Current status and factors of periodontal disease among Japanese high school students: a cross-sectional study.

INTRODUCTION: This study aimed to investigate the prevalence of periodontal disease and the factors of the disease among high school students. METHOD: The participants were all students aged 15-18 years (n = 1202) at a high school in Japan. The data on oral health perceptions and behaviours were collected by a questionnaire survey. The prevalence of periodontal disease among them was investigated with the partial community periodontal index (PCPI). A logistic regression analysis was used to identify the factors associated with the PCPI. RESULTS: A total of 1069 students (88.9%) participated in this study. The prevalence of gingival bleeding, calculus, pocket depth of 4-5 mm, and pocket depth of 6 mm or more were 44.2%, 42.2%, 11.4%, and 1.6%, respectively. Approximately one-third of the students had a fear of dental treatment, and only 28.4% used dental floss. The results of logistic regression analysis, adjusted for sex and school year, showed that not visiting dentists regularly, not using dental floss, brushing teeth for less than 5 min, fear of dental treatment, and drinking sports drinks frequently were positively associated with periodontal conditions. CONCLUSION: This study identified a high prevalence of periodontal disease among Japanese high school students aged 15-18 years and its risk factors, such as poor oral health behaviours and fear of dental treatment.

Effects of root-end filling materials on vascular endothelial cell proliferation and tube formation.

Background/purpose: Regarding root-end filling materials in apical surgery, sealing ability and biocompatibility are useful for treatment. Angiogenesis, which occurs in the process of periapical wound healing, is closely related to bone formation. In this study, we investigated the effects of root-end filling materials on vascular endothelial cell proliferation and angiogenesis. Materials and methods: Mineral trioxide aggregate (MTA), 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin, Super EBA, and CS-BG-multi, bioactive glass-related materials, were used. After curing, each material was soaked in a medium for 1 or 7 days, and then cultured for 1-7 days to investigate the effects on human umbilical vein endothelial cell (HUVEC) proliferation, angiogenesis, and vascular endothelial growth factor receptors (VEGFRs) mRNA expression. Results: In the 1-day soaked sample, there was significantly less proliferation in MTA and Super EBA on day 7 of culture. In the 7-day soaked sample, there was significantly less proliferation in Super EBA and CS-BG-multi on day 7 of culture. Tube formation was significantly high in MTA in both the 1-day and 7-day soaked samples, significantly high in SB in the 1-day soaked sample, and significantly low in Super EBA in both the 1-day and 7-day soaked samples. CS-BG-multi was comparable to the control. VEGFR-1 and VEGFR-2 mRNA expressions showed an upward trend in MTA, and a trend similar to the control in SB. Conclusion: MTA and 4-META/MMA-TBB resin had a higher pro-angiogenic effect while Super EBA had a less pro-angiogenic effect. CS-BG-multi had low toxicity on tube formation of HUVEC.

Development of an error-detection examination for conservative dentistry education.

OBJECTIVE: For dental students, textbooks and lectures provide basic knowledge, and simulated and actual clinical training provide learning in technical and communication skills. At our college, conservative dentistry is taught in the third and fourth years of a 6-year undergraduate degree. Clinical training is undertaken subsequently in the fifth year and includes cavity preparation and composite resin filling tasks. However, despite the clinical importance of a full understanding surrounding these procedures, sixth-year students occasionally provide incorrect answers regarding these procedures in assessments. Although they demonstrated a basic understanding of the procedures, they may have forgotten the acquired knowledge during their clinical training. Therefore, we developed an error-detection examination to evaluate and improve fifth-year students' knowledge. METHODS: Written detailed treatment procedures for standardized, typical, cases were presented to students. Some critical steps were intentionally written incorrectly, and students had to identify and correct these. After correcting the steps, students gave a presentation to their peers on their corrections. This was followed by a summary of the correct answers and a short lecture by the teacher. Students then completed a questionnaire investigating their experience of the examination. RESULTS: Students misunderstood some key treatment steps, such as pretreatment of composite resin filling, amalgam removal, and ceramic inlay fitting. The questionnaire revealed that this method of testing applied knowledge was new to students and helped them to identify knowledge gaps. The test also increased their motivation to study conservative dentistry. Students were open to taking similar tests in different areas. CONCLUSION: Although conservative dentistry is a basic field of dental treatment, mistakes in treatment can lead to early treatment failure or reduce the lifetime of a restored tooth. Therefore, students need to have a deep understanding of procedures. Error-detection examinations may help students identify knowledge gaps and provide useful feedback to teachers to identify areas that they should stress in earlier years.

Enhanced junctional epithelial permeability in TRPV4-deficient mice.

BACKGROUND AND OBJECTIVE: As the interface between the oral cavity and the teeth, the junctional epithelial barrier is critical for gingival defense. The junctional epithelium is subject to mechanical stresses from biting force or external insults such as bacterial attacks, but little is known about the effects of mechanical stimuli on epithelial functions. Transient receptor potential vanilloid 4 (TRPV4) functions as a mechanosensitive nonselective cation channel. In the present study, based on marked expression of TRPV4 in the mouse junctional epithelium, we aimed to clarify the putative links between TRPV4 and junctional complexes in the junctional epithelium. METHODS AND RESULTS: Histological observations revealed that the junctional epithelium in TRPV4-deficient (TRPV4-/- ) mice had wider intercellular spaces than that in wild-type (TRPV4+/+ ) mice. Exogenous tracer penetration in the junctional epithelium was greater in TRPV4-/- mice than in TRPV4+/+ mice, and immunoreactivity for adherens junction proteins was suppressed in TRPV4-/- mice compared with TRPV4+/+ mice. Analysis of a mouse periodontitis model showed greater bone volume loss in TRPV4-/- mice compared with TRPV4+/+ mice, indicating that an epithelial barrier deficiency in TRPV4-/- mice may be associated with periodontal complications. CONCLUSION: The present findings identify a crucial role for TRPV4 in the formation of adherens junctions in the junctional epithelium, which could regulate its permeability. TRPV4 may be a candidate pharmacological target to combat periodontal diseases.

Induction of bone repair in rat calvarial defects using a combination of hydroxyapatite with phosphatidylserine liposomes.

Phosphatidylserine (PS)-normally present on the inner leaflet of the plasma membrane-translocates to the outer leaflet at an early stage of apoptosis. PS-containing liposomes (PSLs) can mimic the effect of apoptotic cells in inducing the secretion of prostaglandin E2 from phagocytes and inhibiting the maturation of dendritic cells and osteoclast precursors. The present study attempted to evaluate the effect of calcium phosphate (in the form of hydroxyapatite [HAP]) in the presence or absence of PSLs for repair of rat calvarial bone defects. The defects, each 5 mm in diameter, were created in the calvaria parietal bone of 8-week-old Wistar rats and subjected to one of the following treatments: no augmentation (Sham), HAP alone, or a mixture of HAP and PSL (HAP+PSL). Micro-computed tomography data showed that the HAP+PSL complexes promoted greater bone regeneration in comparison with either the Sham procedure or HAP alone at 4 and 8 weeks after implantation. The regeneration of calvarial bone defects induced by PSLs was mediated partly through upregulation of the osteogenic marker Alkaline Phosphatase, Type I collagen, osteocalcin, Runx2, and Osterix mRNAs. These data are the first to show that PSLs can influence bone regeneration by regulating osteoblast differentiation.

Milk basic protein supplementation enhances fracture healing in mice.

OBJECTIVES: There is an unmet need for agents that can stimulate bone healing. The goal of this study was to evaluate the effects of basic proteins from milk whey (milk basic protein [MBP]) on fracture healing in mice. METHODS: Closed tibial transverse fractures were generated in 6-wk-old male C3 H/HeJ mice given either tap water or MBP-supplemented water for 3, 7, 14, 28, and 56 d after fracture generation. The tibial tissues were analyzed by radiography, µCT, and a three-point bending test. The expression levels of genes associated with bone metabolism were analyzed by real-time reverse transcription-polymerase chain reaction. RESULTS: Quantitative µCT analysis showed that MBP-treated fractured tibiae had a larger hard callus in the sectional area and a larger volume compared with fractured tibiae without MBP treatment. The expression levels of genes associated with chondrogenesis and osteogenesis showed greater increases in fractured tibiae with MBP treatment. Significant increases in the callus mechanical properties were found in MBP-treated tibiae. CONCLUSIONS: MBP supplementation has the potential to improve fracture healing and bone strength in mouse tibiae. MBP could be a potential safe, low-cost, and easily administered nutritional element to prevent secondary fractures in patients with bone fractures.

Histologic evaluation of the effects of Emdogain gel on injured root apex in rats.

INTRODUCTION: This study investigated the effects of Emdogain gel (EMD) on the injured open apex within periapical lesions. METHODS: Periapical lesions were induced in rats by opening the pulp chambers of the mandibular first molars and filing the apical foramen through the distal root canal with #25 K-files to make an open apex. The teeth were exposed to the oral environment for 7 days. Then we irrigated the distal root canals and divided them into EMD-treated and propylene glycol alginate-treated groups. The rats were killed 7, 14, and 28 days after treatment and examined histochemically. RESULTS: In the EMD-treated rats, more cells expressed transforming growth factor-ß1 or bone morphogenetic protein-2 at 7 days after treatment, and the regeneration of cementum and bone was observed around the root apex at 14 days after treatment. Conversely, in the propylene glycol alginate-treated group, few cells expressed transforming growth factor-ß1 or bone morphogenetic protein-2, and apical periodontal tissue recovery was rarely seen within the periapical lesions throughout the experiment. CONCLUSIONS: These results suggest that EMD does not irritate injured periapical tissue and may create a favorable environment that promotes the healing of destroyed periapical tissues.

Immunohistochemical study of amelogenin and lysosome-associate membrane proteins (LAMPs) in cartilage / Estudio inmunohistoquímico de la amelogenina y proteínas de membrana asociadas a lisosomas (LAMPs) en el cartílago

Amelogenin is one of the enamel matrix proteins secreted by ameloblasts during enamel formation in tooth development. Recent studies showed that the amelogenin is expressed in chondrocyte. Lysosome-associated membrane proteins (LAMPs) have been identified as binding partner proteins to amelogenin and it has been suggested they act as signaling receptors of amelogenin. The purpose of this study is to clarify the localization of amelogenin and LAMPs in growth plate cartilage and cartilaginous nodules in micromass culture. Mouse knee joints including tibia growth plate at 4 weeks old and micromass cultures of limb bud mesenchymal cells after 2 weeks were fixed in paraformaldehyde, routinely processed, sections were cut and immunostained with amelogenin, collagen type II and type X, LAMP-1 and -3. The positive immunoreaction of amelogenin was observed both in proliferation and hypertrophic zone cartilage of growth plate after enzymatic pretreatment in immunostaining. Furthermore, cartilaginous nodules in micromass culture were immunopositive to amelogenin. The chondrocytes in the proliferation zone of the growth plate were immunopositive to LAMP-1 but weakly stained in the chondrocytes of hypertrophic zone. These observations indicate that amelogenin may be present in cartilage matrix produced in vivo and in vitro and amelogenin may involve cartilage formation through the LAMP-1 signaling pathway.

La amelogenina es una de las proteínas de la matriz del esmalte secretadas por ameloblastos durante la formación del esmalte en el desarrollo dentario. Estudios recientes demuestran que la amelogenina se expresa en los condrocitos. Las proteínas de membrana asociadas a lisosomas (LAMPs) se han identificado como proteínas de unión asociadas a la amelogenina; se ha sugerido que actúan como receptores de señalización de la amelogenina. El propósito de este estudio fue aclarar la localización de la amelogenina y las LAMPs en el cartílago de crecimiento y nódulos cartilaginosos en cultivos de micromasa. Articulaciones de la rodilla del ratón, que incluían la placa de crecimiento tibial de 4 semanas de edad y cultivos de micromasa de células mesenquimales del brote del miembro después de 2 semanas se fijaron en paraformaldehído y procesaron rutinariamente. Los cortes fueron sometidos a inmunotinción con amelogenina, colágeno tipo II y X, LAMP-1 y LAMP-3 . Se observó inmunorreacción positiva de amelogenina tanto en la zona proliferación e hipertrófica del cartílago de crecimiento después del pretratamiento enzimático. Además, los nódulos cartilaginosos en el cultivo de micromasa eran inmunopositivos para la amelogenina. Los condrocitos en la zona de proliferación de la placa de crecimiento fueron immunopositivos a LAMP-1, mientras que los condrocitos de la zona hipertrófica se tiñeron débilmente. Estas observaciones indican que la amelogenina puede estar presente en la matriz del cartílago producida tanto in vivo e in vitro, además la amelogenina puede estar implicada en la formación de cartílago mediante la vía de señalización de LAMP-1.

Defective adipose tissue development associated with hepatomegaly in cathepsin E-deficient mice fed a high-fat diet.

Cathepsin E is an intracellular aspartic proteinase, which is predominantly distributed in immune-related and epithelial cells. However, the role of the enzyme in adipose tissues remains unknown. In this study, we investigated the characteristics of cathepsin E-deficient (CatE(-/-)) mice fed a high-fat diet (HFD), as a mouse model of obesity. HFD-fed CatE(-/-) mice displayed reduced body weight gain and defective development of white adipose tissue (WAT) and brown adipose tissue (BAT), compared with HFD-fed wild-type mice. Moreover, fat-induced CatE(-/-) mice showed abnormal lipid accumulation in non-adipose tissues characterized by hepatomegaly, which is probably due to defective adipose tissue development. Detailed pathological and biochemical analyses showed that hepatomegaly was accompanied by hepatic steatosis and hypercholesterolemia in HFD-induced CatE(-/-) mice. In fat-induced CatE(-/-) mice, the number of macrophages infiltrating into WAT was significantly lower than in fat-induced wild-type mice. Thus, the impaired adipose tissue development in HFD-induced CatE(-/-) mice was probably due to reduced infiltration of macrophages and may lead to hepatomegaly accompanied by hepatic steatosis and hypercholesterolemia.

The effect of valproic Acid on mesenchymal pluripotent cell proliferation and differentiation in extracellular matrices.

Valproic acid (2-n-propylpentanoic acid, VPA) is a widely used antiepileptic and anticonvulsant drug. Previous studies have reported that VPA effects osteogenesis in vivo and in vitro, yet it remains unclear whether VPA promotes cell differentiation of osteoblasts derived from mesenchymal cells. The purpose of this study was to clarify the effect of VPA on undifferentiated pluripotent mesenchymal cell proliferation and differentiation into osteoblasts while analyzing the impact of the absence or presence of extracellular matrices (ECMs). Mouse mesenchymal cells were cultured on non-coated plastic, type I collagen-coated, and fibronectin-coated plates in the absence or presence of VPA. A cell proliferation assay was performed in which modified formazan dye content was analyzed and proliferation nuclear antigen (PCNA)-positive cells were counted at various concentrations of VPA. A high concentration of VPA did not clearly alter cell morphology, but large numbers of stress fibers were observed in these cells and the cell proliferation ratio was decreased with positive PCNA counts. In the presence of matrices, the cell proliferation ratio decreased at low VPA concentrations compared with the ratio obtained in the absence of these ECMs. On the other hand, VPA promoted osteoblastic differentiation in the presence of type I collagen. These findings indicate that for undifferentiated mesenchymal cells, VPA promotes a decrease in the cell proliferation rate in the presence of ECMs and promotes osteoblastic differentiation, both of which could provide insight into additional mechanisms of osteoblastic cell differentiation caused by VPA.

Amelogenins: Multi-Functional Enamel Matrix Proteins and Their Binding Partners.

Amelogenins are the most abundant extracellular matrix proteins secreted by ameloblasts during tooth development and are important for enamel formation. Recently, amelogenins have been detected not only in ameloblasts, which are differentiated from the epithelial cell lineage, but also in other tissues, including mesenchymal tissues at low levels, suggesting that amelogenins possess other functions in these tissues. The therapeutic application of an enamel matrix derivative rich in amelogenins resulted in the regeneration of cementum, alveolar bone, and periodontal ligament (PDL) in the treatment of experimental or human periodontitis, indicating the attractive potential of amelogenin in hard tissue formation. In addition, a full-length amelogenin (M180) and leucine-rich amelogenin peptide (LRAP) regulate cementoblast/PDL cell proliferation and migration in vitro. Interestingly, amelogenin null mice show increased osteoclastogenesis and root resorption in periodontal tissues. Recombinant amelogenin proteins suppress osteoclastogenesis in vivo and in vitro, suggesting that amelogenin is involved in preventing idiopathic root resorption. Amelogenins are implicated in tissue-specific epithelial-mesenchymal or mesenchymal-mesenchymal signaling; however, the precise molecular mechanism has not been characterized. In this review, we first discuss the emerging evidence for the additional roles of M180 and LRAP as signaling molecules in mesenchymal cells. Next, we show the results of a yeast two-hybrid assay aimed at identifying protein-binding partners for LRAP. We believe that gaining further insights into the signaling pathway modulated by the multifunctional amelogenin proteins will lead to the development of new therapeutic approaches for treating dental diseases and disorders.

A method for rapid demineralization of teeth and bones.

Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.

The MMP activity in developing rat molar roots and incisors demonstrated by in situ zymography.

Matrix metalloproteinases (MMPs) have been expressed during root development and periodontal tissue formation, whereas it is not known if these MMP molecules are enzymatically active to degrade the extracellular matrices (ECMs). The present study was designed to investigate the gelatinolytic and collagenolytic activity in rat molar root and incisor development. Three-week old rat mandibles were frozen and cut without fixation or decalcification and processed for in situ zymography using substrates gelatin and collagen. The enzymatic activity was assessed according to the intensity of fluorescence due to the lysis of the substrates. Odontoblasts, predentin, cementum, bone and the enamel matrix showed the high activity. The present study demonstrated MMP activity in calcified tissues using in situ zymography for the first time and the possible involvement of the MMP activity in molar root and incisor development and periodontal tissue formation.

Tissue distribution after intravenous dosing of micafungin, an antifungal drug, to rats.

The tissue distribution after an intravenous dose of micafungin (1 mg/kg), a new echinocandin-like lipopeptide antifungal agent, to male rats was investigated. Micafungin in plasma disappeared biexponentially with a terminal half-life of 5.03 h. Micafungin concentrations in liver, kidney, and lung at the first sampling time (5 min) after dosing were 1.15, 1.64, and 2.58-fold higher than the plasma concentration, and the AUC(0- infinity ) were 1.61, 3.42, and 2.89-fold higher than that for plasma. The terminal half-lives for these tissues were 5.14, 4.87, and 5.31 h, respectively, which were comparable to those for plasma. These results suggest that micafungin distributes rapidly and moderately into tissues such as the liver, kidney, and lungs, and that the concentrations in tissues decreased in parallel with the unchanged drug in plasma.

The receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenic pathway is elevated in amelogenin-null mice.

Amelogenins, major components of developing enamel, are predominantly involved in the formation of tooth enamel. Although amelogenins are also implicated in cementogenesis, their precise spatial expression pattern and molecular role are not clearly understood. Here, we report for the first time the expression of two alternate splice forms of amelogenins, M180 and the leucine-rich amelogenin peptide (LRAP), in the periodontal region of mouse tooth roots. Lack of M180 and LRAP mRNA expression correlated with cementum defects observed in the amelogenin-null mice. The cementum defects were characterized by an increased presence of multinucleated cells, osteoclasts, and cementicles. These defects were associated with an increased expression of the receptor activator of the nuclear factor-kappa B ligand (RANKL), a critical regulator of osteoclastogenesis. These findings indicate that the amelogenin splice variants, M180 and LRAP, are critical in preventing abnormal resorption of cementum.