RESUMEN

Evidence has shown that miRNAs could play a role in dental fluorosis, but there is no study has investigated the global expression miRNA profiles of fluoride-exposed enamel organ. In this study, we analysed the differentially expressed (DE) miRNAs between fluoride-treated and control enamel organ for the first time and found several candidate miRNAs and signaling pathways worthy of further research. Thirty Wistar rats were randomly distributed into three groups and exposed to drinking water with different fluoride contents for 10 weeks and during the gestation. The three groups were a control group (distilled water), medium fluoride group (75 mg/L NaF), and high fluoride group (150 mg/L NaF). On the embryonic day 19.5, the mandible was dissected for histological analysis, and the enamel organ of the mandibular first molar tooth germ was collected for miRNA sequencing (miRNA-seq) and quantitative real-time PCR analysis (qRT-PCR). Typical dental fluorosis was observed in the incisors of the prepregnant rats. In addition to the disorganized structure of enamel organ cells, 39 DE miRNAs were identified in the fluoride groups compared with the control group, and good agreement between the miRNA-seq data and qRT-PCR data was found. The functional annotation of the target genes of 39 DE miRNAs showed significant enrichment in metabolic process, cell differentiation, calcium signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway terms. This study provides a theoretical reference for an extensive understanding of the mechanism of fluorosis and potential valuable miRNAs as therapeutic targets in fluorosis.

Asunto(s)

Órgano del Esmalte/efectos de los fármacos , Fluoruros/toxicidad , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , MicroARNs , Animales , Embrión de Mamíferos , Órgano del Esmalte/embriología , Órgano del Esmalte/metabolismo , Femenino , Fluorosis Dental , Ratas Wistar , Transcriptoma/efectos de los fármacos

RESUMEN

Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca2+ signaling. Primary enamel cells and an enamel cell line (LS8) exposed to fluoride showed decreased internal Ca2+ stores and store-operated Ca2+ entry (SOCE). RNA-sequencing analysis revealed changes in gene expression suggestive of endoplasmic reticulum (ER) stress in fluoride-treated LS8 cells. Fluoride exposure did not alter Ca2+ homeostasis or increase the expression of ER stress-associated genes in HEK-293 cells. In enamel cells, fluoride exposure affected the functioning of the ER-localized Ca2+ channel IP3R and the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump during Ca2+ refilling of the ER. Fluoride negatively affected mitochondrial respiration, elicited mitochondrial membrane depolarization, and disrupted mitochondrial morphology. Together, these data provide a potential mechanism underlying dental fluorosis.

Asunto(s)

Calcio/metabolismo , Esmalte Dental/efectos de los fármacos , Fluoruros/farmacología , Mitocondrias/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular , Células Cultivadas , Esmalte Dental/citología , Esmalte Dental/metabolismo , Órgano del Esmalte/citología , Órgano del Esmalte/efectos de los fármacos , Órgano del Esmalte/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Fluorosis Dental/genética , Fluorosis Dental/metabolismo , Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Ratones , Mitocondrias/metabolismo

RESUMEN

OBJECTIVE: The aim of this work is to investigate the possible role of Toll-like receptor 4 (TLR4) during the development of mouse tooth germ. TLR4 is well known to inhibit mineralization and cause inflammation in mature odontoblasts and dental pulp cells. However, unlike these pathological functions of TLR4, little is known about the developmental function(s) of TLR4 during tooth development. MATERIALS AND METHODS: TLR4 expression was studied via Western blot in developing lower mouse incisors from E13.5 to E18.5. To generate functional data about the effects of TLR4, a specific agonist (LPS) was applied to the medium of in vitro tooth germ cultures, followed by Western blot, histochemical staining, ELISA assay, in situ hybridization and RT-qPCR. RESULTS: Increased accumulation of biotin-labelled LPS was detected in the enamel organ and in preodontoblasts. LPS treatment induced degradation of the inhibitor molecule (IκB) of the NF-κB signalling pathway. However, no morphological alterations were detected in cultured tissue after LPS addition at the applied dosage. Activation of TLR4 inhibited the mineralization of enamel and dentin, as demonstrated by alizarin red staining and as decreased levels of collagen type X. mRNA expression of ameloblastin was elevated after LPS administration. CONCLUSION: These results demonstrate that TLR4 may decrease the mineralization of hard tissues of the tooth germ and may trigger the maturation of ameloblasts; it can give valuable information to understand better congenital tooth abnormalities.

Asunto(s)

Transducción de Señal/fisiología , Receptor Toll-Like 4/fisiología , Calcificación de Dientes/fisiología , Germen Dentario/fisiología , Ameloblastos/efectos de los fármacos , Animales , Colágeno Tipo X/análisis , Colágeno Tipo X/efectos de los fármacos , Esmalte Dental/efectos de los fármacos , Esmalte Dental/metabolismo , Proteínas del Esmalte Dental/análisis , Proteínas del Esmalte Dental/efectos de los fármacos , Dentina/efectos de los fármacos , Dentina/metabolismo , Órgano del Esmalte/efectos de los fármacos , Órgano del Esmalte/metabolismo , Proteínas I-kappa B/análisis , Proteínas I-kappa B/efectos de los fármacos , Lipopolisacáridos/farmacología , Ratones , Odontoblastos/efectos de los fármacos , Odontoblastos/metabolismo , Odontogénesis/efectos de los fármacos , Odontogénesis/fisiología , Técnicas de Cultivo de Órganos , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/efectos de los fármacos , Calcificación de Dientes/efectos de los fármacos , Germen Dentario/efectos de los fármacos

RESUMEN

OBJECTIVE: Quantitative real-time PCR (qPCR) is routinely performed for experiments designed to identify the molecular mechanisms involved in the pathogenesis of dental fluorosis. Expression of reference gene(s) is expected to remain unchanged in fluoride-treated cells or in rodents relative to the corresponding untreated controls. The aim of this study was to select optimal reference genes for fluoride experiments performed in vitro and in vivo. DESIGN: Five candidate genes were evaluated: B2m, Eef1a1, Gapdh, Hprt and Tbp. For in vitro experiments, LS8 cells derived from mouse enamel organ were treated with 0, 1, 3 and/or 5mM sodium fluoride (NaF) for 6 or 18 h followed by RNA isolation. For in vivo experiments, six-week old rats were treated with 0 or 100 ppm fluoride as NaF for six weeks at which time RNA was isolated from enamel organs. RNA from cells and enamel organs were reverse-transcribed and stability of gene expression for the candidate reference genes was evaluated by qPCR in treated versus non-treated samples. RESULTS: The most stably expressed genes in vitro according to geNorm were B2m and Tbp, and according to Normfinder were Hprt and Gapdh. The most stable genes in vivo were Eef1a1 and Gapdh. Expression of Ddit3, a gene previously shown to be induced by fluoride, was demonstrated to be accurately calculated only when using an optimal reference gene. CONCLUSIONS: This study identifies suitable reference genes for relative quantification of gene expression by qPCR after fluoride treatment both in cultured cells and in the rodent enamel organ.

Asunto(s)

Expresión Génica/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Fluoruro de Sodio/farmacología , Animales , Línea Celular , Órgano del Esmalte/citología , Órgano del Esmalte/efectos de los fármacos , Fluorosis Dental/etiología , Fluorosis Dental/genética , Perfilación de la Expresión Génica , Masculino , Ratones , Factor 1 de Elongación Peptídica/genética , ARN/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Factor de Transcripción CHOP/genética

RESUMEN

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.

Asunto(s)

Amelogénesis/fisiología , Amelogenina/fisiología , Ameloblastos/química , Ameloblastos/ultraestructura , Amelogénesis/efectos de los fármacos , Amelogenina/genética , Animales , Compuestos Azo , Tampones (Química) , Antiportadores de Cloruro-Bicarbonato/análisis , Cloruros/análisis , Colorantes , Cristalización , Esmalte Dental/química , Esmalte Dental/ultraestructura , Microanálisis por Sonda Electrónica/métodos , Órgano del Esmalte/efectos de los fármacos , Órgano del Esmalte/ultraestructura , Fluoruros/farmacología , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Minerales/análisis , Microtomografía por Rayos X/métodos

RESUMEN

Dental fluorosis is characterized by subsurface hypomineralization and retention of enamel matrix proteins. Fluoride (F(-)) exposure generates reactive oxygen species (ROS) that can cause endoplasmic reticulum (ER)-stress. We therefore screened oxidative stress arrays to identify genes regulated by F(-) exposure. Vitamin E is an antioxidant so we asked if a diet high in vitamin E would attenuate dental fluorosis. Maturation stage incisor enamel organs (EO) were harvested from F(-)-treated rats and mice were assessed to determine if vitamin E ameliorates dental fluorosis. Uncoupling protein-2 (Ucp2) was significantly up-regulated by F(-) (∼1.5 & 2.0 fold for the 50 or 100 ppm F(-) treatment groups, respectively). Immunohistochemical results on maturation stage rat incisors demonstrated that UCP2 protein levels increased with F(-) treatment. UCP2 down-regulates mitochondrial production of ROS, which decreases ATP production. Thus, in addition to reduced protein translation caused by ER-stress, a reduction in ATP production by UCP2 may contribute to the inability of ameloblasts to remove protein from the hardening enamel. Fluoride-treated mouse enamel had significantly higher quantitative fluorescence (QF) than the untreated controls. No significant QF difference was observed between control and vitamin E-enriched diets within a given F(-) treatment group. Therefore, a diet rich in vitamin E did not attenuate dental fluorosis. We have identified a novel oxidative stress response gene that is up-regulated in vivo by F(-) and activation of this gene may adversely affect ameloblast function.

Asunto(s)

Órgano del Esmalte/efectos de los fármacos , Fluoruros/farmacología , Fluorosis Dental/metabolismo , Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Fosfatos/farmacología , Animales , Proteínas del Esmalte Dental/metabolismo , Ratones Endogámicos C57BL , Ratas Sprague-Dawley , Activación Transcripcional , Proteína Desacopladora 2 , Regulación hacia Arriba

RESUMEN

Dental fluorosis is caused by chronic high-level fluoride (F(-)) exposure during enamel development, and fluorosed enamel has a higher than normal protein content. Matrix metalloproteinase 20 cleaves enamel matrix proteins during the secretory stage, and KLK4 further cleaves these proteins during the maturation stage so that the proteins can be reabsorbed from the hardening enamel. We show that transforming growth factor ß1 (TGF-ß1) can induce Klk4 expression, and we examine the effect of F(-) on TGF-ß1 and KLK4 expression. We found that in vivo F(-) inhibits Klk4 but not Mmp20 transcript levels. LacZ-C57BL/6-Klk4 (+/LacZ) mice have LacZ inserted in frame at the Klk4 translation initiation site so that the endogenous Klk4 promoter drives LacZ expression in the same temporal/spatial way as it does for Klk4. KLK4 protein levels in rat enamel and ß-galactosidase staining in LacZ-C57BL/6-Klk4 (+/LacZ) mouse enamel were both significantly reduced by F(-) treatment. Since TGF-ß1 induces KLK4 expression, we tested and found that F(-) significantly reduced Tgf-ß1 transcript levels in rat enamel organ. These data suggest that F(-)-mediated downregulation of TGF-ß1 expression contributes to reduced KLK4 protein levels in fluorosed enamel and provides an explanation for why fluorosed enamel has a higher than normal protein content.

Asunto(s)

Cariostáticos/farmacología , Proteínas del Esmalte Dental/efectos de los fármacos , Fluoruros/farmacología , Calicreínas/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/efectos de los fármacos , Ameloblastos/efectos de los fármacos , Ameloblastos/patología , Amelogenina/análisis , Amelogenina/efectos de los fármacos , Animales , Línea Celular Tumoral , Células Cultivadas , Regulación hacia Abajo , Órgano del Esmalte/efectos de los fármacos , Técnicas de Sustitución del Gen , Calicreínas/análisis , Operón Lac/efectos de los fármacos , Metaloproteinasa 20 de la Matriz/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratas , Ratas Sprague-Dawley , beta-Galactosidasa/análisis

RESUMEN

Excessive intake of fluoride (F) by young children results in the formation of enamel subsurface porosities and pits, called enamel fluorosis. In this study, we used a single high dose of F administered to hamster pups to determine the stage of ameloblasts most affected by F and whether pit formation was related to F-related sub-ameloblastic cyst formation. Hamster pups received a single subcutaneous injection of either 20 mg or 40 mg NaF/kg body weight, were sacrificed 24 h later, and the number of cysts formed in the first molars were counted. Other pups were sacrificed 8 days after F injection, when the first molars had just erupted, to score for enamel defects. All F-injected pups formed enamel defects in the upper half of the cusps in a dose-dependent way. After injection of 20 mg NaF/kg, an average of 2.5 white spots per molar was found but no pits. At 40 mg NaF/kg, almost 4.5 spots per molar were counted as well as 2 pits per molar. The defects in erupted enamel were located in the upper half of the cusps, sites where cysts had formed at the transition stage of ameloblast differentiation. These results suggest that transitional ameloblasts, located between secretory- and maturation-stage ameloblasts, are most sensitive to the effects of a single high dose of F. F-induced cysts formed earlier at the pre-secretory stage were not correlated to either white spots or enamel pits, suggesting that damaged ameloblasts overlying a F-induced cyst regenerate and continue to form enamel.

Asunto(s)

Ameloblastos/efectos de los fármacos , Esmalte Dental/efectos de los fármacos , Órgano del Esmalte/fisiología , Fluorosis Dental/patología , Fluoruro de Sodio/efectos adversos , Ameloblastos/patología , Animales , Cricetinae , Quistes/inducido químicamente , Órgano del Esmalte/efectos de los fármacos , Microtomía , Adhesión en Plástico , Porosidad , Regeneración , Fluoruro de Sodio/administración & dosificación

RESUMEN

Hertwig's epithelial root sheath (HERS), a bilayered epithelial cell sheath located at the cervical loop of the enamel organ in a developing tooth, is at the forefront of root formation. However, little is known about the exact mechanisms that regulate the development of HERS. The neuropeptide vasoactive intestinal peptide (VIP) is involved in the development of various tissues and cells. In this study, we investigated the roles of VIP in HERS development. VIP-immunoreactive nerve fibers were found in the dental pulp and around the root apex of the tooth, while the expression of VIP receptor 1 (VPAC1) was observed in HERS. The expression level of VPAC1 correlated with the development of HERS and was elevated at postnatal days 14 and 21. Using ex vivo cultures of neonatal tooth germs, VIP enhanced the elongation and proliferation of HERS. In vitro, VIP also promoted the proliferation of cells from the HERS-derived cell line, HERS01a cells, and upregulated the mRNA expression of cytokeratin 14 and vimentin (typical molecular markers of HERS) in these cells. These results suggest that VIP may be an essential factor for HERS development.

Asunto(s)

Órgano del Esmalte/citología , Células Epiteliales/metabolismo , Péptido Intestinal Vasoactivo/fisiología , Animales , Línea Celular , Proliferación Celular , Órgano del Esmalte/efectos de los fármacos , Órgano del Esmalte/crecimiento & desarrollo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Queratina-14/genética , Queratina-14/metabolismo , Masculino , Ratones , Ratones SCID , ARN Mensajero/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Raíz del Diente/citología , Raíz del Diente/efectos de los fármacos , Raíz del Diente/crecimiento & desarrollo , Péptido Intestinal Vasoactivo/farmacología , Vimentina/genética , Vimentina/metabolismo

RESUMEN

Fluorosed enamel can be porous, mottled, discolored, hypomineralized, and protein-rich if the enamel matrix is not completely removed. Proteolytic processing by matrix metalloproteinase-20 (MMP20) and kallikrein-4 (KLK4) is critical for enamel formation, and homozygous mutation of either protease results in hypomineralized, protein-rich enamel. Herein, we demonstrate that the lysosomal proteinase cathepsin K is expressed in the enamel organ in a developmentally defined manner that suggests a role for cathepsin K in degrading re-absorbed enamel matrix proteins. We therefore asked if fluoride directly inhibits the activity of MMP20, KLK4, dipeptidyl peptidase I (DPPI) (an in vitro activator of KLK4), or cathepsin K. Enzyme kinetics were studied with quenched fluorescent peptides with purified enzyme in the presence of 0-10 mM NaF, and data were fit to Michaelis-Menten curves. Increasing concentrations of known inhibitors showed decreases in enzyme activity. However, concentrations of up to 10 mM NaF had no effect on KLK4, MMP20, DPPI, or cathepsin K activity. Our results show that fluoride does not directly inhibit enamel proteolytic activity.

Asunto(s)

Proteínas del Esmalte Dental/efectos de los fármacos , Esmalte Dental/enzimología , Fluoruros/farmacología , Péptido Hidrolasas/efectos de los fármacos , Ameloblastos/efectos de los fármacos , Amelogénesis/efectos de los fármacos , Amelogénesis/fisiología , Animales , Catepsina C/análisis , Catepsina C/efectos de los fármacos , Catepsina K/antagonistas & inhibidores , Catepsina K/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Dipéptidos/administración & dosificación , Dipéptidos/farmacología , Relación Dosis-Respuesta a Droga , Órgano del Esmalte/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Calicreínas/antagonistas & inhibidores , Calicreínas/efectos de los fármacos , Leucina/análogos & derivados , Leucina/farmacología , Metaloproteinasa 20 de la Matriz/efectos de los fármacos , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/administración & dosificación , Inhibidores de Proteasas/farmacología , Inhibidores de Serina Proteinasa/administración & dosificación , Inhibidores de Serina Proteinasa/farmacología , Sulfonas/administración & dosificación , Sulfonas/farmacología , Porcinos , Factores de Tiempo

Asunto(s)

Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Odontogénesis/efectos de los fármacos , Germen Dentario/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Ameloblastos/efectos de los fármacos , Amelogenina/análisis , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Órgano del Esmalte/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas de Filamentos Intermediarios/análisis , Cloruro de Litio/farmacología , Factor de Unión 1 al Potenciador Linfoide/análisis , Mesodermo/efectos de los fármacos , Ratones , Diente Molar/efectos de los fármacos , Diente Molar/embriología , Proteínas del Tejido Nervioso/análisis , Nestina , Odontoblastos/efectos de los fármacos , Técnicas de Cultivo de Órganos , Calcificación de Dientes/efectos de los fármacos , Corona del Diente/efectos de los fármacos , Corona del Diente/embriología , Raíz del Diente/efectos de los fármacos , Raíz del Diente/embriología

RESUMEN

Transgenic mice that express dominant-negative RhoA (RhoA(DN) ) in ameloblasts have hypoplastic enamel with defects in molar cusps. ß-catenin and Wnt5a were up-regulated in enamel organs of RhoA(DN) transgenic mice, which indicated that both canonical and non-canonical Wnt pathways are implicated in the process of enamel defect formation. It was hypothesized that expression of RhoA(DN) in ameloblasts interfered with normal enamel development through the pathways that were induced by fluoride. The Wnt and RhoA pathways were further investigated in an ameloblast-lineage cell line (ALC) by treatment with sodium fluoride (NaF). The activities of RhoA and Rho-associated protein kinase (ROCK) II decreased significantly by 8-12 hours, similar to decreased activity in RhoA(DN) transgenic mice. Both canonical and non-canonical Wnt pathways were activated by treatment with NaF, which was verified by western blotting and the ß-catenin-TCF/LEF (T cell factor lymphanoid/enhancer factor) reporter gene (TOPflash) assay. ß-catenin localization to both cytoplasm and nucleus was up-regulated in NaF-treated ALC, while Gsk-3ß, the negative regulator of the Wnt pathway, showed a decreased pattern of expression. The current results indicate that both Wnt and RhoA pathways are implicated in fluoride-induced signaling transductions in the ALC as well as in the development of enamel defects in RhoA(DN) transgenic mice.

Asunto(s)

Ameloblastos/fisiología , Amelogénesis/genética , Hipoplasia del Esmalte Dental/genética , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoA/fisiología , Ameloblastos/efectos de los fármacos , Amelogenina/biosíntesis , Amelogenina/genética , Animales , Línea Celular , Órgano del Esmalte/efectos de los fármacos , Órgano del Esmalte/fisiología , Reguladores de Proteínas de Unión al GTP/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Fluoruro de Sodio/farmacología , Proteínas Wnt/biosíntesis , Proteínas Wnt/genética , Proteína Wnt-5a , beta Catenina/biosíntesis , beta Catenina/genética , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/fisiología , Proteína de Unión al GTP rhoA/genética

RESUMEN

The selective serotonin re-uptake inhibitor (SSRI) fluoxetine is widely used in the treatment of depression in children and fertile women, but its effect on developing tissues has been sparsely investigated. The aim of this study was to investigate if enamel organs and ameloblast-derived cells express serotonin receptors that are affected by peripherally circulating serotonin or fluoxetine. Using RT-PCR and western blot analysis we found that enamel organs from 3-d-old mice and ameloblast-like cells (LS8 cells) express functional serotonin receptors, the rate-limiting enzyme in serotonin synthesis (Thp1), as well as the serotonin transporter (5HTT), indicating that enamel organs and ameloblasts are able to respond to serotonin and regulate serotonin availability. Fluoxetine and serotonin enhanced the alkaline phosphatase activity in the cell culture medium from cultured LS8 cells, whereas the expression of enamelin (Enam), amelogenin (Amel), and matrix metalloproteinase-20 (MMP-20) were all significantly down-regulated. The secretion of vascular endothelial growth factor (VEGF), monocyte chemotactic protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10) was also reduced compared with controls. In conclusion, enamel organs and ameloblast-like cells express functional serotonin receptors. Reduced transcription of enamel proteins and secretion of vascular factors may indicate possible adverse effects of fluoxetine on amelogenesis.

Asunto(s)

Ameloblastos/efectos de los fármacos , Órgano del Esmalte/efectos de los fármacos , Fluoxetina/farmacología , Receptores de Serotonina/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/efectos de los fármacos , Amelogenina/análisis , Amelogenina/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , Línea Celular , Quimiocina CCL2/análisis , Quimiocina CCL2/efectos de los fármacos , Quimiocina CXCL10/análisis , Quimiocina CXCL10/efectos de los fármacos , Medios de Cultivo , Proteínas del Esmalte Dental/análisis , Proteínas del Esmalte Dental/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , L-Lactato Deshidrogenasa/análisis , L-Lactato Deshidrogenasa/efectos de los fármacos , Metaloproteinasa 20 de la Matriz/análisis , Metaloproteinasa 20 de la Matriz/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Receptores de Serotonina/análisis , Serotonina/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/análisis , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Agonistas de Receptores de Serotonina/farmacología , Espectrofotometría Atómica , Triptófano Hidroxilasa/análisis , Triptófano Hidroxilasa/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos

RESUMEN

Traumatic dental injuries are often irreversible, underscoring the need for therapies that protect dental pulp cells and enhance their regeneration. We hypothesized that generation 5 poly amido amine (PAMAM) dendrimers (G5), functionalized with fluorescein isothiocyanate (FL) and αVß3-specific, cyclic arginine-glycine-aspartic acid (RGD) peptides, will bind to dental pulp cells (DPCs) and modulate their differentiation. Dental pulp cells and mouse odontoblast-like cells (MDPC-23) (±) treated with G5-FL-RGD were analyzed via Western blot, RT-PCR, and quantitative PCR. Transcription of dental differentiation markers was as follows: Dentin matrix protein (DMP-1), dentin sialoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) as well as vascular endothelial growth factor (VEGF) all increased via the JNK pathway. Long-term G5-RGD treatment of dental pulp cells resulted in enhanced mineralization as examined via Von Kossa assay, suggesting that PAMAM dendrimers conjugated to cyclic RGD peptides can increase the odontogenic potential of these cells.

Asunto(s)

Dendrímeros/farmacología , Pulpa Dental/efectos de los fármacos , Oligopéptidos/farmacología , Adulto , Secuencia de Aminoácidos , Animales , Western Blotting , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Pulpa Dental/citología , Órgano del Esmalte/citología , Órgano del Esmalte/efectos de los fármacos , Proteínas de la Matriz Extracelular/análisis , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Glicoproteínas/análisis , Humanos , Integrina alfaVbeta3/análisis , Proteínas Quinasas JNK Activadas por Mitógenos/análisis , Ratones , Odontoblastos/citología , Odontoblastos/efectos de los fármacos , Odontogénesis/efectos de los fármacos , Fosfoproteínas/análisis , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sialoglicoproteínas/análisis , Técnicas de Cultivo de Tejidos , Calcificación de Dientes/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/análisis

RESUMEN

PURPOSE: Previous studies have evaluated the presence of serotonin in the dental epithelia and mesenchyme during odontogenesis, suggesting its participation in tooth development. MATERIALS AND METHODS: Here, we used fluoxetine, a selective serotonin re-uptake inhibitor, at a dose of 10 mg/kg, administered for 20 days during pregnancy in 12 Wistar rats to examine the influence of this drug on the development of the enamel organ of the upper first molars of rat fetuses at 17 days of intra-uterine life (i.u.l.), and at one, five and ten days postpartum. The pregnant rats were anesthetized with xylazine at 10 mg/kg and ketamine at 25 mg/kg. The fetuses were removed and beheaded; their jaws were removed, and the upper jaws were exposed. The tissues were fixed in Bouin's fixative, decalcified in 5% nitric acid for 4 - 12 h, conventionally processed for microscopy, and embedded in paraffin. Serial sections of approximately 5 mum were obtained and stained with hematoxylin and eosin, as well as periodic acid-Schiff. RESULTS AND CONCLUSION: Morphological analysis showed no structural changes in the experimental group compared to the controls, suggesting that, at the dose used, fluoxetine does not interfere with serotonin-mediated development of the enamel organ or the process of amelogenesis.

Asunto(s)

Órgano del Esmalte/anatomía & histología , Órgano del Esmalte/efectos de los fármacos , Fluoxetina/administración & dosificación , Inhibidores Selectivos de la Recaptación de Serotonina/administración & dosificación , Amelogénesis/efectos de los fármacos , Amelogénesis/fisiología , Animales , Órgano del Esmalte/crecimiento & desarrollo , Femenino , Modelos Animales , Embarazo , Distribución Aleatoria , Ratas , Ratas Wistar

RESUMEN

The aim of the present study was to assess birefringence of the secretory-stage enamel organic extracellular matrix (ECM) and mechanical properties of mature enamel from rats treated with bisphosphonates. Longitudinal sections were obtained from upper incisors of rats that had been submitted to injections of bisodic etidronate (8 mg/Kg/day), sodium alendronate (30 microg/Kg/day), or sodium chloride as control (8 mg/Kg/day) for 42 days. Sections were immersed in 80% glycerin for 30 min and optical retardation of birefringence brightness in the secretory-stage enamel organic ECM was determined in nanometers. Etidronate-treated rats exhibited extensive morphological changes in the secretory-stage enamel organic ECM inclusive nonbirefringent conspicuous incremental lines, but presented optical retardation values similar to those showed by control rats (p > 0.05). Birefringence of secretory enamel organic ECM from etidronate rats presented an irregular aspect. Alendronate-treated rats did not show morphological alterations in the secretory-stage enamel organic ECM, however, they presented significant reduction in optical retardation of birefringence brightness when compared with control and etidronate rats (p < 0.01). Alendronate and etidronate groups exhibited reductions of approximately 6-10% in mature enamel cross-sectional microhardness when compared with control group (p < 0.01). Scanning electron microscopy analysis showed extensive alterations in mature enamel only from etidronate group with absence of enamel rods. The present work shows that bisphosphonates can affect the birefringence of the secretory-stage enamel organic ECM. The results presented here suggest that alterations in the supramolecular organization of the secretory-stage enamel organic ECM are a plausible mechanism by which environmental factors may cause enamel defects.

Asunto(s)

Alendronato/farmacología , Conservadores de la Densidad Ósea/farmacología , Esmalte Dental/efectos de los fármacos , Órgano del Esmalte/efectos de los fármacos , Ácido Etidrónico/farmacología , Matriz Extracelular/patología , Animales , Birrefringencia , Esmalte Dental/ultraestructura , Órgano del Esmalte/patología , Matriz Extracelular/fisiología , Incisivo , Masculino , Microscopía Electrónica de Rastreo , Microscopía de Polarización , Ratas , Ratas Wistar , Cloruro de Sodio/farmacología

RESUMEN

Purpose: Previous studies have evaluated the presence of serotonin in the dental epithelia and mesenchyme during odontogenesis, suggesting its participation in tooth development. Materials and methods: Here, we used fluoxetine, a selective serotonin re-uptake inhibitor, at a dose of 10 mg/kg, administered for 20 days during pregnancy in 12 Wistar rats to examine the influence of this drug on the development of the enamel organ of the upper first molars of rat fetuses at 17 days of intra-uterine life (i.u.l.), and at one, five and ten days postpartum. The pregnant rats were anesthetized with xylazine at 10 mg/kg and ketamine at 25 mg/kg. The fetuses were removed and beheaded; their jaws were removed, and the upper jaws were exposed. The tissues were fixed in Bouin's fixative, decalcified in 5 percent nitric acid for 4 - 12 h, conventionally processed for microscopy, and embedded in paraffin. Serial sections of approximately 5 mm were obtained and stained with hematoxylin and eosin, as well as periodic acid-Schiff. Results and conclusion: Morphological analysis showed no structural changes in the experimental group compared to the controls, suggesting that, at the dose used, fluoxetine does not interfere with serotonin-mediated development of the enamel organ or the process of amelogenesis.

Asunto(s)

Animales , Femenino , Embarazo , Ratas , Órgano del Esmalte/anatomía & histología , Órgano del Esmalte/efectos de los fármacos , Fluoxetina/administración & dosificación , Inhibidores Selectivos de la Recaptación de Serotonina/administración & dosificación , Amelogénesis/efectos de los fármacos , Amelogénesis/fisiología , Órgano del Esmalte/crecimiento & desarrollo , Modelos Animales , Distribución Aleatoria , Ratas Wistar

RESUMEN

Dental epithelium comprises different cell populations, including ameloblasts and stratum intermedium cells. Ameloblasts are vitamin D targets, and at least five proteins undergo specific modulation of their expression following the addition of 1alpha,25(OH)2 vitamin D3[1alpha,25(OH)2D3]. Stratum intermedium cells have not been studied in any great detail regarding vitamin D impact. Interestingly, in these cells, the tissue non-specific alkaline phosphatase (TNAP) is overexpressed. On the other hand, TNAP is a reliable bone marker of vitamin D action, similar to calbindins in kidney and intestine, previously used for studies of vitamin D activity in ameloblasts. Here, TNAP expression and activity were investigated in vivo in the microdissected epithelium and mesenchyme of mandible incisors. Physiological doses of 1alpha,25(OH)2D3 injected in control rats failed to modify TNAP activity in both dental epithelium and mesenchyme. No significant differences were observed in the steady-state levels of TNAP mRNAs of dental tissues from wild-type and vitamin D nuclear receptor (VDRnuc)-deficient mice of the same litters. These data suggest that, in contrast to ameloblasts, stratum intermedium cells are not sensitive to 1alpha,25(OH)2D3. An explanation for such a responsiveness of stratum intermedium cells to 1alpha,25(OH)2D3 is proposed based on the respective expressions of both vitamin D receptors (VDRnuc and 1,25D3-[MARRS]) and the Dlx2 homeobox gene.

Asunto(s)

Fosfatasa Alcalina/efectos de los fármacos , Órgano del Esmalte/efectos de los fármacos , Germen Dentario/efectos de los fármacos , Vitamina D/farmacología , Vitaminas/farmacología , Ameloblastos/efectos de los fármacos , Amelogénesis/genética , Amelogenina , Animales , Calcitriol/farmacología , Proteínas del Esmalte Dental/efectos de los fármacos , Epitelio/efectos de los fármacos , Proteínas de Homeodominio/genética , Incisivo , Mesodermo/efectos de los fármacos , Ratones , Ratones Transgénicos , ARN Mensajero/análisis , Ratas , Receptores de Calcitriol/deficiencia , Receptores de Calcitriol/genética , Factores de Transcripción/genética

RESUMEN

OBJECTIVE: To investigate the effects of lead exposure at different levels in utero on the teeth eruption and enamel development of rat offsprings. METHODS: 27 pregnant SD rats were divided into three groups randomly: high level lead group (HLG), low level lead group (LLG) and control group with nine rats in each group. The three groups from the gestation day to the end of the gestation were given either deionized water in control group or deionized water containing 200 mg/L Pb2+ as lead acetate through drinking method in high level lead experimental group and 50 mg/L Pb2+ as lead acetate through drinking method in low level lead experimental group. The incisors of newborn rats were marked at the level of the gingival papilla on the 26th day after birth. On the 36th day, the incisors of newborn rats were marked again at the same level. Then the rat offsprings were killed and their blood was collected for lead analysis. The mandible incisors of rat offspring were separated and the content of Pb in incisors was determined by using a graphite furnace atomic absorption spectrometric method. The teeth of rat offspring were observed and the distance between two marks were measured by means of stereomicroscope. The ratio of calcium to phosphate of enamel of rat offspring was compared by electron probe microanalyses. RESULTS: The level of blood lead in 200 mg/L, 50 mg/L treated rat offspring groups was higher than that in control group. The tooth lead of 200 mg/L, 50 mg/L treated rat offspring groups [(77.3 +/- 6.3), (27.8 +/- 4.5) microg/g] were higher than the control [(6.6 +/- 0.8) microg/g, P < 0.01]. Compared with the control group, the teeth of lead exposure experimental groups were smaller and severity of attrition was obvious and pulpal perforations were often observed. These appearances was more distinct in rats of high level lead experimental group. The incisors of lead-treated rat offspring erupted [(0.25 +/- 0.08), (0.30 +/- 0.09) mm/d] more slowly than control ones [(0.39 +/- 0.09) mm/d, P < 0.01]. The ratio of calcium to phosphate (Ca/P) decreased with the increase of lead exposure. It was found that Ca/P in lead exposure experimental groups (1.68 +/- 0.54), (1.37 +/- 0.47) was significantly lower than that in the control group (2.14 +/- 0.33). CONCLUSION: Lead exposure in utero affects the normal eruption of teeth and enamel formation and the degree was related with the lead exposure level.

Asunto(s)

Órgano del Esmalte/efectos de los fármacos , Plomo/efectos adversos , Exposición Materna/efectos adversos , Efectos Tardíos de la Exposición Prenatal/patología , Erupción Dental/efectos de los fármacos , Animales , Femenino , Embarazo , Ratas , Ratas Sprague-Dawley

RESUMEN

OBJECTIVES: The purpose of this paper is to report the development of extensive, gingival eruption cyst formation in neonatal dogs during chronic administration of cyclosporine, which regressed after treatment ceased. MATERIALS AND METHODS: As part of an investigation of the efficacy of adenoviral-mediated gene therapy in a canine model of Duchenne Muscular Dystrophy (DMD), 26 Golden retriever pups were given intramuscular injections of adenoviral gene constructs (at 2-5 days old) either with (15 pups) or without (9 pups) concomitant immunosuppression using oral cyclosporine (from 1 to 3 days old). RESULTS: The daily administration of cyclosporine to Golden Retriever pups from 1 to 3 days of age resulted in the formation of eruption cysts over the incisors after approximately 3 weeks of treatment. The gingiva over the remaining teeth became swollen. However unlike the incisors, part of the crown erupted through the gingiva. Continuous treatment for 60-90 days was associated with persistence and expansion of the cysts over the incisor teeth. The cysts resolved within 1 month if cyclosporine administration was discontinued. Movement of incisors within the jaws was not appreciably affected when compared with age-matched controls. CONCLUSION: Chronic cyclosporine administration to neonatal dogs induces the formation of eruption cysts over the incisors, which is reversible once treatment is discontinued and does not affect intraosseous eruption.

Asunto(s)

Ciclosporina/efectos adversos , Enfermedades de las Encías/inducido químicamente , Inmunosupresores/efectos adversos , Quistes Odontogénicos/inducido químicamente , Adenoviridae/genética , Animales , Animales Recién Nacidos , Diente Premolar/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Diente Canino/efectos de los fármacos , Ciclosporina/sangre , Modelos Animales de Enfermedad , Perros , Órgano del Esmalte/efectos de los fármacos , Órgano del Esmalte/patología , Epitelio/efectos de los fármacos , Epitelio/patología , Femenino , Terapia Genética , Vectores Genéticos , Enfermedades de las Encías/patología , Gingivitis/inducido químicamente , Gingivitis/patología , Inmunosupresores/sangre , Incisivo/efectos de los fármacos , Masculino , Mesodermo/efectos de los fármacos , Mesodermo/patología , Diente Molar/efectos de los fármacos , Distrofias Musculares/terapia , Neutrófilos/patología , Quistes Odontogénicos/patología , Inducción de Remisión , Corona del Diente/efectos de los fármacos , Erupción Dental/efectos de los fármacos