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Patogenesis de la fluorosis dental: mecanismos bioquimicos y celulares.

PATHOGENESIS OF DENTAL FLUOROSIS: BIOCHEMICAL AND CELLULAR MECHANISMS

INTRODUCCION

El uso de fluoruros ha demostrado tener un efecto positivo sobre la prevencion de caries dental y se ha catalogado como una de las medidas de salud publica mas relevantes del siglo XX. (1,2) Su administracion (que puede ser topica o sistemica) tiene el objetivo ultimo de mantener una concentracion constante de iones fluoruro (F-) en la cavidad oral para favorecer la incorporacion de estos a los cristales de la superficie del esmalte erupcionado--disminuyendo la tasa de desmoralizacion y aumentando la tasa de remineralizacion--. (3) Sin embargo, actualmente se conoce que la ingestion excesiva de F- tiene efectos deletereos sobre el esmalte en desarrollo, generando un fenotipo hipomineralizado, poroso y de menor dureza. (4) Ademas de las consecuencias esteticas y funcionales, un estudio in vitro realizado en la Unidad de Investigacion en Caries (UNICA), sobre dientes extraidos con fluorosis moderada provenientes de pacientes colombianos, sugiere que la porosidad del esmalte con fluorosis lo hace mas susceptible a la desmoralizacion. (5)

A diferencia del factor etiologico de la fluorosis dental --que esta plenamente identificado como la exposicion cronica a altas concentraciones de F- entre los 0 y 5 anos--, (6) se conoce poco sobre los mecanismos celulares y moleculares afectados por el F- que llevan al desarrollo de fluorosis. En el caso de Colombia, contamos con reportes epidemiologicos (7) y de identificacion de las fuentes de ingesta de F- posiblemente responsables de la alta prevalencia, (8, 9) pero no se ha investigado sobre la patogenesis del defecto.

Sabemos que la presencia cronica y sostenida del ion F- en el plasma aumenta la probabilidad de su incorporacion a los tejidos en proceso de moralizacion, (10) pero existe la percepcion erronea de que la hipomineralizacion observada en la fluorosis dental es consecuencia unica de la incorporacion excesiva de F- en el esmalte. En esta revision de la literatura, comprenderemos que el fenotipo hipomineralizado de la fluorosis es el desenlace de una serie de efectos que el ion F- puede tener sobre la fisiologia celular y las proteinas responsables de guiar la mineraNzacion del esmalte (biomineralizacion). Primero, realizaremos una breve introduccion al proceso normal de biomineralizacion del esmalte, y posteriormente estudiaremos la evidencia disponible sobre los efectos del F- en momentos claves del proceso.

Amelogenesis y biomineralizacion del esmalte

El esmalte es una bioceramica nanocompuesta, (11) con un 95% de material inorganico, 4% de agua y 1% de materia organica. (12, 13) Para la formacion del esmalte son necesarios cuatro elementos: celulas, iones, proteinas y un compartimento en el que se lleva a cabo la reaccion de mineralizacion (matriz extracelular). (12) El proceso de formacion del esmalte se denomina en conjunto amelogenesis, mientras que el proceso de mineralizacion como tal, que se da entre iones y proteinas secretadas por las celulas, se denomina biomineralizacion. Las celulas formadoras de esmalte se denominan ameloblastos, y durante el proceso atraviesan una serie de cambios resumidos en las siguientes etapas: diferenciacion, secrecion, maduracion y transicion. (14) En la etapa de secrecion de los ameloblastos, estos transportan iones desde el plasma hacia el interior de la celula: el tipo de ion que ingrese dependera, entre otros factores, de su disponibilidad en ese momento en el plasma. (15) Es por este motivo que el mineral del esmalte (que es semejante al mineral puro hidroxiapatita) contiene usualmente una variedad de iones en su estructura, como HP[O.sup.2-.sub.4], C[O.sup.2-.sub.3], [Na.sup.+] y F-. (16)

Ademas de transportar y secretar iones, los ameloblastos sintetizan y secretan una gran cantidad de proteinas, que son el componente mayoritario del esmalte en formacion (> 90%). (17) Dentro de las proteinas secretadas estan, en primer lugar, las de la matriz extracelular (amelogenina, ameloblastina y enamelina) y en segundo lugar las proteasas: metaloproteinasa de matriz 20 (MMP-20) y calicreina 4 (KLK-4). La enamelina y la ameloblastina funcionan como nucleadores, atrayendo iones a su estructura proteica para favorecer el deposito organizado de las sales de fosfato de calcio en forma de cristales. (18)

Por otro lado, la amelogenina asume una organizacion supramolecular superior (nanoesferas de 20 nm) y funciona como un "andamio" que direcciona el crecimiento de los cristales para la formacion de prismas. (19) El papel de la ameloblastina es menos claro, pero se ha encontrado que tiene funciones en la adhesion y el control de la diferenciacion de los ameloblastos. (20) La MMP-20 degrada paulatinamente y de forma selectiva el soporte proteico durante las etapas de secrecion y maduracion, para permitir el ensanchamiento de los cristales de esmalte que previamente crecieron en longitud. (21) En el inicio de la etapa de maduracion, las celulas dejan de producir MMP-20 y comienzan a producir KLK-4, la proteasa que finaliza el proceso de degradacion del material proteico del esmalte. (22) La KLK-4 corta los restos de las proteinas estructurales en peptidos pequenos que pueden procesarse en la celula. (23) Al final de este proceso ordenado de la etapa de maduracion, el componente proteico sera menor al 1% (11) y el esmalte resultante tendra una porosidad minima y una apariencia clinica traslucida, brillante y lisa al tacto.

De la mano de la evidencia sobre los mecanismos normales de formacion del esmalte (resumida anteriormente), se han desarrollado estudios de induccion de fluorosis para comprender que etapas del proceso se ven afectadas por el fluoruro, como se describira a continuacion.

?Que concentraciones de fluoruro se utilizan en experimentos in vivo e in vitro para la induccion de fluorosis dental?

A pesar de la existencia de modelos estandarizados (descritos mas adelante), no existe consenso en cuanto a la concentracion de F- biologicamente relevante para la induccion de fluorosis dental tanto en estudios in vivo como in vitro. Por un lado, una linea de evidencia utiliza en sus estudios concentraciones de F- del orden micromolar ([micron]M) y considera biologicamente relevantes las concentraciones de 2-12 [micron]mol/L. (24-27) Otra linea de evidencia argumenta que, en condiciones normales, existen niveles basales de F- en el fluido del esmalte que se elevan proporcionalmente al aumento de la concentracion de F- en plasma y exponen a los ameloblastos a concentraciones milimolares (mM) de F-.28 Las dificultades en el consenso y la relacion directamente proporcional entre la dosis de F- y las respuestas celulares hacen necesario que los efectos reportados para el F- se examinen siempre acompanados de la concentracion utilizada en los experimentos.

Modelos in vivo e in vitro estandarizados para el estudio de la fluorosis dental

Se han establecido dos modelos in vitro: el primero consiste en una linea de celulas inmortalizadas, similares a ameloblastos, derivadas del organo del esmalte del primer molar de ratones Swiss-Webster recien nacidos. Esta linea se obtuvo con la transfeccion de celulas del epitelio del organo del esmalte con el oncogen del virus SV40 y se denomina LS8. (29) El segundo modelo consiste en cultivos primarios estandarizados de celulas del epitelio del organo del esmalte de fetos humanos de 21 semanas. (30) Este ultimo representa el modelo in vitro ideal para el estudio de fluorosis, por provenir de celulas humanas y expresar mayor numero de marcadores que la linea de raton; sin embargo, la obtencion de las muestras tiene implicaciones eticas y dificultades tecnicas que hacen que el modelo de celulas LS8 sea el mas conveniente y de mayor uso.

En cuanto a los modelos in vivo, se han empleado mamiferos como ratas, (31) ratones, (32) hamsters, (33) conejos (34) y especies mayores, como cerdos (35) y ovejas. (36) El modelo de rata ha demostrado ser el mas apropiado para el estudio de la fluorosis dental, (31) dado que los incisivos de los roedores erupcionan de forma continua, y en un solo diente se pueden apreciar las diferentes etapas del desarrollo del esmalte; ademas, existe evidencia de que en humanos y otros animales, los niveles de F- en plasma requeridos para la aparicion de defectos fluoroticos en esmalte son muy similares. (25, 31 37, 38)

A nivel molecular, la fluorosis dental es consecuencia del retraso en la remocion de las proteinas de la matriz extracelular, principalmente durante la fase de maduracion del esmalte.

La induccion de fluorosis en modelos celulares y animales ha permitido determinar en que medida el F- que circula en el plasma en concentraciones excesivas durante la amelogenesis tiene efectos deletereos sobre las diferentes etapas, incluyendo la etapa de secrecion. (39) En esta etapa se ha reportado que el fluoruro induce alteraciones en el transporte vesicular de los ameloblastos (40) y en la degradacion intracelular de proteinas de la matriz por parte del sistema lisosomal. (41, 42) Sin embargo, los estudios experimentales de fluorosis se han enfocado especificamente en la etapa de maduracion del esmalte (cuando suceden la secuencia ordenada de crecimiento cristalino, la digestion proteolitica por enzimas diferentes y la absorcion de los residuos proteicos), pues ha demostrado ser la mas sensible a los efectos negativos del F-. Esto se fundamenta en estudios in vivo realizados en ratas, en la cuales se demostro que el consumo de altas cantidades de F- retrasa la eliminacion de las proteinas (especialmente de amelogeninas). (24, 25) La capacidad disminuida para la eliminacion de amelogeninas desencadenada por el F- impide el engrosamiento de los cristales de esmalte y lleva a que la mineralizacion se de de forma incompleta. (43)

En la misma etapa de maduracion, en la que los ameloblastos regulan el pH con la secrecion de bicarbonato y el uso de transportadores ionicos para absorber protones de la matriz, (44) la gran cantidad de protones ([H.sup.+]) liberados como consecuencia de la alta tasa de precipitacion de cristales de esmalte genera fluctuaciones de pH (de neutro a acido), (45) y la presencia de altas concentraciones de F- en un ambiente acido tiene efectos deletereos, como presentaremos mas adelante en esta revision. Aunque el efecto del F- sobre la maduracion es critico, su efecto nocivo sobre las demas etapas tampoco es despreciable y podria ser acumulativo, si tenemos en cuenta que la severidad de la fluorosis responde a una exposicion sostenida y prolongada. (43)

Efectos del fluoruro sobre la fisiologia de los ameloblastos

Se ha registrado que a una concentracion de 10 [micron]M de F- ocurre una disminucion en la expresion de MMP-20, (46,47) a concentraciones de entre 10 y 20 [micron]M F- se da un aumento de la apoptosis, y a concentraciones mayores a 1 mM se presenta una alteracion de la proliferacion celular. (47) Ademas, concentraciones de 120 [micron]M de F- reducen la expresion de mensajeros de amelogenina, ameloblastina, enamelina y MMP-20, asi como factores de vascularizacion como el factor de crecimiento endotelial (VEGF), las proteinas quimio-atrayentes de monocitos (MCP-1) y la proteina inducible por interferon (IP-10). (48)

La regulacion del pH es fundamental para el crecimiento cristalino: la precipitacion de los iones durante la maduracion del esmalte libera gran cantidad de protones ([H.sup.+]), despues de lo cual sigue la reaccion [10[Ca.sup.2+] + 6 HP[O.sup.2-.sub.4] + 2[H.sub.2]O [Ca.sub.10] [(P[O.sub.4]).sub.6] [(OH).sub.2] + 8[H.sup.+]], y el pH de la matriz extracelular pasa de neutro a levemente acido. (45) Estos cambios de pH se ven reflejados en la alteracion de la morfologia de estas celulas, que se observan de extremos rugosos ante un pH acido y de extremos lisos ante un pH neutro. En el desarrollo normal del esmalte se presenta una alternancia de estas morfologias; sin embargo, en estudios in vitro se ha observado que la modulacion entre la morfologia lisa y rugosa se retarda, con predominio de la rugosa. (47) Por lo tanto, como consecuencia de la presencia de fluoruro, el pH de la matriz extracelular permanece acido por un tiempo prolongado.

De la exposicion de celulas LS8 a concentraciones de 250-2000 [micro]M de F- se han desprendido observaciones interesantes: el aumento en la concentracion de protones ([H.sup.+]), en presencia de una alta concentracion de iones F-, conduce a la formacion del acido fluorhidrico (HF), que es absorbido por las celulas y causa cambios severos en el metabolismo celular. (49) Estos hallazgos sugieren una hipotesis interesante: el exceso de F-citoplasmatico en el ameloblasto induce estres en el reticulo endoplasmico y la activacion de un mecanismo de defensa llamado "respuesta a proteinas mal plegadas" (UPR, unfolded protein response), que tiene como consecuencia la disminucion de la sintesis y secrecion de KLK-4, indispensable para la eliminacion de la matriz proteica de amelogenina y la maduracion final de los prismas del esmalte. (50-52)

Cuando el F- atraviesa la membrana citoplasmatica hacia el frente de mineralizacion, se registra otra cadena de efectos, demostrada por estudios in vitro recientes, que describen que la llegada masiva de F- al frente de mineralizacion genera una capa hipermineralizada de esmalte que se podria comportar como una barrera fisica que impediria la difusion de iones y proteinas a la subsuperficie del frente de mineralizacion. (53) Este evento molecular podria dificultar el ingreso de la "materia prima" necesaria para la mineralizacion completa de los cristales y, de esta manera, contribuir a la hipomineralizacion del esmalte fluorotico.

Efectos del fluoruro sobre la actividad de las proteasas de la matriz extracelular

Se conoce que el F- es un inhibidor de la actividad de las proteasas de la matriz extracelular del esmalte. Aunque el numero de estudios es limitado, los resultados no han podido demostrar una inhibicion directa de la actividad enzimatica, (54-56) por lo que hasta ahora se descarta al F-como inhibidor de proteasas dentro de la patogenesis de la fluorosis.

Efectos del fluoruro sobre la cinetica de la biomineralizacion

La incorporacion de F- a los cristales de esmalte durante su formacion aumenta la fuerza de union de la amelogenina al cristal (57) y disminuye su tasa de hidrolisis. (58) Esta evidencia se ha recogido en ensayos con amelogenina recombinante unida a hidroxiapatita sintetica con concentraciones de F- similares a las encontradas en dientes humanos con fluorosis. Es posible que la union de las proteinas a cristales con alto contenido de F-desencadene cambios en su conformacion, "ocultando" algunos sitios de corte y disminuyendo el acceso de las proteasas, (57) disminuyendo la velocidad de remocion de las proteinas de la matriz e impidiendo el engrasamiento y la maduracion del cristal. Estos estudios proporcionan evidencia suficiente para sugerir que la fluorosis dental es consecuencia del retraso en la remocion de proteinas durante la etapa de maduracion del esmalte. Ademas, seria posible que las proteinas se retengan en el esmalte erupcionado.

Siguiendo la logica anterior, se han realizado estudios sobre la retencion de amelogenina en el esmalte erupcionado con fluorosis. (58, 59) El bajo contenido proteico del esmalte erupcionado (<1% en el esmalte sano), y las dificultades para la extraccion de proteinas atrapadas en la matriz mineral, han limitado los estudios orientados a extraer, identificar y cuantificar el material proteico del esmalte.

Con el fin de ampliar el estudio de la fluorosis dental en Colombia, en la Unidad de Investigacion en Caries (UNICA) estandarizamos un metodo para la extraccion e identificacion de proteinas del esmalte a traves de cromatografia liquida acoplada a espectrometria de masas, el cual se aplico a una muestra de dientes de pacientes colombianos. (60) Usando este metodo, comparamos las proteinas identificadas en el esmalte erupcionado de dientes sanos y con fluorosis. Con nuestro analisis encontramos amelogenina, ameloblastina y enamelina; esta ultima con mayor frecuencia en el esmalte fluorotico, lo cual sugiere un posible papel de esta proteina en los eventos que desencadenan la fluorosis. Ademas, mediante la cuantificacion relativa de los peptidos identificados de amelogenina, no encontramos diferencias en el contenido proteico entre el esmalte sano y con fluorosis. (11) Los reportes existentes sobre este tema son contradictorios, (58, 59, 61) y a la fecha no podemos hablar de una retencion de proteinas en el esmalte fluorotico, sino de una alteracion en la velocidad de remocion de las mismas, que retrasa el proceso de maduracion del cristal de esmalte. (43)

En la figura 1 se resume la evidencia disponible sobre los mecanismos bioquimicos y celulares reportados a la fecha, posiblemente relacionados con la patogenesis de la fluorosis dental.

CONCLUSIONES Y PERSPECTIVAS

Los mecanismos celulares y moleculares mediante los cuales se produce la fluorosis dental no se han dilucidado por completo. Tampoco existe consenso sobre las concentraciones biologicamente relevantes de F- que generan fluorosis dental en seres humanos. En modelos in vitro e in vivo se ha observado que, cuando el F- se encuentra en altas concentraciones y de forma sostenida, tiene efectos nocivos sobre los ameloblastos. Estos efectos deletereos son proporcionales a las dosis de F- empleadas y tienen como consecuencia la disminucion de la capacidad del ameloblasto para la sintesis y la secrecion de proteinas, especialmente en la etapa de maduracion. La susceptibilidad de esta etapa en especial puede deberse a las fluctuaciones de pH que experimentan los ameloblastos debido a la alta concentracion de protones liberados durante la precipitacion de cristales. Aunque se ha pensado en el F- como inhibidor directo de las proteasas MMP-20 y KLK-4 (como posible causa de la retencion de proteinas), la evidencia disponible a la fecha descarta esa hipotesis. Por ahora, se conoce que el F- afecta la cinetica de la biomineralizacion, disminuye la velocidad de la hidrolisis de las proteinas e interrumpe el proceso de eliminacion de la matriz proteica, desencadenando la mineralizacion incompleta de los cristales de esmalte y dando origen al esmalte poroso caracteristico de la fluorosis dental.

Se espera que futuras investigaciones, orientadas al estudio de la patogenesis de la fluorosis dental, aporten evidencia al estudio de las concentraciones de fluoruro biologicamente relevantes (por ejemplo, fluoruro en plasma de habitantes de zonas endemicas de fluorosis), para asi homologarlas a estudios in vivo e in vitro. Es importante, ademas, bajo dichas concentraciones, realizar estudios sobre otros efectos del F- sobre la fisiologia celular y la cinetica de la biomineralizacion in vitro que permitan dilucidar por completo los mecanismos que conllevan al defecto y replicar estudios que--ante la evidencia contradictoria--confirmen si efectivamente hay o no retencion de proteinas en el esmalte con fluorosis.

AGRADECIMIENTOS

A la doctora Margarita Usuga Vacca, por su revision critica del manuscrito.

CONFLICTO DE INTERESES

Los autores declaran no tener ningun conflicto de interes.

CORRESPONDENCIA

Gina Alejandra Castiblanco Rubio

Unidad de Investigacion en Caries--UNICA

Vicerectoria de Investigaciones

Universidad El Bosque

(+571) 648 9000 Ext 1279

gcastiblanco@unbosque.edu.co

Av. Cra. 9 # 131A-02 Casa XII

Bogota, Colombia

INTRODUCTION

The use of fluorides has proven to have a positive effect on the prevention of tooth decay and has been considered one of the most important public health measures of the 20th century. (1-2) Its administration (which can be topical or systemic) aims to maintain a constant concentration of the fluoride ion (F) in the oral cavity to facilitate the incorporation of these crystals on the surface of the erupted enamel--decreasing demineralization rate and increasing remineralization rate--. (3) However, currently it is known that the excessive ingestion of F- has deleterious effects on enamel development, generating a hypomineralized porous phenotype with reduced hardness. (4) In addition to the aesthetic and functional consequences, an in-vitro study conducted at Unidad de Investigacion en Caries (UNICA) in extracted teeth with moderate fluorosis from Colombian patients, suggests that the porosity of the enamel with fluorosis makes it more susceptible to demineralization. (5)

Unlike the etiologic factor of dental fluorosis--which is fully identified as the chronic exposure to high concentrations of F- between the ages of 0 and 5 years--, (6) little is known about the cellular and molecular mechanisms affected by F- and leading to the development of fluorosis. In Colombia, there are epidemiological reports (7) that identify the sources of intake of F- possibly responsible for the high prevalence, (8,9) but the pathogenesis of the defect has not been properly investigated.

We know that the chronic and sustained presence of the F- ion in plasma increases the likelihood of adhering to tissues in the mineralization process, (10) but there is the misperception that the hypomineralization observed in dental fluorosis is the only consequence of the excessive addition of F- in enamel. In this literature review, we assume that the hypomineralized phenotype of fluorosis is the outcome of a series of possible effects of the F-ion on cell physiology and the proteins responsible for guiding the mineralization of enamel (biomineralization). First, we will make a brief introduction to the normal process of enamel biomineralization, and then we will study the available evidence on the effects of F- during key moments of the process.

Amelogenesis and enamel biomineralization

Enamel is a nano-compound bioceramics (11) with 95% inorganic material, 4% water, and 1% organic matter. (12, 13) Four elements are required for the formation of enamel: cell, ions, proteins, and a compartment where the mineralization reaction takes place (extracellular matrix). (12) The entire process of enamel formation is called amelogenesis, while the mineralization process as such, which takes place between ions and proteins secreted by cells, is called biomineralization. The enamelforming cells are called ameloblasts, and during the process they go through a series of changes that are summarized in the following stages: differentiation, secretion, maturation, and transition. (14) During the phase of secretion of ameloblasts, they carry ions from the plasma into the interior of the cell: the type of ion that enters will depend, among other factors, on its availability in the plasma at the moment. (15) This is why the mineral of enamel (which is similar to the pure mineral hydroxyapatite) usually contains a variety of ions, such as HP[O.sup.2-.sub.4], C[O.sup.2-.sub.3], [Na.sup.+] y F-. (16)

In addition to transporting and secreting ions, ameloblasts synthesize and secrete a large amount of proteins, which are the major component of enamel in formation (> 90%). (17) Among the secreted proteins are first those of the extracellular matrix (amelogenin, ameloblastin, and enamelin) and secondly the proteases: metalloproteinase matrix 20 (MMP-20) and kallikrein 4 (KLK-4). The enamelin and the ameloblast in function as nucleators, attracting ions to their protein structure to favor the organized deposit of crystals of calcium phosphate salts. (18)

On the other hand, the amelogenin takes a higher supramolecular organization (20 nm nanospheres) and works as a "scaffold" that guides the growth of crystals for the formation of prisms. (19) The role of ameloblastin is less clear, but it has been found to have functions in the adhesion and control of ameloblasts differentiation. (20) The MMP-20 gradually and selectively degrades the protein support during the secretion and maturation stages, to allow the widening of the enamel crystals which previously grew in length. (21) At the start of the maturation stage, cells stop producing MMP-20 and begin to produce KLK-4, a protease that completes the process of degradation of enamel protein material. (22) The KLK-4 cuts the remains of structural proteins into small peptides that can be processed in the cell. (23) At the end of this orderly process of the maturation stage, the protein component will be less than 1% (11) and the resulting enamel will have minimum porosity and a translucent shiny look with a smooth feel.

Based on the evidence about the normal mechanisms of enamel formation (summarized above), several studies on the induction of fluorosis have been conducted to understand which steps of the process are affected by fluoride, as described below.

What concentrations of fluoride are used in in vivo and in vitro experiments for the induction of dental fluorosis?

Despite the existence of standardized models (described below), there is no consensus as to the concentration of F- biologically relevant to induce dental fluorosis in in vivo and in vitro studies. On the one hand, a line of evidence in the studies uses micromolar ([micro]M) concentrations of F- and considers that concentrations of 2-12 [micro]mol/L are biologically relevant. (24-27) Another line of evidence argues that, under normal conditions, there are basal levels of F- in the fluid of enamel which proportionally increase in the presence of concentrations of F- in plasma and expose the ameloblasts to milimolar concentrations (mM) of F-. (28) The difficulties for a consensus and the directly proportional relationship between the dose of F- and cellular responses make it necessary to examine the effects reported for Falong with the concentration used in the experiments.

In vivo and in vitro models standardized for the study of dental fluorosis

Two in vitro models have been established: the first one consists of a line of immortalized cells similar to ameloblasts, resulting from the enamel organ of the first molar in newborn Swiss-Webster mice. This line was achieved by transfection of cells in the epithelium of the enamel organ with the oncogene SV40 virus and has been named LS8. (29) The second model consists of standardized primary cultures of epithelial cells of the enamel organ of human fetuses of 21 weeks of age. (30) The latter model represents the ideal in vitro model for the study of fluorosis, as it comes from human cells and expresses more bookmarks than the mouse line; however, sampling has ethical implications and technical difficulties that make the LS8 cell model the more convenient and the most widely used.

As to in vivo models, some mammals have been used, including rats, (31) mice, (32) hamsters, (33) rabbits (34) and higher species, such as pigs (35) and sheep. (36) The rat model has proven to be the most appropriate for the study of dental fluorosis, (31) since the incisors of rodents erupt continuously, and a single tooth can show the different stages of enamel development; in addition, there is evidence that the levels of F-plasma required for the appearance of fluorotic defects in enamel are very similar in humans and other animals. (25, 31,37, 38)

At the molecular level, dental fluorosis is a consequence of the delay in the removal of proteins from the extracellular matrix, mainly during enamel maturation.

The induction of fluorosis in animal and cell models has enabled to determine the extent to which the F-circulating in plasma in excessive concentrations during amelogenesis has deleterious effects on the different stages, including the secretion stage. (39) It has been reported that at this stage fluoride induces alterations in the vesicular transport of ameloblasts (40) and in the intracellular degradation of proteins of the matrix by the lysosomal system. (41, 42) However, experimental studies on fluorosis have focused specifically on the stage of enamel maturation (which includes the orderly sequence of crystal growth, proteolytic digestion by different enzymes, and absorption of protein residues), as it has proven to be the most sensitive to the negative effects of F-. This is based on in vivo studies performed in rats, which have demonstrated that the consumption of high amounts of F- delays the elimination of proteins (especially amelogenins). (24,25) A reduced capacity of amelogenin elimination triggered by F- prevents the thickening of enamel crystals and leads to incomplete mineralization. (43)

In the same stage of maturation, in which the ameloblasts regulate pH by secreting bicarbonate and using ionic transporters to absorb protons from the matrix, (44) the large number of protons ([H.sup.+]) released as a result of the high rate of precipitation of enamel crystals produce fluctuations of pH (from neutral to acid), (45) and the presence of high concentrations of F- in an acidic environment has deleterious effects, as will be discussed later in this review. While the effect of F- on maturation is critical, its adverse effect on the other stages is not negligible and could be cumulative, considering that the severity of the fluorosis is linked to a sustained and prolonged exposure. (43)

Effects of fluoride on the physiology of ameloblasts

It has been reported that a concentration of 10 [micro]M of F- produces a decrease in the expression of MMP20, (46), 47 concentrations of 10 to 20 [micro]M of F- produce an increase in apoptosis, and concentrations above 1 mM produce alterations in cell proliferation. (47) In addition, concentrations of 120 [micro]M of F- reduce the expression of messengers of amelogenin, ameloblastin, enamelin, and MMP-20, as well as factors of vascularization, such as the endothelial growth factor (VEGF), monocyte chemoattractant proteins (MCP-1) and the interferon inducible protein (IP-10). (48)

The pH regulation is fundamental for crystal growth: the precipitation of ions during enamel maturation releases a large amount of protons ([H.sup.+]), followed by reaction [10[Ca.sup.2+] + 6 HP[O.sup.2-.sub.4] + 2[H.sub.2]O [Ca.sub.10] [(P[O.sub.4]).sub.6][(OH).sub.2] + 8[H.sup.+]], and the pH of the extracellular matrix goes from neutral to slightly acidic. (45) These pH changes are reflected in the alteration of the morphology of these cells, which show rough ends in the presence of an acid pH and smooth ends in the presence of a neutral pH. During the normal development of enamel there is an alternation of these morphologies; however, in vitro studies have shown that the modulation between smooth and rough morphology become slow, with predominance of the rough one. (47) therefore, as a result of the presence of fluoride, the pH of the extracellular matrix remains acid for a long time.

The exposure of LS8 cells to concentrations of 250-2000 [micro]M of F- has provided interesting observations: the increase in the concentration of protons ([H.sup.+]), in the presence of a high concentration of ions F-, leads to the formation of hydrofluoric acid (HF), which is absorbed by the cells and causes severe changes in cellular metabolism. (49) These findings suggest an interesting hypothesis: an excess in cytoplasmic F- in ameloblast induces stress in the endoplasmic reticulum and activates a defense called "unfolded protein response" (UPR), which decreases the synthesis and secretion of KLK-4, essential for the elimination of the protein matrix of amelogenin and the final maturation of enamel prisms. (50-52)

When F- passes through the cytoplasmic membrane toward the mineralization front, another chain of effects, demonstrated by recent in vitro studies describing the massive arrival of F- in the mineralization front, produces a hypermineralized layer of enamel which could act as a physical barrier that would prevent the diffusion of ions and proteins to the subsurface of the mineralization front. (53) This molecular event could hinder the entry of "raw material" necessary for full mineralization of crystals and thus contribute to the hypominealization of fluorotic enamel.

Effects of fluoride on the activity of proteases of the extracellular matrix

It is widely known that F- inhibits the activity of proteases of the extracellular matrix of enamel. Since the number of studies is limited, the results have failed to demonstrate a direct inhibition of the enzyme activity, (54-56) and therefore F- has been discarded as an inhibitor of proteases in the pathogenesis of fluorosis.

Effects of fluoride on the kinetics of biomineralization

The incorporation of F- in enamel crystals during their formation increases the binding strength of amelogenin to the crystal (57) and reduces its hydrolysis. (58) This evidence has been gathered in trials with recombinant amelogenin bound to synthetic hydroxyapatite with F- concentrations like those found in human teeth with fluorosis. The binding of proteins to crystals with high content of F- can possibly trigger changes in their conformation, thus "hiding" some cleavage sites and decreasing access to the proteases, (57) reducing the speed of removal of matrix proteins and preventing the thickening and maturation of the crystal. These studies provide sufficient evidence to suggest that dental fluorosis is a consequence of the delay in the removal of proteins during the stage of maturation of enamel. In addition, proteins may possibly be retained in erupted enamel.

Based on this logic, some studies have been conducted on the retention of amelogenin in erupted enamel with fluorosis. (58, 59) The low protein content of erupted enamel (< 1% in healthy enamel) and the difficulties to extract proteins trapped in the mineral matrix have limited the studies aimed at extracting, identifying, and quantifying the protein material of enamel.

In order to expand the study of dental fluorosis in Colombia, at the Unidad de Investigacion en Caries (UNICA) we standardized a method to extract and identify enamel proteins through liquid chromatography along with mass spectrometry. The method was applied to a sample of teeth from Colombian patients. (60) Using this method, we compared the proteins identified in erupted enamel of healthy teeth and teeth with fluorosis. Our analysis showed amelogenin, ameloblastin, and enamelin--the latter more frequently in fluorotic enamel, suggesting a possible role of this protein in the events that trigger fluorosis--. In addition, through the relative quantification of identified peptides of amelogenin, we found no differences in protein content between healthy enamel and enamel with fluorosis. (11) The available reports on this subject are contradictory, (58, 59, 61) and to date we cannot speak of retention of proteins in fluorotic enamel but of an alteration in speed for their removal, which slows down the maturation process of enamel crystal. (43)

Figure 1 summarizes the available evidence on the cellular and biochemical mechanisms reported to date, possibly related to the pathogenesis of dental fluorosis.

CONCLUSIONS AND EXPECTATIONS

The cellular and molecular mechanisms by which dental fluorosis occurs have not been fully explained. Nor is there consensus on the biologically relevant concentrations of F- that produce dental fluorosis in humans. In vitro and in vivo models have shown that high steady concentrations of F- have harmful effects on ameloblasts. These deleterious effects are proportional to the doses of F- used and decreases the capacity of ameloblasts to synthesize and secrete proteins, especially at the maturation stage. The susceptibility of this stage in particular may be due to pH fluctuations experienced by ameloblasts due to a high concentration of protons released during the precipitation of crystals. While F- has been thought to be a direct inhibitor of MMP-20 and KLK-4 proteases (as a possible cause of protein retention), the available evidence to date allows discarding this hypothesis. For now, it is widely known that F- affects the kinetics of biomineralization, slows down the hydrolysis of proteins, and interrupts the process of elimination of the protein matrix, triggering the incomplete mineralization of enamel crystals and producing porous enamel--which is typical of dental fluorosis.

Further studies on the pathogenesis of dental fluorosis are expected to provide evidence to the analysis of biologically relevant concentrations of fluoride (e.g., fluoride in plasma of inhabitants from fluorosis-endemic areas) and thus align them to in vivo and in vitro studies. It is also important, under such concentrations, to carry out studies on other effects of F- on the cellular physiology and the kinetics of biomineralization in vitro to fully elucidate the mechanisms that lead to this defect and to replicate studies that--in the presence of contradictory evidence--confirm whether the enamel with fluorosis shows retention of proteins.

ACKNOWLEDGEMENTS

To Dr. Margarita Usuga Vacca for her critical review of the manuscript.

CONFLICT OF INTEREST

The authors declare not having conflicts of interest.

GINA ALEJANDRA CASTIBLANCO RUBIO [2], STEFANIA MARTIGNON [3], JAIME EDUARDO CASTELLANOS PARRA [4], WILSON ALFONSO MEJIA NARANJO [5]

[1] This project was financed with resources from the Vice Presidency of Research of Universidad El Bosque (PCI 2010-93, PCI-2011-242) and COLCIENCIAS (442-2012).

[2] DMD, Universidad Nacional de Colombia, Bogota, Colombia. Master's Degree in Basic Biomedical Sciences, Universidad El Bosque. Associate Professor. UNICA--Unidad de Investigacion en Caries. Universidad El Bosque.

[3] DMD, Pontificia Universidad Javeriana. Pediatric Dentist, Universidad El Bosque. PhD in Health Sciences, University of Copenhagen. Professor, UNICA--Unidad de Investigacion en Caries.

[4] DMD, Universidad Nacional de Colombia. Master's Degree in Pharmacology and PhD in Sciences-Chemistry, Universidad Nacional de Colombia, Bogota, Colombia. Professor, School of Dentistry, Universidad Nacional de Colombia.

[5] Microbiologist, Universidad de los Andes. Master's Degree in Biochemistry and PhD in Sciences-Chemistry, Universidad Nacional de Colombia. Associate Professor. UNICA--Unidad de Investigacion en Caries. Universidad El Bosque, Bogota, Colombia.

Caption: Figure 1. Possible biochemical and cellular mechanisms involved in the pathogenesis of dental fluorosis, both at the intracellular level and the extracellular matrix

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GINA ALEJANDRA CASTIBLANCO RUBIO [2], STEFANIA MARTIGNON [3], JAIME EDUARDO CASTELLANOS PARRA [4], WILSON ALFONSO MEJIA NARANJO [5]

http://dx.doi.org/ 10.17533/udea.rfo.v28n2a10

[1] Este trabajo fue financiado con recursos de la Vicerrectoria de Investigaciones de la Universidad El Bosque (PCI 2010-93, PCI-2011242) y COLCIENCIAS (442-2012).

[2] Odontologa, Universidad Nacional de Colombia, Bogota, Colombia. Magister en Ciencias Basicas Biomedicas, Universidad El Bosque. Instructora Asociada. UNICA--Unidad de Investigacion en Caries. Universidad El Bosque.

[3] Odontologa, Pontificia Universidad Javeriana. Odontologa Pediatra, Universidad El Bosque. PhD en Ciencias de la Salud, Universidad de Copenhague. Profesora Titular, UNICA--Unidad de Investigacion en Caries.

[4] Odontologo, Universidad Nacional de Colombia. Magister en Farmacologia y PhD en Ciencias-Quimica, Universidad Nacional de Colombia, Bogota, Colombia. Profesor Titular Catedratico, Facultad de Odontologia Universidad Nacional de Colombia.

[5] Microbiologo, Universidad de los Andes. Magister en Bioquimica y PhD en Ciencias-Quimica, Universidad Nacional de Colombia. Profesor Asociado. UNICA--Unidad de Investigacion en Caries. Universidad El Bosque, Bogota, Colombia.

RECIBIDO: JUNIO 9/2016--ACEPTADO: AGOSTO 16/2016

SUBMITTED: JUNE 9/2016--ACCEPTED: AUGUST 16/2016

Caption: Figura 1. Posibles mecanismos bioquimicos y celulares involucrados en la patogenesis de la fluorosis dental, tanto a nivel intracelular como de la matriz extracelular
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Author:Castiblanco Rubio, Gina Alejandra; Martignon, Stefania; Castellanos Parra, Jaime Eduardo; Mejia Nara
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