Reconsideration of Leeway Space Calculated Using Models from Same Patients at Different Growth Stages.

In this study, original leeway space was determined using plaster models prepared from the same individuals over successive years and compared with that reported in an earlier study. The plaster models used for measurement were prepared from a normal dentition group comprising 64 patients and a crowded dentition group comprising 29. The mesiodistal crown width of the deciduous canines, deciduous first molars, and deciduous second molars, as well as that of the canines, first premolars, and second premolars was measured. Leeway space was then calculated and the results compared with that previously reported. The average maxillary and mandibular leeway spaces calculated in the same individuals were 1.46 and 2.66 mm, respectively, whereas those calculated from data collected from different individuals were 0.89 and 2.83 mm, respectively. No direct statistical comparison of leeway space between this and the earlier report was possible. The present results did indicate, however, that it was larger in the maxilla than previously reported. A larger leeway space is advantageous for the normal arrangement of the permanent teeth. While the mesiodistal crown width of some of the lateral segment teeth in the present study was significantly larger in boys than in girls, no significant difference was observed in leeway space between sexes. The mesiodistal width was larger in multiple permanent teeth such as canines in the crowded dentition group, and this difference was significant in boys. On the other hand, no difference was observed in mesiodistal width in the deciduous teeth between the two groups. These results showed a tendency toward larger teeth and less leeway space in the permanent dentition in the crowded dentition group, which may have been a factor in why such crowding occurred.

Melatonin suppresses the antiviral immune response to EMCV infection through intracellular ATP deprivation caused by mitochondrial fragmentation.

Melatonin, a sleep hormone derived from the pineal gland, has an anti-inflammatory effect on the immune system in addition to modulating the brain nervous system. Previous studies have shown that melatonin suppresses signaling pathways downstream of multiple pattern recognition receptors on the innate immune cells during pathogen infection, but the specific mechanism of suppression has not been well understood. Using an encephalomyocarditis virus (EMCV) infection model in macrophages, we investigated the effects of melatonin on the antiviral response in innate immunity and found that melatonin attenuated the uptake of viral particles into macrophages. Furthermore, melatonin suppressed cytoskeletal regulation by decreasing ATP production by mitochondria. Finally, in an in vivo infection experiment, we also found that melatonin administration partially exacerbated the infection in the mouse brain. These results suggest that melatonin may have an inhibitory effect on excessive inflammation by suppressing cytoskeletal regulation in the innate immune system, but also suggest that suppression of inflammation may lead to insufficient protection against EMCV infection in vivo.

The concurrent stimulation of Wnt and FGF8 signaling induce differentiation of dental mesenchymal cells into odontoblast-like cells.

Fibroblast growth factor 8 (FGF8) is known to be a potent stimulator of canonical Wnt/ß-catenin activity, an essential factor for tooth development. In this study, we analyzed the effects of co-administration of FGF8 and a CHIR99021 (GSK3ß inhibitor) on differentiation of dental mesenchymal cells into odontoblasts. Utilizing Cre-mediated EGFP reporter mice, dentin matrix protein 1 (Dmp1) expression was examined in mouse neonatal molar tooth germs. At birth, expression of Dmp1-EGFP was not found in mesenchymal cells but rather epithelial cells, after which Dmp1-positive cells gradually emerged in the mesenchymal area along with disappearance in the epithelial area. Primary cultured mesenchymal cells from neonatal tooth germ specimens showed loss of Dmp1-EGFP positive signals, whereas addition of Wnt3a or the CHIR99021 significantly regained Dmp1 positivity within approximately 2 weeks. Other odontoblast markers such as dentin sialophosphoprotein (Dspp) could not be clearly detected. Concurrent stimulation of primary cultured mesenchymal cells with the CHIR99021 and FGF8 resulted in significant upregulation of odonto/osteoblast proteins. Furthermore, increased expression levels of runt-related transcription factor 2 (Runx2), osterix, and osteocalcin were also observed. The present findings indicate that coordinated action of canonical Wnt/ß-catenin and FGF8 signals is essential for odontoblast differentiation of tooth germs in mice.

Molar incisor hypomineralization: A review and prevalence in Japan.

Molar incisor hypomineralization (MIH) is defined as hypomineralization of systemic origin of one to four first permanent molars, and incisors are also frequently affected. This disorder is a serious concern in pediatric dentistry. Teeth affected by MIH have many dental problems, such as hypersensitivity, poor aesthetics, and rapid progression of dental caries. The prevalence of MIH ranges from 2.8% to 21% among studies with more than 1000 subjects in different countries and age groups. The etiology of MIH is unclear, but genetic and environmental factors have been proposed. This review describes the prevalence, etiology, and clinical management of MIH. A detailed description of MIH prevalence in Japan is also provided.

Caries and Its Association with Infant Feeding and Oral Health-related Behavior in 18-month and 3-year-old Japanese Children.

The primary aim of this study was to determine the prevalence of dental caries and its association with infant feeding and oral health-related behavior in Japanese children between the ages of 18 months and 3 years. A total of 387 infants were initially examined at the age of 18 months (or 19 months in some cases) and then again at 3 years (or at 3 years 1 month in some cases). The primary objective was to identify factors contributing to the prevalence of caries in children aged 18 months. The secondary objective was to follow up children with no caries at the age of 18 months to seek potential correlations between background factors and the increment of caries by the age of 3 years. In an adjusted multivariate logistic regression model, children who continued breastfeeding were approximately 7 times more likely to have dental caries at 18 months of age than those who did not. Infants brought in for an oral examination and consultation at around 12 months of age were less likely to develop dental caries at 18 months of age than those who were not. Multivariate logistic regression analysis revealed that infants who did not receive a dental check-up at 12 months of age showed a significantly higher incidence of dental caries at 3 years of age. The present results suggest that prolonged breastfeeding is a risk factor for early childhood caries at 18 months of age, and that infants in whom a regular oral care program is implemented from the age of 12 months are less likely to develop early childhood caries at 18 months or 3 years.

Investigation of the potential regulator proteins associated with the expression of major surface protein and dentilisin in Treponema denticola.

Objective Treponema denticola is involved in 'chronic' periodontitis pathogenesis. The mechanism underlying the regulation of the expression of its virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine specific protease (dentilisin) is yet to be clarified. We determined the gene expression profiles of Msp- and dentilisin-deficient mutants of T. denticola to identify the regulation network of gene expression concomitant with the inactivation of these virulence genes. Methods Gene expression profiles of T. denticola ATCC 35405 (wild type), dentilisin-deficient mutant K1, and msp-deficient mutant DMSP3 were determined using DNA microarray analysis and quantitative real-time reverse transcription PCR (qRT-PCR). Msp and dentilisin protein levels were determined by immunoblotting and proteolytic activity assays. Results In addition to several differentially expressed genes, dentilisin expression was reduced in DMSP3; msp expression was significantly reduced in K1 (p < 0.05), both at the gene and protein levels. To identify the regulatory system involved, the expression levels of the potential regulators whose expression showed changes in the mutants were evaluated using qRT-PCR. Transcriptional regulators TDE_0127 and TDE_0814 were upregulated in K1, and the potential repressor, TDE_0344, was elevated in DMSP3. Conclusions Dentilisin and Msp expression were interrelated, and gene expression regulators, such as TDE_0127, may be involved in their regulation.

Microbiomes of colored dental biofilms in children with or without severe caries experience.

BACKGROUND: Biofilm coloration can compromise maturation and increase the risk of oral disease in adulthood, though children with colored biofilm do not always demonstrate a poor oral health status. AIM: The microbial compositions of colored and white biofilms in children were compared. DESIGN: Thirty-two dental biofilm samples from 16 children (age < 13 years) were analyzed using 16S rRNA pyrosequencing, then the subjects were divided into severe caries and healthy (caries-free) groups. Correlations between microbiomes and oral health status were also examined. RESULTS: Phylogenetic analysis revealed no distinctly different patterns between colored and white biofilms. In the severe caries group, genus Actinomyces, Cardiobacterium, Kingella, Lautropia, and Veillonella, and family Neisseriaceae were detected, though abundance was significantly different between colored and white biofilm specimens, in contrast to the healthy group. In addition, five colored biofilm samples from the severe caries group contained greater than 15% Actinomyces, which led us to consider that genus to be possibly associated with formation of colored biofilm in children. CONCLUSIONS: Our findings indicate that differences in bacterial composition between colored and white biofilms are higher in individuals with severe caries. Additional research may reveal the significance of colored dental biofilm in children.

High-Level Expression of Alkaline Phosphatase by Adeno-Associated Virus Vector Ameliorates Pathological Bone Structure in a Hypophosphatasia Mouse Model.

Hypophosphatasia (HPP) is a systemic skeletal disease caused by mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). We recently reported that survival of HPP model mice can be prolonged using an adeno-associated virus (AAV) vector expressing bone-targeted TNALP with deca-aspartate at the C terminus (TNALP-D10); however, abnormal bone structure and hypomineralization remained in the treated mice. Here, to develop a more effective and clinically applicable approach, we assessed whether transfection with TNALP-D10 expressing virus vector at a higher dose than previously used would ameliorate bone structure defects. We constructed a self-complementary AAV8 vector expressing TNALP driven by the chicken beta-actin (CBA) promoter (scAAV8-CB-TNALP-D10). The vector was injected into both quadriceps femoris muscles of newborn HPP mice at a dose of 4.5 × 1012 vector genome (v.g.)/body, resulting in 20 U/mL of serum ALP activity. The 4.5 × 1012 v.g./body-treated HPP mice grew normally and displayed improved bone structure at the knee joints in X-ray images. Micro-CT analysis showed normal trabecular bone structure and mineralization. The mechanical properties of the femur were also recovered. Histological analysis of the femurs demonstrated that ALP replacement levels were sufficient to promote normal, growth plate cartilage arrangement. These results suggest that AAV vector-mediated high-dose TNALP-D10 therapy is a promising option for improving the quality of life (QOL) of patients with the infantile form of HPP.

Developmental studies on the acquisition of perception conducting pathways via TRP channels in rat molar odontoblasts using immunohistochemistry and RT-qPCR.

Odontoblasts act as dentin formation and sensory receptors. Recently, it was reported that transient receptor potential ankyrin (TRPA) 1, TRP vanilloid (TRPV) 4 and pannexin 1 (PANX-1) play important roles in odontoblast sensory reception. However, it is not known when odontoblasts begin to possess a sense reception function. The aim of this study was to clarify the development of odontoblasts as sense receptors. Sections of mandibular first molars from postnatal day (PN) 0 to PN12 Wistar rats were prepared for hematoxylin-eosin staining. Immunohistochemically, we used anti-dentin sialoprotein (DSP), anti-TRPA1, anti-TRPV4, anti-PANX-1, and anti-neurofilament (NF) antibodies. In addition, we investigated TRPA1 and TRPV4 expression by reverse transcriptional quantitative polymerase chain reaction (RT-qPCR). At PN0, undifferentiated odontoblasts showed no immunoreaction to anti-DSP, anti-TRPA1, anti-TRPV4, or anti-PANX-1 antibodies. However, immunopositive reactions of these antibodies increased during odontoblast differentiation at PN3 and PN6. An immunopositive reaction of the anti-NF antibody appeared in the odontoblast neighborhood at PN12, when the odontoblasts began to form root dentin, and this appeared later than that of the other antibodies. By RT-qPCR, expression of TRPA1 at PN6 was significantly lower than that at PN0 (p < 0.05) and PN3 (p < 0.01). Expression of TRPV4 at PN6 was significantly lower than that at PN0 (p < 0.01) and PN3 (p < 0.01). The results of this study suggest that odontoblasts may acquire sensory receptor function after beginning to form root dentin, when TRPA1, TRPV4, PANX-1 channels, and nerve fibers are completely formed.

Expression of Cytokeratin in Experimentally Created Inflammatory Cyst in Vivo and in Vitro.

The purpose of this study was to investigate the behavior of epithelial lining derived from Malassez's epithelial rest (MER) cells in experimentally created inflammatory cysts in vivo and in vitro. Porcine MER cells were cultured in vitro with or without interleukin (IL)-1ß (1 ng/ml) or IL-6 (1 ng/ml). Cell proliferation was assessed and expression levels of CK19 and CK13 mRNA determined using RT-PCR. In vivo, a cavity was created in the first molar of Sprague-Dawley male rats and tissue repair observed using immunohistochemical methods. In vitro, treatment with IL-1ß or IL-6 increased proliferation of MER cells and decreased expression of CK19 mRNA, but increased CK13 mRNA at day 1 (p<0.05). In vivo, at 2 weeks, CK19-positive epithelial cells were observed adjacent to the cementum, in the cystic lesion, and in connective tissue. At 3 weeks, they were only detected in cells adjacent to the connective tissue. Cells positive for CK13 were observed throughout the epithelium, except in cells adjacent to connective tissue at weeks 2 and 3. Exposure to IL-1ß and/or IL-6 induced proliferation and differentiation of MER cells.

Treatment of Child with Four Lost Maxillary Incisors due to Traffic Injury.

Tooth injury is frequently encountered in pediatric clinical practice. A clinician may be requested to not only respond at the time of injury, but also properly diagnose how such injury will affect the teeth and dentition. Here, we present a case of a child who lost 4 bilateral maxillary incisors due to a traffic accident, and in whom marked mesial inclination of the bilateral maxillary canines and bilateral maxillary first premolars would subsequently occur. Dental management was provided over an extended period. The boy, aged 9 years 11 months, visited our department with the chief complaint of premature loss of 4 bilateral maxillary incisors and eating disturbance associated with the loss of these teeth. Initially, a denture was fitted. The bilateral maxillary canines subsequently demonstrated marked mesial inclination, however, and erupted from an area equivalent to that of the bilateral central incisors. The bilateral maxillary first premolars also showed mesial inclination and rotation. Taking esthetics into consideration, a Nance holding arch with resin buttons that extended to the maxillary incisors and attached to artificial teeth was used. This appliance was also equipped with a retracting hook, and the bilateral maxillary canines were moved downward and distally. Presently, the removable partial denture is worn for the purposes of retention, esthetics, and mastication. Long-term oral management will be necessary. Therefore, final prosthetic treatment for the missing maxillary incisors is planned for when the patient reaches adulthood.

Difference in Bilateral Timing of Eruption of Permanent Teeth.

The aim of this study was to obtain basic data that might serve as criteria in the diagnosis of delayed eruption of the permanent teeth. The synchronicity of the eruption of corresponding contralateral teeth was determined. Data were obtained on both the deciduous and permanent dentition based on records made every 2 months at the Department of Pediatric Dentistry of Tokyo Dental College. These data were then used to investigate bilateral differences in the timing of eruption of the permanent teeth. Over 80% of incisors, first molars, mandibular canines, and maxillary first premolars erupted within 4 months of their contralateral counterparts, while this occurred in 75% of maxillary canines, mandibular first premolars, and mandibular second molars, 70% of maxillary and mandibular second premolars, and 65% of maxillary second molars. Bilateral differences in the timing of permanent tooth eruption varied depending on type of tooth, and these differences tended to be smaller for teeth erupting at an earlier point in the order of eruption, and greater for those erupting at a later point. These results suggest that the failure of a contralateral tooth to erupt within 4 months of its counterpart might serve as a criterion in a diagnosis of suspected delayed eruption of a permanent tooth.

Development of Jaw and Deciduous Teeth in Japanese Children -Comparing Size of Crown and Alveolar Area between Today and 40 Years Ago.

The purpose of the present study was to investigate change in the size of the jaw and deciduous teeth in Japanese children over the past 40 years by performing measurements on plaster models of the dental arch obtained from children born between 2007 and 2009 (2000s group) and children born between 1968 and 1974 (70s) group. A total of 61 children were enrolled in the 2000s group, comprising 25 boys (average age, 4 years 5 months) and 36 girls (average age, 4 years 4 months), and 93 children in the 70s group, comprising 45 boys (4 years 6 months) and 48 girls (4 years 4 months). The mesiodistal width of the crown, dental arch width, dental arch length, dental arch height, and available arch length in these groups were measured using a 3D measurement system and the sums of the mesiodistal width of the crown and tooth size-arch length discrepancies calculated. The results of the two groups were statistically compared and the following conclusions reached: the size of deciduous teeth has not changed statistically over the last 40 years; the size of the dental arch has decreased in boys (p<0.01), while remaining approximately the same in girls; and the tooth size-arch length discrepancy has decreased, especially in boys (p<0.01). These results indicate that the interdental spaces of the deciduous dentition have decreased, increasing the risk of crowding in Japanese children.

Intentional Partial Pulpotomy for Treatment of Immature Permanent Maxillary Incisor with Talon Cusp.

We report the successful morphological correction of a maxillary lateral incisor with a talon cusp and the preservation of vital pulp by employing an intentional partial pulpotomy. A talon cusp is a comparatively rare morphological tooth anomaly that may cause multiple clinical problems. An 11-year-old girl was referred to our department for morphological correction of a maxillary lateral incisor with a talon cusp prior to orthodontic treatment. Following a detailed CT examination of the interior morphology, the abnormal cusp was resected and an intentional partial pulpotomy performed. Five years postoperatively, the tooth showed no abnormalities. These results indicate that an intentional partial pulpotomy following a detailed CT examination is effective in correcting complicated morphological anomalies such as a talon cusp containing extended dental pulp.

Relationship between Interdental Spacing and Colonization by Streptococcus mutans in Children.

The purpose of this study was to investigate the relationship between interdental spacing in the upper incisors and other associated factors with colonization by Streptococcus mutans in children. A total of 47 children aged between 24 and 36 months with no decayed cavities were enrolled in the study. Models were obtained to evaluate interdental spacing in the upper anterior region. The relationships between S. mutans colonization and number of erupted teeth, interdental spacing, diet, and oral hygiene behavior were investigated. S. mutans was detected in the saliva of 14 children (29.8%). No sociodemographic variable was associated with colonization by S. mutans. Neither number of erupted teeth nor children's age showed an influence on colonization by S. mutans. However, children with interdental spacing in the upper incisors were less likely to show colonization by S. mutans (15.8%) than those without (39.3%). Furthermore, children who ceased breast-and/or bottle-feeding by 15 months of age were less likely to show colonization by S. mutans (22.7%) than those who continued feeding until 19 months of age or more (46.2%).

Alveolar Growth in Japanese Infants : A Comparison between Now and 40 Years ago.

To investigate differences in alveolar growth during the primary dentition period among different age groups, dental arch width, length, and height were measured in 93 dental arch plaster models obtained from 4-year-olds born between 1968 and 1974, 88 from 5-year-olds born between 1968 and 1974 (70s group), 61 from 4-year-olds born between 2007 and 2009, and 56 from 5-year-olds born between 2006 and 2008 (2000s group). A comparison was also performed to determine whether there was any difference in change between 4 and 5 years of age. The results showed age- and sex-dependent differences in growth patterns in primary dental arch width, length, and height. The amount of change was greater in the 2000s group than in the 70s group, suggesting that yearly growth between 4 and 5 years of age has increased over the last 40 years.

Influence of lyophilization factors and gelatin concentration on pore structures of atelocollagen/gelatin sponge biomaterial.

This study aimed to investigate influences of lyophilization factors and gelatin concentration on pore structures of ACG sponge. ACG sponges of different freezing temperatures (-30, -80 and -196oC), freezing times (1, 2 and 24 h), gelatin concentrations (0.6%AC+0.15%G, 0.6%AC+0.6%G and 0.6%AC+2.4%G), and with 500 µM fluvastatin were fabricated. Pore structures including porosity and pore size were analyzed by scanning electron microscopy and ImageJ. The cytotoxic effects of ACG sponges were evaluated in vitro. Freezing temperature did not affect porosity while high freezing temperature (-30oC) increased pore size. The high gelatin concentration group (0.6%AC+2.4%G) had decreased porosity and pore size. Freezing time and 500 µM fluvastatin did not affect pore structures. The cytotoxicity and cell proliferation assays revealed that ACG sponges had no cytotoxic effects on human mesenchymal stromal cell growth and proliferation. These results indicate that ACG sponge may be a good biomaterial scaffold for bone regeneration.

Influence of Lifestyle Factors on Risk of Dental Caries among Children Living in Urban China.

The prevalence of dental caries has been decreasing among kindergarten children in Shanghai, China, over recent years, although it still remains at an unacceptably high level. The purpose of this study was to identify which factors were important in providing oral health guidance and achieving further improvement in the oral health status of kindergarten children in urban China. A survey was conducted on dental caries in 128 Japanese and 368 Chinese kindergarten children and a questionnaire given to their parents/guardians on each child's lifestyle and dietary habits from birth to the present. Correlations between responses to each questionnaire item and the status of dental caries were statistically analyzed. The dft index score (p=0.0016), prevalence of dental caries (p=0.0002), and percentages of children with decayed (untreated caries-affected) teeth (p<0.0001) were significantly higher in the Chinese than in the Japanese children. Many differences were observed in lifestyle factors between the two groups. The percentage of parents failing to control the child's snacking habits between meals was higher in China, and weaning was significantly delayed in China compared with in Japan. These lifestyle factors were considered to be associated closely with the high risk of dental caries in Chinese kindergarten children. These findings indicate that oral health guidance for kindergarten children in Shanghai, China, should focus on control of dietary habits, including control of inter-meal snacking, and breastfeeding practices. The results of this study may help improve the status of dental caries among Chinese children.

Characterization of a potential ABC-type bacteriocin exporter protein from Treponema denticola.

BACKGROUND: Treponema denticola is strongly associated with the development of periodontal disease. Both synergistic and antagonistic effects are observed among bacterial species in the process of biofilm formation. Bacteriocin-related genes have not yet been fully characterized in periodontopathic bacteria. The aim of this study was to detect and characterize bacteriocin-associated proteins in T. denticola. METHODS: The whole genome sequence of T. denticola ATCC 35405 was screened with a Streptococcus mutans bacteriocin immunity protein (ImmA/Bip) sequence. The prevalence of homologous genes in T. denticola strains was then investigated by Southern blotting. Expression of the genes was evaluated by qRT-PCR. RESULTS: In the genome sequence of T. denticola, an amino acid sequence coded by the open reading frame TDE_0719 showed 26 % identity with the S. mutans ImmA. Furthermore, two protein sequences encoded by TDE_0425 and TDE_2431 in T. denticola ATCC 35405 showed ~40 % identity with that coded by TDE_0719. Therefore, TDE_0425, TDE_0719, and TDE_2431 were designated as tepA1, A2, and A3, respectively. Open reading frames showing similarity to the HlyD family of secretion proteins were detected downstream of tepA1, A2, and A3. They were designated as tepB1, B2, and B3, respectively. A gene harboring a bacteriocin-like signal sequence was detected upstream of tepA1. The prevalence of tepA1 and A2 differed among Treponema species. Susceptibility to chloramphenicol and ofloxacin was slightly decreased in a tepA2 mutant while that to kanamycin was increased. Expression of tepA3-B3 was increased in the tepA2 mutant. CONCLUSION: These results indicate that T. denticola ATCC 35405 has three potential bacteriocin export proteins and that the presence of these genes differs among the Treponema strains. TepA3-B3 of the corresponding proteins may be involved in resistance to chloramphenicol.

Dentilisin involvement in coaggregation between Treponema denticola and Tannerella forsythia.

Periodontitis arises from a biofilm consisting of gram-negative anaerobic rods and spirochetes. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, termed the Red complex, have been co-isolated with high frequency from chronic periodontitis lesions, and these microorganisms are thought to be major pathogens of the disease. Coaggregation is an important strategy in the colonization of dental plaque biofilm by these bacteria. In the present study, we investigated the coaggregation of T. denticola strains with T. forsythia ATCC 43037 by use of visual grading or spectrophotometry. T. denticola ATCC 35405 coaggregated with T. forsythia, reaching a plateau at approximately 60 min. This coaggregation was inhibited by heat treatment of T. denticola ATCC 35405, but not of T. forsythia. Disaccharides such as sucrose, maltose, and lactose inhibited coaggregation by approximately 50%. The coaggregation reaction varied among T. denticola strains. There was somewhat less coaggregation between T. denticola ATCC 33520 and T. forsythia than between T. denticola ATCC 35405 and T. forsythia, although this difference was not statistically significant; T. denticola ATCC 33521 showed a trace level of coaggregation with T. forsythia. The magnitude of coaggregation among the three T. denticola strains was proportional to their dentilisin activities. Inactivation of dentilisin abolished coaggregation activity, but inactivation of the major outer sheath protein did not. In addition, phenylmethylsulfonyl fluoride did not affect coaggregation. These results indicate that dentilisin is involved indirectly in the coaggregation between T. denticola and T. forsythia, because its proteolytic activity is not required, possibly via ligand maturation.