Effect of viscosity of experimental universal adhesive on bond strength to dentin prepared with Er:YAG laser.

The aim of this study was to clarify the effect of universal adhesive (UA) viscosity on the bond strength of resin composite to dentin prepared with Er:YAG laser. Four experimental UAs (SI-1, SI-2, SI-3, and SI-4) were developed by adding 1, 2, 3, and 4 wt/% nanosilica to BeautyBond Xtreme (Shofu), respectively. BeautyBond Xtreme was used as a control (SI-0). The viscosities of experimental UAs were measured using a B-type viscometer. After bovine mandibular anterior teeth were ground with #600 emery paper to obtain the flattened dentin surfaces, the dentin surfaces were cut thinly by irradiating the Er:YAG laser. Specimens were prepared using the respective UA and flowable resin composite and subjected to the microtensile bond strength (µTBS) test. The data from viscosity measurement and the µTBS test were statistically analyzed using the Kruskal-Wallis test. The mean values of viscosity significantly differed among the all experimental groups (p < 0.01). The µTBS of SI-1 and SI-2 was significantly higher than that of SI-0, SI-3, and SI-4 (p < 0.001). The µTBS of SI-0 was significantly lower than that of SI-4 (p < 0.001). The viscosities of the experimental universal adhesives significantly affected their bond strength to laser-cut dentin.

Establishing protein expression profiles involved in tooth development using a proteomic approach.

Various growth and transcription factors are involved in tooth development and developmental abnormalities; however, the protein dynamics do not always match the mRNA expression level. Using a proteomic approach, this study comprehensively analyzed protein expression in epithelial and mesenchymal tissues of the tooth germ during development. First molar tooth germs from embryonic day 14 and 16 Crlj:CD1 (ICR) mouse embryos were collected and separated into epithelial and mesenchymal tissues by laser microdissection. Mass spectrometry of the resulting proteins was carried out, and three types of highly expressed proteins [ATP synthase subunit beta (ATP5B), receptor of activated protein C kinase 1 (RACK1), and calreticulin (CALR)] were selected for immunohistochemical analysis. The expression profiles of these proteins were subsequently evaluated during all stages of amelogenesis using the continuously growing incisors of 3-week-old male ICR mice. Interestingly, these three proteins were specifically expressed depending on the stage of amelogenesis. RACK1 was highly expressed in dental epithelial and mesenchymal tissues during the proliferation and differentiation stages of odontogenesis, except for the pigmentation stage, whereas ATP5B and CALR immunoreactivity was weak in the enamel organ during the early stages, but became intense during the maturation and pigmentation stages, although the timing of the increased protein expression was different between the two. Overall, RACK1 plays an important role in maintaining the cell proliferation and differentiation in the apical end of incisors. In contrast, ATP5B and CALR are involved in the transport of minerals and the removal of organic materials as well as matrix deposition for CALR.

Visual evaluation for the elasticity of suprahyoid muscles using sonographic elastography during tongue pressure measurement: A pilot study.

BACKGROUND: Suprahyoid muscles behavior during the tongue lifting movement has not yet been elucidated. OBJECTIVE: The purpose of this study was to investigate the potential of elastography imaging to examine developmental oral dysfunction in children and oral hypofunction in older adults using sonography. METHODS: Tongue pressure was measured using a manometer with a probe. The tongue pressure was measured with simultaneously scanning the geniohyoid muscle (GHM) and the anterior belly of the digastric muscle (DGM) using sonographic elastography. Sagittal images of the GHM and coronal images of the DGM were used for the strain ratio measurement. The strain ratio of the muscles was measured three times for each subject with the tongue pressure values of 0-30 kPa. RESULTS: The strain ratio of the GHM were higher than those of the DGM at tongue pressure of 10, 20 and 30 kPa. The strain ratio of the GHM increased as the tongue pressure increased in all participants. In contrast, the strain ratio of the DGM tended to slowly decrease as tongue pressure increased in female participants. CONCLUSION: Sonographic elastography is useful for visual and quantitative evaluation of elastic properties in suprahyoid muscles during tongue lifting movements.

Quantitative evaluation of elasticity of lower orbicularis oris muscle during the lip closing measurement using sonographic elastography.

BACKGROUND: Lip closing functions have never been evaluated from the viewpoint of elastic properties. OBJECTIVES: The purpose of the present study was to investigate the behavior of the lower orbicularis oris muscle during a button-pull exercise to measure lip closing force and quantitatively evaluate its elastic properties using sonographic elastography. METHODS: Appropriate compression loads for elastography were randomly measured on one of three types of acoustic couplers on three examiners. Compression tests were performed on three types of acoustic couplers within the appropriate compression load. Using the acoustic coupler with the lowest elastic modulus, the strain ratio of the lower orbicularis oris muscle during lip closing was measured, and elastography was performed on six males under tension loads of 0-8 N. RESULTS: The intraclass correlation coefficient (1, 3) for the tension load of 0 N was 0.81. Elastography showed that the strain ratio values increased significantly (p < 0.05) as the tension load increased. CONCLUSIONS: Combining the data obtained from lip closing test devices and sonographic elastography enabled the muscle performance to be evaluated objectively and accurately.

Analysis of salivary factors related to the oral health status in children.

Early detection of oral disease is important to reduce its severity and increase the likelihood of successful treatment. This study aimed to perform a quantitative assessment of the saliva components as a first stage of the research to screen oral homeostasis. Here, saliva secretions collected from children were evaluated, and their constituents were analyzed to investigate the potential correlations between the buffering capacity and a range of salivary factors. Subjects aged 3-16 years in the primary, mixed, or permanent dentition stage, were selected for this study. The following salivary factors were analyzed: flow rate, total protein, total sugar quantifications, and constituent analyses using RT-PCR and western blotting. The associations between each factor and the buffering capacity were then analyzed using multiple regression analysis. Flow rate, BPIFA2 RNA level, histatin 1 and BPIFB1 protein levels as well as female sex were positively associated with buffering capacity. In contrast, total sugar concentration and MUC7 RNA levels showed a negative relationship with the buffering capacity. Some of these constituents may indicate oral homeostasis and are therefore potential biomarkers of oral health status. These results suggest that the analyses of the correlations between oral homeostasis and salivary factors are an effective strategy for identifying the susceptibility to oral diseases.

Evaluation of permanent and primary enamel and dentin mineral density using micro-computed tomography.

OBJECTIVES: The present study was performed to investigate the mineral density distribution in enamel and dentin for both permanent and primary teeth and to establish the standard density per tooth type using micro-computed tomography (CT). METHODS: Fifty-seven extracted human teeth (37 permanent, 20 primary) were evaluated in the present study. The enamel and dentin mineral densities in the extracted teeth were measured using micro-CT. Cubic regression curves were used to determine the mineral density distribution in the enamel and dentin for each tooth type. RESULTS: The mean values, distributions, and regression equations of the mineral densities were obtained. The mean mineral density values for permanent enamel and dentin were significantly higher than those for their primary counterparts for each tooth type. CONCLUSIONS: In the present study, we demonstrated the distribution of mineral density in sound enamel and dentin and attempted to determine the standard mineral density for each tooth type using micro-CT. The mineral density distributions found in this study contribute to our understanding of the mechanical properties of enamel and dentin. A positive correlation suggests that the systemic bone mineral density could be predicted based on the analysis of exfoliated teeth, such as in patients with hypophosphatasia. The present results may be useful in establishing a numerical standard for the mechanism involved in root fracture and for early detection of root fracture risk.

Mesenchymal Wnt/ß-catenin signaling limits tooth number.

Tooth agenesis is one of the predominant developmental anomalies in humans, usually affecting the permanent dentition generated by sequential tooth formation and, in most cases, caused by mutations perturbing epithelial Wnt/ß-catenin signaling. In addition, loss-of-function mutations in the Wnt feedback inhibitor AXIN2 lead to human tooth agenesis. We have investigated the functions of Wnt/ß-catenin signaling during sequential formation of molar teeth using mouse models. Continuous initiation of new teeth, which is observed after genetic activation of Wnt/ß-catenin signaling in the oral epithelium, was accompanied by enhanced expression of Wnt antagonists and a downregulation of Wnt/ß-catenin signaling in the dental mesenchyme. Genetic and pharmacological activation of mesenchymal Wnt/ß-catenin signaling negatively regulated sequential tooth formation, an effect partly mediated by Bmp4. Runx2, a gene whose loss-of-function mutations result in sequential formation of supernumerary teeth in the human cleidocranial dysplasia syndrome, suppressed the expression of Wnt inhibitors Axin2 and Drapc1 in dental mesenchyme. Our data indicate that increased mesenchymal Wnt signaling inhibits the sequential formation of teeth, and suggest that Axin2/Runx2 antagonistic interactions modulate the level of mesenchymal Wnt/ß-catenin signaling, underlying the contrasting dental phenotypes caused by human AXIN2 and RUNX2 mutations.

The Role of Genetic Factors in the Outbreak Mechanism of Dental Caries.

OBJECTIVE: The aim of the present study was to investigate the relationships between cariogenic bacterial infection and single nucleotide polymorphisms (SNPs) in candidate genes associated with dental caries, and to explore the factors related to caries in children. STUDY DESIGN: Children aged 3 to 11 years were selected. Detection of cariogenic bacteria (Streptococcus mutans, Streptococcus oralis, Streptococcus sobrinus and Lactobacillus) from the plaque of each patient, and SNP analyses of five candidate genes (MBL2, TAS2R38, GLUT2, MMP13 and CA6) were performed using DNA isolated from buccal mucosal cells. The dental caries experience in primary and permanent teeth was determined using the decayed, missing and filled teeth (DMFT) index, and the effects of the observed factors on the DMFT value were analyzed by multiple regression analysis. RESULTS: The results of the multiple regression analysis showed that the DMFT value significantly increased in the presence of S. mutans or S. sobrinus (p < 0.001), while the dmft/DMFT value decreased in the presence of nucleobase C in MBL2 (p < 0.05). CONCLUSION: These results suggest that the MBL2 gene is related to the pathogenesis of dental caries.

Presence of BPIFB1 in saliva from non-obese diabetic mice.

We previously showed that mRNA expression of BPIFB1 (Bpifb1), an antibacterial protein in the palate, lung, and nasal epithelium clone protein family, was increased in parotid acinar cells in non-obese diabetic (NOD, NOD/ShiJcl) mice, which is an animal model for Sjögren's syndrome. However, we did not previously assess the protein levels. In this report, we confirmed the expression of BPIFB1 protein in the parotid glands of NOD mice. Immunoblotting of subcellular fractions revealed that BPIBB1 was localised in secretory granules in parotid glands from NOD mice, and was almost not in parotid glands from the control mice. BPIFB1 had N-linked glycan that reacted with Aleuria aurantia lectin, which caused two types of spots with a slightly different pI and molecular weight. The expression of BPIFB1 protein was also demonstrated by immunohistochemistry. BPIFB1 was detected in the saliva from NOD mice but not in the saliva from the control mice, indicating individual constitution. BPIFB1 in saliva may be applied to other research as a diagnostic marker.

Characterization of a PTH1R missense mutation responsible for Jansen type metaphyseal chondrodysplasia.

Parathyroid hormone and parathyroid hormone-related peptide (PTHrP), and its receptor (PTH1R) play an important role in differentiation of bone and cartilage in the developing stages. Constitutive dimers of PTH1R are believed to be dissociated by ligand binding, and monomeric PTH1R is capable of activating G protein. Jansen type metaphyseal chondrodysplasia is caused by missense mutations in PTH1R, which are constitutively active even without the presence of the ligands. However, the underlying pathomechanisms remained largely unknown. In this study, we have attempted to further characterize a PTH1R missense mutation H223R responsible for Jansen type metaphyseal chondrodysplasia. cDNAs encoding wild-type (Wt)- and H223R mutant (Mut)-PTH1R were transfected into HEK293T cells, and as a consequence of western blots, both the Wt- and Mut-PTH1R proteins showed several fragments between 55 and 65 kDa in size, while the patterns of N-glycosylation were distinct between them. Then we hypothesized that the Mut-PTH1R might physically interact with the Wt-PTH1R, leading to affect the downstream cAMP accumulation. Co-immunoprecipitation assays clearly showed that interaction occurred not only between the Wt-PTH1R themselves, but also between the Wt- and Mut-PTH1R. Furthermore, we performed CRE reporter assays to investigate cAMP accumulation. Constitutive, ligand-independent cAMP accumulation was observed in HEK293T cells expressing the Mut-PTH1R. Interestingly, there was a statistically lower constitutive activity in HEK293T cells co-expressing the Wt- and Mut-PTH1R proteins. Summarizing, it seems likely that Mut-PTH1R may be, at least in part, co-localized with Wt-PTH1R by forming a heterodimer, leading to affect the function to each other.

Nondestructive Microcomputed Tomography Evaluation of Mineral Density in Exfoliated Teeth with Hypophosphatasia.

Most cases of hypophosphatasia (HPP) exhibit early loss of primary teeth. Results of microcomputed tomography (micro-CT) analysis of teeth with HPP have rarely been reported. The purpose of the present study was to describe the mineral density distribution and mapping of exfoliated teeth from an HPP patient using micro-CT. Four exfoliated teeth were obtained from a patient with HPP. Enamel and dentin mineral densities of exfoliated teeth were measured on micro-CT. The mean values of enamel and dentin mineral densities in mandibular primary central incisors with HPP were 1.61 and 0.98 g/cm3, respectively. The corresponding values in the mandibular primary lateral incisors were 1.60 and 0.98 g/cm3, respectively. Enamel hypoplasia was seen in the remaining teeth, both maxillary and mandibular primary canines and first and second molars. Micro-CT enables nondestructive, noninvasive evaluation and is useful for studying human hard tissues obtained from patients.

Characteristics of alveolar bone associated with physiological movement of molar in mice: a histological and histochemical study.

Mouse molars undergo distal movement, during which new bone is formed at the mesial side of the tooth root whereas the preexisting bone is resorbed at the distal side of the root. However, there is little detailed information available regarding which of the bones that surround the tooth root are involved in physiological tooth movement. In the present study, we therefore aimed to investigate the precise morphological differences of the alveolar bone between the bone formation side of the tooth root, using routine histological procedures including silver impregnation, as well as by immunohistochemical analysis of alkaline phosphatase and tartrate-resistant acid phosphatase activity, and immunohistochemical analysis of the expression of the osteocyte markers dentin matrix protein 1, sclerostin, and fibroblast growth factor 23. Histochemical analysis indicated that bone formation by osteoblasts and bone resorption by osteoclasts occurred at the bone formation side and the bone resorption side, respectively. Osteocyte marker immunoreactivity of osteocytes at the surface of the bone close to the periodontal ligament differed at the bone formation and bone resorption sides. We also showed different specific features of osteocytic lacunar canalicular systems at the bone formation and bone resorption sides by using silver staining. This study suggests that the alveolar bone is different in the osteocyte nature between the bone formation side and the bone resorption side due to physiological distal movement of the mouse molar.

Identification of marker proteins by orthodontic treatment: relationship of RANKL in the gingival crevicular fluid and of amylase in whole saliva with orthodontic treatment.

Orthodontic medical treatment is performed to move a tooth to the optimal position to obtain optimal occlusion. Orthodontic treatment is accompanied by mechanical stress due to orthodontic force and by psychological stress that is experienced as pain or displeasure. The purpose of this study was to identify stress marker proteins during orthodontic treatment. Levels of receptor activator of NFκB (RANKL) and heat shock protein 70 (HSP70) in the gingival crevicular fluid (GCF) were analyzed as markers of mechanical stress, and levels of chromogranin A (CgA) and amylase in whole saliva were analyzed as markers of psychological stress. GCF was collected from control and experimental teeth at initiation of treatment and 24 h after treatment. Whole saliva was collected before treatment, at initiation of treatment and 24 h after treatment. RANKL was expressed at 24 h after treatment in the experimental GCF, but not in the control GCF. HSP70 appeared to be constitutively expressed in GCF, and its levels showed no major change between the control and experimental groups from initiation of treatment to 24 h after treatment. Amylase activity in whole saliva was enhanced at 24 h after treatment compared to control, but CgA levels showed little change between the groups. These results indicated that RANKL and amylase may be the candidate markers for mechanical and psychological stress, respectively, during orthodontic treatment, even though the total protein concentration and amylase activity displayed a large standard deviation among subjects. Further studies are therefore required to establish these markers for clinical use.

Prevalence of cariogenic and periodontopathic bacteria in Japanese children in the primary and mixed dentitions.

AIM: The purpose of the present study was to evaluate the relationship between infection with cariogenic bacteria or periodontal pathogens and the oral condition of children in the primary and mixed dentition stages. METHOD: Children aged 3 to 11 years were selected Detection of cariogenic and periodontopathic bacteria and single nucleotide polymorphism (SNP) analyses were performed, and the prevalence of infection with cariogenic bacteria or periodontal pathogens based on caries experience and dental stage was compared. RESULTS: The prevalence of Streptococcus mutans in both stages was significantly higher in the caries group than in the caries-free group. The prevalence of Streptococcus sobrinus was significantly higher in the caries group only in the mixed dentition stage. The prevalence ofperiodontal pathogens was significantly higher in the mixed dentition stage than in the primary dentition stage, regardless of caries experience. However, there were no significant differences in the prevalence of the periodontal pathogens between the primary dentition and mixed dentition stages, based on caries experience. CONCLUSION: Our data suggested that cariogenic and periodontopathic bacteria have different infection patterns, and that the period of infection with these bacteria also differs.

Tannerella forsythia and the HLA-DQB1 allele are associated with susceptibility to periodontal disease in Japanese adolescents.

Periodontal disease is a multiple factor disease caused by genetic factors, environmental factors, and periodontal bacteria (periodontal pathogens). The present study aimed to elucidate the risk factors for periodontal disease in Japanese adolescents. Subjects (11-16 years old) were classified into three groups: localized aggressive periodontitis (LAP), periodontal attachment loss (PAL), and periodontally healthy (PH) groups. Genomic DNA isolated from the buccal mucosa was used for single-nucleotide polymorphism analyses of the candidate genes (interleukin-1alpha-889; interleukin-1alpha +4845; interleukin-1beta +3954; an immunoglobulin G Fc gamma receptor, FcgammaRIIa-R/H131; and a human leukocyte antigen class II allele, HLA-DQB1) of aggressive periodontitis. Subgingival plaque samples obtained from the same subjects were used for 16S rRNAbased polymerase chain reaction analysis of five important periodontal pathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, and Tannerella forsythia). Tannerella forsythia was detected in the deepest periodontal pockets in all subjects in the LAP and PAL groups. The prevalence of an atypical BamHI restriction site in HLA-DQB1 of the LAP group was significantly higher than that in the PH and PAL groups. Furthermore, all subjects who had the atypical BamHI restriction site in HLA-DQB1 had T. forsythia infection. These results suggested that T. forsythia is associated with periodontal disease in Japanese adolescents and also suggested that HLA-DQB1 is related to LAP and is associated with T. forsythia infection.

Rat wild-type parathyroid hormone receptor (PTH-R) and mutant PTH-R(P132L) show the different intracellular localization in vitro.

A replacement of proline with leucine at position 132 of the receptor for parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP), i.e., PTH-R, has been discovered in human Blomstrand's lethal chondrodysplasia. As skeletal deformities in this type of chondrodysplasia appear to compromise the receptor binding to its ligands, we examined the possibility that rat PTH-R carrying P132L mutation (PTH-R(P132L)) would result in abnormal intracellular localization. Osteoblastic MC3T3-E1 cells were transfected with expression vectors containing cDNAs encoding either wild-type PTH-R or mutant PTH-R(P132L). The cells expressing the wild-type PTH-R produced a receptor protein with a molecular mass of 66.3 kDa, which localized its immunoreactivity mainly on the cell surfaces. In contrast, the PTH-R(P132L) was hardly detected on the cell surfaces, but accumulated within the rough-surfaced endoplasmic reticulum. Consistent with this localization, the cells expressing the mutant receptor failed to generate cyclic AMP in response to PTH. Furthermore, a remarkably weaker intensity of the 66.3 kDa band compared with the wild-type counterpart suggests that PTH-R(P132L) is prone to degradation in the transfected cells. In summary, these findings indicate that defective transport of PTH-R(P132L) to the cell surface would be a molecular basis for Blomstrand's chondrodysplasia.