Prospective association between maternal bonding disorders and child toothbrushing frequency: A cross-sectional study of the Japan Environment and Children's Study.

BACKGROUND: Daily toothbrushing prevents early childhood caries, but reinforcement depends on facilitative parenting behaviours. Mother-to-infant bonding, the maternal affection towards the infant, is an environmental factor that strongly influences parenting. AIM: This study examined the association between maternal bonding and children's daily toothbrushing frequency. DESIGN: The sample consisted of 83 954 mother-infant pairs at two years postpartum, derived from the initial sample of JECS (cohort study), which included 104 062 foetuses. Maternal bonding disorders were assessed using the Mother-to-Infant Bonding Scale (MIBS). After multiple imputation for missing data, a multinomial logistic regression analysis was conducted with adjustments for several maternal (eg, age at delivery) and child-related (eg, self-performed toothbrushing) variables. RESULTS: The odds ratio (95% confidence interval) for the association of maternal bonding disorders with the low (once per day) and the very low child toothbrushing frequency (<1 per day) was 1.12 (1.07-1.17) and 1.23 (0.91-1.66), respectively, after covariate adjustments. Furthermore, the univariate general linear model showed that the mean MIBS scores significantly decreased as the daily child toothbrushing frequency increased. CONCLUSIONS: The prevalence of maternal bonding disorders at one year postpartum was prospectively associated with a lower frequency of child toothbrushing at two years of age.

Excessive Game Playing Is Associated with Poor Toothbrushing Behavior among Athletic Children: A Cross-Sectional Study in Miyagi, Japan.

Dental problems among athletes have been cautioned due to negative impacts not only on their oral health but also on athletic performance. Acquirement of appropriate oral health behavior mainly composed of toothbrushing in childhood can be one of the most important strategies for advancing children's athletic possibilities. Although habits of screen viewing, including game playing, and TV viewing have direct impacts on children's health and behavioral development, little is known about the association between these habits and toothbrushing frequency. A cross-sectional survey examining sports activities was conducted using a self-report questionnaire among school-aged athletic children belonging to the Miyagi Amateur Sports Association (n = 6,658). All statistical analyses were performed with SPSS, and P-values less than 0.05 were considered statistically significant. The association between a lower brushing frequency (< 2 times a day) and screen-viewing behavior was examined using multivariate logistic models after adjusting for sex, age, body mass index (BMI), studying time, and sleep duration. After adjustment for all covariates, longer game playing (> 2 hrs a day), but not TV viewing, significantly correlated with lower brushing frequency (P for trend < 0.001). Importantly, longer game-playing behavior was also associated with unhealthy dental behavior defined as a lower brushing frequency regardless of the awareness of dental caries (P for trend < 0.001). In conclusion, this is the first study indicating a type-specific unfavorable impact of screen viewing on oral health behavior among athletic children. Excessive game playing may adversely affect oral health literacy more strongly than TV viewing.

Factors associated with sports-related dental injuries among young athletes: a cross-sectional study in Miyagi prefecture.

BACKGROUND: Sports-related dental injuries, such as tooth fracture, loosening, and avulsion, are a major concern among young athletes because they directly impair oral function. Although the preventive efficacy of mouthguards has been well established, the prevalence of sports-related dental injuries remains high among young athletes. The aim of this study is to identify the variables contributing to the risk of sports-related dental injuries by conducting a survey on large population of young athletes in Miyagi prefecture. METHODS: A cross-sectional study was conducted with school-aged athletes (aged 6-15 years, n = 5735) using a self-reported questionnaire. The questionnaire examined general variables, including sex, age, and body mass index; sports-related variables, including sports-type, team level, activity schedule, break time, and verbal/physical abuse by coaches; and lifestyle variables related to free time, including screen-time and sleep duration. Their associations with sports-related dental injuries were examined using multivariate logistic regression models. RESULTS: The prevalence of sports-related dental injuries was 13.3% (763 of 5735 young athletes) and was higher in males (14.3%, 592 of 4132) than in females (10.7%, 171 of 1603; adjusted odds ratios [ORs] and 95% confidence intervals [CIs]: 1.48 [1.22-1.79], p < 0.001). After stratification according to sex, significant associations with the prevalence of sports-related dental injuries were evident for three variables-insufficient break time, verbal abuse, and physical punishment-in males (adjusted ORs [95% CI]: 1.35 [1.03-1.77], p = 0.032; 1.31 [1.05-1.62], p = 0.015; and 1.36 [1.06-1.75], p = 0.016, respectively) but not in females (adjusted ORs [95% CI]: 0.88 [0.53-1.47], p = 0.623; 1.29 [0.87-1.91], p = 0.206; and 0.97 [0.57-1.63], p = 0.894, respectively). CONCLUSIONS: Although our results might be based on the individual athlete's self-perception to the sports-related variables, our results suggest that insufficient break time, verbal abuse, and physical punishment from coaches are positively associated with the prevalence of sports-related dental injuries in young male athletes.

Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells.

Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through ß-catenin-dependent canonical and ß-catenin-independent noncanonical pathways. Canonical Wnt/ß-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of ß-catenin as well as ß-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the ß-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells.

A Strategy against the Osteonecrosis of the Jaw Associated with Nitrogen-Containing Bisphosphonates (N-BPs): Attempts to Replace N-BPs with the Non-N-BP Etidronate.

Bisphosphonate (BP)-related osteonecrosis of the jaw (BRONJ) can occur when enhanced bone-resorptive diseases are treated with nitrogen-containing BPs (N-BPs). Having previously found, in mice, that the non-N-BP etidronate can (i) reduce the inflammatory/necrotic effects of N-BPs by inhibiting their intracellular entry and (ii) antagonize the binding of N-BPs to bone hydroxyapatite, we hypothesized that etidronate-replacement therapy (Eti-RT) might be useful for patients with, or at risk of, BRONJ. In the present study we examined this hypothesis. In each of 25 patients receiving N-BP treatment, the N-BP was discontinued when BRONJ was suspected and/or diagnosed. After consultation with the physician-in-charge and with the patient's informed consent, Eti-RT was instituted in one group according to its standard oral prescription. We retrospectively compared this Eti-RT group (11 patients) with a non-Eti-RT group (14 patients). The Eti-RT group (6 oral N-BP patients and 5 intravenous N-BP patients) and the non-Eti-RT group (5 oral N-BP patients and 9 intravenous N-BP patients) were all stage 2-3 BRONJ. Both in oral and intravenous N-BP patients (particularly in the former patients), Eti-RT promoted or tended to promote the separation and removal of sequestra and thereby promoted the recovery of soft-tissues, allowing them to cover the exposed jawbone. These results suggest that Eti-RT may be an effective choice for BRONJ caused by either oral or intravenous N-BPs and for BRONJ prevention, while retaining a level of anti-bone-resorption. Eti-RT may also be effective at preventing BRONJ in N-BP-treated patients at risk of BRONJ. However, prospective trials are still required.

Periodontal Disease Is Associated with Insomnia among Victims of the Great East Japan Earthquake: A Panel Study Initiated Three Months after the Disaster.

In March 2011, the Great East Japan Earthquake (GEJE), which was followed by a devastating tsunami, destroyed the societal and the public hygiene systems in Japanese coastal areas. Insomnia, the greatest issue among disaster victims, has detrimental effects on both physical and psychological health. Periodontitis causes chronic discomfort and inflammation, and little is known about its impact on insomnia. Three months after the earthquake, a health panel survey was conducted over four surveys, till September 2013, in which information regarding 8,015 adults was collected and used. In addition to the heath-related questionnaire, other variables including subjective symptoms of oral diseases were recorded, and the Athens Insomnia Scale was used to evaluate the severity of insomnia. The association between insomnia and periodontal disease was examined using multilevel logistic models on the panel data, after adjusting for sex, age, economic status, comorbidities, body mass index, post-traumatic stress reactions, habitual smoking and alcohol drinking, and the Kessler Psychological Distress Scale score. In addition to the higher prevalence of insomnia among GEJE victims, significant association was revealed between insomnia and gum problems (OR = 2.16, 95% CI = 1.43-3.26), and difficulty chewing (OR = 2.22, 95% CI = 1.40-3.51), after adjusting for all covariates. The present study revealed significant association between insomnia and periodontal disease among GEJE victims. This indicated that together, integrated oral health care for disaster victims would contribute not only to prevention of oral infectious diseases, but may also help alleviate other problems caused by these harmful events.

Roles played by histamine in strenuous or prolonged masseter muscle activity in mice.

Bruxism and/or clenching, resulting in fatigue or dysfunction of masseter muscles (MM), may cause temporomandibular disorders. Functional support of the microcirculation is critical for prolonged muscle activity. Histamine is a regulator of the microcirculation and is supplied by release from its stores and/or by de novo production via the induction of histidine decarboxylase (HDC). Interleukin (IL)-1, a cytokine involved in temporomandibular disorders, is an inducer of HDC. In the present study, we examined the roles of histamine, HDC and IL-1 in MM activity. Experiments were conducted using our R+G+ model. A mouse restrained (R+) inside a narrow cylinder (front end blocked with a thin plastic strip) gnaws away (G+) the strip to escape, with the weight reduction in the strip serving as an index of MM activity. Fexofenadine (a peripherally acting histamine H1 receptor antagonist) reduced MM activity in normal mice. Both H1 receptor-deficient and HDC-deficient mice exhibited low MM activity. Prolonged R+G+ induced HDC activity in MM. Mast cell-deficient mice exhibited strikingly low HDC induction in MM (and also in the quadriceps femoris muscle) in response to muscle activity or IL-1ß. Mast cells were present around blood vessels and nerves in the epimysium and perimysium of MM. These results, together with others reported previously, suggest that: (i) peripheral histamine supports strenuous MM activity; (ii) strenuous MM activity stimulates mast cells to release histamine and to induce HDC (which replenishes the histamine pool in mast cells, possibly mediated by IL-1); and (iii) peripheral histamine H1 receptor antagonists may be effective in treating temporomandibular disorders or preventing prolonged clenching and/or bruxism.

Influence of maternal postpartum depression on children's toothbrushing frequency.

OBJECTIVES: Daily toothbrushing behaviour is very effective for the prevention of early childhood dental caries (ECC), but is mostly dependent upon parental management. Intrapersonal association between lower toothbrushing frequency and depression is well known; however, the impact of maternal postpartum depression on child toothbrushing behaviour within the mother-child dyad remains unknown. The aim of this study was to determine the association between the prevalence of maternal postpartum depression and lower toothbrushing frequency in children aged two years. METHODS: A secondary analysis of a data set from the Japan Environment and Children's Study was performed. A total of 104 062 fetuses were enrolled after obtaining informed written parental consent, in which 84 533 mother-infant pairs were included after applying exclusion criteria. The Edinburgh Postpartum Depression Scale (EPDS) was used to evaluate maternal postpartum depression (a total score of ≥9 in EPDS) at one and six months postpartum. Indeed, the participants were classified based on the persistence of postpartum depression: 'Resilient' (no prevalence); 'Improving' (prevalence only at one month postpartum); 'Emergent' (prevalence only at six months postpartum); and 'Chronic' (prevalence at both time points). The association between postpartum depression and a toothbrushing frequency in children (the reference group: more than once per day, the low group: once per day, and the very-low group: less than once per day) was examined using Poisson regression models with adjustments for maternal and child characteristics after multiple imputations for missing data. RESULTS: The prevalence of maternal postpartum depression at one and six months postpartum was 13.9% and 11.4%, respectively; the proportions of each persistence group were 81.1% ('Resilient'), 7.5% ('Improving'), 5.0% ('Emergent') and 6.4% ('Chronic'). Concerning children's toothbrushing frequency, 51.6% and 0.5% of participants self-reported frequencies of once per day and less than once per day, respectively. The association of maternal postpartum depression with a lower toothbrushing frequency in children consistently had higher relative risks (RRs). However, these associations were weakened when adjusting for whether the child could self-perform toothbrushing or whether this was done under parental supervision. A key result is that participants with persistent postpartum depression at both one and six month(s) postpartum showed the highest adjusted RRs (95% CI) for lower toothbrushing frequency in children (1.08 [1.04-1.12] with a decrease in children's toothbrushing frequency). CONCLUSION: Maternal mental health provides valuable screening information for children with lower toothbrushing frequency for the purpose of preventing ECC.

Prospective association of short sleep duration in newborns with bruxism behavior in children: The Japan Environment and Children's Study (JECS).

BACKGROUND: Bruxism is defined as a repetitive masticatory muscle activity, characterized by clenching or grinding of the teeth and/or by bracing or thrusting of the mandible. Although the bruxism etiology can be bidirectionally described with sleep disorders, there are few studies available to understand the association of sleep duration with bruxism behavior in early childhood. METHODS: A dataset of children (n = 90,148) from the Japan Environment and Children's Study (JECS) - an ongoing nationwide, prospective birth cohort study - was used to investigate the prospective association of the infant's sleep duration with bruxism behavior, which were acquired using self-reported questionnaire. After multiple imputation of data, the association between sleep duration and bruxism behavior in early childhood was examined using a binomial logistic regression analysis. It was conducted with adjustments for several maternal (e.g., age at delivery and sleep duration) and child-related (e.g., sex and sleep position) variables. RESULTS: The prevalence of bruxism behavior at the ages of two and four were 16.2% and 22.5%, respectively. Using the shortest sleep duration group's participants (≤13 h) as the reference, the odds ratio (95% confidence interval) for prevalence of bruxism behavior decreased with longer sleep duration at one month of age, but not at 36 months of age, after covariate adjustments. Furthermore, in comparison with the impacts between daytime and nighttime sleep, nighttime sleep with longer duration were dominantly associated with bruxism behavior. CONCLUSION: The sleep duration in infant stage, especially during newborn stage was associated with the prevalence of bruxism behavior in children.

Synthesis and intracellular transportation of type I procollagen during functional differentiation of odontoblasts.

The expression of type I collagen, the most component of dentin extracellular matrix proteins (ECMs) in odontoblast is correlated with the activity of dentin formation. Since odontoblast possesses a distinct cellular process for protein transport into the dentinal tubule, it is important to examine the intracellular protein localization. However, a study focusing on odontoblast processes has not been performed. Type I collagen is synthesized as procollagen, which is immediately converted to collagen upon secretion. After characterization of antiserum to rat type I procollagen, we investigated the intracellular localization of type I procollagen in odontoblasts during and after dentinogenesis, using immunohistochemistry and in situ hybridization. The level of mRNA expression decreased during dentinogenesis, whereas the intracellular localization of type I procollagen in odontoblast processes become more distinct. The percentage of dentinal tubules with type I procollagen increased significantly with aging. Odontoblasts in pulp horn, in particular, showed moderate expression of type I procollagen after dentinogenesis. Since loss of occlusion also caused a significant decrease in type I procollagen, we concluded that occlusal stimulation activated type I procollagen synthesis in odontoblasts. We also suggest that analysis of intracellular transport of type I procollagen via odontoblast processes may be a new approach to evaluation of odontoblast function.

Dynamic changes in bone metabolism in the rat temporomandibular joint after molar extraction using bone scintigraphy.

Functional adaptation with reformation of bone tissue structure occurs after changes in mechanical stress distribution. To examine how occlusal changes affect the dynamics of bone metabolism in the mandibular condyle, bone scintigraphy of rat condyles was taken using (99m)technetium-methylene-diphosphonate (99mTc-MDP) after extraction of maxillary molars resulting in unilateral loss of occlusal support. Accumulation of 99mTc-MDP was significantly higher in the condyles on the extracted side than on the intact side 3 d after molar extraction. In addition, bone mineral density (BMD) and osteoprotegerin expression in extracted-side condyles were significantly increased while osteoclast numbers were significantly decreased when compared with intact-side condyles. These differences were not detected 28 d after molar extraction. These findings suggest that occlusal change transiently results in changes in the dynamics of bone metabolism at the mandibular condyles through the downregulation of osteoclastogenesis. These changes may be involved in functional adaptation of the temporomandibular joint.

Transforming growth factor-beta1 expression is up-regulated in maturation-stage enamel organ and may induce ameloblast apoptosis.

Transforming growth factor-beta1 (TGF-beta1) regulates a variety of cellular responses that are dependent on the developmental stage and on the origins of the cell or the tissue. In mature tissues, and especially in tissues of epithelial origin, TGF-beta1 is generally considered to be a growth inhibitor that may also promote apoptosis. The ameloblast cells of the enamel organ epithelium are adjacent to and responsible for the developing enamel layer on unerupted teeth. Once the enamel layer reaches its full thickness, the tall columnar secretory-stage ameloblasts shorten, and a portion of these maturation-stage ameloblasts become apoptotic. Here we investigate whether TGF-beta1 plays a role in apoptosis of the maturation-stage ameloblasts. We demonstrate in vitro that ameloblast lineage cells are highly susceptible to TGF-beta1-mediated growth arrest and are prone to TGF-beta1-mediated cell death/apoptosis. We also demonstrate in vivo that TGF-beta1 is expressed in the maturation-stage enamel organ at significantly higher levels than in the earlier secretory-stage enamel organ. This increased expression of TGF-beta1 correlates with an increase in expression of the enamel organ immediate-early stress-response gene and with a decrease in the anti-apoptotic Bcl2 : Bax expression ratio. We conclude that TGF-beta1 may play an important role in ameloblast apoptosis during the maturation stage of enamel development.

High prevalence of toothache among Great East Japan Earthquake survivors living in temporary housing.

OBJECTIVES: The Great East Japan Earthquake and subsequent tsunami cause large-scale destruction in the north-eastern coastal areas in Japan, and forced many survivors to relocate to prefabricated temporary housing, a typical emergency accommodation. Based on the hypothesis that higher toothache prevalence among the disaster survivors is associated with postdisaster distress, we determined the impact of temporary residential environment as potential stressor on the subjective toothache prevalence. METHODS: A repeated cross-sectional health survey based on self-reported questionnaire was conducted in 2776 disaster survivors, of whom 1446 participants underwent dental examination by dentists. Housing type was categorized into three groups including the same housing as before the earthquake, temporary housing and rented/new housing. The association of housing type with subjective toothache prevalence was examined using multivariate logistic regression analysis in all subjects and subgroup analysis in dental examination applicants. Stratified analysis by survey wave was applied with inclusion of covariates such as the socio-demographic factors, and presence of insomnia and psychological distress. In subgroup analysis, presence of dental caries and gum problems in dental examination were included as factors of direct exposure to subjective toothache. RESULTS: In the first survey wave, the participants living in the temporary housing had significantly higher odds ratio (OR) for toothache prevalence compared to the participants living in the same housing (OR: 3.76, 95% confidence interval [CI]: 1.85-7.65, P < 0.001); whereas in all other survey waves, there was no significant difference. Subgroup analysis of dental examination applicants confirmed the presence of significant association of subjective toothache prevalence in the temporary housing group alone (OR: 3.27, 95% CI: 1.38-7.76, P = 0.004), but not in the rented/new housing group (OR: 1.50, 95% CI: 0.57-3.91, P = 0.411), even after adjusting for covariates related to oral findings. CONCLUSION: Temporary housing may be a factor to increase the risk of subjective toothache among disaster survivors only at postdisaster acute phase.

Fluoride induces endoplasmic reticulum stress and inhibits protein synthesis and secretion.

BACKGROUND: Exposure to excessive amounts of fluoride (F(-)) causes dental fluorosis in susceptible individuals; however, the mechanism of F(-)-induced toxicity is unclear. Previously, we have shown that high-dose F(-) activates the unfolded protein response (UPR) in ameloblasts that are responsible for dental enamel formation. The UPR is a signaling pathway responsible for either alleviating endoplasmic reticulum (ER) stress or for inducing apoptosis of the stressed cells. OBJECTIVES: In this study we determined if low-dose F(-) causes ER stress and activates the UPR, and we also determined whether F(-) interferes with the secretion of proteins from the ER. METHODS: We stably transfected the ameloblast-derived LS8 cell line with secreted alkaline phosphatase (SEAP) and determined activity and localization of SEAP and F(-)-mediated induction of UPR proteins. Also, incisors from mice given drinking water containing various concentrations of F(-) were examined for eucaryotic initiation factor-2, subunit alpha (eIF2alpha) phosphorylation. RESULTS: We found that F(-) decreases the extracellular secretion of SEAP in a linear, dose-dependent manner. We also found a corresponding increase in the intracellular accumulation of SEAP after exposure to F(-). These changes are associated with the induction of UPR proteins such as the molecular chaperone BiP and phosphorylation of the UPR sensor PKR-like ER kinase, and its substrate, eIF2alpha. Importantly, F(-)-induced phosphorylation of eIF2alphawas confirmed in vivo. CONCLUSIONS: These data suggest that F(-) initiates an ER stress response in ameloblasts that interferes with protein synthesis and secretion. Consequently, ameloblast function during enamel development may be impaired, and this may culminate in dental fluorosis.

Transfer of the bone morphogenetic protein 4 gene into rat periodontal ligament by in vivo electroporation.

OBJECTIVE: Regulation of alveolar bone metabolism is required in clinical dentistry. The aim of the present study was to establish a method for gene transfer into the periodontal ligament (PDL) by in vivo electroporation with a plasmid vector and to investigate the effects of BMP-4 transfer into the PDL. DESIGN: Plasmids containing mouse BMP-4 cDNA (pCAGGS-BMP4) were transfected into cultured rat PDL cells by in vitro electroporation, and BMP-4 production and secretion were detected by immunocytochemistry and western blotting. Next, pCAGGS-BMP4 was injected into the PDL of rats, and electroporation was performed in vivo, using original paired-needle electrodes. BMP-4 expression was examined by immunohistochemical staining 3, 7, 14, 21, and 28days after electroporation. Control groups were injected with pCAGGS by electroporation, injected with pCAGGS-BMP4 without electroporation, or subjected to neither injection nor electroporation. RESULTS: In vitro-transfected rat PDL cells exhibited production and secretion of the mature-form BMP-4. After in vivo electroporation of pCAGGS-BMP4, site-specific BMP-4 expression peaked on day 3, gradually decreased until day 14, and was absent by day 21. We observed no unfavorable effects such as inflammation, degeneration, or necrosis. CONCLUSIONS: Gene transfer by electroporation with plasmid DNA vectors has several advantages over other methods, including the non-viral vector, non-immunogenic effects, site-specific expression, simplicity, cost-effectiveness, and limited histological side effects. Our results indicate that the method is useful for gene therapy targeting the periodontal tissue, which regulates alveolar bone remodeling.

Involvement of peripheral artemin signaling in tongue pain: possible mechanism in burning mouth syndrome.

Burning mouth syndrome is characterized by altered sensory qualities, namely tongue pain hypersensitivity. We found that the mRNA expression of Artemin (Artn) in the tongue mucosa of patients with burning mouth syndrome was significantly higher than that of control subjects, and we developed a mouse model of burning mouth syndrome by application of 2,4,6-trinitrobenzene sulfonic acid (TNBS) diluted with 50% ethanol to the dorsum of the tongue. TNBS treatment to the tongue induced persistent, week-long, noninflammatory tongue pain and a significant increase in Artn expression in the tongue mucosa and marked tongue heat hyperalgesia. Following TNBS treatment, the successive administration of the transient receptor potential vanilloid 1 (TRPV1) antagonist SB366791 or neutralizing anti-Artn antibody completely inhibited the heat hyperalgesia. The number of glial cell line-derived neurotrophic factor family receptor α3 (GFRα3)-positive and TRPV1-positive trigeminal ganglion (TG) neurons innervating the tongue significantly increased following TNBS treatment and was significantly reduced by successive administration of neutralizing anti-Artn antibody. The capsaicin-induced current in TG neurons innervating the tongue was enhanced following TNBS treatment and was inhibited by local administration of neutralizing anti-Artn antibody to the tongue. These results suggest that the overexpression of Artn in the TNBS-treated tongue increases the membrane excitability of TG neurons innervating the tongue by increasing TRPV1 sensitivity, which causes heat hyperalgesia. This model may be useful for the study of tongue pain hypersensitivity associated with burning mouth syndrome.

Extracellular ATP inhibits IL-1-induced MMP-1 expression through the action of CD39/nucleotidase triphosphate dephosphorylase-1 on human gingival fibroblasts.

Extracellular adenosine triphosphate (ATP) is sequentially dephosphorylated by two ectoenzymes: CD39/nucleotidase triphosphate dephosphorylase (ENTPD) and CD73/5'-ectonucleotidase (5'-NT). Adenosine, its notable metabolite, may elicit potent anti-inflammatory responses. We examined whether the CD39-adenosinergic axis may exist in gingival fibroblasts and have an effect on the expression of matrix metalloproteinase (MMP)-1, the excess production of which leads to pathological matrix degradation. We showed that transcripts of CD39, CD73, and adenosine receptors A1, A2a, and A2b, but not A3, were expressed by human gingival fibroblasts by RT-PCR. We also identified the expression of CD39 in fibroblastic cells in rat gingiva by immunohistochemistry. ATP inhibited the expression of MMP-1 triggered by interleukin-1 at gene and protein levels. However, ATP-γS, a stable ATP analog, did not. The ATP-mediated MMP-1 inhibition was restored in the presence of POM-1, a specific ENTPD inhibitor, suggesting that CD39/ENTPD was involved in the MMP-1 inhibition. ATP metabolites including adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), and adenosine inhibited MMP-1 expression, but ADP-ßS, a stable ADP, did not, suggesting that adenosine converted from ATP by the action of CD39/ENTPD and CD73/5'-NT may contribute to MMP-1 inhibition. Adenosine-mediated MMP-1 inhibition was restored in the presence of H89, a protein kinase A (PKA) inhibitor. Conversely, forskolin, an enhancer of intracellular cAMP, mimicked the effect of adenosine, suggesting that the cAMP/PKA signaling pathway is involved in adenosine-mediated MMP-1 inhibition. The present findings suggest the existence of an endogenous anti-tissue destructive mechanism in gingival tissue via the CD39-adenosinergic axis.

Physiological distal drift in rat molars contributes to acellular cementum formation.

Occlusal forces may induce the physiological teeth migration in humans, but there is little direct evidence. Rat molars are known to migrate distally during aging, possibly caused by occlusal forces. The purpose of this study was to determine if a reduction in occlusion would decrease teeth migration and affect associated periodontal structures such as cementum. To reduce occlusal forces, the right upper first molar (M1) in juvenile rats was extracted. The transition of the position of upper second molar (M2) and formation of M2 cementum was followed during aging. From the cephalometric analyses, upper M2 was located more anterior compared with the original position with aging after M1 extraction. Associated with this "slowing-down" of the physiological drift, cementum thickness on distal surface, but not on mesial surface, of M2 root was significantly increased. The accumulation of alizarin red as vital stain indicative of calcification, was observed in the distal cementum of M2 root only on the side of M1 extraction. Extraction of M1 that results in less functional loading, distinctly attenuates the physiological drift only in the upper dentition. The decreased physiological drift appears to activate acellular cementum formation only on distal surface of M2 root, perhaps due to reduced mechanical stress associated with the attenuated distal drift. In conclusion, the physiological distal drift in rat molars appears to be largely driven by the occlusal force and also affects the formation of acellular cementum. These findings provide additional direct evidence for an important role of occlusal forces in tooth migration.

Measurement of fluoride-induced endoplasmic reticulum stress using Gaussia luciferase.

Endoplasmic reticulum (ER) stress and its consequent activation of the unfolded protein response (UPR) signaling pathway have been implicated in several pathophysiologic disorders as well as in drug resistance to treatment of tumors. Several techniques have been devised that qualitatively and quantitatively demonstrate the presence of ER stress and the activation of the UPR; however, most of these methods cannot be used to measure ER stress in real time. Here we describe the use of cells stably transduced with a secreted reporter, Gaussia luciferase (Gluc), to measure fluoride-induced ER stress. Factors that affect ER homeostasis, such as high-dose fluoride, will cause decreased Gluc secretion that can be measured as a decrease in Gluc activity in the culture medium supernatant. Gluc catalyzes the oxidative decarboxylation of coelenterazine (CTZ) to coeleneteramide, resulting in blue bioluminescence (λ(max) 485 nm). Therefore, Gluc activity can be easily quantified by mixing a small aliquot of the medium supernatant with CTZ and measuring the resulting bioluminescence in a luminometer. Among the various reporters used so far, Gluc is regarded as the most sensitive indicator of ER stress. A second advantage for using Gluc is its ability to function in a wide pH range. This is especially useful for studying fluoride-mediated toxicity as fluoride-induced stress is enhanced under acidic conditions. Since Gluc can be measured in a noninvasive manner, it has been used in several in vitro and in vivo applications. In this chapter, we detail our methodology for using Gluc to monitor fluoride-induced ER stress.

The acid test of fluoride: how pH modulates toxicity.

BACKGROUND: It is not known why the ameloblasts responsible for dental enamel formation are uniquely sensitive to fluoride (F(-)). Herein, we present a novel theory with supporting data to show that the low pH environment of maturating stage ameloblasts enhances their sensitivity to a given dose of F(-). Enamel formation is initiated in a neutral pH environment (secretory stage); however, the pH can fall to below 6.0 as most of the mineral precipitates (maturation stage). Low pH can facilitate entry of F(-) into cells. Here, we asked if F(-) was more toxic at low pH, as measured by increased cell stress and decreased cell function. METHODOLOGY/PRINCIPAL FINDINGS: Treatment of ameloblast-derived LS8 cells with F(-) at low pH reduced the threshold dose of F(-) required to phosphorylate stress-related proteins, PERK, eIF2alpha, JNK and c-jun. To assess protein secretion, LS8 cells were stably transduced with a secreted reporter, Gaussia luciferase, and secretion was quantified as a function of F(-) dose and pH. Luciferase secretion significantly decreased within 2 hr of F(-) treatment at low pH versus neutral pH, indicating increased functional toxicity. Rats given 100 ppm F(-) in their drinking water exhibited increased stress-mediated phosphorylation of eIF2alpha in maturation stage ameloblasts (pH<6.0) as compared to secretory stage ameloblasts (pH approximately 7.2). Intriguingly, F(-)-treated rats demonstrated a striking decrease in transcripts expressed during the maturation stage of enamel development (Klk4 and Amtn). In contrast, the expression of secretory stage genes, AmelX, Ambn, Enam and Mmp20, was unaffected. CONCLUSIONS: The low pH environment of maturation stage ameloblasts facilitates the uptake of F(-), causing increased cell stress that compromises ameloblast function, resulting in dental fluorosis.