Jiro Miura

Basolateral Mg2+ extrusion via CNNM4 mediates transcellular Mg2+ transport across epithelia: a mouse model.

Transcellular Mg2+ transport across epithelia, involving both apical entry and basolateral extrusion, is essential for magnesium homeostasis, but molecules involved in basolateral extrusion have not yet been identified. Here, we show that CNNM4 is the basolaterally located Mg2+ extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg2+ extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg2+ by exchanging intracellular Mg2+ with extracellular Na+. Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg2+ extrusion activity. These results demonstrate the crucial importance of Mg2+ extrusion by CNNM4 in organismal and topical regulation of magnesium.

Biomedical applications of a real-time terahertz color scanner

A real-time THz color scanner has the potential to further expand the application scope of THz spectral imaging based on its rapid image acquisition rate. We demonstrated three possible applications of a THz color scanner in the biomedical field: imaging of pharmaceutical tablets, human teeth, and human hair. The first application showed the scanner’s potential in total inspection for rapid quality control of pharmaceutical tablets moving on a conveyor belt. The second application demonstrated that the scanner can be used to identify a potential indicator for crystallinity of dental tissue. In the third application, the scanner was successfully used to visualize the drying process of wet hairs. These demonstrations indicated the high potential of the THz color scanner for practical applications in the biomedical field.

Effects of the tea catechin epigallocatechin gallate on Porphyromonas gingivalis biofilms.

The aim of this study was to investigate the effects of tea catechin epigallocatechin gallate (EGCg) on established biofilms and biofilm formation by Porphyromonas gingivalis, a major pathogen of periodontal disease.

Multiscale analysis of stress distribution in teeth under applied forces

OBJECTIVES The present study aimed to clarify stress distribution in teeth under an external applied load using microindentation and multiscale simulation. METHODS Anisotropic material properties of enamel and dentin were measured using a microindentation method with a dynamic ultra microhardness tester. The numerical model was constructed using the finite element mesh superposition method with a microscale (enamel rods), mesoscale (teeth), and macroscale (bone). Multiscale simulation was used to analyze different scale structures. A concentrated load (10 N) was applied to the labial surface of the central incisor. RESULTS The results revealed stresses of 5 MPa (normal stress) and 6.5 MPa (shear stress) in the enamel sheath at the center of the labial region compared to 1.4 MPa (normal stress) and 21.4 MPa (shear stress) in the cervical region. SIGNIFICANCE These results suggest that shear stresses in the enamel sheath may play significant roles in stress transmission.

Comparative Analysis of Mouse-Induced Pluripotent Stem Cells and Mesenchymal Stem Cells During Osteogenic Differentiation In Vitro

Induced pluripotent stem cells (iPSCs) can differentiate into mineralizing cells and are, therefore, expected to be useful for bone regenerative medicine; however, the characteristics of iPSC-derived osteogenic cells remain unclear. Here, we provide a direct in vitro comparison of the osteogenic differentiation process in mesenchymal stem cells (MSCs) and iPSCs from adult C57BL/6J mice. After 30 days of culture in osteogenic medium, both MSCs and iPSCs produced robustly mineralized bone nodules that contained abundant calcium phosphate with hydroxyapatite crystal formation. Mineral deposition was significantly higher in iPSC cultures than in MSC cultures. Scanning electron microscopy revealed budding matrix vesicles in early osteogenic iPSCs; subsequently, the vesicles propagated to exhibit robust mineralization without rich fibrous structures. Early osteogenic MSCs showed deposition of many matrix vesicles in abundant collagen fibrils that became solid mineralized structures. Both cell types demonstrated increased expression of osteogenic marker genes, such as runx2, osterix, dlx5, bone sialoprotein (BSP), and osteocalcin, during osteogenesis; however, real-time reverse transcription-polymerase chain reaction array analysis revealed that osteogenesis-related genes encoding mineralization-associated molecules, bone morphogenetic proteins, and extracellular matrix collagens were differentially expressed between iPSCs and MSCs. These data suggest that iPSCs are capable of differentiation into mature osteoblasts whose associated hydroxyapatite has a crystal structure similar to that of MSC-associated hydroxyapatite; however, the transcriptional differences between iPSCs and MSCs could result in differences in the mineral and matrix environments of the bone nodules. Determining the biological mechanisms underlying cell-specific differences in mineralization during in vitro iPSC osteogenesis may facilitate the development of clinically effective engineered bone.

Accumulation of advanced glycation end-products in human dentine.

Cross-linking of collagen by Advanced Glycation End-products (AGEs) occurs by non-enzymatic glycation (Maillard reaction). The purpose of this study was to examine whether AGEs are formed in human dentinal collagen, and to consider any possible influence of AGEs on dentinal physiology. Mechanical characteristics, fluorescence spectra and immunohistochemical analyses of demineralized dentine sections from young subjects were compared with those of aged ones. The same investigations were performed with young dentine artificially glycated by incubation in 0.1M ribose solution. Indentation measurement indicated that the sections from aged dentine were mechanically harder than those from young dentine. The hardness of young dentine increased after incubation in ribose solution. Fluorescence peak wavelength of the young dentine was shorter than that of the aged one, but shifted towards the peak wavelength of the aged one after incubation in ribose solution. These changes were considered to be due to accumulation of AGEs. Existence of AGEs in dentinal collagen was confirmed by immunohistochemical analysis. The obtained results suggest that AGEs accumulation occurs in dentinal collagen and is affected by both human age and physiological conditions such as glucose level in blood because dentinal collagen receives nourishment via dental pulp and tubules.

Detection of Dentinal Microcracks Using Infrared Thermography

INTRODUCTION It is difficult to make a definite diagnosis of a cracked tooth solely based on an inspection within the root canal, especially in case of microcracks. At present, there seems to be no established method to detect dentinal microcracks in roots; therefore, the current detection techniques need to be improved. Vibrothermography (VibroIR) helps to detect microcracks by the friction heat generated from ultrasonic vibration. The purpose of this study was to establish a novel method using VibroIR to detect dentinal microcracks. METHODS The root canals of 20 roots with cracks and control roots were prepared after removing the tooth crowns. A tapered indenter was inserted into the root canal and pressed until a microcrack was created under an optical microscope. Using VibroIR, the detection trials for dentinal microcracks were performed with an ultrasonic vibration power ranging from 0.43 to 1.48 W at an angle of 0°, 30°, 45°, 60°, and 90° between the ultrasonic vibration point and the microcrack line. After the detection test, the microcrack width was measured with an optical microscope. RESULTS Frictional heat was detected in the microcracks with thermography at 0.89 to 1.48 W and at an ultrasonic vibration point angle less than 60° from the crack line for 10 seconds. Microcracks with a width of 4 to 35.5 μm were detected with this method. CONCLUSIONS VibroIR may be an effective method for the diagnosis of root dentinal microcracks.

Simple observation of Streptococcus mutans biofilm by scanning electron microscopy using ionic liquids.

Scanning electron microscopy (SEM) has been successfully used to image biofilms because of its high resolution and magnification. However, conventional SEM requires dehydration and metal coating of biological samples before observation, and because biofilms consist mainly of water, sample dehydration may influence the biofilm structure. When coated with an ionic liquid, which is a kind of salt that exists in the liquid state at room temperature, biological samples for SEM observation do not require dehydration or metal coating because ionic liquids do not evaporate under vacuum conditions and are electrically conductive. This study investigates the ability of ionic liquids to allow SEM observation of Streptococcus mutans biofilms compared with conventional coating methods. Two hydrophilic and two hydrophobic ionic liquids, all of which are electronic conductors, are used. Compared with samples prepared by the conventional method, the ionic-liquid-treated samples do not exhibit a fibrous extracellular matrix structure and cracking on the biofilm surface. The hydrophilic ionic liquids give clearer images of the biofilm structure than those of the hydrophobic ionic liquids. This study finds that ionic liquids are useful for allowing the observation of biofilms by SEM without preparation by dehydration and metal coating.

Mechanical and morphological evaluation of the bond-dentin interface in direct resin core build-up method

OBJECTIVES The purpose of this study was to evaluate the interfacial adhesion between resin and root canal dentin in the direct resin core build-up method in terms of microtensile bond strength (μTBS) and dentin micro morphology. METHODS Single-rooted human teeth were decoronated at the cementoenamel junction and endodontically treated. Post spaces were prepared in the roots to a depth of 10mm. The spaces were then treated with a dual-cure bonding system, and filled with dual-cure resin composite. After 24-h storage in water at 37 °C, they were trimmed into approximately 1.0-mm(2) beams for μTBS. Bond strength was analyzed with one-way ANOVA and Tukeys test. The fractured surfaces were examined by scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDX). Sectioned specimens were observed by ultra-high-voltage transmission electron microscopy. RESULTS The bond strength of root dentin decreased gradually from the coronal to apical side, and the bond strength of the coronal section was significantly higher than that of the radicular section. Moreover, the failure modes in the coronal and apical sides of the specimens differed. The apical specimens fractured within the core material, while the coronal specimens fractured at the bonding layer. SEM and EDX analyses revealed that the core material penetrated into dentinal tubules in the apical region. SIGNIFICANCE In the direct resin core build-up method, the interfacial adhesion of resin to root canal dentin may be insufficient in the apical region of the root canal due to poor polymerization.

Temporal dynamics of bacterial microbiota in the human oral cavity determined using an in situ model of dental biofilms

Numerous studies on oral biofilms have been performed in vitro, although it is difficult to mimic the oral environment. Here we used an in situ model to conduct a quantitative analysis and comprehensive identification of bacterial communities over time by performing deep sequencing of 16S rRNA genes. We show here that the number of viable bacteria in supragingival biofilms increased in two steps. Using scanning and transmission electron microscopy, as well as confocal laser scanning microscopy, we detected gram-positive cocci during the first 8 h. The biofilm was subsequently covered with a thick matrix-like structure composed of different bacterial morphotypes that diversified as the number of bacteria increased. Streptococcus accounted for >20% of the population until 16 h, and obligate anaerobes such as Fusobacterium, Prevotella and Porphyromonas predominated after 48 h, and this increase was statistically significant after 96 h (P<0.05). Together, our data demonstrate that an initial population of facultative anaerobic bacteria was replaced with a population of gram-negative anaerobic bacteria during oral biofilm formation. This study, therefore, contributes to a comprehensive understanding of the composition of the bacterial microbiota involved in the health of the human oral cavity.