RESUMEN
Hypomineralization of developing enamel is associated with changes in ameloblast modulation during the maturation stage. Modulation (or pH cycling) involves the cyclic transformation of ruffle-ended (RE) ameloblasts facing slightly acidic enamel into smooth-ended (SE) ameloblasts near pH-neutral enamel. The mechanism of ameloblast modulation is not clear. Failure of ameloblasts of Cftr-null and anion exchanger 2 ( Ae2)-null mice to transport Cl- into enamel acidifies enamel, prevents modulation, and reduces mineralization. It suggests that pH regulation is critical for modulation and for completion of enamel mineralization. This report presents a review of the major types of transmembrane molecules that ameloblasts express to transport calcium to form crystals and bicarbonates to regulate pH. The type of transporter depends on the developmental stage. Modulation is proposed to be driven by the pH of enamel fluid and the compositional and/or physicochemical changes that result from increased acidity, which may turn RE ameloblasts into SE mode. Amelogenins delay outgrowth of crystals and keep the intercrystalline space open for diffusion of mineral ions into complete depth of enamel. Modulation enables stepwise removal of amelogenins from the crystal surface, their degradation, and removal from the enamel. Removal of matrix allows slow expansion of crystals. Modulation also reduces the stress that ameloblasts experience when exposed to high acid levels generated by mineral formation or by increased intracellular Ca2+. By cyclically interrupting Ca2+ transport by RE ameloblasts and their transformation into SE ameloblasts, proton production ceases shortly and enables the ameloblasts to recover. Modulation also improves enamel crystal quality by selectively dissolving immature Ca2+-poor crystals, removing impurities as Mg2+ and carbonates, and recrystallizing into more acid-resistant crystals.
Asunto(s)
Ameloblastos/fisiología , Amelogénesis/fisiología , Transporte Iónico/fisiología , Ameloblastos/metabolismo , Animales , Antiportadores de Cloruro-Bicarbonato/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Esmalte Dental/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , RatonesRESUMEN
Exposure of forming enamel to fluoride results into formation of hypomineralized enamel. We tested whether enamel hypomineralization was caused by lower expression of the NCKX4/SLC24A4 Ca2+-transporter by ameloblasts. Three commercial antibodies against NCKX4 were tested on enamel organs of wild-type and Nckx4-null mice, one of which (a mouse monoclonal) was specific. This antibody gave a prominent staining of the apical plasma membranes of maturation ameloblasts, starting at early maturation. The layer of immuno-positive ameloblasts contained narrow gaps without immunostaining or with reduced staining. In fluorotic mouse incisors, the quantity of NCKX4 protein in ameloblasts as assessed by western blotting was not different from that in non-fluorotic ameloblasts. However, immunostaining of the apical plasma membranes of fluorotic ameloblasts was strongly reduced or absent suggesting that trafficking of NCKX4 to the apical membrane was strongly reduced. Exposure to fluoride may reduce NCKX4-mediated transport of Ca2+ by maturation stage ameloblasts which delays ameloblast modulation and reduces enamel mineralization.
Asunto(s)
Ameloblastos/metabolismo , Antiportadores/metabolismo , Membrana Celular/metabolismo , Esmalte Dental/metabolismo , Amelogénesis/fisiología , Animales , Fluoruros/metabolismo , Ratones Endogámicos C57BL , Sodio en la Dieta/metabolismo , Calcificación de Dientes/fisiologíaRESUMEN
Formation and growth of hydroxyapatite crystals during amelogenesis generate a large number of protons that must be neutralized, presumably by HCO3 (-)ions transported from ameloblasts into the developing enamel matrix. Ameloblasts express a number of transporters and channels known to be involved in HCO3 (-)transport in other epithelia. However, to date, there is no functional evidence for HCO3 (-)transport in these cells. To address questions related to HCO3 (-)export from ameloblasts, we have developed a polarized 2-dimensional culture system for HAT-7 cells, a rat cell line of ameloblast origin. HAT-7 cells were seeded onto Transwell permeable filters. Transepithelial resistance was measured as a function of time, and the expression of transporters and tight junction proteins was investigated by conventional and quantitative reverse transcription polymerase chain reaction. Intracellular pH regulation and HCO3 (-)transport were assessed by microfluorometry. HAT-7 cells formed epithelial layers with measureable transepithelial resistance on Transwell permeable supports and expressed claudin-1, claudin-4, and claudin-8-key proteins for tight junction formation. Transport proteins previously described in maturation ameloblasts were also present in HAT-7 cells. Microfluorometry showed that the HAT-7 cells were polarized with a high apical membrane CO2 permeability and vigorous basolateral HCO3 (-)uptake, which was sensitive to Na(+)withdrawal, to the carbonic anhydrase inhibitor acetazolamide and to H2DIDS inhibition. Measurements of transepithelial HCO3 (-)transport showed a marked increase in response to Ca(2+)- and cAMP-mobilizing stimuli. Collectively, 2-dimensional HAT-7 cell cultures on permeable supports 1) form tight junctions, 2) express typical tight junction proteins and electrolyte transporters, 3) are functionally polarized, and 4) can accumulate HCO3 (-)ions from the basolateral side and secrete them at the apical membrane. These studies provide evidence for a regulated, vectorial, basolateral-to-apical bicarbonate transport in polarized HAT-7 cells. We therefore propose that the HAT-7 cell line is a useful functional model for studying electrolyte transport by ameloblasts.
Asunto(s)
Ameloblastos/metabolismo , Bicarbonatos/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/análogos & derivados , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/antagonistas & inhibidores , Acetazolamida/farmacología , Animales , Calcio/farmacología , Dióxido de Carbono/metabolismo , Inhibidores de Anhidrasa Carbónica/farmacología , Proteínas Portadoras/análisis , Técnicas de Cultivo de Célula , Línea Celular , Permeabilidad de la Membrana Celular/fisiología , Polaridad Celular/fisiología , Claudina-1/análisis , Claudina-4/análisis , Claudinas/análisis , AMP Cíclico/farmacología , Proteínas del Esmalte Dental/análisis , Impedancia Eléctrica , Fluorometría/métodos , Concentración de Iones de Hidrógeno , Calicreínas/análisis , Ratas , Sodio/farmacología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/fisiologíaRESUMEN
Ameloblasts express transmembrane proteins for transport of mineral ions and regulation of pH in the enamel space. Two major transporters recently identified in ameloblasts are the Na(+)K(+)-dependent calcium transporter NCKX4 and the Na(+)-dependent HPO4 (2-) (Pi) cotransporter NaPi-2b. To regulate pH, ameloblasts express anion exchanger 2 (Ae2a,b), chloride channel Cftr, and amelogenins that can bind protons. Exposure to fluoride or null mutation of Cftr, Ae2a,b, or Amelx each results in formation of hypomineralized enamel. We hypothesized that enamel hypomineralization associated with disturbed pH regulation results from reduced ion transport by NCKX4 and NaPi-2b. This was tested by correlation analyses among the levels of Ca, Pi, Cl, Na, and K in forming enamel of mice with null mutation of Cftr, Ae2a,b, and Amelx, according to quantitative x-ray electron probe microanalysis. Immunohistochemistry, polymerase chain reaction analysis, and Western blotting confirmed the presence of apical NaPi-2b and Nckx4 in maturation-stage ameloblasts. In wild-type mice, K levels in enamel were negatively correlated with Ca and Cl but less negatively or even positively in fluorotic enamel. Na did not correlate with P or Ca in enamel of wild-type mice but showed strong positive correlation in fluorotic and nonfluorotic Ae2a,b- and Cftr-null enamel. In hypomineralizing enamel of all models tested, 1) Cl(-) was strongly reduced; 2) K(+) and Na(+) accumulated (Na(+) not in Amelx-null enamel); and 3) modulation was delayed or blocked. These results suggest that a Na(+)K(+)-dependent calcium transporter (likely NCKX4) and a Na(+)-dependent Pi transporter (potentially NaPi-2b) located in ruffle-ended ameloblasts operate in a coordinated way with the pH-regulating machinery to transport Ca(2+), Pi, and bicarbonate into maturation-stage enamel. Acidification and/or associated physicochemical/electrochemical changes in ion levels in enamel fluid near the apical ameloblast membrane may reduce the transport activity of mineral transporters, which results in hypomineralization.
Asunto(s)
Ameloblastos/fisiología , Amelogénesis/fisiología , Ameloblastos/metabolismo , Animales , Antiportadores/fisiología , Western Blotting , Calcificación Fisiológica/fisiología , Antiportadores de Cloruro-Bicarbonato/fisiología , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Esmalte Dental/crecimiento & desarrollo , Microanálisis por Sonda Electrónica , Ratones , Potasio/metabolismo , Sodio/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/fisiologíaRESUMEN
Formation of apatite crystals during enamel development generates protons. To sustain mineral accretion, maturation ameloblasts need to buffer these protons. The presence of cytosolic carbonic anhydrases, the basolateral Na(+) bicarbonate cotransporter Nbce1, and the basolateral anion exchanger Ae2a,b in maturation ameloblasts suggests that these cells secrete bicarbonates into the forming enamel, but it is unknown by which mechanism. Solute carrier (Slc) family 26A encodes different anion exchangers that exchange Cl(-)/HCO3 (-), including Slc26a3/Dra, Slc26a6/Pat-1, and Slc26a4/pendrin. Previously, we showed that pendrin is expressed in ameloblasts but is not critical for enamel formation. In this study, we tested the hypothesis that maturation ameloblasts express Dra and Slc26a6 to secrete bicarbonate into the enamel space in exchange for Cl(-). Real-time polymerase chain reaction detected mRNA transcripts for Dra and Slc26a6 in mouse incisor enamel organs, and Western blotting confirmed their translation into protein. Both isoforms were immunolocalized in ameloblasts, principally at maturation stage. Mice with null mutation of either Dra or Slc26a6 had a normal dental or skeletal phenotype without changes in mineral density, as measured by micro-computed tomography. In enamel organs of Slc26a6-null mice, Dra and pendrin protein levels were both elevated by 52% and 55%, respectively. The amount of Slc26a6 protein was unchanged in enamel organs of Ae2a,b- and Cftr-null mice but reduced in Dra-null mice by 36%. Our data show that ameloblasts express Dra, pendrin, or Slc26a6 but each of these separately is not critical for formation of dental enamel. The data suggest that in ameloblasts, Slc26a isoforms can functionally compensate for one another.
Asunto(s)
Ameloblastos/fisiología , Antiportadores/fisiología , Ameloblastos/metabolismo , Animales , Proteínas de Transporte de Anión/metabolismo , Proteínas de Transporte de Anión/fisiología , Western Blotting , Esmalte Dental/crecimiento & desarrollo , Esmalte Dental/metabolismo , Esmalte Dental/fisiología , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Transportadores de Sulfato , Microtomografía por Rayos XRESUMEN
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/métodosRESUMEN
During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.
Asunto(s)
Órgano del Esmalte/citología , Órgano del Esmalte/embriología , Simportadores de Sodio-Bicarbonato/metabolismo , Ameloblastos/citología , Ameloblastos/metabolismo , Amelogénesis , Animales , Western Blotting , Calcificación Fisiológica/genética , Antiportadores de Cloruro-Bicarbonato/metabolismo , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Órgano del Esmalte/diagnóstico por imagen , Órgano del Esmalte/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Incisivo/metabolismo , Mandíbula/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Simportadores de Sodio-Bicarbonato/deficiencia , Simportadores de Sodio-Bicarbonato/genética , Regulación hacia Arriba/genética , Microtomografía por Rayos XRESUMEN
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.
Asunto(s)
Antiportadores de Cloruro-Bicarbonato/efectos de los fármacos , Fluoruros/efectos adversos , Fluorosis Dental/etiología , Ameloblastos/efectos de los fármacos , Ameloblastos/patología , Amelogénesis/efectos de los fármacos , Amelogénesis/genética , Animales , Bicarbonatos/análisis , Antiportadores de Cloruro-Bicarbonato/análisis , Antiportadores de Cloruro-Bicarbonato/genética , Cloruros/análisis , Colorantes , Esmalte Dental/química , Esmalte Dental/efectos de los fármacos , Esmalte Dental/patología , Proteínas del Esmalte Dental/análisis , Difusión , Femenino , Fluorosis Dental/genética , Fluorosis Dental/patología , Heterocigoto , Homocigoto , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Ratones , Ratones Noqueados , Minerales/análisis , Fenotipo , Ratas , Ratas Wistar , Calcificación de Dientes/efectos de los fármacos , Calcificación de Dientes/genéticaRESUMEN
OBJECTIVES: The objectives of this study were to establish a bisphosphonate-related osteonecrosis of the jaw (BRONJ) rat model and to analyse the effects of teriparatide (TP) on this model. METHODS: Sprague-Dawley rats were divided into three groups: I-zoledronic acid (ZA, n = 10); II-ZA and teriparatide (ZA + TP, n = 10); III-control (n = 10). Osteonecrosis was induced by administering zoledronic acid to groups ZA and ZA + TP. A week after the injections, rats underwent extraction of the first left mandibular molar. Following a four week period, TP was administered to the ZA + TP group for 28 days. Upon killing, extraction sockets were examined clinically, radiologically and histopathologically. RESULTS: Clinical examination revealed necrotic bone exposure in none of the animals. MicroCT (µCT) examination showed that bone mineral density of the newly formed bone in the extraction socket was lower in the ZA group than in the ZA + TP group (p < 0.05). Histopathological examination revealed that only the ZA and ZA + TP groups developed osteonecrosis, and the osteonecrotic bone area in the ZA group was larger than that in the ZA + TP group (p < 0.05). Tartrate-resistant acid phosphatase (TRAcP) enzyme histochemistry revealed that the number of detached and large osteoclasts were higher in the ZA group than in other groups, whereas the number of apoptotic osteoclasts in both ZA and ZA + TP groups were higher than in the control group (p < 0.05). CONCLUSIONS: Our data indicate that bisphosphonate-related osteonecrosis of the jaw model used in the present study is an attractive model to investigate treatment modalities and that TP might be an effective treatment in BRONJ.
Asunto(s)
Osteonecrosis de los Maxilares Asociada a Difosfonatos/prevención & control , Conservadores de la Densidad Ósea/uso terapéutico , Teriparatido/uso terapéutico , Fosfatasa Ácida/análisis , Proceso Alveolar/efectos de los fármacos , Proceso Alveolar/patología , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/análisis , Osteonecrosis de los Maxilares Asociada a Difosfonatos/patología , Densidad Ósea/efectos de los fármacos , Conservadores de la Densidad Ósea/administración & dosificación , Conservadores de la Densidad Ósea/efectos adversos , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Adhesión Celular/efectos de los fármacos , Recuento de Células , Difosfonatos/administración & dosificación , Difosfonatos/efectos adversos , Difosfonatos/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Imidazoles/administración & dosificación , Imidazoles/efectos adversos , Imidazoles/uso terapéutico , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Isoenzimas/análisis , Diente Molar/cirugía , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Osteogénesis/efectos de los fármacos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Fosfatasa Ácida Tartratorresistente , Teriparatido/administración & dosificación , Extracción Dental , Alveolo Dental/efectos de los fármacos , Alveolo Dental/patología , Microtomografía por Rayos X , Ácido ZoledrónicoRESUMEN
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 & dosificaciónRESUMEN
Enamel biomineralization results in a release of protons into the enamel matrix, causing an acidification of the local microenvironment. This acidification, which may be enhanced by more rapid mineral deposition in the presence of fluoride, must be neutralized by the overlying ameloblasts. The electrogenic sodium bicarbonate co-transporter NBCe1 has been localized in mouse ameloblasts, and has been proposed to have a role in matrix pH regulation. In this study, transcript analysis by PCR showed NBCe1-A present in human ameloblasts, whereas mouse ameloblasts expressed NBCe1-B. In situ hybridization and qPCR in mouse and fetal human incisors showed that NBCe1 mRNA was up-regulated as ameloblasts differentiated. Ingestion of 50 ppm fluoride resulted in an up-regulation of NBCe1 mRNA in maturation-stage mouse ameloblasts in vivo, as compared with controls. NBCe1 expression was up-regulated by low pH, but not by fluoride, in human ameloblast-lineage cells in vitro. The up-regulation of NBCe1 in vivo as enamel maturation and mineralization progressed provides evidence that NBCe1 participates in pH modulation during enamel formation. Up-regulation of NBCe1 in fluorosed maturation ameloblasts in vivo, with no effect of fluoride in vitro, supports the hypothesis that fluoride-enhanced mineral deposition results in acidification of the mineralizing enamel matrix.
Asunto(s)
Ameloblastos/metabolismo , Amelogénesis/genética , Fluoruros/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Simportadores de Sodio-Bicarbonato/genética , Amelogénesis/efectos de los fármacos , Animales , Células Cultivadas , Matriz Extracelular/metabolismo , Femenino , Fluoruros/farmacología , Humanos , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos , Simportadores de Sodio-Bicarbonato/metabolismo , Calcificación de Dientes , Regulación hacia ArribaRESUMEN
White opacities and pits are developmental defects in enamel caused by high intake of fluoride (F) during amelogenesis. We tested the hypothesis that these enamel pits develop at locations where F induces the formation of sub-ameloblastic cysts. We followed the fate of these cysts during molar development over time. Mandibles from hamster pups injected with 20mg NaF/kg at postnatal day 4 were excised from 1h after injection till shortly after tooth eruption, 8 days later. Tissues were histologically processed and cysts located and measured. Cysts were formed at early secretory stage and transitional stage of amelogenesis and detected as early 1h after injection. The number of cysts increased from 1 to almost 4 per molar during the first 16h post-injection. The size of the cysts was about the same, i.e., 0.46±0.29×10(6)µm(3) at 2h and 0.50±0.35×10(7)µm(3) at 16h post-injection. By detachment of the ameloblasts the forming enamel surface below the cyst was cell-free for the first 16h post-injection. With time new ameloblasts repopulated and covered the enamel surface in the cystic area. Three days after injection all cysts had disappeared and the integrity of the ameloblastic layer restored. After eruption, white opaque areas with intact enamel surface were found occlusally at similar anatomical locations as late secretory stage cysts were seen pre-eruptively. We conclude that at this moderate F dose, the opaque sub-surface defects with intact surface enamel (white spots) are the consequence of the fluoride-induced cystic lesions formed earlier under the late secretory-transitional stage ameloblasts.
Asunto(s)
Amelogénesis/efectos de los fármacos , Cariostáticos/efectos adversos , Hipoplasia del Esmalte Dental/etiología , Enfermedades Mandibulares/inducido químicamente , Quistes Odontogénicos/inducido químicamente , Fluoruro de Sodio/efectos adversos , Germen Dentario/efectos de los fármacos , Ameloblastos/efectos de los fármacos , Ameloblastos/patología , Animales , Animales Recién Nacidos , Cricetinae , Mandíbula , Enfermedades Mandibulares/complicaciones , Diente Molar , Quistes Odontogénicos/complicacionesRESUMEN
Intake of excess amounts of fluoride during tooth development cause enamel fluorosis, a developmental disturbance that makes enamel more porous. In mild fluorosis, there are white opaque striations across the enamel surface, whereas in more severe cases, the porous regions increase in size, with enamel pitting, and secondary discoloration of the enamel surface. The effects of fluoride on enamel formation suggest that fluoride affects the enamel-forming cells, the ameloblasts. Studies investigating the effects of fluoride on ameloblasts and the mechanisms of fluorosis are based on in vitro cultures as well as animal models. The use of these model systems requires a biologically relevant fluoride dose, and must be carefully interpreted in relation to human tooth formation. Based on these studies, we propose that fluoride can directly affect the ameloblasts, particularly at high fluoride levels, while at lower fluoride levels, the ameloblasts may respond to local effects of fluoride on the mineralizing matrix. A new working model is presented, focused on the assumption that fluoride increases the rate of mineral formation, resulting in a greater release of protons into the forming enamel matrix.
Asunto(s)
Ameloblastos/efectos de los fármacos , Cariostáticos/farmacología , Fluoruros/farmacología , Fluorosis Dental/etiología , Amelogénesis/efectos de los fármacos , Animales , Cariostáticos/efectos adversos , Cariostáticos/análisis , Células Cultivadas , Esmalte Dental/efectos de los fármacos , Modelos Animales de Enfermedad , Fluoruros/efectos adversos , Fluoruros/sangre , Fluorosis Dental/patología , Humanos , Modelos Biológicos , Odontogénesis/efectos de los fármacos , Calcificación de Dientes/efectos de los fármacosRESUMEN
Sclerostin is an inhibitor of bone formation expressed by osteocytes. We hypothesized that sclerostin is expressed by cells of the same origin and also embedded within mineralized matrices. In this study, we analyzed (a) sclerostin expression using immunohistochemistry, (b) whether the genomic defect in individuals with van Buchem disease (VBD) was associated with the absence of sclerostin expression, and (c) whether this was associated with hypercementosis. Sclerostin was expressed by cementocytes in mouse and human teeth and by mineralized hypertrophic chondrocytes in the human growth plate. In individuals with VBD, sclerostin expression was absent or strongly decreased in osteocytes and cementocytes. This was associated with increased bone formation, but no overt changes in cementum thickness. In conclusion, sclerostin is expressed by all 3 terminally differentiated cell types embedded within mineralized matrices: osteocytes, cementocytes, and hypertrophic chondrocytes.
Asunto(s)
Proteínas Morfogenéticas Óseas/biosíntesis , Proteínas Morfogenéticas Óseas/deficiencia , Osteocitos/metabolismo , Osteosclerosis/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Animales , Niño , Condrocitos/metabolismo , Cemento Dental/metabolismo , Femenino , Marcadores Genéticos , Placa de Crecimiento/metabolismo , Humanos , Anomalías Maxilomandibulares/etiología , Masculino , Maloclusión/etiología , Ratones , Persona de Mediana Edad , Osteosclerosis/complicaciones , Osteosclerosis/diagnóstico por imagen , Radiografía Panorámica , Anomalías Dentarias/etiología , Adulto JovenRESUMEN
Mechanosensitive osteocytes are essential for bone remodeling. Nitric oxide, an important regulator of bone remodeling, is produced by osteocytes through the activity of constitutive endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS). We hypothesized that these enzymes regulate the tissue response to orthodontic force, and therefore we investigated eNOS and iNOS expression in osteocytes during orthodontic force application. The upper rat molars were moved mesially by NiTi coil springs (10 cN, 120 hrs) in a split-mouth design. Immunohistochemical staining revealed that, in the tension area, eNOS-positive osteocytes increased from 24 hrs on, while iNOS-positive osteocytes remained largely constant. In the compression area, iNOS-positive osteocytes increased after 6 hrs, while eNOS- positive osteocytes increased after 24 hrs. This suggests that eNOS mediates bone formation in the tension area, while iNOS mediates inflammation-induced bone resorption in the compression area. Both eNOS and iNOS seem to be important regulators of bone remodeling during orthodontic force application.
Asunto(s)
Remodelación Ósea/fisiología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Osteocitos/enzimología , Técnicas de Movimiento Dental , Animales , Resorción Ósea/fisiopatología , Recuento de Células , Aleaciones Dentales , Inmunohistoquímica , Masculino , Diente Molar , Níquel , Óxido Nítrico Sintasa de Tipo II/análisis , Óxido Nítrico Sintasa de Tipo III/análisis , Métodos de Anclaje en Ortodoncia/instrumentación , Alambres para Ortodoncia , Osteogénesis/fisiología , Distribución Aleatoria , Ratas , Ratas Wistar , Estrés Mecánico , Factores de Tiempo , Titanio , Técnicas de Movimiento Dental/instrumentaciónRESUMEN
Whether low intensity pulsed ultrasound therapy stimulates osteogenesis in mandibular distraction was investigated in a double-blind trial. Nine patients underwent a vertical mandibular distraction over a distance of 5.1+/-1.2mm. Ultrasound or placebo therapy was started daily from the first day of distraction. After 46+/-8.1 days consolidation, two endosseous implants were inserted and a transmandibular biopsy was taken. Ultrasonographs were taken regularly to follow osteogenesis inside the gap. There were no complications during the 44+/-7.1 months of follow-up. Microradiographic measurements of the biopsies revealed no differences in the area of mineralized tissue in the distraction gap. The cranially distracted bone segment appeared significantly more radiolucent than the caudal bone. Histological examination showed large lacunae inside the cranially distracted bone segment, filled with clusters of osteoclasts and surrounded by clear tetracycline double labels. Within the distraction gap, woven bone was present, with no apparent differences between the treatment groups. Ultrasonographic follow-up revealed that osteogenesis inside the distraction gap progresses from 4 to 20 weeks post distraction, with no differences between the ultrasound and the placebo groups. In summary, ultrasound treatment does not appear to stimulate bone formation in the severely resorbed vertical distracted mandible.
Asunto(s)
Pérdida de Hueso Alveolar/rehabilitación , Regeneración Ósea/fisiología , Avance Mandibular/métodos , Osteogénesis por Distracción/métodos , Terapia por Ultrasonido/métodos , Anciano , Pérdida de Hueso Alveolar/cirugía , Callo Óseo/citología , Callo Óseo/fisiología , Implantación Dental Endoósea , Implantes Dentales , Método Doble Ciego , Humanos , Arcada Edéntula/rehabilitación , Estudios Longitudinales , Mandíbula/citología , Mandíbula/fisiología , Persona de Mediana EdadRESUMEN
One of the mechanisms by which epithelial cells regulate intracellular pH is exchanging bicarbonate for Cl(-). We tested the hypothesis that in ameloblasts the anion exchanger-2 (Ae2) is involved in pH regulation during maturation stage amelogenesis. Quantitative X-ray microprobe mineral content analysis, scanning electron microscopy, histology, micro-computed tomography and Ae2 immuno-localisation analyses were applied to Ae2-deficient and wild-type mouse mandibles. Immuno-localisation of Ae2 in wild-type mouse incisors showed a very strong expression of Ae2 in the basolateral membranes of the maturation stage ameloblasts. Strikingly, zones of contiguous ameloblasts were found within the maturation stage in which Ae2 expression was extremely low as opposed to neighbouring cells. Maturation stage ameloblasts of the Ae2(a,b)(-/-) mice failed to stain for Ae2 and showed progressive disorganisation as enamel development advanced. Maturation stage enamel of the Ae2(a,b)(-/-) mice contained substantially less mineral and more protein than wild-type enamel as determined by quantitative X-ray microanalysis. Incisor enamel was more severely affected than molar enamel. Scanning electron microscopy revealed that the rod-inter-rod structures of the Ae2(a,b)(-/-) mice incisor enamel were absent. Mineral content of dentine and bone of Ae2(a,b)(-/-) mice was not significantly different from wild-type mice. The enamel from knockout mouse teeth wore down much faster than that from wild-type litter mates. Basolateral bicarbonate secretion via the anionic exchanger Ae2 is essential for mineral growth in the maturation stage enamel. The observed zonal expression of Ae2 in the maturation stage ameloblasts is in line with a model for cyclic proton secretion during maturation stage amelogenesis.
Asunto(s)
Amelogénesis/fisiología , Proteínas de Transporte de Anión/fisiología , Antiportadores/fisiología , Esmalte Dental/crecimiento & desarrollo , Diente/crecimiento & desarrollo , Ameloblastos/metabolismo , Amelogénesis/genética , Animales , Proteínas de Transporte de Anión/genética , Antiportadores/genética , Huesos/química , Esmalte Dental/metabolismo , Esmalte Dental/ultraestructura , Dentina/química , Incisivo/metabolismo , Ratones , Ratones Endogámicos , Ratones Noqueados , Microscopía Electrónica de Rastreo , Minerales/análisis , Modelos Biológicos , Diente Molar/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Proteínas SLC4A , Diente/metabolismo , Calcificación de Dientes/genética , Calcificación de Dientes/fisiologíaRESUMEN
In the late seventies of the previous century the culture conditions to grow rodent tooth germs in vitro in order to investigate de novo formation and mineralization of dentine and enamel, were improved. This tooth model was then used for basic studies of mineralization processes in enamel and dentine. A review on experiments aimed at elucidating the function of osteocalcin, the predominant non-collagenous protein found in all mineralising type I connective tissues, in dentine formation is presented. Studies with tooth organ cultures and analysis of osteocalcin-deficient mouse dentin failed to disclose a function of osteocalcin in dentin formation.
Asunto(s)
Amelogénesis/fisiología , Esmalte Dental/metabolismo , Dentina/metabolismo , Dentinogénesis/fisiología , Osteocalcina/fisiología , Animales , Humanos , Minerales/metabolismo , Modelos Animales , Técnicas de Cultivo de Órganos , Osteocalcina/metabolismo , RoedoresRESUMEN
Bone tissue can adapt to orthodontic load. Mechanosensing in bone is primarily a task for the osteocytes, which translate the canalicular flow resulting from bone loading into osteoclast and osteoblast recruiting signals. Apoptotic osteocytes attract osteoclasts, and inhibition of osteocyte apoptosis can therefore affect bone remodeling. Since TNF-alpha is a pro-inflammatory cytokine with apoptotic potency, and elevated levels are found in the gingival sulcus during orthodontic tooth movement, we investigated if mechanical loading by pulsating fluid flow affects TNF-alpha-induced apoptosis in chicken osteocytes, osteoblasts, and periosteal fibroblasts. During fluid stasis, TNF-alpha increased apoptosis by more than two-fold in both osteocytes and osteoblasts, but not in periosteal fibroblasts. One-hour pulsating fluid flow (0.70 +/- 0.30 Pa, 5 Hz) inhibited (-25%) TNF-alpha-induced apoptosis in osteocytes, but not in osteoblasts or periosteal fibroblasts, suggesting a key regulatory role for osteocyte apoptosis in bone remodeling after the application of an orthodontic load.
Asunto(s)
Apoptosis/fisiología , Remodelación Ósea/fisiología , Osteoblastos/fisiología , Osteocitos/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Adaptación Fisiológica , Animales , Células Cultivadas , Embrión de Pollo , Regulación hacia Abajo , Líquido Extracelular/fisiología , Fibroblastos/fisiología , Periostio/citología , Periostio/fisiología , Flujo Pulsátil , Resistencia al Corte , Cráneo/citología , Estrés Mecánico , Técnicas de Movimiento Dental , Soporte de PesoRESUMEN
We tested the hypothesis that the sensitivity of forming dental enamel to fluoride (F-) is ameloblast developmental stage-dependent and that enamel mineralization disturbances at the surface of fluorotic enamel are caused by damage to late-secretory- and transitional-stage ameloblasts. Four-day-old hamsters received a single intraperitoneal dose of 2.5-20 mg NaF/kg body weight and were examined, 24 h later, by histology and histochemistry. A single dose of >or=5 mg of NaF/kg induced the formation of a hyper- followed by a hypomineralized band in the secretory enamel, without changing the ameloblast structure. At 10 mg of NaF/kg, cystic lesions became apparent under isolated populations of distorted late-secretory- and transitional-stage ameloblasts. Staining with von Kossa stain showed that the enamel under these lesions was hypermineralized. At 20 mg of NaF/kg, cystic lesions containing necrotic cells were also found in the early stages of secretory amelogenesis and were also accompanied with hypermineralization of the enamel surface. We concluded that the sensitivity to F- is ameloblast developmental stage-dependent. Groups of transitional ameloblasts are most sensitive, followed by those at early secretory stages. These data suggest that a F-induced increase in cell death in the transitional-stage ameloblasts accompanies the formation of cystic lesions, which may explain the formation of enamel pits seen clinically in erupted teeth.