Morihiko Oka

Effects of ovariectomy and/or dietary calcium deficiency on bone dynamics in the rat hard palate, mandible and proximal tibia

The effects of ovariectomy and/or dietary calcium deficiency on bone dynamics were examined by comparing the histomorphometric changes in these bones. Five groups of rats were studied, (1) unoperated basal controls; (2) sham-operated, fed on a normal-calcium diet; (3) ovariectomized, fed on a normal-calcium diet; (4) sham-operated, fed on a calcium-deficient diet; (5) ovariectomized, fed on a calcium-deficient diet. The basal controls were killed at 6 weeks of age, and the remaining groups were killed at 18 weeks of age. The hard palate, mandible and proximal tibia were processed undemineralized for quantitative bone histomorphometry. Bone volume, eroded surface, osteoid surface and bone-formation ratio were calculated. A significant age-related increase in bone volume and a significant decrease in bone formation were observed in the hard palate and mandible, whereas no significant age-related increase in bone volume could be found in the tibia. In the hard palate, ovariectomy neither inhibited age-related increases in bone volume nor affected bone dynamics, while both the combined ovariectomy and dietary calcium deficiency and dietary calcium deficiency alone led to bone loss and increased bone turnover. In contrast, in the mandible and proximal tibia, ovariectomy alone as well as dietary calcium deficiency led to bone loss and increased bone turnover. Ovariectomy, therefore, produced no significant changes in the hard palate, but affected bone dynamics in the mandible and tibia. However, dietary calcium deficiency induced bone loss and increased bone turnover in the hard palate, mandible and proximal tibia, independently of ovariectomy.

Gene Expression and Distribution of Connective Tissue Growth Factor (CCN2/CTGF) During Secondary Ossification Center Formation

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metalloproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.

Distribution, gene expression, and functional role of EphA4 during ossification

EphA4 receptor tyrosine kinase has been shown to be critically involved in neural tissue development. Here, we found EphA4 was also distributed among hypertrophic chondrocytes and osteoblasts in the growth plate of developing mouse long bones. In vitro evaluation revealed that ephA4 expression was elevated upon hypertrophic differentiation of chondrocytes and that markedly stronger expression was observed in osteoblastic SaOS-2 than chondrocytic HCS-2/8 cells. Of note, RNAi-mediated silencing of ephA4 in SaOS-2 cells resulted in the repression of osteocalcin gene expression and alkaline phosphatase activity. Interestingly, confocal laser-scanning microscopic analysis revealed the presence of EphA4 molecules in the nucleus as well as on the surface of SaOS-2 cells. These findings are the first indication of a critical role of EphA4 in ossification, especially at the final stage in which osteoblasts and hypertrophic chondrocytes play major roles.

Effect of occlusal rehabilitation on spatial memory and hippocampal neurons after long‐term loss of molars in rats

Experimental loss of occlusal support caused by the extraction or grinding of molar teeth has been reported to foment the impairment of learning and memory in laboratory animals. The purpose of this study was to examine the effect of occlusal reconstruction after long-term loss of molars on spatial memory by using 8-arm radial maze and by assessing histopathological changes of neuron density in the hippocampus. Experimental dentures were inserted into the oral cavities of molarless rats to recover the occlusal support. Age-matched groups of control, molarless and denture-wearing rats were trained to perform the maze tasks. The difference of the error incidence in the maze task was evaluated between three groups. The difference of neuron density between three groups was also evaluated at the end of the maze task. Serum corticosterone levels were also measured to estimate the chronic stress, which could be caused by extraction, insertion of the experimental denture or any experimental procedure. The error incidence in the denture-wearing group was significantly higher than that of the control group, but significantly lower than that of the molarless group. Significant differences of neuron density were observed between three groups in each of the hippocampal CA1, CA3 and DG subfields. No significant difference of the serum corticosterone levels between three groups could be observed. From the results of this study, it was suggested that the recovery of occlusal support would bring amelioration of cognitive impairment concomitant with long period loss of molars in rats.

A Tumor Suppressor Gene Product, Platelet‐Derived Growth Factor Receptor‐Like Protein Controls Chondrocyte Proliferation and Differentiation

The platelet‐derived growth factor receptor‐like (PDGFRL) gene is regarded as a tumor suppressor gene. However, nothing is known about the molecular function of PDGFRL. In this study, we initially clarified its function in chondrocytes. Among all cell lines examined, the PDGFRL mRNA level was the highest in chondrocytic HCS‐2/8 cells. Interestingly, the proliferation of chondrocytic HCS‐2/8 cells was promoted by PDGFRL overexpression, whereas that of the breast cancer‐derived MDA‐MB‐231 cells was inhibited. Of note, in PDGFRL‐overexpressing HCS‐2/8 cells, the expression of chondrocyte differentiation marker genes, SOX9, ACAN, COL2A1, COL10A1, and ALP, was decreased. Moreover, we confirmed the expression of PDGFRL mRNA in normal cartilage tissue and chondrocytes. Eventually, the expression of PDGFRL mRNA in condrocytes except in the case of hypertrophic chondrocytes was demonstrated in vivo and in vitro. These findings suggest that PDGFRL plays the different roles, depending upon cell types. Particularly, in chondrocytes, PDGFRL may play a new and important role which is distinct from the function previously reported. J. Cell. Biochem. 118: 4033–4044, 2017.

New theoretical model to measure pressure produced during impression procedure for complete dentures—Visual inspection of impression material flow

OBJECTIVE A theoretical model, based on fluid dynamics, was developed to measure impression pressure. The purpose of this study was to evaluate the validity of this theoretical model by comparing its theoretical analysis against actual pressure measurements conducted using an impression tray and edentulous oral mucosa analog embedded with pressure sensors. METHODS In the theoretical model, a hollow tube was mounted onto an impression tray by penetrating through the tray. When force was applied to the tray, pressure was produced which then caused the impression material to flow into the hollow tube. Length of impression material which flowed into tube was denoted as l. In the calculation formula for theoretical model, pressure impulse I was expressed as a function of impression flow length l. For actual pressure measurements, four electric pressure sensors were embedded in an experimental edentulous arch. To visually observe and measure length of impression material flow, four transparent silicon tubes were mounted vertically at different positions on tray. During tray seating, impression material flowed into tubes and pressure which caused material flow movement was measured by the embedded sensor at each tubes position. RESULTS Based on actual pressure measurements under one experimental condition, regression analysis of pressure data acquired from electric sensors yielded the formula, Y=0.056X²+0.124X. Based on theoretical analysis using a particular viscosity value, the numerical formula yielded was Y=0.057X², which resembled that of the regression formula. SIGNIFICANCE Theoretical model presented in this paper augured well for clinical application as an easy and economical means to examine magnitude and distribution of impression pressure by measuring lengths of impression material flow in tubes fixed to impression tray.