Tadashi Honjo

A three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy

Osteocytes are the most numerous cells in bone, embedded within the mineralized bone matrix. Their slender cytoplasmic processes form a complex intercellular network. In addition, these processes are thought to be important structures in the response to mechanical stress. This study provides an extensive analysis of the three-dimensional structure of the osteocyte and its processes in 16-day-old embryonic chick calvariae, based on nondestructive subsurface histotomography using both confocal laser scanning (CLS) microscopy and differential interference contrast (DIC) microscopy. OB7.3, a chicken osteocyte-specific monoclonal antibody, and Texas Red-X-conjugated phalloidin were used to confirm the osteocyte phenotype and to identify whole cells in the calvariae, respectively. Serial CLS images revealed morphological changes in bone cells up to 20 microm in depth. Osteocytes had widely spread their processes into the osteoblast layer, and we found for the first time that some of these processes had elongated to the vascular-facing surface of the osteoblast layer. Furthermore, stereotype images reconstructed from CLS images could show the three-dimensional distribution of these processes. Using the stereopair image, we could evaluate the frequency of processes between osteocytes and osteoblasts. Complementation of DIC microscopy revealed canaliculi and lacunae with high contrast. The distributional pattern of canaliculi generally coincided with that of the osteocyte processes. We consider that the combination method of CLS microscopy and DIC microscopy using a laser scanning microscope is a very useful new technical approach for investigating osteocytes in bone.

Terminal Differentiation of Osteoblasts to Osteocytes Is Accompanied by Dramatic Changes in the Distribution of Actin-Binding Proteins

Immunofluorescence staining of actin‐binding proteins in osteoblasts and osteocytes was performed. α‐Actinin, myosin, and tropomyosin showed similar organization in both osteoblastic stress fibers and osteocyte processes. However, fimbrin, villin, filamin, and spectrin showed dramatic differences in distribution between osteoblasts and osteocytes. This study suggested that terminal differentiation of osteoblasts to osteocytes is accompanied by highly dramatic changes in the distribution of actin‐binding proteins.

Fluid Shear Stress Induces Less Calcium Response in a Single Primary Osteocyte Than in a Single Osteoblast: Implication of Different Focal Adhesion Formation†

The immediate calcium response to fluid shear stress was compared between osteocytes and osteoblasts on glass using real‐time calcium imaging. The osteoblasts were responsive to fluid shear stress of up to 2.4 Pa, whereas the osteocytes were not. The difference in flow‐induced calcium may be related to differences in focal adhesion formation.

Hormonal, pH, and Calcium Regulation of Connexin 43–Mediated Dye Transfer in Osteocytes in Chick Calvaria

Gap junctional intercellular communication among osteocytes in chick calvaria, their natural 3D environment, was examined using FRAP analysis. Cell–cell communication among osteocytes in chick calvaria was mediated by Cx43 and was regulated by extracellular pH, extracellular calcium ion concentration, and PTH.

Analysis of pain level in cases treated with Invisalign aligner: comparison with fixed edgewise appliance therapy.

BackgroundThe aim of this study was to evaluate and compare the difference in the level of pain using the visual analog scale (VAS) between cases treated with the edgewise appliance and Invisalign. In addition, the cause of pain and discomfort in the Invisalign cases was identified.MethodsThe sample consisted of 145 cases for the edgewise group (EG; n = 55), Invisalign group (IG; n = 38), and edgewise and Invisalign group (EIG; n = 52). VAS scores were collected during the first three stages (first stage: 0 to 7 days, second stage: 14 to 21 days, and third stage: 28 to 35 days) and at the end of the treatment (overall VAS score). Evaluation of the cause of pain was categorized into three different types of problem (category 1: non-smoothed marginal ridge or missing materials, category 2: deformation of attachments, and Category 3: deformation of the tray). Statistical comparison of VAS scores between groups was performed by two-way analysis of variance.ResultsA significantly higher VAS score was observed at 3 and 4 days after, at 1, 2, and 3 days after, and at 2 and 3 days after in stages 1, 2, and 3, respectively, in EG compared to EIG and IG. A significant difference was observed in overall VAS scores between EG and IG in intensity of pain, number of days that pain lasted, and discomfort level. Only intensity of pain resulted in a significant difference between EG and EIG. Most of the causes of problem in the Invisalign cases were deformation of the tray.ConclusionsInvisalign may offer less pain compared to the edgewise appliance during the initial stages of treatment. In the use of Invisalign, deformation of tray must be carefully checked to avoid pain and discomfort for the patients.

Runx1 is involved in the fusion of the primary and the secondary palatal shelves

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.

Promotion of Ccn2 expression and osteoblastic differentiation by actin polymerization, which is induced by laminar fluid flow stress

Fluid flow stress (FSS) is a major mechanical stress that induces bone remodeling upon orthodontic tooth movement, whereas CCN family protein 2 (CCN2) is a potent regenerator of bone defects. In this study, we initially evaluated the effect of laminar FSS on Ccn2 expression and investigated its mechanism in osteoblastic MC3T3-E1 cells. The Ccn2 expression was drastically induced by uniform FSS in an intensity dependent manner. Of note, the observed effect was inhibited by a Rho kinase inhibitor Y27632. Moreover, the inhibition of actin polymerization blocked the FSS-induced activation of Ccn2, whereas inducing F-actin formation using cytochalasin D and jasplakinolide enhanced Ccn2 expression in the same cells. Finally, F-actin formation was found to induce osteoblastic differentiation. In addition, activation of cyclic AMP-dependent kinase, which inhibits Rho signaling, abolished the effect of FSS. Collectively, these findings indicate the critical role of actin polymerization and Rho signaling in CCN2 induction and bone remodeling provoked by FSS.

A Case Report of Multidisciplinary Treatment of an Adult Patient with Bilateral Cleft Lip and Palate

This is a case report about the successful orthodontic treatment of a bilateral cleft lip and palate patient by using a combination of bone grafting and subsequent prosthodontic rehabilitation. An adult patient with a bilateral cleft lip and palate presented with a concave profile, anterior and lateral crossbite, a markedly deep overbite, and residual bilateral alveolar clefts. His jaw movement patterns were unstable and irregular due to his collapsed bite. Orthodontic treatment with bilateral bone grafting improved his concave profile by downward and backward rotation of the mandible within the freeway space, and optimum occlusion and functionally stable and smooth jaw movements were obtained. After a 6-year retention period, no skeletal relapse could be detected, and his occlusal stability was satisfactory.

Runx/Cbfb signaling regulates postnatal development of granular convoluted tubule in the mouse submandibular gland

Background: The rodent salivary gland is not fully developed at birth and the cellular definitive differentiation takes place postnatally. However, little is known about its molecular mechanism. Results: Here we provide the loss‐of‐function genetic evidence that Runx signaling affects postnatal development of the submandibular gland (SMG). Core binding factor β (Cbfb) is a cotranscription factor which forms a heterodimer with Runx proteins. Cbfb was specifically expressed in the duct epithelium, specifically in the SMG. Epithelial Cbfb deficiency resulted in decrease in the size of the SMG and in the saliva secretion on postnatal day 35. The Cbfb mutant SMG specifically exhibited involution of the granular convoluted tubules (GCT), with a down‐regulated expression of its marker genes, such as Klk1, Ngf, and Egf. The induction of GCT is under the control of androgens, and the Cbfb mutant SMG demonstrated down‐regulated expression of Crisp3, an androgen‐dependent transcript. Because the circulating testosterone or tissue dihydrotestosterone levels were not affected in the Cbfb mutants, it appears that Runx/Cbfb signaling regulate androgen receptor pathway, but does not affect the circulating testosterone levels or the enzymatic conversion to DHT. Conclusions: Runx signaling is important in the postnatal development of androgen‐dependent GCT in the SMG. Developmental Dynamics 244:488–496, 2015.

Quantitative assessment of post-graduate orthodontic program in Okayama University

Abstract Recently, the importance of evaluation of dental education has been emphasized. In order to achieve and maintain the high level of graduate student quality, quantitative assessment is essential for university educational programs. Especially in orthodontics, basic science is extremely important from clinical point of view. However, there has been no report that has quantitatively evaluated the outcome of post-graduate seminar in orthodontic program. Total of 18 graduate students in orthodontic graduate program in Okayama University from year 2002 to 2004 participated in this study. Quantitative assessment before and after the Proffits reading seminar was evaluated among different chapters. As a result, significant improvement (approximately 30% increase) in the total average scores was observed in all 3 years. Furthermore, there seem to be common subjects that the students have difficulty in understanding. In conclusion, significant increase in scores after the seminar was observed in all examined 3 years, and the progress of the basic knowledge of orthodontics was clearly demonstrated by quantitative assessment. Furthermore, we may have to consider what subjects we need to concentrate in teaching the post-graduate students in the future.