between hPDL cells has not yet been elucidated. In this study, we investigated functional gap-junction-mediated intercellular communication in isolated primary hPDL cells. SEM images indicated that the cells were in contact with each other via dendritic processes, and also showed high anti-connexin43 (Cx43) immunoreactivity on these processes. Gap-junctional intercellular communication (GJIC) among hPDL cells was assessed by fluorescence recovery after a photobleaching (FRAP) analysis, which exhibited dye coupling between hPDL cells, and was remarkably down-regulated when the cells were treated with a GJ blocker. Additionally, we examined GJs under hypoxic stress. The fluorescence recovery and expression levels of Cx43 decreased time-dependently under the hypoxic condition. Exposure to GJ inhibitor or hypoxia increased RANKL expression, and decreased OPG expression. This study shows that GJIC is responsible for hPDL cells and that its activity is reduced under hypoxia. This is consistent with the possible role of hPDL cells in regulating the biochemical reactions in response to changes in the hypoxic environment.

Sensitive detection of drugs using SERS in a wet system was developed for forensic drug analysis.