O papel de proteínas das vias WNT/ß-Catenina, TGF-ß/BMP4 e SHH na odontogênese e odontomas / The role of proteins from the WNT/ß-Catenin, TGF-ß/BMP4 and SHH pathways in odontogenesis and odontomas

O desenvolvimento do dente depende de uma série de interações sinalizadoras recíprocas entre o epitélio oral (EO) e o ectomesênquima derivado da crista neural, a via WNT com o TGF-ß e BMP4 tem sido implicada na tumorigênese. A via de sinalização tipo Wingless (Wnt) / ß-catenina é essencial para a ativação precoce da odontogênese e no desenvolvimento de tumores odontogênicos. O TGF-ß e as BMPs tem sido associadas aos processos de dentinogênese reacionária e reparadora. A sinalização de Shh pode regular a proliferação celular no ectomesênquima dentário, controlando assim a morfogênese dentária. O objetivo da pesquisa foi investigar a atuação de algumas proteínas das vias na odontogênese e na formação de odontomas e tumores odontogênicos mistos benignos, para isto, foi desenvolvido um estudo seccional restrospectivo e imuno-histoquímico contendo 23 odontomas compostos, 21 odontomas complexos, 17 germes dentários, 05 fibro-odontomas ameloblásticos e 01 fibroma ameloblástico. Os resultados encontrados demonstraram maiores imunoexpressões da via WNT/ß-catenina no epitélio dos germes dentários (p<0,001) e no fibroma ameloblástico, enquanto que, esteve no ectomesênquima dos odontomas (p<0,001) e fibro-odontomas ameloblásticos. A via WNT/ßcatenina correlacionou-se moderadamente e significativamente com a CK14 no epitélio (p = 0,007) dos odontomas. A BMP4 foi imunoexpressa, especialmente, no ectomesênquima dos odontomas complexos (mediana = 33,7; p<0,001). A via Shh foi mais imunoexpressa no epitélio dos germes dentários (p<0,001) e no ectomesênquima dos odontomas complexos (p=0,029). De forma similar, o TGFß apresentou maior imunoexpressão no epitélio dos germes dentários (p<0,001) e no ectomesênquima dos odontomas complexos (p = 0,002). O dente em desenvolvimento exibiu maiores concentrações para estas proteínas no epitélio odontogênico nas fases de botão e capuz e a expressão diferencial ocorreu, principalmente, no ectomesênquima dos tumores, o que indica que esse componente é de fato mais proliferativo (AU).

Tooth development depends on a series of reciprocal signaling interactions between oral epithelium (EO) and neural crest-derived ectomesenchyme, the WNT pathway with TGF-ß and BMP4 has been implicated in tumorigenesis. The Wingless (Wnt)/ß-catenin signaling pathway is essential for the early activation of odontogenesis and the development of odontogenic tumors. TGF-ß and BMPs have been associated with reactionary and reparative dentinogenesis processes. Shh signaling can regulate cell proliferation in dental ectomesenchyme, thus controlling dental morphogenesis. The objective of the research was to investigate the role of some proteins in the pathways in odontogenesis and in the formation of odontomas and benign mixed odontogenic tumors. tooth germs, 05 ameloblastic fibro-odontomas and 01 ameloblastic fibroma. The results found showed higher immunoexpressions of the WNT/ß-catenin pathway in the epithelium of tooth germs (p<0.001) and in ameloblastic fibroma, while it was in the ectomesenchyme of odontomas (p<0.001) and ameloblastic fibroodontomas. The WNT/ß-catenin pathway correlated moderately and significantly with CK14 in the epithelium (p = 0.007) of odontomas. BMP4 was immunoexpressed, especially in the ectomesenchyme of complex odontomas (median = 33.7; p<0.001). The Shh pathway was more immunoexpressed in the epithelium of tooth germs (p<0.001) and in the ectomesenchyme of complex odontomas (p=0.029). Similarly, TGF-ß showed higher immunoexpression in the epithelium of tooth germs (p<0.001) and in the ectomesenchyme of complex odontomas (p = 0.002). The developing tooth exhibited higher concentrations of these proteins in the odontogenic epithelium in the bud and cap phases and the differential expression occurred mainly in the ectomesenchyme of the tumors, which indicates that this component is in fact more proliferative (AU).

Caracterización microscópica de la dentina de dientes temporales / Microscopic characterization of the dentin of temporary teeth

La dentina se compone de un mineral de fosfato de calcio identificado como dahllita, que se dispone en pequeños cristales de hidroxiapatita carbonatada con dimensiones de 36 × 25 × 4 nm, y por una fase orgánica cuyo principal componente es el colágeno tipo 1 en 90%, que se orienta en forma de malla. Esta conformación corresponde a los dientes permanentes. Dentro de las estructuras, encontramos túbulos dentinarios que miden, aproximadamente, entre 0.5-1 µm de diámetro en la periferia y hasta 3-5 µm cerca de la pulpa. En el presente estudio, realizado en dentina de dientes temporales, el lumen de dichos túbulos es más grande cuando se encuentra cerca de la pulpa dental. Asimismo, se encontraron cambios elementales importantes de acuerdo con las diferentes profundidades en las que se observó, encontrando un aumento en el peso porcentual de carbono cuando se encuentra a mayor profundidad, lo que indica una composición orgánica mayor en la dentina pulpar. En estudios de dientes permanentes esta composición es disminuida y con mayor concentración en la dentina cercana a la unión amelodentinaria. En dentina de dientes temporales se encontraron diferencias en el recuento de túbulos dentinarios por mm2, comparado a la dentina de dientes permanentes, donde el número de túbulos no varía mucho (AU)

Dentin is composed of a calcium phosphate mineral identified as dahllite, which is arranged in small crystals of carbonated hydroxyapatite with dimensions of 36 × 25 × 4 nm, and by an organic phase whose main component is type l collagen in 90%, which is oriented in the form of a mesh. This conformation corresponds to permanent teeth. Within the structures, we find dentin tubules that measure approximately 0.5-1 µm in diameter at the periphery and up to 3-5 µm near the pulp. In the present study, carried out in dentin of primary teeth, the lumen of these tubules is larger when it is close to the dental pulp. Likewise, important elemental changes were found according to the different depths in which it was observed, finding an increase in the percentage weight of carbon when it is at a greater depth, indicating a greater organic composition in the pulp dentin. In studies of permanent teeth, this composition is decreased and with a higher concentration in the dentin near the amelodentinal junction. In dentin of primary teeth, differences were found in the count of dentin tubules per mm2, compared to dentin of permanent teeth, where the number of tubules did not vary much (AU)

Evaluation of dentinogenesis inducer biomaterials: an in vivo study

Abstract When exposure of the pulp to external environment occurs, reparative dentinogenesis can be induced by direct pulp capping to maintain pulp tissue vitality and function. These clinical situations require the use of materials that induce dentin repair and, subsequently, formation of a mineralized tissue. Objective: This work aims to assess the effect of tricalcium silicate cements and mineral trioxide aggregate cements, including repairing dentin formation and inflammatory reactions over time after pulp exposure in Wistar rats. Methodology: These two biomaterials were compared with positive control groups (open cavity with pulp tissue exposure) and negative control groups (no intervention). The evaluations were performed in three stages; three, seven and twenty-one days, and consisted of an imaging (nuclear medicine) and histological evaluation (H&E staining, immunohistochemistry and Alizarin Red S). Results: The therapeutic effect of these biomaterials was confirmed. Nuclear medicine evaluation demonstrated that the uptake of 99mTc-Hydroxymethylene diphosphonate (HMDP) showed no significant differences between the different experimental groups and the control, revealing the non-occurrence of differences in the phosphocalcium metabolism. The histological study demonstrated that in mineral trioxide aggregate therapies, the presence of moderate inflammatory infiltration was found after three days, decreasing during follow-ups. The formation of mineralized tissue was only verified at 21 days of follow-up. The tricalcium silicate therapies demonstrated the presence of a slight inflammatory infiltration on the third day, increasing throughout the follow-up. The formation of mineralized tissue was observed in the seventh follow-up day, increasing over time. Conclusions: The mineral trioxide aggregate (WhiteProRoot®MTA) and tricalcium silicate (Biodentine™) present slight and reversible inflammatory signs in the pulp tissue, with the formation of mineralized tissue. However, the exacerbated induction of mineralized tissue formation with the tricalcium silicate biomaterial may lead to the formation of pulp calcifications

Alteraciones de la dentina con el envejecimiento / Dentin alterations with aging

Diferentes estudios han demostrado que después de la tercera década de vida hay una transición en la microestructura de la dentina. Dada la importancia de ésta como sustrato para la adhesión de materiales de restauración utilizados en operatoria y rehabilitación oral, ha sido objetivo de este trabajo realizar una búsqueda bibliográfica de las publicaciones existentes en inglés y español de los últimos 15 años, haciendo uso de buscadores científicos como Pubmed, Google Schoolar y LILACS que permitieran actualizar la información existente ayudando a entender los efectos biológicos del envejecimiento de la dentina, identificando los cambios de mayor relevancia a nivel histológico. De la búsqueda realizada se concluye que el envejecimiento de la dentina está asociado con aumento de dentina secundaria, formación de dentina esclerótica opaca, variaciones en la composición química de la matriz y disminución del número y actividad de los odontoblastos. Los conocimientos sobre el envejecimiento de la dentina deben tenerse en cuenta frente a estudios relacionados con materiales de restauración dental, ya que los cambios en la microestructura y capacidad funcional de la dentina con el envejecimiento requieren que éstos se adapten a dichas variaciones (AU)

Different studies have shown that aafter the third term of life there is a transition in the microstructure of dentin. Given the importance of dentin as a substrate for the adhesion of restorative materials used in operative and oral rehabilitation, the aim of the present work was to conduct a search of the scientific literature in English and Spanish, published in the last 15 years, using search engines such as Pubmed, Google Schoolar and LILACS, for an update on the biological effects of dentin aging, to identify the most relevant age-related histological changes in dentina. The data obtained from the literature review allow concluding that dentin aging is associated with an increase in secondary dentina, opaque sclerotic dentin formation, variations in the chemical composition of the matrix and a decrease in odontoblast number and activity. Updated information on dentin aging should be taken into account in studies on dental restoration materials, since the latter must adapt to aging-related changes in the microstructure and functional capacity of dentin (AU)

The functional significance of dentin sialoprotein-phosphophoryn and dentin sialoprotein / 国际口腔科学杂志·英文版

Phosphophoryn (PP) and dentin sialoprotein (DSP) are the most dominant non-collagenous proteins in dentin. PP is an extremely acidic protein that can function as a mineral nucleator for dentin mineralization. DSP was first identified in 1981, yet its functional significance is still controversial. Historically, these two proteins were considered to be independently synthesized and secreted by dental pulp cells into the developing dentin matrix. However, with the identification of the DSP coding sequence in 1994, followed 2 years later by the finding that the PP coding sequence was located immediately downstream from the DSP sequence, it became immediately clear that DSP and PP proteins were derived from a single DSP-PP (i.e., dentin sialophosphoprotein, DSPP) transcript. Since DSPP cDNA became available, tremendous progress has been made in studying DSP-PP mRNA distribution and DSP generation from the DSP-PP precursor protein at specific cleavage sites by protease tolloid-related-1 (TLR1) or bone morphogenetic protein 1 (BMP1). The functions of DSP-PP and DSP were investigated via DSP-PP knockout (KO) and DSP knockin in DSP-PP KO mice. In addition, a number of in vitro studies aimed to elucidate DSPP and DSP function in dental pulp cells.

Efeito de flavonoides sobre microrganismos de interesse endodôntico e sua influência na expressão de marcadores de mineralização / Effect of flavonoids on endodontic microorganisms and their influence on the expression of mineralization markers

O tratamento endodôntico de dentes permanentes jovens representa um grande desafio clínico devido a presença de paredes dentinárias finas, divergentes ou paralelas e ápice aberto, o que dificulta a desinfecção e a execução dos procedimentos convencionais. Terapias de regeneração endodôntica envolvem o uso de materiais capazes de promover uma desinfecção eficaz sem causar citotoxicidade, além de induzir a diferenciação de células-tronco ou bioestimular células remanescentes do tecido pulpar mesmo após a injúria. Nesse contexto, os flavonoides, polifenóis presentes em frutas e vegetais, poderiam ser agentes interessantes para o tratamento endodôntico de dentes imaturos devido a sua amplitude terapêutica. Dessa forma, o objetivo do presente trabalho foi avaliar o efeito antimicrobiano, citotóxico e indutor de mineralização de flavonoides com finalidade de aplicação endodôntica. Este trabalho de tese foi dividido em três capítulos. O capítulo 1 avaliou a toxicidade dos flavonoides taxifolina, crisina, pinocembrina e galangina sobre fibroblastos pelo ensaio de MTT, a atividade antimicrobiana pela determinação da concentração inibitória e bactericida mínima, e a ação antibiofilme do flavonoide com melhor efeito antimicrobiano, por meio de ensaios em placas de poliestireno e em dentina radicular bovina por meio da análise por microscopia confocal. Os resultados mostraram que o flavonoide taxifolina não foi tóxico para os fibroblastos em nenhuma das concentrações analisadas, enquanto que os flavonoides crisina, pinocembrina e galangina apresentaram efeitos citotóxicos. Crisina, pinocembrina e galangina não apresentaram efeito antimicrobiano frente E. faecalis and S. mutans nas concentrações testadas. A taxifolina foi capaz de inibir todas as bactérias testadas, eliminar biofilmes de E. faecalis e S. mutans em placas de poliestireno e reduzir significantemente o biofilme de E. faecalis em túbulos dentinários. O capítulo 2 avaliou a citotoxicidade do flavonoide taxifolina e o seu potencial sobre a indução de marcadores de mineralização dentinária (produção de fosfatase alcalina - ALP, nódulos de mineralização ­ NM e expressão dos genes DSPP ­ sialofosfoproteína dentinária e DMP-1 ­ proteína da matriz dentinária - 1) em células semelhantes a odontoblastos MDPC-23, após tratamentos de 24, 72h e contínuo. A taxifolina não apresentou citotoxicidade em nenhum dos três tipos de tratamento analisados. Todas as concentrações do tratamento de 24h e as concentrações de 10 e 5µM do tratamento de 72h aumentaram a atividade de ALP. A formação de NM aumentou com os tratamentos de taxifolina à 10µM em ambos os tratamentos de 24 e 72h, e à 5µM no tratamento de 24h. A expressão de DMP-1 aumentou com o tratamento de taxifolina em ambos os tratamentos de 24 e 72h, enquanto que a de DSPP aumentou apenas com o tratamento de 72h na concentração de 5µM. O capítulo 3 avaliou a citotoxicidade da taxifolina, e o seu potencial sobre a indução de marcadores de mineralização óssea (ALP, NM e expressão dos genes ALP e colágeno 1 - Col-1) em células semelhantes a osteoblastos Saos-2, após tratamentos de 24, 72h e contínuo. Os resultados mostraram que os tratamentos com taxifolina nas concentrações de 10, 5 e 1µM não foram citotóxicos em nenhum dos períodos analisados. O tratamento de 72h da taxifolina à 10µM foi capaz de aumentar a atividade de ALP e a formação de NM, além de aumentar a expressão de Col-1 após 13 dias. O tratamento de 24h da taxifolina na concentração de 10µM aumentou a expressão de ALP após 6 dias. Conclui-se que a taxifolina é um flavonoide com potencial uso para o tratamento endodôntico de dentes permanentes jovens, devido à sua ação antimicrobiana/antibiofilme, baixa citotoxicidade e capacidade de estimular a mineralização em odontoblastos e osteoblastos(AU)

The endodontic treatment of young permanent teeth represents a great clinical challenge due to the presence of thin, divergent or parallel dentin walls and the open apex that makes it difficult to disinfect and perform conventional endodontic procedures. Endodontic regeneration therapies involve the use of materials capable of promoting effective disinfection without causing cytotoxicity, in addition to inducing differentiation of stem cells or biostimulating remaining pulp tissue cells even after injury. In this context, the flavonoids, polyphenols present in fruits and vegetables, could be interesting agents for the endodontic treatment of immature teeth due to the wide therapeutic use. Thus, the objective of the present study was to evaluate the antimicrobial, cytotoxic effects and capacity of mineralization induction of flavonoids for endodontic application. This thesis was divided into three chapters. The chapter 1 evaluated the toxicity of taxifolin, chrysin, pinocembrin and galangin flavonoids on fibroblasts by the MTT method, antimicrobial activity by determining the minimum inhibitory and bactericidal concentrations and analyzed the antibiofilm action of the flavonoid with the best antimicrobial effect, by means of the biofilm assays in polystyrene plates and in bovine root dentin and confocal microscopy analysis. The results showed that the flavonoid taxifolin was not toxic on fibroblasts in any tested concentration, while chrysin, pinocembrin and galangin flavonoids showed cytotoxic effects. Chrysin, pinocembrin and galangin showed no antimicrobial effect against E. faecalis and S. mutans in any tested concentrations. Taxifolin was able to inhibit all tested bacteria, to eliminate E. faecalis and S. mutans biofilms on polystyrene plates and significantly reduce E. faecalis biofilms from the dentin tubules. The chapter 2 evaluated the cytotoxicity of taxifolin and its potential on the induction of dentin mineralization markers (alkaline phosphatase production - ALP, mineralization nodules - MN and expression of genes DSPP - dentin sialophosphoprotein and DMP-1 - dentin matrix protein - 1) on odontoblast-like cells MDPC-23, after treatments of 24, 72h and continuous. Taxifolin did not present cytotoxicity at any of the three types of treatments analyzed. All concentrations of 24h-treatment and 10 and 5µM of 72htreatment increased ALP activity. NM formation increased with taxifolin treatments at 10µM in both 24 e 72h treatments, and at 5µM in the 24h-treatment. Expression of DMP-1 increased with taxifolin in both 24 e 72h-treatments, whereas DSPP expression increased only with 72h-treatment at 5µM. The chapter 3 evaluated the cytotoxicity of taxifolin and its potential on the induction of bone mineralization markers (ALP, NM and expression of ALP and collagen 1 -Col-1 genes) on Saos-2 osteoblast-like cells, after treatments of 24, 72h and continuous. The results showed that taxifolin treatments at 10, 5 and 1µM were not cytotoxic in any of the periods analyzed. The 72h-treatment of taxifolin at 10µM was able to increase ALP activity and NM formation, in addition to increasing Col-1 expression after 13 days. The 24h-treatment of taxifolin at 10µM increased ALP expression after 6 days. It is concluded that taxifolin is a flavonoid with potential use for endodontic treatment of young permanent teeth due to its antimicrobial/antibiofilm action, low cytotoxicity and ability to stimulate mineralization in odontoblasts and osteoblasts(AU)

Influência do peróxido de hidrogênio sobre a diferenciação celular e a mineralização do tecido pulpar após procedimento clareador: estudo histológico e imunoistoquímico / Influence of hydrogen peroxide on cell differentiation and mineralization of pulp tissue after bleaching: histological and immunohistochemical study

Introdução Através de modelo experimental caracterizado por nosso grupo de pesquisa, e protocolo clareador adaptado, verificamos que o peróxido de hidrogênio (H2O2) contido no gel clareador pode gerar efeitos ao tecido pulpar, que ainda não estão completamente compreendidos. Outros estudos mostram uma indução à mineralização, levando à posterior calcificação de grande parte do tecido pulpar e à formação de nódulos. Objetivos Os objetivos deste trabalho foram divididos em duas etapas: 1 ­ Verificar os efeitos do H2O2 na expressão de marcadores da mineralização no tecido pulpar, por meio da imunomarcação de osteocalcina (OCN) e osteopontina (OPN); e a presença de resposta celular específica ao estresse oxidativo, por meio de imunomarcação com anticorpo para espécies reativas de oxigênio (EROs); 2 ­ Determinar a capacidade de resposta ao estresse oxidativo gerado pelo H2O2 no tecido pulpar, por meio da imunomarcação de Heme-oxigenase-1 (HO-1); investigar os efeitos do gel clareador sobre a diferenciação odontoblástica, por meio da imunomarcação do fator de transcrição Jun-D; e a influência do estresse oxidativo gerado pelo H2O2 na identificação de células-tronco mesenquimais (CTMs) do tecido pulpar, por meio da técnica de imunofluorescência, com identificação concomitante de positividade celular para CD90, CD73, CD105 e negatividade para CD45. Materiais e métodos Foram utilizados 60 ratos Wistar que tiveram os molares superiores direitos ou esquerdos clareados com 0,01 mL de H2O2 35%, em uma aplicação de 30 minutos, de forma randomizada. Os molares do lado não clareado serviram de controle. Após 0 horas, 2, 3, 7, 15 e 30 dias (n=10), os animais foram mortos e as maxilas processadas para avaliação histológica, imunoistoquímica (OCN, OPN, EROs, HO-1 e Jun-D) e de imunofluorescência (CD90, CD73, CD105, CD45). Os resultados foram submetidos a testes estatísticos específicos (p<0,05). Resultados No tempo de 0 horas, houve necrose em toda a polpa coronária (p<0,05), e aos 2 e 3 dias, no terço oclusal (p<0,05); aos 7, 15 e 30 dias, não houve inflamação, assim como no controle (p>0,05). Dentina terciária estava presente aos 7 dias, aumentando em 15 e 30 dias (p<0,05). Em relação aos marcadores de mineralização, OCN foi ausente imediatamente após procedimento clareador, aumentando ao longo dos períodos, se tornando significativa aos 15 e 30 dias (p<0,05); OPN apresentou maior imunomarcação aos 7 e 15 dias no grupo clareado (p<0,05). A imunomarcação de EROs foi significativa em todos os terços da polpa coronária no grupo clareado aos 7 e 15 dias, e no terço cervical aos 2 e 30 dias, comparada ao controle (p<0,05). HO-1 revelou maior imunomarcação no grupo clareado nos terços médio e cervical da polpa coronária aos 2 e 3 dias, em todos os terços aos 7 dias, e no terço oclusal aos 15 dias, quando comparado ao grupo controle (p<0,05). Imunomarcação nuclear para Jun-D foi significativa no grupo clareado no terço cervical da polpa coronária aos 2 e 3 dias, e nos terços oclusal e médio aos 7 dias, quando comparado ao grupo controle (p<0,05), reduzindo nos demais períodos (p>0,05). Poucas células CD90+/CD73+/CD105+/CD45- foram observadas no tecido pulpar do grupo controle e do grupo clareado em todos os períodos de análise (p>0,05). Conclusões Pode-se concluir que: 1 ­ A redução da inflamação e o processo de reparo pulpar após procedimento clareador está associado com o aumento de OCN, e OPN participa durante o processo de reparo; EROs está presente no processo de defesa celular contra o estresse oxidativo decorrente do H2O2. 2 ­ As células pulpares apresentam capacidade de resposta ao estresse oxidativo expressando HO-1 nos períodos onde há inflamação, até o início do reparo; Jun-D é presente no tecido pulpar durante a redução do processo inflamatório e início da produção de dentina terciária; a presença de estresse oxidativo não influencia o número de células CD90+/CD73+/CD105+/CD45- identificadas in vivo no tecido pulpar(AU)

Introduction Through an experimental model characterized by our research group, and adapted bleaching protocol, we verified that the hydrogen peroxide (H2O2) of bleaching gel can generate effects on the pulp tissue, which are not yet completely understood. Studies show an induction to mineralization, leading to subsequent calcification of a large part of the pulp tissue and to the formation of nodules. Objectives The objectives of this study were divided into two stages: 1 ­ To verify the effects of H2O2 on the expression of mineralization markers in pulp tissue, through of immunolabelling of the osteocalcin (OCN) and osteopontin (OPN); and the presence of specific cellular response to oxidative stress, by immunolabeling with antibody to reactive oxygen species (ROS); 2 ­ To determine the capacity of response to oxidative stress generated by H2O2 in pulp tissue, through of immunolabelling of Heme-oxigenase-1 (HO-1); to investigate the effects of the bleaching gel on the odontoblastic differentiation, through the immunolabelling of the transcription factor Jun-D; and the influence of the oxidative stress generated by H2O2 on the identification of mesenchymal stem cells (MSCs) of the pulp tissue by the immunofluorescence technique, with the concomitant identification of cellular positivity for CD90, CD73, CD105 and negativity for CD45. Materials and methods Sixty Wistar rats were used, and the right or left upper molars were bleached with 0.01 mL of 35% H2O2, in a direct application of 30 minutes, randomly. The molars on the unbleached side served as controls. After 0 hours, 2, 3, 7, 15 and 30 days (n=10), the animals were killed and the jaws processed for histological, immunohistochemical (OCN, OPN, ROS, HO-1 and JunD) and immunofluorescence (CD90, CD73, CD105, CD45) analysis. The results were submitted to specific statistical tests (P<0.05). Results At 0 hours, there was necrosis throughout the coronary pulp (P<0.05), and at 2 and 3 days, in the occlusal third (P<0.05); at 7, 15 and 30 days, there was no inflammation, as well as in the control (P>0.05). Tertiary dentin was present at 7 days, increasing in 15 and 30 days (P<0.05). In relation to mineralization markers, OCN was absent immediately after bleaching procedure, increasing over the periods, becoming significant at 15 and 30 days (P<0.05); OPN has higher immunolabelling at 7 and 15 days in the bleached group (P<0.05). Immunolabelling of ROS was significant in all thirds of the coronary pulp in the bleached group at 7 and 15 days, and in the cervical third at 2 and 30 days, compared to the control group (P<0.05). HO-1 showed higher immunolabelling in the bleached group in the middle and cervical thirds of the coronary pulp at 2 and 3 days, in all thirds at 7 days, and in the occlusal third at 15 days, when compared to the control group (P<0.05). Nuclear immunolabelling for Jun-D was significant in the bleached group in the cervical third of the coronary pulp at 2 and 3 days, and in the occlusal and middle thirds at 7 days, when compared to the control group (P<0.05), reducing in the other periods (P>0.05). Low number of CD90+/CD73+/CD105+/CD45- cells were observed in the pulp tissue of the control group and the bleached group in all periods of analysis (P>0.05). Conclusions It is concluded that: 1 ­ The reduction of inflammation and the pulp repair process after bleaching is associated with increased of OCN, and OPN participates during the repair process; ROS are present in the cellular defence process against oxidative stress by H2O2. 2 ­ The pulp cells had capacity to respond to oxidative stress expressing HO-1 in the periods where there is inflammation, until the beginning of the repair; Jun-D is present in the pulp tissue during the reduction of the inflammatory process and the beginning of the production of tertiary dentin; the presence of oxidative stress does not influence the number of CD90+/CD73+/CD105+/CD45- cells identified in vivo in the pulp tissue(AU)

Dentinogenesis imperfecta type II: A case report with 17 years of follow-up

Dentinogenesis imperfecta is a dominant autosomal hereditary disorder of dentin formation that affects the deciduous and permanent teeth. Its etiology is characterized by inadequate cell differentiation during odontogenesis. The clinical characteristics of dentinogenesis imperfecta are discolored teeth with a translucency that varies from gray to brown or amber. Radiographically, the teeth exhibit pulp obliteration, thin and short roots, bell-shaped crowns, and periapical bone rarefaction. The aim of this report was to present a case of dentinogenesis imperfecta type II that was followed up over a 17-year period. This report also presents scanning electron microscopy images of the enamel and dentin, showing that both were altered in the affected teeth. The disease characteristics and the treatments that were administered are reported in this study to guide dentists with respect to the need for early diagnosis and adequate follow-up to avoid major sequelae.

The role of nuclear factor I-C in tooth and bone development

Nuclear factor I-C (NFI-C) plays a pivotal role in various cellular processes such as odontoblast and osteoblast differentiation. Nfic-deficient mice showed abnormal tooth and bone formation. The transplantation of Nfic-expressing mouse bone marrow stromal cells rescued the impaired bone formation in Nfic(-/-) mice. Studies suggest that NFI-C regulate osteogenesis and dentinogenesis in concert with several factors including transforming growth factor-β1, Krüppel-like factor 4, and β-catenin. This review will focus on the function of NFI-C during tooth and bone formation and on the relevant pathways that involve NFI-C.

As principais alterações dentárias de desenvolvimento / The main changes of dental development

Introdução: os distúrbios no desenvolvimento das estruturas dentárias podem ter origem hereditária, congênita ou adquirida. Tais alterações podem afetar tanto a forma, tamanho, número, posição, constituição e\ou função dos dentes, portanto é de extrema importância à atuação do cirurgião dentista no diagnóstico e tratamento. Objetivo: discorrer sobre as anomalias dentárias de desenvolvimento, apresentando as alterações quanto ao número, tamanho, forma e estrutura dos dentes, ressaltando a importância do cirurgião dentista em conhecer essas anomalias, saber identificá-las e estabelecer a melhor conduta terapêutica. Método: foi realizado uma revisão sistemática da literatura nas bases de dados eletrônicos: PubMED/Medline, Lilacs, Portal Capes-Periódicos, Scielo, Bireme, Scopus e livros específicos sobre a temática da pesquisa. Conclusão: alterações dentárias do desenvolvimento, são postas diante do cirurgião-dentista geralmente por insatisfação estética ou descobertas em consulta de rotina, então cabe ao profissional saber diagnosticar, e traçar o plano de tratamento mais adequado a cada caso.

Introduction: the disturbances in the development of dental structures can be hereditary, congenital or acquired. Such changes can affect both the shape, size, number, position, constitution and \ or function of the teeth, so it is of utmost importance the role of the dentist in the diagnosis and treatment. Objective: this study aims to discuss the dental anomalies of development, addressing the changes in the number, size, shape and structure of the teeth, emphasizing the importance of dental surgeons to meet these anomalies, learn to identify them and establish the best treatment. Method: it was conducted a systematic review of literature in electronic databases: PubMed / Medline, Lilacs, Capes-Journal Portal, Scielo, Bireme, Scopus and specific books on the subject of research. Conclusion: dental developmental changes are usually presented to the dental surgeon due to aesthetic dissatisfaction or findings in routine consultation, so it is up to the professional to know how to diagnose, and to draw the most adequate treatment plan for each case.

Requirement of Smad4-mediated signaling in odontoblast differentiation and dentin matrix formation / 대한해부학회지

Dentin is the major part of tooth and formed by odontoblasts. Under the influence of the inner enamel epithelium, odontoblasts differentiate from ectomesenchymal cells of the dental papilla and secrete pre-dentin which then undergo mineralization into dentin. Transforming growth factor-beta (TGF-β)/bone morphogenetic protein (BMP) signaling is essential for dentinogenesis; however, the precise molecular mechanisms remain unclear. To understand the role of TGF-β/BMP signaling in odontoblast differentiation and dentin formation, we generated mice with conditional ablation of Smad4, a key intracellular mediator of TGF-β/BMP signaling, using Osr2 or OC-Cre mice. Here we found the molars of Osr2(Cre)Smad4 mutant mice exhibited impaired odontoblast differentiation, and normal dentin was replaced by ectopic bone-like structure. In Osr2(Cre)Smad4 mutant mice, cell polarity of odontoblast was lost, and the thickness of crown dentin was decreased in later stage compared to wild type. Moreover, the root dentin was also impaired and showed ectopic bone-like structure similar to Osr2(Cre)Smad4 mutant mice. Taken together, our results suggest that Smad4-dependent TGF-β/BMP signaling plays a critical role in odontoblast differentiation and dentin formation during tooth development.

The Role of Autonomous Wntless in Odontoblastic Differentiation of Mouse Dental Pulp Cells

PURPOSE: Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. Deletion of the Wntless (Wls) gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation. However, it remains unclear if autonomous Wnt ligands are necessary for differentiation of dental pulp cells into odontoblast-like cells to induce reparative dentinogenesis, one of well-known feature of pulp repair to form tertiary dentin. MATERIALS AND METHODS: To analyze the autonomous role of Wls for differentiation of dental pulp cells into odontoblast-like cells, we used primary dental pulp cells from unerupted molars of Wls-floxed allele mouse after infection with adenovirus for Cre recombinase expression to knockout the floxed Wls gene or control GFP expression. The differentiation of dental pulp cells into odontoblast-like cells was analyzed by quantitative real-time polymerase chain reaction. RESULT: Proliferation rate was significantly decreased in dental pulp cells with Cre expression for Wls knockout. The expression levels of Osterix (Osx), runt-related transcription factor 2 (Runx2), and nuclear factor I-C (Nfic) were all significantly decreased by 0.3-fold, 0.2-fold, and 0.3-fold respectively in dental pulp cells with Wls knockout. In addition, the expression levels of Bsp, Col1a1, Opn, and Alpl were significantly decreased by 0.7-fold, 0.3-fold, 0.8-fold, and 0.6-fold respectively in dental pulp cells with Wls knockout. CONCLUSION: Wnt ligands produced autonomously are necessary for proper proliferation and odontoblastic differentiation of mouse dental pulp cells toward further tertiary dentinogenesis.

Chitosan-collagen biomembrane embedded with calcium-aluminate enhances dentinogenic potential of pulp cells

Abstract The development of biomaterials capable of driving dental pulp stem cell differentiation into odontoblast-like cells able to secrete reparative dentin is the goal of current conservative dentistry. In the present investigation, a biomembrane (BM) composed of a chitosan/collagen matrix embedded with calcium-aluminate microparticles was tested. The BM was produced by mixing collagen gel with a chitosan solution (2:1), and then adding bioactive calcium-aluminate cement as the mineral phase. An inert material (polystyrene) was used as the negative control. Human dental pulp cells were seeded onto the surface of certain materials, and the cytocompatibility was evaluated by cell proliferation and cell morphology, assessed after 1, 7, 14 and 28 days in culture. The odontoblastic differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, total protein production, gene expression of DMP-1/DSPP and mineralized nodule deposition. The pulp cells were able to attach onto the BM surface and spread, displaying a faster proliferative rate at initial periods than that of the control cells. The BM also acted on the cells to induce more intense ALP activity, protein production at 14 days, and higher gene expression of DSPP and DMP-1 at 28 days, leading to the deposition of about five times more mineralized matrix than the cells in the control group. Therefore, the experimental biomembrane induced the differentiation of pulp cells into odontoblast-like cells featuring a highly secretory phenotype. This innovative bioactive material can drive other protocols for dental pulp exposure treatment by inducing the regeneration of dentin tissue mediated by resident cells.

Dentin matrix protein 1 and phosphate homeostasis are critical for postnatal pulp, dentin and enamel formation / 国际口腔科学杂志·英文版

Deletion or mutation of dentin matrix protein 1 (DMP1) leads to hypophosphatemic rickets and defects within the dentin. However, it is largely unknown if this pathological change is a direct role of DMP1 or an indirect role of phosphate (Pi) or both. It has also been previously shown that Klotho-deficient mice, which displayed a high Pi level due to a failure of Pi excretion, causes mild defects in the dentinal structure. This study was to address the distinct roles of DMP1 and Pi homeostasis in cell differentiation, apoptosis and mineralization of dentin and enamel. Our working hypothesis was that a stable Pi homeostasis is critical for postnatal tooth formation, and that DMP1 has an antiapoptotic role in both amelogenesis and dentinogenesis. To test this hypothesis, Dmp1-null (Dmp1(-/-)), Klotho-deficient (kl/kl), Dmp1/Klotho-double-deficient (Dmp1(-/-)/kl/kl) and wild-type (WT) mice were killed at the age of 6 weeks. Combinations of X-ray, microcomputed tomography (μCT), scanning electron microscopy (SEM), histology, apoptosis and immunohistochemical methods were used for characterization of dentin, enamel and pulp structures in these mutant mice. Our results showed that Dmp1(-/-) (a low Pi level) or kl/kl (a high Pi level) mice displayed mild dentin defects such as thin dentin and a reduction of dentin tubules. Neither deficient mouse line exhibited any apparent changes in enamel or pulp structure. However, the double-deficient mice (a high Pi level) displayed severe defects in dentin and enamel structures, including loss of dentinal tubules and enamel prisms, as well as unexpected ectopic ossification within the pulp root canal. TUNEL assay showed a sharp increase in apoptotic cells in ameloblasts and odontoblasts. Based on the above findings, we conclude that DMP1 has a protective role for odontoblasts and ameloblasts in a pro-apoptotic environment (a high Pi level).

The use of dental papillae cells and millipore filter for bioengineered dentin / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To observe the ability of SD rat dental papillae cells forming dentin-like structure induced by millipore filter combined with transforming growth factor-β(1) (TGF-β(1)).</p><p><b>METHODS</b>The first passage SD rat dental papillae cells were enzymatically dissociated and centrifuged to obtain a cell mass. The cell mass was seeded on the millipore filter combined with TGF-β(1). The complex was incubated for 6 d in vitro or transplanted under the renal capsule for 2 weeks. Then the differentiation of dental papillae cells on the filter and the formation of mineral tissue on the implant were analyzed.</p><p><b>RESULTS</b>A layer of polarized columnar cells were observed along the surface of the millipore filter, with cell processes extending into the porous media. Dentin sialoprotein (DSP) and dentin matrix protein-1 (DMP-1) were positive in these cells. After 2 weeks, tubular dentin matrix was deposited on the surface of the aligned cells. Scanning electron microscopy showed that the thickness of newly formed tubular dentin was consistent. DSP and DMP-1 were expressed in columnar cells, tubular matrix and the dental papillae cells adjacent to the filter.</p><p><b>CONCLUSIONS</b>The millipore filter combined with TGF-β(1) could effectively recruit progenitors onto its surface and induce odontoblast differentiation, secrete matrix in a homogenous manner, leading to dentinogenesis.</p>

Dentification ability of inbred strain mice tooth germs homologically transplanted into oral submucosa / 中南大学学报(医学版)

OBJECTIVE@#To establish a suitable environment for the bioengineered teeth in vivo by observing the dentification ability of BALB/C mice tooth germs homologically implanted into the oral submucosa.@*METHODS@#The first molar tooth germs of BALB/C mice 4 days after birth were transplanted into the oral submucosa of BALB/C male mice, and then recycled for regular histological observation after 1, 2, 3, and 6 week transplantation.@*RESULTS@#The tooth germs in the oral submucosa grew well with continuing developing enamelum and pulpodentinal complex, and the dentinal tubules were clear.@*CONCLUSION@#The environment of the BALB/C male mice oral submucosa is favorable for the growth of tooth germs in inbred strain BALB/C mice, and it can provide a new environment for the development of bioengineered teeth in vivo.

The comparison of gene expression from human dental pulp cells and periodontal ligament cells / 대한치과보존학회지

The purpose of this study was to characterize functional distinction between human dental pulp cells(PC) and periodontal ligament cells(PDLC) using cDNA microarray assay and to confirm the results of the microarray assay using RT-PCR. 3 genes out of 51 genes which were found to be more expressed(>2 fold) in PC were selected, and 3 genes out of 19 genes which were found to be more expressed(>2 fold) in PDLC were selected for RT-PCR as well. According to this study, the results were as follows: 1. From the microarray assay, 51 genes were more expressed (2 fold) from PC than PDLC. 2. RT-PCR confirmed that ITGA4 and TGF beta2 were more expressed in PC than in PDLC 3. From the microarray assay, 19 genes were more expressed (2 fold) from PDLC than PC. 4. RT-PCR confirmed that LUM, WISP1, and MMP1 were more expressed in PDLC than in PC. From the present study, different expression of the genes between the PC and PDLC were characterized to show the genes which play an important role in dentinogenesis were more expressed from PC than PDLC, while the genes which were related with collagen synthesis were more expressed from PDLC than PC.

Dentin permeability: the basis for understanding pulp reactions and adhesive technology

Permeability involves the passage of fluids, ions, molecules, particulate matter and bacteria into and through a substance or tissue under different and varying conditions. The permeability of the dentin is essential to support the physiology and reaction patterns of the pulp-dentin organ. Nutrients and impulses are transported from the pulp via the odontoblast process and the contents of its tubules maintain the dentin as a vital tissue. However, the main interest of this paper focuses on penetration from the outside towards the pulp rather than from the pulp towards the outside. The present overview centers on the dentinal tubules; how they are formed and how they change as a result of normal and abnormal function, age, and pathological processes and the effect of these processes on the permeability of dentin. Particular attention is focused on the patency of the dentinal tubules.This overview is largely based on the author’s own research, clinical insights and active participation in continuing dental education over the last 50 years. It is not a review of the literature related to the permeability of dentin. Rather it presents interpretation of results related to the permeability of dentin based on experience and opinions acquired over a lifetime in dental research.

A permeabilidade envolve a passagem de fluidos, íons, moléculas, material particulado e bactérias para o interior e através de uma substância ou tecido sob diferentes e variáveis condições. A permeabilidade da dentina é essencial para a fisiologia e para os padrões de reação do órgão dentino-pulpar. Nutrientes e impulsos são transportados a partir da polpa por meio dos processos odontoblásticos e o conteúdo dos seus túbulos mantém a vitalidade da dentina. Entretanto, o principal interesse deste artigo concentra-se na penetração do exterior para a polpa e não da polpa para o exterior. O presente artigo centra-se essencialmente nos túbulos dentinários; como eles são formados e como eles mudam em razão da função normal e anormal, idade e processos patológicos, e o efeito destes processos na permeabilidade da dentina. Especial atenção é dada à patência dos túbulos dentinários. Este artigo apresenta uma visão geral do assunto e baseia-se amplamente nas pesquisas e percepções clinicas do próprio autor e em sua ativa participação na educação odontológica continuada ao longo dos últimos 50 anos. Não se trata de uma revisão da literatura relacionada à permeabilidade dentinária, mas sim da interpretação de resultados relacionados à permeabilidade dentinária com base na experiência e opiniões formadas ao longo de toda uma vida dedicada à pesquisa odontológica.

Mecanismos envolvidos na diferenciação de células-tronco de dentes decíduos exfoliados humanos (SHED) em odontoblastos e células endoteliais / Mechanisms underlying the differentiation of stem cells from human exfoliated deciduous teeth (SHED) into odontoblasts and endothelial cells

A engenharia de tecido pulpar tem como objetivo substituir a polpa dentária inflamada ou necrosada por um tecido saudável e funcional, capaz de formar nova dentina para reparar a estrutura dentária perdida. Assim, os objetivos deste trabalho foram: avaliar a habilidade de diferenciação de células tronco de dentes decíduos exfoliados humanos (SHED) em odontoblastos funcionais, demonstrando a formação de tecido mineralizado in vivo; e estudar o efeito de VEGF em SHED com relação à estimulação de vias de sinalização celular (STAT3, AKT e ERK), proliferação, migração, formação de estruturas tubulares e diferenciação em células endoteliais. O início do processo de mineralização de SHED tratadas com dexametasona, ácido ascórbico 'beta' - glicerofosfato pôde ser detectado por meio da produção da enzima fosfatase alcalina a partir da segunda semana de cultura, mas a expressão de RNAm para DSPP só foi observada após 28 dias de indução. Utilizando-se o modelo de fatias de dentes e matrizes condutivas implantadas no dorso de camundongos imunodeprimidos, demonstrou-se a diferenciação de SHED em células semelhantes a odontoblastos, as quais tiveram imunomarcação positiva com o anticorpo DMP-1. A deposição de dentina, seguindo um ritmo centrípeto de crescimento, numa taxa de 14,1 µm por dia também foi demonstrada por meio da marcação com tetraciclina. O tratamento das SHED com VEGF estimulou a fosforilação de ERK e AKT e a diminuiu a fosforilação de STAT3 em um período de uma hora, provavelmente por meio de sua ligação com os receptores VEGFR-1 e NP-1 presentes nestas células. Além disso, VEGF intensificou a organização das SHED em estruturas tubulares, havendo diferença estatisticamente significativa entre os grupos tratado e não tratado a partir do 5o dia de tratamento. Entretanto, VEGF não estimulou a proliferação nem a migração destas células. Os resultados de RT-PCR mostraram que SHED cultivadas em fatias de dentes e ...

Dental pulp tissue engineering aims to replace the inflamed or necrotic pulp by a healthy and functionally competent tissue able to form new dentin in order to repair lost structure. The purposes of this work were: to evaluate the differentiation ability of stem cells from human exfoliated deciduous teeth (SHED) into functional odontoblasts, showing the formation of mineralized tissue in vivo; and to study the effect of VEGF on SHED with regards to the stimulation of cell signaling pathways (STAT3, AKT and ERK), the proliferation, migration, capillary sprouting, and the differentiation into endothelial cells. The beginning of the mineralization process of SHED treated with dexamethasone, ascorbic acid and beta-glycerophosphate could be detected through the production of alkaline phosphatase after the second week of culture, but the expression of DSPP mRNA was only observed after 28 days of induction. Using the tooth slice and scaffold model implanted in the dorsum of immunocompromised mice, the differentiation of SHED into odontoblast-like cells, which were immunostained with DMP-1 antibody, was demonstrated. Dentin deposition following a centripetal rhythm, in a rate of 14.1 µm per day, was also shown through the tetracycline labeling. VEGF treatment of SHED stimulated the ERK and AKT phosphorilation, and decreased the phosphorilation of STAT3 over 1 hour period, presumably due to its binding to VEGFR-1 and NP-1 receptors in these cells. In addition, VEGF enhanced SHED organization into tubular structures, with statistically significant difference between the treated group and the non-treated one after the 5th day of treatment. However, VEGF did not stimulate proliferation and migration of these cells. RT-PCR results demonstrated that SHED seeded in the tooth slices and scaffolds expressed VEGFR-2 after the first day of VEGF stimulation...

Effects of nimodipine on human dentinogenesis / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Studies have showed that L type calcium channel plays an important role in dentin calcification and affects tooth development and tooth reparation after injury. The objective of this article is to study the effects of nimodipine, blocking agent of L type calcium channel, on human dentinogenesis using human tooth slice organ culture in vitro.</p><p><b>METHODS</b>Young healthy human premolars were collected, and cut into 2 mm-thick transverse slices by low speed diamond saw. Agarose beads dipped in nimodipine solution and PBS weresy minetrically placed on tooth slices, and the slices were then embedded in a semisolid agarose-based medium and cultured with organ culture method for 1 week. Fluorescent band of tetracycline, Von-Kossa staining, immunohistochemical staining of the slices and transmission electron microscopy (TEM) of odontoblasts were observed to evaluate dentinogenesis changes of the slices.</p><p><b>RESULTS</b>Tooth slices were successfully cultured in vitro for 1 week and the odontoblasts could maintain their original morphology. After treatment with nimodipine, the fluorescent band of tetracycline was narrow and weak, and globular calcification in predentine was decreased compared with the control. TEM showed that secretory vesicles in odontoblast were somewhat increased, hut iminunohistochemical staining for collagen I showed no difference between the two groups.</p><p><b>CONCLUSION</b>Nimodipine can influence the calcification of dentine, but has no obvious influence on the synthesis and secretion of dentine matrix. The results show that L type calcium channel is important in dentin calcification.</p>