Ippei Watari

The effect of diabetes mellitus on rat mandibular bone formation and microarchitecture

The aim of this study was to assess the effect of type 1 diabetes mellitus (DM) on the structure of mandibular bone and on the changes of alveolar/jaw bone formation. Experimental DM was induced in 3-wk-old male Wistar rats by a single dose of 60 mg/kg body weight of streptozotocin. All rats were injected with calcein on days 21 and 28. The rats were killed when 8 wk of age. Bone structure was analyzed by bone histomorphometry, microcomputed tomography (micro-CT), and histological section. Histomorphometric analysis showed that the mineral apposition and the bone formation rates in most of the mandibular regions were significantly decreased in the DM group compared with the control group. Micro-CT analysis showed significant deterioration of the bone quality in rats with DM. For a histometric measure of bone resorption, the number of osteoclasts along the distal surface of the alveolar wall was counted. The number of osteoclasts was significantly lower in the rats with DM than in the controls. These findings suggest that uncontrolled DM decreases mandibular bone formation, reduces the rate of bone turnover in the alveolar wall surrounding the root, and affects the quality of bone structure resulting in retardation of its skeletal development.

Effect of experimental diabetes on craniofacial growth in rats

OBJECTIVES The goal of this study was to assess the effect of type 1 diabetes mellitus (T1DM) on the craniofacial growth and skeletal maturation using the STZ-DM rat model. DESIGN Experimental T1DM was induced in 3-week-old male Wistar rats by a single dose of 60 mg/kg body weight of streptozotocin (STZ). Lateral and dorsoventral X-rays of the head were taken at the age of 7 weeks. The X-rays were scanned, digitised and selected linear distances were measured and analysed statistically. RESULTS In STZ-DM statistical analysis of results revealed a reduction in growth of most of the linear measurements in the neurocranium and mandible by X-ray analysis, and all measurements were significantly lower in viscerocranium. CONCLUSIONS Uncontrolled T1DM reduces craniofacial growth, resulting in retardation of skeletal development.

Expression of glucagon-like peptide-1 receptor and glucose‑dependent insulinotropic polypeptide receptor is regulated by the glucose concentration in mouse osteoblastic MC3T3-E1 cells

Glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon-like peptide-1 receptor (GLP‑1R) are incretin receptors that play important roles in regulating insulin secretion from pancreatic β cells. Incretin receptors are also thought to play a potential role in bone metabolism. Osteoblasts in animals and humans express GIPR; however, the presence of GLP-1R in these cells has not been reported to date. Thus, the aim of this study was to determine whether GLP-1R and GIPR are expressed in osteoblastic cells, and whether their expression levels are regulated by the extracellular glucose concentration. Mouse osteoblastic MC3T3-E1 cells were cultured in medium containing normal (5.6 mM) or high (10, 20 or 30 mM) glucose concentrations, with or without bone morphogenetic protein-2 (BMP-2). RT-PCR, western blot analysis and immunofluorescence were carried out to determine GIPR and GLP-1R mRNA and protein expression levels. Cell proliferation was also assessed. The GLP-1R and GIPR mRNA expression levels were higher in the MC3T3-E1 cells cultured in medium containing high glucose concentrations with BMP-2 compared with the cells cultured in medium containing normal glucose concentrations with or without BMP-2. GLP-1R protein expression increased following culture in high-glucose medium with BMP-2 compared with culture under normal glucose conditions. However, the cellular localization of GLP-1R was not affected by either glucose or BMP-2. In conclusion, our data demonstrate that the expression of GLP-1R and GIPR is regulated by glucose concentrations in MC3T3-E1 cells undergoing differentiation induced by BMP-2. Our results reveal the potential role of incretins in bone metabolism.

Intermittent posterior displacement of the rat mandible in the growth period affects the condylar cancellous bone

OBJECTIVE To examine whether intermittent posterior condylar displacement causes changes in cancellous bone in the mandibular condyle during the growth period. MATERIALS AND METHODS Sixteen 5-week-old male Wistar rats were divided into experimental and control groups. In the experimental group, an appliance was attached to the maxillary incisors to induce posterior displacement of the condyles in the occluded condition. Untreated rats served as the control group. Animals were sacrificed at 14 days, and the condyles were removed to analyze the three-dimensional cancellous bone structure by microcomputed tomography (micro-CT). Serial sagittal paraffin sections of the condyles were used for hematoxylin and eosin staining to investigate histomorphological changes and for tartrate-resistant acid phosphatase (TRAP) staining to identify osteoclastic cells. RESULTS Micro-CT analysis showed that in the experimental group, the bone volume fraction and the degree of anisotropy were significantly decreased compared with those in the control group in the anterior region of the condyle. Moreover, the number of TRAP-positive cells was significantly greater in the same region in the experimental group than in the control group. CONCLUSION Intermittent posterior displacement of the mandible can cause region-specific changes in the profile and microarchitecture of the condylar cancellous bone.

Unilateral maxillary molar extraction influences AQP5 expression and distribution in the rat submandibular salivary gland

OBJECTIVE Mastication has been regarded as a crucial factor for maintaining the morphology and function of secretion in salivary glands. Although it is known that occlusion affects mastication, the detailed process for how occlusal changes affect the secretory function of salivary glands is still unknown. Aquaporin 5 (AQP5) is a membrane protein that forms water channels, and plays an important role in water transport. In this study, we investigated the structural changes and alterations in the expression and distribution of AQP5 in the rat submandibular salivary gland (SMG) under occlusal hypofunction after unilateral molar extraction. METHODS Seven-week-old male Wistar rats (n = 36) were used in the study. In the experimental group, all of the right maxillary molars were extracted. Rats with no molar extraction were used as the control group. The rats were euthanized at 7, 14 or 28 days after the procedure, and the right SMGs were isolated and subjected to histological analyses. The expression and distribution of AQP5 were detected by immunohistochemistry. RESULTS Morphological analyses revealed hypertrophic changes in the acinar cells in the experimental group. Immunohistochemical staining of AQP5 was detected in the apical membrane (APM) and intercellular secretory canaliculi of acinar cells in both groups. On the other hand, the AQP5 expression in the APM and intercellular secretory canaliculi of acinar cells was less prominent in the experimental group than in the control group. CONCLUSIONS The present findings suggest that unilateral molar extraction has significant influences on the function of water transport in the rat SMG.

Effect of functional shift of the mandible on lubrication of the temporomandibular joint

Lubrication of synovial joints reduces the coefficient of friction of the articular cartilage surface. To investigate the effect of malocclusion on the lubrication of the temporomandibular joint (TMJ), we evaluated lubricin expression in the rat TMJ immunohistochemically, under conditions of functional lateral shift of the mandible, during period of growth. Thirty 5-week-old male Wistar rats were divided into experimental, recovery, and control groups. Each rt in the experimental and recovery groups was fitted with an acrylic-plate guiding appliance. The rats in the experimental and control groups were killed at 14 and 28 days after the appliance was attached. Each rat in the recovery group was detached from the appliance at 14 days, and was killed 14 days after the appliance was removed. In the experimental group, the expression of lubricin staining in TMJ cartilage was significantly decreased during the experimental period. In the recovery group, the expression of lubricin staining in TMJ cartilage was significantly greater than in the experimental group, and there was no significant difference at 28 days between the control and recovery groups. Analysis of these data suggests that a functional lateral shift of the mandible during the growth period influences lubrication of the TMJ.

Calcitonin and vitamin D3 have high therapeutic potential for improving diabetic mandibular growth

The goal of this study was to assess the effect of the intermittent combination of an antiresorptive agent (calcitonin) and an anabolic agent (vitamin D3) on treating the detrimental effects of Type 1 diabetes mellitus (DM) on mandibular bone formation and growth. Forty 3-week-old male Wistar rats were divided into four groups: the control group (normal rats), the control C+D group (normal rats injected with calcitonin and vitamin D3), the diabetic C+D group (diabetic rats injected with calcitonin and vitamin D3) and the diabetic group (uncontrolled diabetic rats). An experimental DM condition was induced in the male Wistar rats in the diabetic and diabetic C+D groups using a single dose of 60 mg·kg−1 body weight of streptozotocin. Calcitonin and vitamin D3 were simultaneously injected in the rats of the control C+D and diabetic C+D groups. All rats were killed after 4 weeks, and the right mandibles were evaluated by micro-computed tomography and histomorphometric analysis. Diabetic rats showed a significant deterioration in bone quality and bone formation (diabetic group). By contrast, with the injection of calcitonin and vitamin D3, both bone parameters and bone formation significantly improved (diabetic C+D group) (P < 0.05). These findings suggest that these two hormones might potentially improve various bone properties.

Influence of liquid diet feeding on calcitonin gene-related peptide-like immunoreactive nerve fibers in rat temporomandibular joints during growth period

Abstract Biomechanical factors of masticatory functions are related to the development of the temporomandibular joint (TMJ) components during growth. However, the effects on nerve fibers have not been fully clarified. We investigated the influence of masticatory muscle activity on nerve fibers in the TMJ during growth by changing the consistency of diet. Twenty 3-week-old male Wistar rats were divided into two groups. The experimental group was fed a liquid diet, while the control group had a normal hard diet. Rats were sacrificed at 6-, and 9-week-old. We investigated the expression of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibers, which are most abundant in the TMJ and have a nociceptive function, using immunohistochemistry for CGRP. No significant difference in the number of CGRP-LI nerve fibers was detected in 6-week-old rats, but in 9-week-old ones the number in the experimental group was significantly greater than that in the control. We concluded that masticatory muscle activity in the TMJ closely correlates with peripheral sensory nerve fibers during growth.

Degeneration of fungiform and circumvallate papillae following molar extraction in rats

Abstract Objective. Proper occlusion facilitates food intake and gustatory function is indispensable for the enjoyment of food. Although an interaction between dentoalveolar and gustatory afferent neurons has been suggested by previous studies, the relationship between occlusion and gustation remains unclear. This study investigated the effect of upper molar extraction which diminished occlusal support on peripheral gustatory receptors in rats. Materials and methods. Thirty-six 7-week-old male Wistar rats were randomly assigned to either the experimental or the control group. All maxillary molars were extracted from rats in the experimental group under anesthesia, while a sham operation was conducted in the control group. The rats were euthanized 7, 14 or 28 days after the procedure. The morphology of the circumvallate papillae and taste buds using immunohistochemical methods and the fungiform papillae were visualized with 1% methylene blue. Results. Defects in the gustatory epithelium were observed after maxillary molar extraction. Rats in the experimental group had significantly fewer fungiform papillae, narrower circumvallate papillae, shallower trench depth, smaller trench area, smaller taste bud area, lower ratios of taste bud area to trench area and fewer taste buds than those in the control group. Conclusions. The findings indicate that molar extraction would affect peripheral gustatory receptors. This is the first study to characterize changes in rat fungiform and circumvallate papillae after maxillary molar extraction. This study suggests a possible synergic relationship between dentoalveolar perception and gustatory function, which has clinical implications that occlusion is closely correlated with gustatory perception.

Expression of glucose-dependent insulinotropic polypeptide and its receptor in the rat major salivary glands.

Glucose-dependent insulinotropic polypeptide receptors (GIPR) are expressed throughout the body. The expression of its ligand, glucose-dependent insulinotropic polypeptide (GIP) however, has only been reported in a limited numbers of organs. Although the rat submandibular salivary gland (SMG) has been found to express GIP, its biological role is still not understood. Moreover, nothing is known about the expression of GIP in other types of salivary glands, i.e. the parotid (PG) and sublingual (SLG) glands. We detected the expression of GIP mRNA in the rat PG, SMG and SLG. Immunohistochemical analyses revealed that GIP and GIPR were expressed only in the ductal area of all types of major salivary glands, and no immunostaining was found in the acini area. We also found GIP expression in the rat SMG to be age dependent, with 8-week-old rats showing 2-3-fold higher than those of 9- and 11-week-old rats, respectively. This is the first study to indicate both GIP and GIPR expression in the rat major salivary glands, as well as its variation in the rat SMG during the growth period. These findings are crucial for a better understanding of the physiological function of GIP in rat major salivary gland.