Expression of enamel proteins in rough endoplasmic reticulum and golgi complex in human dental germs / Expresión de proteínas del esmalte en retículo endoplásmico rugoso y complexo golgiensis en gérmenes dentales humanos

Tooth enamel is the hardest tissue in the body. The organic matrix configuration is provided by the main proteins amelogenin, ameloblastin and enamelysin (MMP20), an enzyme that helps to shape the matrix. The aim of this study was to determine by histochemistry the expression of amelogenin and enamelysin through the rough endoplasmic reticulum in the late stages of amelogenesis, and its expression in the Complexus golgiensis (Golgi complex / Golgi apparatus) in the early stages in human fetuses. In early stages a colocalization of both proteins inside the Golgi apparatus was found, being more evident the relationship between Golgi and amelogenin (99.92 %). In the late stage, a colocalization of both proteins and rugged endoplasmic reticulum was found. With enamelysin being more evident in relation with rough endoplasmic reticulum (99.95 %). Our findings demonstrated the presence of amelogenin and enamelysin in odontoblast and ameloblast. However, the presence of these two proteins in odontoblast remains unknown.

El esmalte dental es el tejido más duro del cuerpo. La configuración de la matriz orgánica es proporcionada por las proteínas principales amelogenina, ameloblastina y enamelisina (MMP20), una enzima que ayuda a dar forma a la matriz. El objetivo de este estudio fue determinar mediante histoquímica la expresión de amelogenina y enamelisina a través del retículo endoplasmático rugoso en las últimas etapas de la amelogénesis , y su expresión en el Complexo golgiensis en las primeras etapas de formación en fetos humanos. En las primeras etapas se observó colocalización de ambas proteínas en el interior del Complexo golgiensis, siendo más evidente la relación entre Golgi y amelogenina (99,92 %). En la última etapa, se identificó una colocalización de ambas proteínas y retículo endoplásmico rugoso. Resulto más evidente la enamelisina en relación con el retículo endoplasmático rugoso (99,95 %). Nuestros resultados demostraron la presencia de amelogenina y enamelisina en odontoblastos y ameloblastos, sin embargo se desconoce la presencia de estas dos proteínas en odontoblastos.

Medium modification with bone morphogenetic protein 2 addition for odontogenic differentiation

Abstract The aim of this study was to evaluate whether medium modification improves the odontogenic differentiation of human dental pulp stem cells (DPSC) in vitro and in vivo. DPSC isolated from human impacted third molar teeth were analysed for clusters of differentiation with flow cytometry. Odontogenic differentiation was stimulated by medium modification with the addition of bone morphogenetic protein 2 (BMP2). The expression of dentin sialophosphoprotein, dentin matrix protein 1, enamelysin/matrix metalloproteinase 20 and the phosphate-regulating gene with homologies to endopeptidases on the X chromosome of the cells were analysed with RT-PCR at 7, 14 and 21 days. Then, DPSC were transplanted on the back of immunocompromised mice via a hydroxyapatite tricalcium phosphate scaffold, and the structure of the formed tissue was investigated. The cells were identified as mesenchymal stem cells with a 98.3% CD73 and CD90 double-positive cell rate. The increase in mineralization capacity and expression of human enamel-dentin specific transcripts proportional to the culture period were determined after differentiation. Six weeks after transplantation, an osteo-dentin matrix was formed in the group in which odontogenic differentiation was stimulated, and the odontogenic characteristics of the matrix were confirmed by histological examination and RT-PCR analysis. Odontogenic differentiation of the isolated and characterized human DPSC was improved with medium modification by the addition of BMP2 in vitro and in vivo. The defined medium and applied technique have a potential use for forming reparative dentin in the future, but the effects of the method should be investigated in long-term studies.

In vivo performance of different scaffolds for dental pulp stem cells induced for odontogenic differentiation

Abstract This study was designed to determine the in vivo performance of three different materials as scaffolds for dental pulp stem cells (DPSC) undergoing induced odontogenic differentiation. An odontogenic medium modified by the addition of recombinant human bone morphogenetic protein 2 was used in the experimental groups to induce differentiation. Mesenchymal stem cell medium was used in the control groups. DPSC were transplanted onto the backs of mice via three scaffolds: copolymer of L-lactide and DL-lactide (PLDL), copolymer of DL-lactide (PDL) and hydroxyapatite tricalcium phosphate (HA/TCP). The expression levels of dentin sialo-phosphoprotein (DSPP), dentin matrix protein-1 (DMP1), enamelysin/matrix metalloproteinase 20 (MMP20) and phosphate-regulating gene with homologies to endopeptidases on X chromosome (PHEX) were analysed using RT-PCR. The expressions in the experimental groups were compared to those in the control groups. The transcript expressions at 6 and 12 weeks were significantly different for all scaffolds (p < 0.05), except for the expression of DSPP in the PLDL group with regard to the time variable. Although there was a decrease in the expression of enamelysin/MMP20 in PLDL and HA/TCP at 12 weeks, all other expressions increased and reached their highest level at 12 weeks. The highest DSPP expression was in the PDL group (p < 0.05). The highest expression of DMP1 was detected in the HA/TCP group (p < 0.05). The highest expression of PHEX was in the PLDL group (p < 0.05). Consequently, PLDL and PDL seemed to be promising scaffold candidates for odontogenic regeneration at least as HA-TCP, when they were applied with the DPSC induced for odontogenic differentiation.

The role of enamelysin (MMP-20) in tooth development: systematic review / El papel de la enamelisina (MMP-20) en el desarrollo dentario: revisión sistemática

ABSTRACT. Introduction: ameloblasts are cells responsible for the production and mineralization of the organic matrix of enamel through several stages: pre-secretory, secretory, transition, and maturation. The organic matrix components are produced in the secretory phase. In the maturation phase, the organic component is removed and the mineralization process starts. This process requires the involvement of matrix metalloproteinase 20 (MMP-20), also called enamelysin. Several studies have shown the presence of MMP-20 in tooth development and its relationship to alterations in enamel formation. The objective was: to classify the different studies and laboratory techniques used to demonstrate the involvement of enamelysin in tooth development and its relation to pathologies during enamel formation. Methods: a systematic review was conducted with the following bibliographic databases: PubMed, Science-Direct, Hinari, and SciELO, in order to classify the different studies related to the involvement of MMP-20 in tooth development and the methods to detect its expression, between the years of 2009 and 2014. Results and conclusions: 11 in vitro models show that MMP-20 has specific cleavage sites for enamel matrix proteins. This process is altered by chemical composition, ions, and the presence of hydroxyapatite. Enamel morphology is altered in the knockout models. In human studies, MMP-20 has been associated with increased susceptibility to dental caries, enamel thickness, and dental agenesis.

RESUMEN. Introducción: el ameloblasto es la célula encargada de la producción y mineralización de la matriz orgánica del esmalte. Atraviesa varias etapas: la fase pre-secretora, secretora, de transición y maduración. En la fase secretora se producen los componentes de la matriz orgánica. En la fase de maduración se elimina el componente orgánico y se inicia el proceso de mineralización. Este proceso requiere de la participación de la metaloproteinasa de matriz 20 (MMP-20) o también llamada enamelisina. Diversos estudios demuestran la presencia de MMP-20 en el desarrollo dentario y su relación con alteraciones en la formación del esmalte. El objeto fue clasificar los diferentes estudios y técnicas de laboratorio empleadas que demuestren la participación de enamelisina en el desarrollo dentario y su relación con patologías en la formación del esmalte. Métodos: se realizó una revisión sistemática de la literatura con las siguientes bases bibliográficas: PubMed, Science-Direct, Hinari y SciELO, con el fin de clasificar los diferentes estudios relacionados con la participación de MMP-20 en el desarrollo dental y los métodos utilizados para detectar su expresión, entre los años de 2009 a 2014. Resultados y conclusiones: los modelos in vitro evidencian que MMP-20 tiene sitios específicos de escisión para las proteínas de matriz de esmalte. Este proceso se ve alterado por la composición química, iones, y la presencia de hidroxiapatita. En los modelos knockout la morfología del esmalte está alterada. En los estudios en humanos, se ha relacionado la MMP-20 con una mayor susceptibilidad de caries dental, el grosor completo de esmalte y agenesias dentales.

Expression of Mmp-20 in dental germs of human fetus / Expresion de Mmp-20 en gérmenes dentales de fetos humanos

During experiments in animal studies, it has been observed that enamelysin (MMP-20) is expressed during tooth development in the late secretory stage of amelogenesis but not in the mature enamel.The aim of this research was to determine the location of MMP-20 in human tooth germs in the different structures of the enamel organ.The detection of MMP-20 was performed by immunohistochemistry in 20 specimens obtained from human fetuses. Immunostaining of MMP-20 was observed from the presecretor stadium in stellate reticulum and intermediate stratum and in the basal portion of ameloblasts in the secretory stage in stellate reticulum, stratum intermedium, secretory ameloblasts, odontoblasts and dental papilla. The results of this research show the location of MMP-20 in tooth germ development in humans and provides the foundation for future research about the process of dental organ formation.

En estudios realizados en animales de experimentación se ha observado que la enamelisina (MMP-20) se expresa durante el desarrollo dental durante el estadio de secreción tardío de la amelogénesis pero no en el esmalte maduro. El objetivo de la presente investigación fue determinar la localización de MMP-20 en gérmenes dentarios humanos en las diferentes estructuras del órgano del esmalte. Se analizaron 20 especímenes obtenidos de fetos humanos, efectuando la detección de MMP-20 por Inmunohistoquímica. Se observó inmunolocalización de MMP-20 desde el estadio presecretor en retículo estrellado y estrato intermedio, así como en porción basal de ameloblastos; en el estadio secretor en retículo estrellado, estrato intermedio, ameloblastos secretores, odontoblastos y papila dental. Los resultados de la presente investigación muestran la localización de la MMP-20 en el desarrollo del germen dentario en humanos y aporta las bases para futuras investigaciones acerca del proceso de formación de los órganos dentales.

Efeito da amoxicilina e do fluoreto no desenvolvimento do esmalte dentário de ratos / Effect of amoxicillin and fluoride on enamel development in rat teeth

O uso da amoxicilina durante a primeira infância tem sido relacionado ao desenvolvimento de defeitos de esmalte denominados Hipomineralização Molar-Incisivo (HMI). Ademais, há relatos de possível potencialização dos efeitos do fluoreto no esmalte pela amoxicilina. Objetivo: A proposta do presente estudo foi avaliar o efeito da amoxicilina, e da associação amoxicilina com fluoreto no desenvolvimento do esmalte dentário de ratos. Metodologia: O experimento foi dividido em três capítulos. Capítulo 1 - Quinze ratas prenhas (Rattus norvegicus, albinus, Holtzman) foram aleatoriamente distribuídas em três grupos que receberam, a cada 24 h, dose intragástrica de amoxicilina 100 mg/kg/dia (grupo GA100), amoxicilina 500 mg/kg/dia (grupo GA500) ou soro fisiológico (grupo GS), a partir do 130 dia de prenhez. Após o nascimento, doze filhotes de cada grupo receberam o mesmo tratamento das respectivas mães durante os períodos de 7 dias (n=6) e 12 dias (n=6) de vida. Após a eutanásia, as cabeças dos ratos foram removidas, fixadas e processadas para inclusão em parafina. Os cortes frontais da cabeça exibindo os primeiros molares superiores foram em parte corados com H&E e em parte submetidos à reação imuno-histoquímica para detecção de amelogenina e metaloproteinases da matriz-20 (MMP-20). Os cortes corados com H&E foram utilizados para análise morfológica e mensuração da espessura da camada de esmalte. A imunoexpressão para amelogenina e MMP-20 foram avaliados por análise semiquantitativa (H-score). Os dados foram analisados estatisticamente pelo teste Tukey com nível de significância de 5%. Capítulo 2 - Quinze ratas prenhas (Rattus norvegicus, albinus, Holtzman) foram aleatoriamente distribuídas em três grupos, que receberam, amoxicilina 100 mg/kg/dia (grupo GA100), amoxicilina 500 mg/kg/dia (grupo GA500) ou soro fisiológico (grupo GS), conforme descrito no capítulo 1. Após o nascimento, dez filhotes por grupo receberam o mesmo tratamento do 10 ao 400 dia de vida. Aos 40 dias, os animais sofreram eutanásia, os incisivos e molares foram extraídos, e submetidos às análise do conteúdo de cálcio, pelo método de titulação com EDTA, de fósforo por método colorimétrico usando espectrofotômetro. E a densidade eletrônica do esmalte nos primeiros molares foi analisada pela tomografia micro-computadorizada (XRM). Capítulo 3 - Quarenta ratos foram distribuídos, aleatoriamente, em quatro grupos, expostos ao fluoreto na água (100 ppm (mg F/L) ou à administração intragástrica de amoxicilina (500 mg/Kg peso), segundo as combinações: grupo controle (GS); grupo amoxicilina (GA), grupo fluoreto (GF) e grupo amoxicilina com fluoreto (GA+F). Após 60 dias, amostras de plasma e uma tíbia foram coletados e analisados quanto a concentração de fluoreto por meio de eletrodo de íon seletivo. Os incisivos foram submetidos à quantificação do grau de fluorose (por um software de análise de imagens, Dental fluorosis by Image Analysis ­ DFIA), a análise de conteúdo de cálcio, pelo método de titulação com EDTA, de fósforo por método colorimétrico usando espectrofotômetro, e determinações da espessura do esmalte e de sua microdureza. Resultados: Capítulo 1 - Aos 7 dias, diversas estruturas vacuolares foram observadas na porção citoplasmática distal dos ameloblastos dos ratos dos grupos GA100 e GA500; além disso, a espessura do esmalte foi significantemente menor nos grupos GA100 e GA500 quando comparado ao GS (p<0,001). Em contraposição, diferenças estatisticamente significantes não foram observadas entre os grupos no período de 12 dias (p=0,111). Diferenças entre os grupos também não foram observadas na intensidade de imunomarcação exibida pelos ameloblastos à anti-amelogenina (p=0,818) e à anti-MMP-20 (p=0,855). Capítulo 2 - Não foram observadas diferenças estatisticamente significante no conteúdo de cálcio (p=0,180), fósforo (p=0,054) e densidade eletrônica (p=0,150) entre os grupos. Capítulo 3 - Observou-se que a espessura do esmalte (p=0,228) e a concentração de cálcio (p=0,592) e fósforo (p=0,409) nos incisivos não diferiram estatisticamente entre os grupos. No entanto, a concentração de fluoreto nos tecidos dentário, ósseo e plasma foi maior nos grupos expostos em GF e GA+F (p<0.001). A análise fotográfica pelo índice DFIA e a microdureza mostraram diferenças significativas entre os grupos (p<0.001), sendo que GF apresentou maior severidade de hipomineralização do esmalte, seguido por GA+F e GA. Houve um aumento linear na dureza do esmalte quando nas profundidades de 10 µm a 30 µm, sendo esse aumento entre 4 a 5 KHN por µm de profundidade nos GA+F e GF, e entre a 7 a 8 KHN por µm nos grupos GA e GS. Os achados desse estudo mostraram que os animais expostos cronicamente ao fluoreto desenvolveram hipomineralização de esmalte, e a associação da amoxicilina não potencializou a severidade da hipomineralização. Conclusão: Capítulo 1 - Conclui-se que a amoxicilina pode provocar alterações durante a amelogênese, as alterações foram observadas no estágio secretor da matriz do esmalte, sugerindo atraso na aposição de matriz do esmalte. Capítulo 2 - Conclui-se que a amoxicilina não influenciou o conteúdo mineral dos incisivos e molares de ratos. Capítulo 3 - Concluímos que a amoxicilina não influenciou o desenvolvimento do esmalte, bem como não potencializou o efeito do fluoreto

Background: Amoxicillin use in early childhood has been associated with molarincisor hypomineralization. Moreover, it has been supposed an association between amoxicillin and fluoride on enamel defects. Aim. This study aimed to evaluate in vivo the effect of amoxicillin and amoxicillin associated with fluoride on the enamel development of rat tooth. Materials and Methods: The research was divided into three studies. Chapter 1 ­ Fifteen pregnant Holtzman rats (Rattus norvegicus albinus) were divided randomly into three groups to receive saline (SG), 100 mg/kg/day amoxicillin (A100G), or 500 mg/kg/day amoxicillin (A500G), intragastrically, from days 13 to 22 of pregnancy. Twelve offsprings per group got the same dose until day 12. After 7 and 12 days, the specimens were fixed and embedded in paraffin. In the HEstained sections, the thickness of enamel matrix of upper molar germs was evaluated. Moreover, detection of amelogenin on day 7 and matrix metalloproteinase 20 (MMP -20) on day 12 by immunohistochemistry were carried out. Chapter 2 - Fifteen rats (Rattus norvegicus, Albinus,Holtzman) were randomly divided into three groups to receive saline (SG), 100 mg/kg/day amoxicillin (A100G), or 500 mg/kg/day amoxicillin (A500G), as described in Chapter 1. Ten offspring per group received the same dose until day 40. The animals were euthanized on day 40. A total of 60 upper incisors and 60 upper first molars from 30 animals were dissected. Calcium (Ca) determination was performed by the compleximetric titration method and phosphorus (Pi) determination by colorimetric analysis using a spectrophotometer at 680 nm. A total of 30 lower first molars from 15 animals were analyzed under X-ray microtomography (XMT). Chapter 3 - Forty rats were, randomly, divided into four groups exposed to fluoride in water (100 ppm (mg F/L) or intragastric administration of amoxicillin (500 mg /kg body weight), according to the combinations: control group (SG); amoxicillin group (AG), fluoride group (FG) and amoxicillin with fluoride group (A+FG.) After 60 days, the animals were euthanized. The concentrations of fluoride in the plasma, tibia and incisors of the rats were determined. The incisors were also analysed by the fluorosis severity (by software image analysis, Dental fluorosis by Image analysis - DFIA), calcium content by compleximetric titration method and phosphorus content by colorimetric method, enamel thickness, and its hardness. Results: Chapter 1 - A100G and A500G groups showed lower thickness of the enamel matrix than GS (p<0.001) on day 7, but not on day 12 (p=0.111). On day 7, the treated groups showed morphological changes in ameloblasts, represented by vacuoles. The amelogenin (p=0.818) and MMP20 (p=0.855) immunostaining intensity did not differ among groups. Chapter 2 - There were no statistically significant differences in Ca content (p=0.180), Pi content (p=0.054), or XMT electron density (p=0.150) among the groups. Chapter 3 - The thickness of the enamel (p=0.228), Ca content (p= 0.592) and Pi content (p=0.409) were not statistically different among the groups. However, the concentration of fluoride in the tooth, bone, and plasma was higher in the FG and A+FG (p< 0.001) than AG and SG. The DFIA index and microhardness showed significant differences among groups (p<0.001), and FG presented greater severity hypomineralization enamel than SG, followed by A+FG and AG. The hardness increased linearly in the 10 µm a 30 µm depths, being from 4 to 5 KHN per mm in depth in the AG and A+FG, and from 7 to 8 KHN mm in depth in the AG and SG. The findings of this study showed that animals chronically exposed to fluoride developed enamel hypomineralization, and the association of amoxicillin had no role in the hypomineralization severity. Conclusion: Chapter 1 - Amoxicillin can cause changes only during secretory stage, suggesting a delay in ameloblasts differentiation. Chapter 2 ­ Amoxicillin did not affect the mineral content and electron density of rat teeth. Chapter 3 ­ Amoxicillin did not influence the development of the enamel, and did not potentiate the effect of fluoride

No evidence for association between Amelogenesis imperefecta and candidate genes

Amelogenesis imperfecta [AI] is an inherited tooth disorder. Despite the fact that up to now, several gene mutations in MMP20, ENAM, AMELX and KLK4 genes have been reported to be associated with AI, many other genes suggested to be involved. The main objective of this study was to find the mutations in three major candidate genes including MMP20, ENAM and KLK4 responsible for AI from three Iranian families with generalized hypoplastic phenotype in all teeth. All exon/intron boundaries of subjected genes were amplified by polymerase chain reaction and subjected to direct sequencing. One polymorphisms was identified in KLK4 exon 2, in one family a homozygous mutation was found in the third base of codon 22 for serine [TCG>TCT], but not in other families. Although these base substitutions have been occurred in the signaling domain, they do not seem to influence the activity of KLK4 protein. Our results might support the further evidence for genetic heterogeneity; at least, in some AI cases are not caused by a gene in these reported candidate genes