Evaluation of Enamel Acid Resistance and Whitening Effect of the CAP System.

This study aimed to compare the effectiveness of a novel professional tooth-strengthening system and a conventional caries-prevention method that involved the use of high fluoride concentrations, to determine whether the system has a whitening effect. Bovine tooth-enamel samples were treated with fluoride gel (conventional APF method) or a mixture of citric acid gel, calcium phosphate (α-TCP), and fluoride gel, referred to as the CAP system; these treatments were performed to generate an acid-resistant layer on the enamel surface. For the evaluation of the acid resistance, a cyclic experiment, involving a 1-h remineralization and a 24-h acid treatment, was conducted thrice after the treatments. The height profiles were observed using a 3D-measuring laser microscope and the hardness was evaluated by Vickers hardness test. The morphological changes in the surface and cross-section of the enamel were observed by scanning electron microscopy. To evaluate the whitening effect, the enamel was ground until the color of the underlying dentin was recognizable; the CAP system was applied once, and the color change was measured using a color difference meter. As a result, it was confirmed that an acid-resistant layer was formed on the tooth surfaces treated with the CAP system, and a whitening effect was obtained.

Characterization of Living Dental Pulp Cells in Direct Contact with Mineral Trioxide Aggregate.

Mineral trioxide aggregate (MTA) was introduced as a material for dental endodontic regenerative therapy. Here, we show the dynamics of living dental pulp cells in direct contact with an MTA disk. A red fluorescence protein (DsRed) was introduced into immortalized porcine dental pulp cells (PPU7) and cloned. DsRed-PPU7 cells were cultured on the MTA disk and cell proliferation, chemotaxis, the effects of growth factors and the gene expression of cells were investigated at the biological, histomorphological and genetic cell levels. Mineralized precipitates formed in the DsRed-PPU7 cells were characterized with crystal structural analysis. DsRed-PPU7 cells proliferated in the central part of the MTA disk until Day 6 and displayed a tendency to move to the outer circumference. Both transforming growth factor beta and bone morphogenetic protein promoted the proliferation and movement of DsRed-PPU7 cells and also enhanced the expression levels of odontoblastic gene differentiation markers. Mineralized precipitates formed in DsRed-PPU7 were composed of calcium and phosphate but its crystals were different in each position. Our investigation showed that DsRed-PPU7 cells in direct contact with the MTA disk could differentiate into odontoblasts by controlling cell-cell and cell-substrate interactions depending on cell adhesion and the surrounding environment of the MTA.

Overactivation of the NF-κB pathway impairs molar enamel formation.

OBJECTIVE: Hypohidrotic ectodermal dysplasia (HED) is a hereditary disorder characterized by abnormal structures and functions of the ectoderm-derived organs, including teeth. HED patients exhibit a variety of dental symptoms, such as hypodontia. Although disruption of the EDA/EDAR/EDARADD/NF-κB pathway is known to be responsible for HED, it remains unclear whether this pathway is involved in the process of enamel formation. EXPERIMENTAL SUBJECTS AND METHODS: To address this question, we examined the mice overexpressing Ikkß (an essential component required for the activation of NF-κB pathway) under the keratin 5 promoter (K5-Ikkß). RESULTS: Upregulation of the NF-κB pathway was confirmed in the ameloblasts of K5-Ikkß mice. Premature abrasion was observed in the molars of K5-Ikkß mice, which was accompanied by less mineralized enamel. However, no significant changes were observed in the enamel thickness and the pattern of enamel rods in K5-Ikkß mice. Klk4 expression was significantly upregulated in the ameloblasts of K5-Ikkß mice at the maturation stage, and the expression of its substrate, amelogenin, was remarkably reduced. This suggests that abnormal enamel observed in K5-Ikkß mice was likely due to the compromised degradation of enamel protein at the maturation stage. CONCLUSION: Therefore, we could conclude that the overactivation of the NF-κB pathway impairs the process of amelogenesis.

A new method for estimating time since death by analysis of substances deposited on the surface of dental enamel in a body immersed in seawater.

The present investigation was performed with the objective of developing a method to estimate how long a corpse had been immersed in water after death (the time since death). Accurate determination of the time elapsed since death may lead to identification of the place of drowning, and therefore, serves not only as a piece of information useful for determination of the cause of death but also leads to prompt identification of the body. The results showed that diatoms attached to the surface of dental enamel increased with prolongation of immersion time in water. Further, as the immersion time increased, the quantity of O, Si, Mg, K, Al, and S detected on the surface of dental enamel increased, while the quantity of the main dental components (Ca and P) that were detected gradually decreased. Based on these results, we calculated a regression formula to estimate the immersion time. Our method is considered to be a breakthrough technique for evaluating the time since death more objectively, compared to the conventional method of determination based on the degree of decomposition of the corpse.

FAM83H and casein kinase I regulate the organization of the keratin cytoskeleton and formation of desmosomes.

FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts.

Ultrastructural Analyses of Alveolar Bone in a Patient With Osteomyelitis Secondary to Osteopetrosis: A Review of the Literature.

Osteopetrosis is a generic term for generalized sclerotic conditions caused by rare genetic disorders. Decreased osteoclastic activities disturb bone remodeling, resulting in greater mineral density and greater compressive strength; therefore, bone fracture is a major physical symptom of osteopetrosis. Osteomyelitis of the maxilla or mandible is a common and well-documented complication of osteopetrosis. Local infection, such as odontogenic infection, is more likely to lead to osteomyelitis, and treatment strategies can be challenging. However, detailed ultrastructural analyses of bone from patients with osteopetrosis and odontogenic infection are limited. This report describes a case of osteomyelitis of the maxilla and mandible secondary to osteopetrosis in an adult patient and presents ultrastructural data of alveolar bone tissue analyzed by contact microradiography, electron probe microanalysis, and x-ray diffraction. Cases of osteomyelitis of the jaw secondary to osteopetrosis also are reviewed.

Identification, characterization, and expression of dentin matrix protein 1 gene in Xenopus laevis.

Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein expressed mainly in bone and dentin, and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family. The DMP1 gene, however, appears to evolve rapidly in comparison with other SIBLING genes, even though such functionally important molecules usually evolve more slowly than less important ones. The purpose of this study was to identify and characterize an ortholog of the DMP1 gene in an amphibian (Xenopus laevis; X. laevis) to clarify molecular evolutionary alterations in DMP1 associated with calcified tissues in tetrapods. Furthermore, we analyzed the mRNA expression of this gene to elucidate its functional change in bone and developing tooth germ in comparison with amniote DMP1s. The similarities of the deduced amino acid sequence of X. laevis DMP1 to that of the corresponding amniote proteins were low, although they did share several unique features specific to DMP1 and have similar properties. Expression of X. laevis DMP1 mRNA was predominant in osteocytes and odontoblasts, but only transiently observed in ameloblasts, as in amniotes. These results suggest that DMP1 has conserved several functions during tetrapod evolution. This indicates that continuity of biochemical properties has been more important in maintaining DMP1 functionality than that of the sequence of amino acid residues, which has undergone change over the course of molecular evolution.

Immunolocalization of TAK1, TAB1, and p38 in the developing rat molar.

In tooth development, transforming growth factor beta (TGF-ß) and bone morphogenetic protein (BMP) are involved in cell differentiation and matrix protein production. TGF-ß and BMP have two signaling pathways: the Smad pathway and the non-Smad pathway. However, only a few studies have focused on the non-Smad pathway in tooth development. TGF-ß-activated kinase 1 (TAK1) is activated by TGF-ß or BMP and binds to TAK1-binding protein (TAB1), activating p38 or c-Jun N-terminal kinase (JNK), forming the non-Smad signaling pathway. In this study, we examined the distribution of these kinases, TGF-ß receptor 1 (TGF-ß-R1), BMP receptor-1B (BMPR-1B) and Smad4 in cells of the rat molar germ histochemically, in order to investigate the signaling pathway in each type of cell. Immunostaining for TGF-ß-R1, BMPR-1B, Smad4, TAK1, TAB1 and phosphorylated-p38 (p-p38) showed similar reactions. In the cervical loop, reactions were clearer than in other enamel epithelium. In the inner enamel epithelium, signal increased with differentiation into ameloblasts, became strongest in the secretory stage, and decreased rapidly in the maturation stage. Signal also increased upon differentiation from preodontoblasts to odontoblasts. In Hertwig's epithelial sheath, with the exception of BMPR-1B, reactions were stronger in the later stage, showing more enamel protein secretion than in the early stage. However, no clear reaction corresponding to phosphorylated-JNK was observed in any type of cell. These results suggest that TGF-ß or BMP is involved in the induction of differentiation of inner enamel epithelium cells into ameloblasts, and preodontoblast differentiation into odontoblasts, the regulation of cervical loop cell proliferation, the elongation or regulation of the epithelial sheath, and the secretion of enamel protein and dentin matrix protein through the non-Smad signaling pathway via TAK1, TAB1 and p38 as well as Smad signaling pathways in the rat molar germ.

Surface observation of thin hydroxyapatite-coated implants at 80 months after insertion.

We observed surfaces and cross sections of thin hydroxyapatite (HA)-coated implants produced by the thermal decomposition method in a patient attending our clinic who underwent implant removal at 80 months due to fracture of the implants. On the implant surfaces of the removed sample, most of the HA had dissolved, and extensive osseointegration was observed where Ti had closely bonded to bone. This indicated that the HA coated on the implant surfaces had disappeared and osseointegration had been established where Ti directly bonded to the bone. In addition, calcium titanate (CaTiO(3)) and HA layers formed by the thermal decomposition method showed no desorption. The results clearly indicate the positive clinical potential of thin HA-coating by the thermal decomposition method.

Transforming growth factor ß inducible apoptotic cascade in epithelial cells during rat molar tooth eruptions.

In tooth eruptions, the presence of apoptotic epithelial cells at the eruption site has been reported, but the factors that induce apoptosis in these cells remain to be elucidated, as do the induction pathways. In this study, we focused our attention on transforming growth factor beta (TGF-beta), which is known to induce apoptosis during embryonic development. Oral epithelium and dental lamina of maxillary first molars in 8- and 15-day-old rats were used to investigate the induction pathway of apoptosis by performing the immunohistochemical tests outlined below and assessing the characteristics of cells that undergo apoptosis by transmission electron microscopy in rats 8 and 15 days after birth. We examined TGF-beta-receptor 1, TGF-beta inducible transcription factor 1 (TIEG1), NADPHoxidase 4 (Nox4), cytochrome c, caspase-3 (active form and pro-enzyme), apoptosis-inducing protein Daxx, apoptosis signal-regulating kinase 1 (ASK1), glycogen synthase kinase-3 beta phosphorylated on serine 9 (p-GSK-3beta), and beta-catenin. We also performed periodic acid Schiff (PAS) reaction and terminal deoxynucleotidyl transferase-mediated dUTD nick end labeling (TUNEL) staining. At eruption sites 8 days after birth, reactions to TGF-beta-receptor 1, TIEG1, Nox4, cytochrome c, caspase-3, p-GSK-3beta, and beta-catenin, and PAS-positive cells were observed in areas close to the basal layer of oral epithelium through to the center of the dental lamina, but no reaction to Daxx or ASK1 was noted at these sites. Electron microscopy revealed the accumulation of glycogen granules in the cells that showed reactions to the above-mentioned markers as well as in the spaces among them. In the rats 15 days after birth (immediately before tooth eruption), the PAS-positive cells that showed reactions to the above antibodies remained on the buccal side of the epithelium, and high-electron-density apoptotic bodies and TUNEL-positive bodies were noted. Therefore, during tooth eruption, TGF-beta may induce apoptosis of cells rich in glycogen granules, and cytochrome c and caspase-3 may function to induce apoptosis. In addition, reactive oxygen species may be involved in this induction pathway via TIEG1 and Nox4 without involvement of Daxx and ASK1. Moreover, overexpression of p-GSK-3beta and beta-catenin may also contribute to apoptosis of oral epithelium at the eruption site and dental lamina cells. Glycogen storage mediated by p-GSK-3beta and crosstalk between the TGF-beta and Wnt signaling pathways may participate in the formation of tooth eruption passage.

Ultrastructural study of tissues surrounding replanted teeth and dental implants.

OBJECTIVES: The aim of this study was to describe the ultrastructure of the dentogingival border at replanted teeth and implants. MATERIAL AND METHODS: Wistar rats (8 weeks old) were divided into groups for replantation and implantation experiments. In the former, the upper right first molars were extracted and then immediately replanted. In the latter, pure titanium implants were used. All tissues were fixed, demineralized and embedded in epoxy resin for ultrastructural observations. RESULTS: One week after replantation, the junctional epithelium was lost, and the oral sulcular epithelium covered the enamel surface. The amount of the epithelium increased in 2 weeks, and resembled the junctional epithelium, and the internal basal lamina and hemidesmosomes were formed in 4 weeks. One week after implantation, peri-implant epithelium was formed, and in 2 and 4 weeks, this epithelium with aggregated connective tissue cells were observed. In 8 weeks, the peri-implant epithelium receded, and aligned special cells with surrounding elongated fibroblasts and bundles of collagen fibers appeared to seal the implant interface. CONCLUSION: In replantation of the tooth, the internal basal lamina remained at the surface of the enamel of the replanted tooth, which is likely to be related to regeneration of the junctional epithelium and the attachment apparatus at the epithelium-tooth interface. Following implantation, a layer of cells with characteristics of connective tissue cells, but no junctional epithelium and attachment apparatus, was formed to seal the site of the implant.

Detailed consideration of physicochemical properties of CO3apatites as biomaterials in relation to carbonate content using ICP, X-ray diffraction, FT-IR, SEM, and HR-TEM.

CO3apatites with different carbonate contents were synthesized at 60 +/- 1 degrees C and pH 7.4 +/- 0.2 under different carbonate concentrations (0-0.3 mol/L) in the supplied solutions. Their physicochemical properties were analyzed using various methods. Inductively coupled plasma gave accurate chemical analysis data for calcium and phosphate contents. X-ray diffraction analysis showed a clear chemical shift at high carbonate content. A CO3(2-) absorption peak area approximately proportional to carbonate content was observed through Fourier transmission infrared spectroscopy. Scanning electron microscopy and high-resolution transmission electron microscopy revealed a dramatic change of the crystal shape. Osteoblast proliferation at the surface of each CO3apatite-collagen sponge indicated that osteoblasts deformed to expand and cover the surface of the sponge, and appeared to adhere well to the sponge.

SEM observation of collagen fibrils secreted from the body surface of osteoblasts on a CO3apatite-collagen sponge.

The secretion of collagen by osteoblasts was observed by scanning electron microscopy (SEM). Osteoblast-like cells were cultured on a CO3apatite-collagen sponge reinforced with a porous HAp frame. After three days' incubation, a number of newly created matrix fibrils, forming a network structure, were observed at the cell surface. SEM also showed that osteoblasts secreted collagen fibrils from their membrane, and that the collagen fibrils were twisted together. When collagen in an aqueous sol solution was sprayed onto the extremely smooth surface of a mica plate to support the secretion of osteoblasts, a collagen network structure could be clearly observed with atomic force microscopy (AFM). With this in vitro phenomenon, we could confirm the formation of collagen network structure without biological function. Therefore, it was suggested that the CO3apatite-collagen sponge used in this study is a favorable scaffold biomaterial, on which osteoblasts could produce the unmistakable, characteristic extracellular matrix for mineralization. For therapeutic use of hard tissue biomaterials, collagen formation as an extracellular matrix (ECM) is very important because mineralization is subsequent to ECM.

Adhesion of 4-META/MMA-TBB resin to heated dentin: effects of pre-treatments with FeCl3 and/or HEMA.

The purpose of this study was to compare the tensile bond strengths (TBSs) and failure mode of 4-META/MMA-TBB resin to 60 degrees C-heated and unheated bovine dentin, especially to investigate the influence of ferric chloride contained in citric acid pre-conditioning. In addition, the effect of HEMA priming for heated dentin was also evaluated. The TBSs to heated dentin were significantly lower than those to unheated dentin. Adhesive failures were observed in most specimens of the heated and HEMA-non primed group. HEMA application to heated dentin significantly increased the TBSs in each acid conditioning, which were also significantly higher than those of the unheated and ferric chloride-contained citric acid-conditioned group. It was clarified that heating dentin decreased the bond strength without HEMA priming even if the dentin surfaces were acid conditioned with 10-3, while HEMA priming after acid conditioning recovered the bond strength.

Remineralization effects of xylitol on demineralized enamel.

We morphologically determined the effects of xylitol on the remineralization of artificially demineralized enamel. The samples were demineralized and then immersed in a remineralizing solution with or without 20% xylitol at 37 degrees C for 2 weeks. Samples were observed using contact microradiography, a multipurpose image processor (MIP) and a high-resolution electron microscope (HRTEM). Contact microradiography indicated that remineralization occurred in the surface and deep layers of samples immersed in a non-xylitol solution. Samples immersed in a xylitol solution demonstrated less mineralization in the outer 10 microm of the outermost surface layers, but more mineralization in the middle and deep layers, than was observed in the non-xylitol samples. The MIP evaluation indicated that remineralization was more prominent in layers at depths of 50-60 microm in the xylitol samples than in the non-xylitol samples. Observation of the xylitol samples by HRTEM revealed crystals of various sizes and irregular shapes with unclear crystal angles in the outermost surface layers. In the middle layers, they had thickened and angles of crystals are clear. These results indicate that xylitol can induce remineralization of deeper layers of demineralized enamel by facilitating Ca2+ movement and accessibility.

High-resolution electron microscopy of enamel-crystal demineralization and remineralization in carious lesions.

Although dental caries destroys dental hard tissues, both demineralization as a destructive process and remineralization as a restorative process take place in carious lesions. These conflicting processes occur constantly either simultaneously or alternately. In crystallographic terms, demineralization is crystal dissolution, and remineralization is restoration of partially dissolved crystals, new crystal formation and growth of surviving crystals. In this review, we employ high-resolution transmission electron microscopy to demonstrate demineralization and remineralization of enamel crystals obtained from cross-cut sections of the c-axes of crystals from carious lesions.

Fluoride and apatite formation in vivo and in vitro.

In recent years, the biomineralization process has attracted much interest from academics and industries for potential technological application. The rule in biomineralization is to have a variety of interfaces and surfaces which can act as nucleators. The ultimate step in any biomineralization process, i.e. the deposition of mineral, must conform to the driving forces operating on the system. A new paradigm in the assessment of the driving force for biomineralization is that a variety of ions existing in the mineralizing milieu are not a bystander, but are instead an active player that directly regulates the precipitation process and nature of biogenic apatites. Thus, the most putative stoichiometric model of a biomineral is (Ca)(5-x)(Mg)q(Na)u(HPO4)v(CO3)w(PO4)(3-y)(OH,F)(1-z). Fluoride participates in many aspects of calcium phosphate formation in vivo and has enormous effects on its process and on the nature and properties of the final products. In the development of biogenic apatites, fluoride ion in the mineralizing media is supposed to accelerate the hydrolysis of acidic precursor(s) and increase the growth rates by augmenting the driving force for precipitation. Inhibitory activities of ions and molecules are related to their adsorption onto the apatite surfaces. From theoretical and practical points of view, it is of paramount importance to elucidate and predict the effect and outcome of fluoride (accelerator) and inhibitors of biological relevance, because of their use in combination for healthcare in dentistry and medicine, e.g. prevention of dental caries and calculus deposition and in the formulation of antiosteoporosis treatments.