ABSTRACT
Caries and dental erosion are common oral diseases. Traditional treatments involve the mechanical removal of decay and filling but these methods are not suitable for cases involving large-scale enamel erosion, such as hypoplasia. To develop a noninvasive treatment, promoting remineralisation in the early stage of caries is of considerable clinical significance. Therefore, biomimetic mineralisation is an ideal approach for restoring enamel. Biomimetic mineralisation forms a new mineral layer that is tightly attached to the surface of the enamel. This review details the state-of-art achievements on the application of amelogenin and non-amelogenin, amorphous calcium phosphate, ions flow and other techniques in the biomimetic mineralisation of enamel. The ultimate goal of this review was to shed light on the requirements for enamel biomineralisation. Hence, herein, we summarise two strategies of biological minimisation systems for in situ enamel restoration inspired by amelogenesis that have been developed in recent years and compare their advantages and disadvantages.
Subject(s)
Biomimetic Materials , Dental Enamel/drug effects , Dental Restoration, Permanent , Amelogenesis/drug effects , Amelogenin/chemistry , Amelogenin/pharmacology , Animals , Biomimetic Materials/chemical synthesis , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Biomimetics/methods , Calcification, Physiologic/drug effects , Calcification, Physiologic/physiology , Calcium Phosphates/chemistry , Calcium Phosphates/pharmacology , Dental Enamel/chemistry , Dental Restoration, Permanent/instrumentation , Dental Restoration, Permanent/methods , HumansABSTRACT
PURPOSE: To investigate the amelogenesis-inductive effects of surface structures at the nanoscale. For this purpose, variable nanostructured titanium dioxide (TiO2) surfaces were used as a framework to regulate the amelogenic behaviors of ameloblasts with the administration of retinoic acid (RA)/dexamethasone (DEX). MATERIALS AND METHODS: TiO2 nanotubular (NT) surfaces were fabricated via anodization. Mouse ameloblast-like LS-8 cells were seeded and cultured on NT surfaces in the presence or absence of RA/DEX for 48 h. The amelogenic behaviors and extracellular matrix (ECM) mineralization of LS-8 cells on nanostructured Ti surfaces were characterized using field emission scanning electron microscope, laser scanning confocal microscope, quantitative polymerase chain reaction, MTT assay, and flow cytometry. RESULTS: TiO2 NT surfaces (tube size ~30 and ~80 nm) were constructed via anodization at 5 or 20 V and denoted as NT5 and NT20, respectively. LS-8 cells exhibited significantly increased spread and proliferation, and lower rates of apoptosis and necrosis on NT surfaces. The amelogenic gene expression and ECM mineralization differed significantly on the NT20 and the NT5 and polished Ti sample surfaces in standard medium. The amelogenic behaviors of LS-8 cells were further changed by RA/DEX pretreatment, which directly drove maturation of LS-8 cells. CONCLUSION: Controlling the amelogenic behaviors of ameloblast-like LS-8 cells by manipulating the nanostructure of biomaterials surfaces represents an effective tool for the establishment of a systemic framework for supporting enamel regeneration. The administration of RA/DEX is an effective approach for driving the amelogenesis and maturation of ameloblasts.
Subject(s)
Amelogenesis/drug effects , Cell Differentiation/drug effects , Dexamethasone/pharmacology , Nanostructures/chemistry , Titanium/pharmacology , Tretinoin/pharmacology , Ameloblasts/cytology , Ameloblasts/drug effects , Ameloblasts/metabolism , Amelogenesis/genetics , Animals , Apoptosis/drug effects , Calcification, Physiologic/drug effects , Cell Adhesion/drug effects , Cell Line , Cell Proliferation/drug effects , Cell Shape/drug effects , Cells, Cultured , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Gene Expression Regulation/drug effects , Mice , Surface PropertiesABSTRACT
Cancer is the second most frequent cause of death in children. Because the prognosis for childhood malignancies has improved, attention has now focused on long-term consequences of cancer treatment. The immediate effects of chemotherapy on soft tissues have been well described; however, there is less information about long-term effects of chemotherapy on the development of dental tissues. To test the association between the effect of chemotherapy on enamel development, we examined two groups of rats: one that had received an intraperitoneal dose of 200 mg/kg of irinotecan, whereas the other (control) group had received vehicle only. Rats were killed at 6, 48 and 96 hr post-injection; the mandibles dissected out, fixed for histological evaluation and scanned for mineralization defects by Micro-CT. Our results showed structural changes in the ameloblast layer along with a significant reduction in mineralization and thickness of enamel at 96 hr after chemotherapy. These data demonstrate that irinotecan induces structural changes in forming enamel that become apparent after anticancer chemotherapy treatment.
Subject(s)
Ameloblasts/drug effects , Amelogenesis/drug effects , Antineoplastic Agents/adverse effects , Dental Enamel/drug effects , Incisor/drug effects , Irinotecan/adverse effects , Ameloblasts/pathology , Animals , Calcification, Physiologic/drug effects , Dental Enamel/diagnostic imaging , Dental Enamel/growth & development , Dental Enamel/pathology , Female , Incisor/diagnostic imaging , Incisor/growth & development , Incisor/pathology , Injections, Intraperitoneal , Mandible/diagnostic imaging , Mandible/drug effects , Mandible/growth & development , Mandible/pathology , Rats , Rats, Wistar , X-Ray MicrotomographyABSTRACT
We report here a novel biomimetic approach to the regeneration of human enamel. The approach combines the use of inorganic pyrophosphate (PPi) to control the onset and rate of enamel regeneration and the use of leucine-rich amelogenin peptide (LRAP), a nonphosphorylated 56-amino acid alternative splice product of amelogenin, to regulate the shape and orientation of growing enamel crystals. This study builds on our previous findings that show LRAP can effectively guide the formation of ordered arrays of needle-like hydroxyapatite (HA) crystals in vitro and on the known role mineralization inhibitors, like PPi, play in the regulation of mineralized tissue formation. Acid-etched enamel surfaces of extracted human molars, cut perpendicular or parallel to the direction of the enamel rods, were exposed to a PPi-stabilized supersaturated calcium phosphate (CaP) solution containing 0 to 0.06 mg/mL LRAP for 20 h. In the absence of LRAP, PPi inhibition was reversed by the presence of etched enamel surfaces and led to the formation of large, randomly distributed plate-like HA crystals that were weakly attached, regardless of rod orientation. In the presence of 0.04 mg/mL LRAP, however, densely packed mineral layers, comprising bundles of small needle-like HA crystals, formed on etched surfaces that were cut perpendicular to the enamel rods. These crystals were strongly attached, and their arrangement reflected to a significant degree the underlying enamel prism pattern. In contrast, under the same conditions with LRAP, little to no crystal formation was found on enamel surfaces that were cut parallel to the direction of the enamel rods. These results suggest that LRAP preferentially interacts with ab surfaces of mature enamel crystals, inhibiting their directional growth, thus selectively promoting linear growth along the c-axis of enamel crystals. The present findings demonstrate a potential for the development of a new approach to regenerate enamel structure and properties.
Subject(s)
Amelogenesis/drug effects , Biomimetics , Dental Enamel Proteins/pharmacology , Acid Etching, Dental , Animals , Calcium Phosphates/pharmacology , Crystallization , Humans , Hydrogen-Ion Concentration , In Vitro Techniques , Microscopy, Electron, Scanning , Surface Properties , SwineABSTRACT
OBJECTIVES: The present study explored the histological and chemical effects of infantile thiamine deficiency (ITD) on enamel development through the examination of exfoliated deciduous teeth from a patient who had been fed during his first year of life with a thiamine-deficient milk substitute. MATERIALS AND METHODS: Ground sections derived from six exfoliated primary teeth were examined. Slices from a light microscope were photographed for histological analysis. We calculated the time when the amelogenesis insults occurred, and the data were cross-examined with the patient's medical history. We then measured the enamel content of calcium, phosphate, oxygen, carbon, and magnesium on two lines from the dentino-enamel junction (DEJ) to the outer surface using an energy dispersive X-ray spectrometer. RESULTS: Carbon (organic matter) concentration in postnatal enamel was 2.37 times higher in ITD, phosphate levels were lower, and magnesium and calcium levels tended to be higher in ITD teeth. CONCLUSION: Chemical and histological analysis enabled us to confirm that thiamine deficiency in infancy impaired postnatal amelogenesis and resulted in less calcified enamel with a higher level of organic matter. Higher postnatal enamel carbon and magnesium concentration found in ITD may derive from either impaired mineralization caused by disturbed cellular metabolism or indirect damage to the ameloblasts due to the physical condition. Ca/P mean ratio in ITD teeth was higher than the mean ratio in the control displaying a damaged mineralization process. CLINICAL RELEVANCE: This is probably the first description of infantile thiamine deficiency effect on amelogenesis resulting in less calcified enamel.
Subject(s)
Amelogenesis/drug effects , Dental Enamel/chemistry , Dentin/chemistry , Infant Food/analysis , Thiamine Deficiency/complications , Tooth, Deciduous , Calcium/analysis , Child , Humans , Magnesium/analysis , Male , Phosphates/analysisABSTRACT
Endocrine-disrupting chemicals (EDCs) that interfere with the steroid axis can affect amelogenesis, leading to enamel hypomineralization similar to that of molar incisor hypomineralization, a recently described enamel disease. We investigated the sex steroid receptors that may mediate the effects of EDCs during rat amelogenesis. The expression of androgen receptor (AR), estrogen receptor (ER)-α, and progesterone receptor was dependent on the stage of ameloblast differentiation, whereas ERß remained undetectable. AR was the only receptor selectively expressed in ameloblasts involved in final enamel mineralization. AR nuclear translocation and induction of androgen-responsive element-containing promoter activity upon T treatment, demonstrated ameloblast responsiveness to androgens. T regulated the expression of genes involved in enamel mineralization such as KLK4, amelotin, SLC26A4, and SLC5A8 but not the expression of genes encoding matrix proteins, which determine enamel thickness. Vinclozolin and to a lesser extent bisphenol A, two antiandrogenic EDCs that cause enamel defects, counteracted the actions of T. In conclusion, we show, for the first time, the following: 1) ameloblasts express AR; 2) the androgen signaling pathway is involved in the enamel mineralization process; and 3) EDCs with antiandrogenic effects inhibit AR activity and preferentially affect amelogenesis in male rats. Their action, through the AR pathway, may specifically and irreversibly affect enamel, potentially leading to the use of dental defects as a biomarker of exposure to environmental pollutants. These results are consistent with the steroid hormones affecting ameloblasts, raising the issue of the hormonal influence on amelogenesis and possible sexual dimorphism in enamel quality.
Subject(s)
Ameloblasts/drug effects , Ameloblasts/metabolism , Endocrine Disruptors/toxicity , Amelogenesis/drug effects , Animals , Benzhydryl Compounds/toxicity , Cell Line , Chloride-Bicarbonate Antiporters/genetics , Chloride-Bicarbonate Antiporters/metabolism , Dental Enamel/drug effects , Dental Enamel/metabolism , Dental Enamel Proteins/genetics , Dental Enamel Proteins/metabolism , Fluorescent Antibody Technique , Gene Expression Profiling , Kallikreins/genetics , Kallikreins/metabolism , Male , Monocarboxylic Acid Transporters/genetics , Monocarboxylic Acid Transporters/metabolism , Oxazoles/toxicity , Phenols/toxicity , Rats , Rats, Wistar , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Receptors, Progesterone/genetics , Receptors, Progesterone/metabolism , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Sulfate TransportersABSTRACT
Dental fluorosis has recently been diagnosed in wild marsupials inhabiting a high-fluoride area in Victoria, Australia. Information on the histopathology of fluorotic marsupial enamel has thus far not been available. This study analyzed the developmental and post-eruptive defects in fluorotic molar enamel of eastern grey kangaroos (Macropus giganteus) from the same high-fluoride area using light microscopy and backscattered electron imaging in the scanning electron microscope. The fluorotic enamel exhibited a brownish to blackish discolouration due to post-eruptive infiltration of stains from the oral cavity and was less resistant to wear than normally mineralized enamel of kangaroos from low-fluoride areas. Developmental defects of enamel included enamel hypoplasia and a pronounced hypomineralization of the outer (sub-surface) enamel underneath a thin rim of well-mineralized surface enamel. While the hypoplastic defects denote a disturbance of ameloblast function during the secretory stage of amelogenesis, the hypomineralization is attributed to an impairment of enamel maturation. In addition to hypoplastic defects, the fluorotic molars also exhibited numerous post-eruptive enamel defects due to the flaking-off of portions of the outer, hypomineralized enamel layer during mastication. The macroscopic and histopathological lesions in fluorotic enamel of M. giganteus match those previously described for placental mammals. It is therefore concluded that there exist no principal differences in the pathogenic mechanisms of dental fluorosis between marsupial and placental mammals. The regular occurrence of hypomineralized, opaque outer enamel in the teeth of M. giganteus and other macropodids must be considered in the differential diagnosis of dental fluorosis in these species.
Subject(s)
Dental Enamel/drug effects , Dental Enamel/pathology , Environmental Exposure/adverse effects , Fluorides/adverse effects , Fluorosis, Dental/etiology , Fluorosis, Dental/pathology , Tooth Eruption/drug effects , Age Factors , Amelogenesis/drug effects , Animals , Bone and Bones/metabolism , Female , Fluorides/metabolism , Fluorosis, Dental/diagnostic imaging , Macropodidae , Male , Molar , RadiographyABSTRACT
BACKGROUND: The use of amoxicillin during early childhood has been associated with molar incisor hypomineralization. AIM: The objective of this study was to determine whether the use of amoxicillin interferes with enamel development, during secretion and early mineralization stages. DESIGN: Fifteen pregnant rats were randomly assigned to three groups that received physiological solution (sham group), 100 mg/kg/day amoxicillin (A100G), and 500 mg/kg/day amoxicillin (A500G). After birth, the pups in each group received the same treatment until post-natal day 7 or 12. The upper first molars were analyzed histomorphometrical and immunostaining with amelogenin on day 7, and MMP-20 on day 12 was performed using a semiquantitative method (H-score). RESULTS: At 7 days, several vacuolar structures were observed in the ameloblasts in the A100G and A500G groups. A significant reduction of the enamel thickness (P < 0.001) was found in amoxicillin-treated rats compared with the sham group. Significant differences were not observed in enamel thickness (P > 0.05) between the groups of 12-day-old rats. Moreover, significant differences were not observed in the number of amelogenin- and MMP-20-immunolabeled ameloblasts (P > 0.05) between groups. CONCLUSION: The present results suggest that amoxicillin interferes with the initial stages of amelogenesis by causing structural changes in the ameloblasts and a reduction of the enamel matrix.
Subject(s)
Ameloblasts/drug effects , Amelogenesis/drug effects , Amoxicillin/toxicity , Dental Enamel Proteins/drug effects , Animals , Dental Enamel/drug effects , Humans , RatsABSTRACT
Amoxicillin used in early childhood may be associated with enamel hypomineralization. Our aim was to assess disturbances of amelogenesis in mice lower incisors induced by chronic administration of amoxicillin/clavulanic acid (AMC). Twenty-eight C57BL/6 male mice, of similar age, randomly divided into a control and 3 treatment groups (n = 7) received subcutaneous injection, once per day, for 60 days: 50, 100, and 150 mg/kg BW of AMC. Scanning electron microscopy/energy dispersive X-ray spectroscopy analysis in AMC treatment groups showed higher content in F and a decrease in P and Ca. Morphology changes ranged from scratched patterns, and small isolated pits-like enamel loss, to generalized demineralized enamel surface, giving a rough, foamy, scaly, or even cracked eggshell appearance to the affected areas. Histological analysis showed disturbances of maturation ameloblasts, which were less organized, with increased amounts of clear vacuoles in the cytoplasm and slightly more elongated and less condensed nucleus. Additionally, they were often detached from the enamel matrix. Transitional ameloblasts formed underlying the cysts of varied sizes. In conclusion, AMC dose-dependently affect ameloblast functions especially in the maturation phase, causing hypomineralized enamel formation with quantitative and/or qualitative defects.
Subject(s)
Amelogenesis/drug effects , Amoxicillin-Potassium Clavulanate Combination/toxicity , Dental Enamel/drug effects , Dental Enamel/growth & development , Animals , Dental Enamel/pathology , Mice , Mice, Inbred C57BL , Microscopy, Electron, Scanning , Toxicity Tests, ChronicABSTRACT
Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX(-/-)) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX(-/-) mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX(-/-) mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX(-/-) mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX(-/-) mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation.
Subject(s)
Amelogenesis/physiology , Amelogenin/physiology , Ameloblasts/chemistry , Ameloblasts/ultrastructure , Amelogenesis/drug effects , Amelogenin/genetics , Animals , Azo Compounds , Buffers , Chloride-Bicarbonate Antiporters/analysis , Chlorides/analysis , Coloring Agents , Crystallization , Dental Enamel/chemistry , Dental Enamel/ultrastructure , Electron Probe Microanalysis/methods , Enamel Organ/drug effects , Enamel Organ/ultrastructure , Fluorides/pharmacology , Hydrogen-Ion Concentration , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron , Minerals/analysis , X-Ray Microtomography/methodsABSTRACT
Genetic factors influence the effects of fluoride (F) on amelogenesis and bone homeostasis but the underlying molecular mechanisms remain undefined. A label-free proteomics approach was employed to identify and evaluate changes in bone protein expression in two mouse strains having different susceptibilities to develop dental fluorosis and to alter bone quality. In vivo bone formation and histomorphometry after F intake were also evaluated and related to the proteome. Resistant 129P3/J and susceptible A/J mice were assigned to three groups given low-F food and water containing 0, 10 or 50 ppmF for 8 weeks. Plasma was evaluated for alkaline phosphatase activity. Femurs, tibiae and lumbar vertebrae were evaluated using micro-CT analysis and mineral apposition rate (MAR) was measured in cortical bone. For quantitative proteomic analysis, bone proteins were extracted and analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free semi-quantitative differential expression analysis. Alterations in several bone proteins were found among the F treatment groups within each mouse strain and between the strains for each F treatment group (ratio ≥1.5 or ≤0.5; p<0.05). Although F treatment had no significant effects on BMD or bone histomorphometry in either strain, MAR was higher in the 50 ppmF 129P3/J mice than in the 50 ppmF A/J mice treated with 50 ppmF showing that F increased bone formation in a strain-specific manner. Also, F exposure was associated with dose-specific and strain-specific alterations in expression of proteins involved in osteogenesis and osteoclastogenesis. In conclusion, our findings confirm a genetic influence in bone response to F exposure and point to several proteins that may act as targets for the differential F responses in this tissue.
Subject(s)
Amelogenesis/drug effects , Amelogenesis/genetics , Bone and Bones/drug effects , Bone and Bones/physiology , Fluorides/pharmacology , Homeostasis/drug effects , Homeostasis/genetics , Alkaline Phosphatase/blood , Animals , Bone and Bones/metabolism , Collagen Type I/metabolism , Fluorosis, Dental/prevention & control , Gene Expression Regulation/drug effects , Mice , Osteogenesis/drug effects , Phenotype , Proteomics , Species SpecificityABSTRACT
Enamel fluorosis is characterized by hypomineralization, and forkhead box O1 (Foxo1) is essential for mouse enamel biomineralization. This study investigated the effect of fluoride on Foxo1 expression and its implications for enamel fluorosis. Mandibular incisors were extracted from Sprague Dawley rats treated for 3 months with water containing 0, 50, or 100 p.p.m. Fâ». Immunohistochemistry was used to localize and quantify FOXO1 expression in dental epithelial layer cells of the incisors. The effect of fluoride on expression of Foxo1, kallikrein-4 (Klk4), and amelotin (Amtn) mRNAs was analyzed by real-time RT-PCR, and western blotting was used to measure total and nuclear FOXO1 protein levels in mature dental epithelial cells. The results revealed that nuclear FOXO1 was mainly localized in the transition and the mature ameloblasts and exhibited weaker expression in the rats exposed to fluoride. In addition to the reduced levels of Foxo1, Klk4, and AmtnmRNAs, the protein levels of total and nuclearFOXO1 were decreased in the mature dental epithelial cells exposed to fluoride. Thus, excessive fluoride may have an effect on the expression levels of Foxo1 in dental epithelial cells and thereby affect hypomineralization of the enamel during fluorosis.
Subject(s)
Cariostatic Agents/toxicity , Fluorides/toxicity , Forkhead Transcription Factors/drug effects , Incisor/cytology , Nerve Tissue Proteins/drug effects , Ameloblasts/drug effects , Amelogenesis/drug effects , Animals , Cell Nucleus/drug effects , Cytoplasm/drug effects , Dental Enamel/drug effects , Dental Enamel Proteins/drug effects , Epithelial Cells/drug effects , Female , Fluorosis, Dental/etiology , Incisor/drug effects , Kallikreins/drug effects , Male , Random Allocation , Rats , Rats, Sprague-DawleyABSTRACT
Dental fluorosis is characterized by subsurface hypomineralization and increased porosity of enamel, associated with a delay in the removal of enamel matrix proteins. To investigate the effects of fluoride on ameloblasts, A/J mice were given 50 ppm sodium fluoride in drinking water for four weeks, resulting serum fluoride levels of 4.5 µM, a four-fold increase over control mice with no fluoride added to drinking water. MicroCT analyses showed delayed and incomplete mineralization of fluorosed incisor enamel as compared to control enamel. A microarray analysis of secretory and maturation stage ameloblasts microdissected from control and fluorosed mouse incisors showed that genes clustered with Mmp20 appeared to be less downregulated in maturation stage ameloblasts of fluorosed incisors as compared to control maturation ameloblasts. One of these Mmp20 co-regulated genes was the global chromatin organizer, special AT-rich sequence-binding protein-1 (SATB1). Immunohistochemical analysis showed increased SATB1 protein present in fluorosed ameloblasts compared to controls. In vitro, exposure of human ameloblast-lineage cells to micromolar levels of both NaF and AlF3 led to a significantly increase in SATB1 protein content, but not levels of Satb1 mRNA, suggesting a fluoride-induced mechanism protecting SABT1 from degradation. Consistent with this possibility, we used immunohistochemistry and Western blot to show that fluoride exposed ameloblasts had increased phosphorylated PKCα both in vivo and in vitro. This kinase is known to phosphorylate SATB1, and phosphorylation is known to protect SATB1 from degradation by caspase-6. In addition, production of cellular diacylglycerol (DAG) was significantly increased in fluorosed ameloblasts, suggesting that the increased phosphorylation of SATB1 may be related to an effect of fluoride to enhance Gαq activity of secretory ameloblasts.
Subject(s)
Ameloblasts/metabolism , Amelogenesis/drug effects , Fluorosis, Dental/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , Matrix Attachment Region Binding Proteins/genetics , Sodium Fluoride/pharmacology , Ameloblasts/pathology , Amelogenesis/genetics , Animals , Caspase 6/genetics , Caspase 6/metabolism , Dental Enamel/metabolism , Dental Enamel/pathology , Diglycerides/metabolism , Female , Fluorosis, Dental/metabolism , Fluorosis, Dental/pathology , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Gene Expression Profiling , Gene Expression Regulation , Humans , Incisor/metabolism , Incisor/pathology , Male , Matrix Attachment Region Binding Proteins/metabolism , Matrix Metalloproteinase 20/genetics , Matrix Metalloproteinase 20/metabolism , Mice , Oligonucleotide Array Sequence Analysis , Phosphorylation , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal TransductionABSTRACT
There has been increasing concerns over last 20 years about the potential adverse effects of endocrine disruptors (EDs). Bisphenol A (BPA), genistein (G) and vinclozolin (V) are three widely used EDs having similar effects. Tooth enamel has recently been found to be an additional target of BPA that may be a causal agent of molar incisor hypomineralization (MIH). However, populations are exposed to many diverse EDs simultaneously. The purpose of this study was therefore to assess the effects of the combination of G, V and BPA on tooth enamel. Rats were exposed daily in utero and after birth to low doses of EDs mimicking human exposure during the critical fetal and suckling periods when amelogenesis takes place. The proportion of rats presenting opaque areas of enamel hypomineralization was higher when rats were treated with BPA alone than with a combination of EDs. The levels of mRNAs encoding the main enamel proteins varied with BPA treatment alone and did not differ significantly between controls and combined treatment groups. In vitro, rat ameloblastic HAT-7 cells were treated with the three EDs. BPA induced enamelin and reduced klk4 expression, G had no such effects and V reduced enamelin expression. These findings suggest that combinations of EDs may affect enamel less severely than BPA alone, and indicate that enamel hypomineralization may differ according to the characteristics of the ED exposure.
Subject(s)
Amelogenesis/drug effects , Benzhydryl Compounds/pharmacology , Dental Enamel/drug effects , Endocrine Disruptors/pharmacology , Phenols/pharmacology , Tooth Demineralization/chemically induced , Tooth/drug effects , Animals , Dental Enamel Proteins/pharmacology , Rats, WistarABSTRACT
Bisphenol A (BPA) is a widespread endocrine disrupting chemical (EDC) strongly suspected to have adverse health effects. Numerous tissues and cells are affected by BPA, and we showed recently that BPA targets include ameloblasts and enamel. We therefore investigated the effects of BPA on ameloblasts and the possible involvement of the estrogen signaling pathway. Rats were exposed daily to low-dose BPA, and developed enamel hypomineralization similar to human molar incisor hypomineralization (MIH). BPA increased ameloblast proliferation in vivo and in vitro. The proliferation of the rat dental epithelial cell line HAT-7 was also increased by estrogen (E2). Ameloblasts express ERα but not ERß both in vivo and in vitro. The ER antagonist ICI 182,780 was used to inactivate ERα and abolished the effects of E2 on cell proliferation and transcription, but only partially reduced the effects of BPA. In conclusion, we show, for the first time, that: 1) BPA has ER-dependent and ER-independent effects on ameloblast proliferation and gene transcription; 2) the estrogen signaling pathway is involved in tooth development and the enamel mineralization process; and 3) BPA impacts preferentially amelogenesis in male rats. These results are consistent with the steroid hormones having effect on ameloblasts, raising the issues of the hormonal influence on amelogenesis and possible differences in enamel quality between sexes.
Subject(s)
Ameloblasts/drug effects , Benzhydryl Compounds/pharmacology , Cell Proliferation/drug effects , Dental Enamel/drug effects , Endocrine Disruptors/pharmacology , Estrogens/pharmacology , Phenols/pharmacology , Ameloblasts/cytology , Ameloblasts/metabolism , Amelogenesis/drug effects , Animals , Dental Enamel/cytology , Dental Enamel/metabolism , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/genetics , Estrogen Receptor beta/metabolism , Male , Rats , Rats, WistarABSTRACT
Enamel fluorosis is an irreversible structural enamel defect following exposure to supraoptimal levels of fluoride during amelogenesis. We hypothesized that fluorosis is associated with excess release of protons during formation of hypermineralized lines in the mineralizing enamel matrix. We tested this concept by analyzing fluorotic enamel defects in wild-type mice and mice deficient in anion exchanger-2a,b (Ae2a,b), a transmembrane protein in maturation ameloblasts that exchanges extracellular Cl(-) for bicarbonate. Defects were more pronounced in fluorotic Ae2a,b (-/-) mice than in fluorotic heterozygous or wild-type mice. Phenotypes included a hypermineralized surface, extensive subsurface hypomineralization, and multiple hypermineralized lines in deeper enamel. Mineral content decreased in all fluoride-exposed and Ae2a,b(-/-) mice and was strongly correlated with Cl(-). Exposure of enamel surfaces underlying maturation-stage ameloblasts to pH indicator dyes suggested the presence of diffusion barriers in fluorotic enamel. These results support the concept that fluoride stimulates hypermineralization at the mineralization front. This causes increased release of protons, which ameloblasts respond to by secreting more bicarbonates at the expense of Cl(-) levels in enamel. The fluoride-induced hypermineralized lines may form barriers that impede diffusion of proteins and mineral ions into the subsurface layers, thereby delaying biomineralization and causing retention of enamel matrix proteins.
Subject(s)
Chloride-Bicarbonate Antiporters/drug effects , Fluorides/adverse effects , Fluorosis, Dental/etiology , Ameloblasts/drug effects , Ameloblasts/pathology , Amelogenesis/drug effects , Amelogenesis/genetics , Animals , Bicarbonates/analysis , Chloride-Bicarbonate Antiporters/analysis , Chloride-Bicarbonate Antiporters/genetics , Chlorides/analysis , Coloring Agents , Dental Enamel/chemistry , Dental Enamel/drug effects , Dental Enamel/pathology , Dental Enamel Proteins/analysis , Diffusion , Female , Fluorosis, Dental/genetics , Fluorosis, Dental/pathology , Heterozygote , Homozygote , Hydrogen-Ion Concentration , Indicators and Reagents , Mice , Mice, Knockout , Minerals/analysis , Phenotype , Rats , Rats, Wistar , Tooth Calcification/drug effects , Tooth Calcification/geneticsABSTRACT
RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up-regulated. Transgenic mice were generated that express a dominant-negative RhoA transgene in ameloblasts using amelogenin gene-regulatory sequences. Transgenic and wild-type (WT) molar tooth germs were incubated with sodium fluoride (NaF) or sodium chloride (NaCl) in organ culture. Filamentous actin (F-actin) stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared with WT ameloblasts treated with NaCl or with transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/Rho-associated protein kinase (ROCK) pathway in the transgenic mice. Little difference in quantitative fluorescence (an estimation of fluorosis) was observed between WT and transgenic incisors from mice provided with drinking water containing NaF. We subsequently found reduced transgene expression in incisors compared with molars. Transgenic molar teeth had reduced amelogenin, E-cadherin, and Ki67 compared with WT molar teeth. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein, amelogenin, and E-cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited.
Subject(s)
Ameloblasts/metabolism , Amelogenesis/drug effects , Dental Enamel/drug effects , Sodium Fluoride/pharmacology , Tooth Germ/growth & development , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , Ameloblasts/drug effects , Amelogenin/metabolism , Animals , Dental Enamel/abnormalities , Dental Enamel Hypoplasia/metabolism , Fluorosis, Dental/metabolism , Gene Expression , Incisor/pathology , Mice , Mice, Transgenic , Microscopy, Fluorescence , Molar/pathologyABSTRACT
Endocrine-disrupting chemicals (EDCs), including bisphenol A (BPA), are environmental ubiquitous pollutants and associated with a growing health concern. Anecdotally, molar incisor hypomineralization (MIH) is increasing concurrently with EDC-related conditions, which has led us to investigate the effect of BPA on amelogenesis. Rats were exposed daily to BPA from conception until day 30 or 100. At day 30, BPA-affected enamel exhibited hypomineralization similar to human MIH. Scanning electron microscopy and elemental analysis revealed an abnormal accumulation of organic material in erupted enamel. BPA-affected enamel had an abnormal accumulation of exogenous albumin in the maturation stage. Quantitative real-time PCR, Western blotting, and luciferase reporter assays revealed increased expression of enamelin but decreased expression of kallikrein 4 (protease essential for removing enamel proteins) via transcriptional regulation. Data suggest that BPA exerts its effects on amelogenesis by disrupting normal protein removal from the enamel matrix. Interestingly, in 100-day-old rats, erupting incisor enamel was normal, suggesting amelogenesis is only sensitive to MIH-causing agents during a specific time window during development (as reported for human MIH). The present work documents the first experimental model that replicates MIH and presents BPA as a potential causative agent of MIH. Because human enamel defects are irreversible, MIH may provide an easily accessible marker for reporting early EDC exposure in humans.
Subject(s)
Benzhydryl Compounds/toxicity , Dental Enamel Hypoplasia/chemically induced , Endocrine Disruptors/toxicity , Phenols/toxicity , Prenatal Exposure Delayed Effects/chemically induced , Amelogenesis/drug effects , Animals , Biomarkers/metabolism , Blotting, Western , Dental Enamel Hypoplasia/metabolism , Dental Enamel Proteins/metabolism , Female , Humans , Kallikreins/metabolism , Male , Microscopy, Electron, Scanning , Pregnancy , Prenatal Exposure Delayed Effects/metabolism , Random Allocation , Rats , Rats, Wistar , Real-Time Polymerase Chain ReactionABSTRACT
OBJECTIVE: Excess fluoride intake during tooth development is known to cause dental fluorosis. It has also been suggested that amoxicillin use in early childhood is associated with enamel hypomineralization. The aim was to investigate separate and combined effects of sodium fluoride (NaF) and amoxicillin on enamel formation in vitro. DESIGN: Mandibular molar tooth germs of E18 mouse embryos were cultured for 10 days in a medium containing NaF (10, 12 or 15µM) and/or amoxicillin (0.5, 1, 2 or 3.6mg/mL) or sodium clavulanate (0.07mg/mL) alone or in combination with 0.5mg/mL of amoxicillin. Morphological changes were studied from the whole tooth photographs and histological tissue sections with light microscope. RESULTS: Only with the highest concentrations of NaF or amoxicillin alone the extent of enamel in the first molars measured as the vertical enamel height/crown height ratio was reduced (p<0.01, p<0.001, respectively). At lower concentrations, combination of NaF (12µM) and amoxicillin (2mg/mL) significantly reduced enamel extent compared with the controls (p<0.001). Histologically, the ameloblasts were still columnar but poorly organized and the nascent enamel was often non-homogeneous. Enamel formation was not seen in any second molars exposed to 12µM NaF and 2mg/mL of amoxicillin (or higher concentrations) compared with the presence of enamel in half of the controls (p<0.001). CONCLUSIONS: Amoxicillin and NaF dose dependently affect developing enamel of mouse molars in vitro and the effects are potentiative. The clinical significance of the results remains to be studied.