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1.
Polymers (Basel) ; 13(22)2021 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-34833250

RESUMEN

Ectomesenchymal stem cells derived from the dental pulp are of neural crest origin, and as such are promising sources for cell therapy and tissue engineering. For safe upscaling of these cells, microcarrier-based culturing under dynamic conditions is a promising technology. We tested the suitability of two microcarriers, non-porous Cytodex 1 and porous Cytopore 2, for culturing well characterized dental pulp stem cells (DPSCs) using a shake flask system. Human DPSCs were cultured on these microcarriers in 96-well plates, and further expanded in shake flasks for upscaling experiments. Cell viability was measured using the alamarBlue assay, while cell morphology was observed by conventional and two-photon microscopies. Glucose consumption of cells was detected by the glucose oxidase/Clark-electrode method. DPSCs adhered to and grew well on both microcarrier surfaces and were also found in the pores of the Cytopore 2. Cells grown in tissue culture plates (static, non-shaking conditions) yielded 7 × 105 cells/well. In shake flasks, static preincubation promoted cell adhesion to the microcarriers. Under dynamic culture conditions (shaking) 3 × 107 cells were obtained in shake flasks. The DPSCs exhausted their glucose supply from the medium by day seven even with partial batch-feeding. In conclusion, both non-porous and porous microcarriers are suitable for upscaling ectomesenchymal DPSCs under dynamic culture conditions.

2.
Front Pharmacol ; 12: 682654, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34149428

RESUMEN

Background: Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized. Ameloblasts secrete enamel matrix proteins and Ca2+, and also regulate extracellular pH as the formation of hydroxyapatite crystals generates large quantities of protons. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects such as hypomineralization. Aims: Our aims were to optimize the culture conditions for the three-dimensional growth of ameloblast-derived HAT-7 cells and to test the effects of fluoride exposure on HAT-7 spheroid formation. Methods: To generate 3D HAT-7 structures, cells were dispersed and plated within a Matrigel extracellular matrix scaffold and incubated in three different culture media. Spheroid formation was then monitored over a two-week period. Ion transporter and tight-junction protein expression was investigated by RT-qPCR. Intracellular Ca2+ and pH changes were measured by microfluorometry using the fluorescent dyes fura-2 and BCECF. Results: A combination of Hepato-STIM epithelial cell differentiation medium and Matrigel induced the expansion and formation of 3D HAT-7 spheroids. The cells retained their epithelial cell morphology and continued to express both ameloblast-specific and ion transport-specific marker genes. Furthermore, like two-dimensional HAT-7 monolayers, the HAT-7 spheroids were able to regulate their intracellular pH and to show intracellular calcium responses to extracellular stimulation. Finally, we demonstrated that HAT-7 spheroids may serve as a disease model for studying the effects of fluoride exposure during amelogenesis. Conclusion: In conclusion, HAT-7 cells cultivated within a Matrigel extracellular matrix form three-dimensional, multi-cellular, spheroidal structures that retain their functional capacity for pH regulation and intracellular Ca2+ signaling. This new 3D model will allow us to gain a better understanding of the molecular mechanisms involved in amelogenesis, not only in health but also in disorders of enamel formation, such as those resulting from fluoride exposure.

3.
Int J Mol Sci ; 22(8)2021 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-33924361

RESUMEN

TRPM7 plays an important role in cellular Ca2+, Zn2+ and Mg2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.


Asunto(s)
Ameloblastos/metabolismo , Calcio/metabolismo , Canales Catiónicos TRPM/metabolismo , Ameloblastos/citología , Ameloblastos/efectos de los fármacos , Anilidas/farmacología , Animales , Línea Celular , Humanos , Concentración de Iones de Hidrógeno , Incisivo/citología , Activación del Canal Iónico/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Mibefradil/farmacología , Ratones , Modelos Biológicos , Naltrexona/análogos & derivados , Naltrexona/farmacología , Ratas , Tiadiazoles/farmacología
4.
Environ Health ; 19(1): 38, 2020 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-32248806

RESUMEN

BACKGROUND: Previous studies have shown a correlation between fluoride concentrations in urine and community water fluoride concentrations. However, there are no studies of the relationship between community water fluoridation, urine, serum, and amniotic fluid fluoride concentrations in pregnant women in the US. The aim of this study was to determine the relationship between maternal urine fluoride (MUF), maternal urine fluoride adjusted for specific gravity (MUFSG), maternal serum fluoride (MSF), amniotic fluid fluoride (AFF) concentrations during pregnancy, and community water fluoridation in Northern California. METHODS: Archived samples of urine, serum and amniotic fluid collected from second trimester pregnant women in Northern California from 47 different communities in Northern California and one from Montana (n = 48), were analyzed for fluoride using an ion specific electrode following acid microdiffusion. Women's addresses were matched to publicly reported water fluoride concentrations. We examined whether fluoride concentrations in biospecimens differed by fluoridation status of the community water, and determined the association between water fluoride concentrations and biospecimen fluoride concentrations using linear regression models adjusted for maternal age, smoking, Body Mass Index (BMI), race/ethnicity, and gestational age at sample collection. RESULTS: Fluoride concentrations in the community water supplies ranged from 0.02 to 1.00 mg/L. MUF, MSF , and AFF concentrations were significantly higher in pregnant women living in communities adhering to the U.S. recommended water fluoride concentration (0.7 mg/L), as compared with communities with less than 0.7 mg/L fluoride in drinking water. When adjusted for maternal age, smoking status, BMI, race/ethnicity, and gestational age at sample collection, a 0.1 mg/L increase in community water fluoride concentration was positively associated with higher concentrations of MUF (B = 0.052, 95% CI:0.019,0.085), MUFSG (B = 0.028, 95% CI: -0.006, 0.062), MSF (B = 0.001, 95% CI: 0.000, 0.003) and AFF (B = 0.001, 95% CI: 0.000, 0.002). CONCLUSIONS: We found universal exposure to fluoride in pregnant women and to the fetus via the amniotic fluid. Fluoride concentrations in urine, serum, and amniotic fluid from women were positively correlated to public records of community water fluoridation. Community water fluoridation remains a major source of fluoride exposure for pregnant women living in Northern California.


Asunto(s)
Líquido Amniótico/química , Fluoruración , Fluoruros/metabolismo , Exposición Materna/estadística & datos numéricos , Adulto , California , Agua Potable/química , Femenino , Feto/química , Fluoruros/sangre , Fluoruros/orina , Humanos , Montana , Embarazo , Segundo Trimestre del Embarazo , Adulto Joven
5.
Physiol Behav ; 206: 76-83, 2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-30904570

RESUMEN

Fluoride ingestion has been linked to changes in behavior in mice and rats, related to dose, sex of the animal, and the timing of exposure. Previous studies have shown the behavior of female rats to be most affected by postnatal fluoride exposure, and in this study we determined the effects of postnatal fluoride exposure on anxiety related behavior and serotonin. Mice given 50 ppm fluoride in drinking water had increased entries in the open arms of the elevated plus maze, suggesting reduced anxiety. Both peripheral and central serotonin was increased in the fluoride treated mice. In a cohort of children drinking water containing 2.5 ppm fluoride, serum serotonin was also increased as compared to controls. The mechanisms by which fluoride results in an increase peripheral and central serotonin are not well understood, but warrant further study, as these effects may also be relevant to prenatal fluoride related changes in behavior in both mice and humans.


Asunto(s)
Conducta Animal/efectos de los fármacos , Fluoruros/administración & dosificación , Aprendizaje por Laberinto/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Serotonina/sangre , Conducta Social , Administración Oral , Animales , Química Encefálica , Femenino , Fluoruros/análisis , Ratones
6.
Front Physiol ; 9: 750, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30013481

RESUMEN

During enamel development, formation of hydroxyapatite crystals and regulation of pH in the enamel matrix require massive transport of ions. Both ameloblasts and adjacent dental epithelial cells in the stellate reticulum co-express several transmembrane cotransporters/ion-exchangers for transport of ions across plasma membranes. Gap junctions (GJs) enable intercellular exchanges of ions between neighboring cells. This suggests that the ameloblasts and other cell layers of the enamel organ, form a functional unit. During the bell stage of tooth formation, the non-ameloblast dental epithelium highly expresses the Na-K-Cl cotransporter (Nkcc1). Nkcc1-null mice are associated with enamel hypomineralization and increased expression of GJ protein connexin 43 (Cx43), suggesting that reduced ion transport in the Nkcc1-null mouse is in part compensated by increased intercellular ion transport through GJs. To understand the role of GJs in ion transport and its effect on pH regulation, we examined in a mouse strain in which Cx43 was ablated selectively in DMP1 expressing cells (Cx43flox/flox mice crossed with DMP1-8kb-Cre mice), including ameloblasts. Micro-CT analysis showed that the mineral density at late maturation stage incisal enamel of the Cx43-null mice was 10% less than in controls, whereas that in dentin was unchanged. Maturation stage ameloblasts of mice lacking the pH regulating sodium/bicarbonate transporter NBCe1 (Nbce1-null), or chloride channel Cftr (Cftr-null) were found to have increased Cx43-immunostaining. These results support the possibility that GJs in the ameloblast-papillary complex at the maturation stage contribute to ion transport by enabling passage of ions directly from cells of the papillary layer into ameloblast layer. Increasing the number of GJs may partly compensate the reduction of ion-cotransporters and ion exchangers in dental epithelium.

7.
Front Physiol ; 8: 924, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29209227

RESUMEN

Na+:K+:2Cl- cotransporters (NKCCs) belong to the SLC12A family of cation-coupled Cl- transporters. We investigated whether enamel-producing mouse ameloblasts express NKCCs. Transcripts for Nkcc1 were identified in the mouse dental epithelium by RT-qPCR and NKCC1 protein was immunolocalized in outer enamel epithelium and in the papillary layer but not the ameloblast layer. In incisors of Nkcc1-null mice late maturation ameloblasts were disorganized, shorter and the mineral density of the enamel was reduced by 10% compared to wild-type controls. Protein levels of gap junction protein connexin 43, Na+-dependent bicarbonate cotransporter e1 (NBCe1), and the Cl--dependent bicarbonate exchangers SLC26A3 and SLC26A6 were upregulated in Nkcc1-null enamel organs while the level of NCKX4/SLC24A4, the major K+, Na+ dependent Ca2+ transporter in maturation ameloblasts, was slightly downregulated. Whole-cell voltage clamp studies on rat ameloblast-like HAT-7 cells indicated that bumetanide increased ion-channel activity conducting outward currents. Bumetanide also reduced cell volume of HAT-7 cells. We concluded that non-ameloblast dental epithelium expresses NKCC1 to regulate cell volume in enamel organ and provide ameloblasts with Na+, K+ and Cl- ions required for the transport of mineral- and bicarbonate-ions into enamel. Absence of functional Nkcc1 likely is compensated by other types of ion channels and ion transporters. The increased amount of Cx43 in enamel organ cells in Nkcc1-null mice suggests that these cells display a higher number of gap junctions to increase intercellular communication.

8.
Int J Oral Sci ; 9(11): e3, 2017 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-29165423

RESUMEN

The Encouraging Novel Amelogenesis Models and Ex vivo cell Lines (ENAMEL) Development workshop was held on 23 June 2017 at the Bethesda headquarters of the National Institute of Dental and Craniofacial Research (NIDCR). Discussion topics included model organisms, stem cells/cell lines, and tissues/3D cell culture/organoids. Scientists from a number of disciplines, representing institutions from across the United States, gathered to discuss advances in our understanding of enamel, as well as future directions for the field.


Asunto(s)
Amelogénesis , Esmalte Dental/fisiología , Animales , Técnicas de Cultivo de Célula , Línea Celular , Humanos , Células Madre/fisiología
10.
Arch Oral Biol ; 83: 214-221, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28806687

RESUMEN

OBJECTIVE: Fluoride excess of 0.05-0.07mgF/kgbw/day in water or food additives like salt is the principal cause of endemic dental fluorosis. How fluoride causes these defects is not clear yet. Recent studies in rodents suggest that development of enamel fluorosis is associated with insufficient neutralization of protons released during the formation of hypermineralized lines. DESIGN: Here we examined whether hypermineralization could also be assessed by MicroCT in developing molar enamel of humans exposed to fluoride. RESULT: Micro-CT analysis of hypomineralized enamel from human fluorotic molars graded by the Thylstrup-Fejerskov (TF) Index as III-IV showed weak hypermineralized lines and hypermineralized patches not seen in TF-I/II grade enamel. The mesio-distal sides of these molar teeth were significantly smaller (∼18%, p=0.02) than in TF-I/II teeth. CONCLUSION: The patterns of changes observed in human fluorotic teeth were similar to those in fluorotic rodent incisors. The data are consistent with the hypothesis that also in developing human teeth fluoride-stimulated local acidification of enamel could be a mechanism for developing fluorotic enamel.


Asunto(s)
Fluorosis Dental/diagnóstico por imagen , Incisivo/diagnóstico por imagen , Diente Molar/diagnóstico por imagen , Desmineralización Dental/diagnóstico por imagen , Diente Impactado/diagnóstico por imagen , Adolescente , Adulto , Animales , Modelos Animales de Enfermedad , Femenino , Fluorescencia , Humanos , Incisivo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Diente Molar/patología , Desmineralización Dental/patología , Diente Impactado/patología , Microtomografía por Rayos X
11.
J Biol Chem ; 292(33): 13531-13540, 2017 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-28673966

RESUMEN

Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Esmalte Dental/metabolismo , Inducción Enzimática , Subunidad 1 del Complejo Mediador/metabolismo , Receptor Notch1/agonistas , Transducción de Señal , Calcificación de Dientes , Fosfatasa Alcalina/química , Animales , Línea Celular Transformada , Esmalte Dental/ultraestructura , Genes Reporteros , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Inmunoprecipitación , Subunidad 1 del Complejo Mediador/antagonistas & inhibidores , Subunidad 1 del Complejo Mediador/genética , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Regiones Promotoras Genéticas , Multimerización de Proteína , Proteolisis , Interferencia de ARN , Receptor Notch1/metabolismo , Elementos de Respuesta
12.
Front Physiol ; 8: 940, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29375389

RESUMEN

We have recently developed a novel in vitro model using HAT-7 rat ameloblast cells to functionally study epithelial ion transport during amelogenesis. Our present aims were to identify key transporters of bicarbonate in HAT-7 cells and also to examine the effects of fluoride exposure on vectorial bicarbonate transport, cell viability, and the development of transepithelial resistance. To obtain monolayers, the HAT-7 cells were cultured on Transwell permeable filters. We monitored transepithelial resistance (TER) as an indicator of tight junction formation and polarization. We evaluated intracellular pH changes by microfluorometry using the fluorescent indicator BCECF. Activities of ion transporters were tested by withdrawal of various ions from the bathing medium, by using transporter specific inhibitors, and by activation of transporters with forskolin and ATP. Cell survival was estimated by alamarBlue assay. Changes in gene expression were monitored by qPCR. We identified the activity of several ion transporters, NBCe1, NHE1, NKCC1, and AE2, which are involved in intracellular pH regulation and vectorial bicarbonate and chloride transport. Bicarbonate secretion by HAT-7 cells was not affected by acute fluoride exposure over a wide range of concentrations. However, tight-junction formation was inhibited by 1 mM fluoride, a concentration which did not substantially reduce cell viability, suggesting an effect of fluoride on paracellular permeability and tight-junction formation. Cell viability was only reduced by prolonged exposure to fluoride concentrations greater than 1 mM. In conclusion, cultured HAT-7 cells are functionally polarized and are able to transport bicarbonate ions from the basolateral to the apical fluid spaces. Exposure to 1 mM fluoride has little effect on bicarbonate secretion or cell viability but delays tight-junction formation, suggesting a novel mechanism that may contribute to dental fluorosis.

13.
Eur J Oral Sci ; 124(5): 415-425, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27422589

RESUMEN

Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non-fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator-deficient (Cftr null), anion exchanger-2-deficient (Ae2a,b null), and amelogenin-deficient (Amelx null) mice, was determined by quantitative X-ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated-matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl- ) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl- content, the stronger the negative effects. In Amelx-null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein-4-deficient (Klk4-null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation.


Asunto(s)
Ameloblastos , Amelogénesis/fisiología , Protones , Amelogenina , Animales , Esmalte Dental/metabolismo , Proteínas del Esmalte Dental , Concentración de Iones de Hidrógeno , Ratones , Minerales
14.
J Periodontol ; 87(7): 820-7, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27043257

RESUMEN

BACKGROUND: Extracts of enamel matrix proteins are used to regenerate periodontal tissue; amelogenin, the most abundant enamel protein, plays an important role in this regeneration. Studies have demonstrated that amelogenin fragments promote tissue regeneration, but the bioactive site of amelogenin remains unclear. This study explores the functional domain of amelogenin by investigating effects of four amelogenin species on cementoblast proliferation. METHODS: Four amelogenin species based on amelogenin cleavage products were investigated: 1) recombinant human full-length amelogenin (rh174); 2) amelogenin cleavage product lacking the C-terminal (rh163); 3) amelogenin cleavage product lacking the N-terminal (rh128); and 4) the C-terminal region of rh174 (C11 peptide), which was synthesized and purified. Human cementoblast-like cell line (HCEM) cells were cultured and treated with rh174, rh163, rh128, or C11 peptide. Cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium assay and cell proliferation enzyme-linked immunosorbent assay. Mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) (MAPK-ERK) pathway was examined by Western blot analysis. RESULTS: Proliferation of HCEM cells was significantly enhanced on treatment with rh174, rh128, or C11 peptide. However, rh163 had no effect compared with the untreated control group. Western blot analysis revealed enhanced phosphorylated ERK1/2 signaling after addition of rh128 or C11 peptide and reduced phosphorylated ERK1/2 signaling after blocking with a specific MAPK inhibitor (U0126). CONCLUSION: C-terminal amelogenin cleavage product increased proliferation of HCEM through MAPK-ERK signaling pathway, indicating possible application of C11 peptide for periodontal tissue regeneration.


Asunto(s)
Amelogenina/farmacología , Proliferación Celular/efectos de los fármacos , Cementogénesis , Cemento Dental/citología , Humanos , Péptidos , Transducción de Señal
15.
Eur J Oral Sci ; 123(1): 9-16, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25557910

RESUMEN

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr-null mice by quantitative electron probe microanalysis. Maturation-stage enamel from Cftr-null mice contained less chloride and calcium than did wild-type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr-null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr-deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/metabolismo , Esmalte Dental/química , Calcificación de Dientes/fisiología , Ameloblastos/metabolismo , Amelogénesis/fisiología , Animales , Bicarbonatos/análisis , Tampones (Química) , Calcio/análisis , Cariostáticos/farmacología , Cloruros/análisis , Cloruros/metabolismo , Esmalte Dental/efectos de los fármacos , Microanálisis por Sonda Electrónica , Fluoresceínas , Colorantes Fluorescentes , Fluoruros/análisis , Fluoruros/sangre , Fluoruros/farmacología , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Ratones , Ratones Endogámicos CFTR , Microtomografía por Rayos X/métodos
16.
PLoS One ; 9(7): e102893, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25047033

RESUMEN

Human deciduous and permanent teeth exhibit different developmental processes, morphologies, histological characteristics and life cycles. In addition, their pulp tissues react differently to external stimuli, such as the pulp sensitivity test, dental trauma and pulp therapy materials. These suggest differences in gene expression and regulation, and in this study we compared gene-expression profiles of the human dental pulp from deciduous and permanent teeth. Pulp tissues from permanent premolars and deciduous molars aged 11-14 years were extirpated and mRNA was isolated for cDNA microarray analysis, and quantitative real-time PCR (qPCR). Other teeth were used for immunohistochemical analysis (IHC). Microarray analysis identified 263 genes with a twofold or greater difference in expression level between the two types of pulp tissue, 43 and 220 of which were more abundant in deciduous and permanent pulp tissues, respectively. qPCR analysis was conducted for eight randomly selected genes, and the findings were consistent with the cDNA microarray results. IHC confirmed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was broadly expressed in deciduous dental pulp tissue, but minimally expressed in permanent dental pulp tissue. Immunohistochemical analysis showed that calbindin 1 (CALB1), leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and gamma-aminobutyric acid A receptor beta 1 (GABRB1) were abundantly expressed in permanent predentin/odontoblasts, but only minimally expressed in deciduous dental pulp tissue. These results show that deciduous and permanent pulp tissues have different characteristics and gene expression, suggesting that they may have different functions and responses to therapies focused on pulp or dentin regeneration.


Asunto(s)
Pulpa Dental/metabolismo , Diente/metabolismo , Adolescente , Calbindina 1/genética , Calbindina 1/metabolismo , Niño , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Diente Primario/metabolismo
17.
Odontology ; 102(2): 147-53, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23794061

RESUMEN

Supra-optimal intake of sodium fluoride (NaF) during early childhood results in formation of irreversible enamel defects. Monofluorophosphate (MFP) was considered as less toxic than NaF but equally cariostatic. We compared the potency of MFP and NaF to induce pre-eruptive sub-ameloblastic cysts and post-eruptive white spots and pits in developing hamster enamel. Hamster pups were injected subcutaneously with either NaF or MFP in equimolar doses of either 9 mg or 18 mg F/kg body weight. At 9 mg F/kg, MFP induced more but smaller sub-ameloblastic cysts with a collective cyst volume twice as large as that induced by NaF. Eight days after F injection, all F-injected groups had formed 4-6 white spots per molar, with an additional 2 pits per molar in the low MFP group. Twenty-eight days after injection, most white spots had turned into pits (5-6 per molar) and only the high MFP group still contained 2 white spots per molar. We conclude that parenterally applied MFP is more potent in inducing enamel defects than NaF. Most white spots formed turn into pits by functional use of the dentition. The higher potency of parenteral MFP may be associated with sustained elevated F levels in the enamel organ by enzymatic hydrolysis of MFP by alkaline phosphatase activity.


Asunto(s)
Esmalte Dental/efectos de los fármacos , Fluoruros/administración & dosificación , Fluorosis Dental/etiología , Fosfatos/administración & dosificación , Fluoruro de Sodio/administración & dosificación , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Cricetinae , Esmalte Dental/enzimología , Esmalte Dental/patología , Fluoruros/farmacología , Fluorosis Dental/patología , Infusiones Parenterales , Fosfatos/farmacología , Fluoruro de Sodio/farmacología
18.
Cells Tissues Organs ; 198(2): 99-110, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24192998

RESUMEN

The development of cell-based therapeutic strategies to bioengineer tooth tissue is a promising approach for the treatment of lost or damaged tooth tissue. The lack of a readily available cell source for human dental epithelial cells (ECs) severely constrains the progress of tooth bioengineering. Previous studies in model organisms have demonstrated that developing dental mesenchyme can instruct nondental epithelium to differentiate into enamel-forming epithelium. In this study, we characterized the ability of fetal and adult human dental mesenchyme to promote differentiation of human embryonic stem cell (hESC)-derived ECs (ES-ECs) into ameloblast-lineage cells. ES-ECs were co-cultured either with human fetal dental mesenchymal cells (FDMCs) or with adult dental mesenchymal cells (ADMCs) in either a three-dimensional culture system, or in the renal capsules of SCID mice. When co-cultured with FDMCs in vitro, ES-ECs polarized and expressed amelogenin. Tooth organ-like structures assembled with epithelium and encased mesenchyme and developing enamel-like structures could be detected in the complexes resulting from in vitro and ex vivo co-culture of ES-ECs and FDMCs. In contrast, co-cultured ES-ECs and ADMCs formed amorphous spherical structures and occasionally formed hair. Transcription factors were significantly upregulated in FDMCs compared to ADMCs including MSX1, GLI1, LHX6, LHX8,LEF1 and TBX1. In summary, FDMCs but not ADMCs had the capacity to induce differentiation of ES-ECs into ameloblast lineage cells. Further characterization of the functional differences between these two types of dental mesenchyme could enable reprogramming of ADMCs to enhance their odontogenic inductive competence.


Asunto(s)
Diferenciación Celular , Mesodermo/embriología , Diente/embriología , Adulto , Ameloblastos/citología , Ameloblastos/metabolismo , Amelogenina/metabolismo , Animales , Linaje de la Célula , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Epiteliales/citología , Epitelio/embriología , Feto/citología , Humanos , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Masculino , Mesodermo/citología , Ratones , Ratones SCID , Odontogénesis/genética , Fenotipo , Reacción en Cadena de la Polimerasa , Diente/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Regulación hacia Arriba/genética
19.
Matrix Biol ; 32(7-8): 432-42, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23747796

RESUMEN

Highly mineralized tooth enamel develops from an extracellular matrix chiefly comprised of amelogenins formed by splicing of 7 (human) or 9 (rodent) exons secreted from specialized epithelial cells known as ameloblasts. Here we examined the role of the 59 amino acid alternatively spliced amelogenin known as leucine rich amelogenin peptide (LRAP) on enamel formation, using transgenic murine models in which LRAP overexpression is driven by an amelogenin promoter (TgLRAP). Beginning in the secretory stage of mouse amelogenesis, we found a reduced thickness of enamel matrix and a loss of Tomes' processes, followed by upregulated amelogenin mRNA expression, inhibited amelogenin secretion and loss of cell polarity. In the presecretory stage (P0) amelogenin m180 mRNA expression was increased 58 fold along with a 203 fold increase in MMP-20 expression and 3.5 and 3.2 fold increased in respectively enamelin and ameloblastin. When LRAP was overexpressed on an amelogenin knockout mouse model, the ameloblasts were not affected. Further, expression of the global chromatin organizer and transcription factor SATB1 was reduced in secretory stage TgLRAP ameloblasts. These findings identify a cellular role for LRAP in enamel formation that is not directly related to directing enamel crystal formation as is reported to be the primary function of full length amelogenins. The effect of LRAP overexpression in upregulating amelogenins, MMP-20 and SATB1, suggests a role in protein regulation critical to ameloblast secretion and matrix processing, to form a mineralized enamel matrix.


Asunto(s)
Ameloblastos/fisiología , Amelogénesis/fisiología , Diferenciación Celular/efectos de los fármacos , Proteínas del Esmalte Dental/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ameloblastos/efectos de los fármacos , Animales , Diferenciación Celular/fisiología , Cartilla de ADN/genética , Regulación de la Expresión Génica/fisiología , Técnicas Histológicas , Inmunohistoquímica , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Metaloproteinasa 20 de la Matriz/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Microtomografía por Rayos X
20.
Int J Oral Sci ; 5(1): 1-6, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23538640

RESUMEN

This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.


Asunto(s)
Ameloblastos/fisiología , Células Madre Embrionarias/fisiología , Amelogénesis/genética , Amelogenina/análisis , Proteína Morfogenética Ósea 4/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula , Células Madre Embrionarias/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Factor 8 de Crecimiento de Fibroblastos/análisis , Proteínas Hedgehog/análisis , Proteínas de Homeodominio/análisis , Humanos , Queratinas/análisis , Queratinas/clasificación , Cloruro de Litio/farmacología , Factor de Transcripción MSX1/análisis , Mucosa Bucal/citología , Fenotipo , Regeneración/fisiología , Piel/citología , Factores de Transcripción/análisis , Tretinoina/farmacología , Proteína del Homeodomínio PITX2
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