Effects of RhoA on the adherens junction of murine ameloblasts / 北京大学学报(医学版)

OBJECTIVE@#To investigate the regulation mechanism of RhoA signaling pathway during the enamel formation by using the EGFP-RhoADominant Negative (EGFP-RhoADN) transgenic mice model, from the aspect of adherens junctions, and to provide a theory basis for mechanism of enamel development defects.@*METHODS@#The enamel thickness of mandibular first molars of EGFP-RhoADN transgenic mice and wild type (WT) mice were observed by scanning electronic microscopy at 20 kV, and the enamel thickness of the distal face of the central cusp was measured at 10 locations via analysis by ImageJ (Rasband, 1997-2009). The enamel organs from mandibular first molars from postnatal-4-day (P4) EGFP-RhoADN mice and wild type mice were isolated, and the total RNA and protein were extracted from the epithelium of the enamel organs. The expression level of the adherens junctions components in ameloblasts layer of the postnatal-4-day EGFP-RhoADN transgenic mice and wild type mice mandibular first molars were detected by real-time PCR and Western blot assay.@*RESULTS@#The EGFP-RhoADN transgenic mice had decreased enamel thickness in their bilateral mandibular first molars versus those of control group (n=20), and enamel thickness was (84.60±0.20) μm vs. (106.24±0.24) μm, P<0.05. The protein expressions of E-cadherin, α-E-catenin and pan-cadherin in ameloblasts layer of postnatal-4-day EGFP-RhoADN transgenic mice molars were down-regulated, and the protein level of β-catenin in ameloblasts layer of P4 EGFP-RhoADN transgenic mice molars was up-regulated. The mRNA level of E-cadherin in ameloblasts layer of P4 EGFP-RhoADN transgenic mice molars was down-regulated versus that of WT mice, and the gene expression of E-cadherin was 0.93±0.01 vs. 1.00±0.02, P<0.05. The mRNA level of β-catenin in ameloblasts layer of P4 EGFP-RhoADN transgenic mice molars was up-regulated versus that of WT mice, and the gene expression of β-catenin was 1.23±0.03 vs. 1.00±0.05, P<0.05.@*CONCLUSION@#In the mandibular first molars of EGFP-RhoADN transgenic mice, the enamel formation was disrupted and the adherens junctions of EGFP-RhoADN transgenic mice ameloblasts were implicated during amelogenesis. RhoA signaling pathway may play a critical role in enamel development by altering the adherens junctions in ameloblasts.

Osteocalcin Expression and Mineralization in Developing Tooth of Xenopus laevis

Osteocalcin (OC) is the most abundant noncollagenous protein of extracellular matrix in the bone. In an OC deficient mouse, bone formation rates are increased in cancellous and cortical bones. OC is known as a negative regulator of mineral apposition. OC is also expressed in the tooth of the rat, bovine, and human. However, little is known about OC during tooth development in Xenopus. The purpose of this study is to compare the expression of OC with mineralization in the developing tooth of Xenopus, by using von Kossa staining and in situ hybridization. At stage 56, the developmental stage of tooth germ corresponds to the cap stage, and an acellular zone was apparent between the dental papilla and the enamel organ. From stage 57, calcium deposition was revealed by von Kossa staining prior to OC expression, and the differentiated odontoblasts forming predentin were located at adjoining predentin. At stage 58, OC transcripts were detected in the differentiated odontoblasts. At stage 66, OC mRNA was expressed in the odontoblasts, which was aligned in a single layer at the periphery of the pulp. These findings suggest that OC may play a role in mineralization and odontogenesis of tooth development in Xenopus.

Bivalent histone modifications during tooth development / 国际口腔科学杂志·英文版

Histone methylation is one of the most widely studied post-transcriptional modifications. It is thought to be an important epigenetic event that is closely associated with cell fate determination and differentiation. To explore the spatiotemporal expression of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) epigenetic marks and methylation or demethylation transferases in tooth organ development, we measured the expression of SET7, EZH2, KDM5B and JMJD3 via immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis in the first molar of BALB/c mice embryos at E13.5, E15.5, E17.5, P0 and P3, respectively. We also measured the expression of H3K4me3 and H3K27me3 with immunofluorescence staining. During murine tooth germ development, methylation or demethylation transferases were expressed in a spatial-temporal manner. The bivalent modification characterized by H3K4me3 and H3K27me3 can be found during the tooth germ development, as shown by immunofluorescence. The expression of SET7, EZH2 as methylation transferases and KDM5B and JMJD3 as demethylation transferases indicated accordingly with the expression of H3K4me3 and H3K27me3 respectively to some extent. The bivalent histone may play a critical role in tooth organ development via the regulation of cell differentiation.

Current Concepts and Occurrence of Epithelial Odontogenic Tumors: I. Ameloblastoma and Adenomatoid Odontogenic Tumor

Ameloblastomas and adenomatoid odontogenic tumors (AOTs) are common epithelial tumors of odontogenic origin. Ameloblastomas are clinico-pathologically classified into solid/multicystic, unicystic, desmoplastic, and peripheral types, and also divided into follicular, plexiform, acanthomatous, granular types, etc., based on their histological features. Craniopharyngiomas, derived from the remnants of Rathke's pouch or a misplaced enamel organ, are also comparable to the odontogenic tumors. The malignant transformation of ameloblastomas results in the formation of ameloblastic carcinomas and malignant ameloblastomas depending on cytological dysplasia and metastasis, respectively. AOTs are classified into follicular, extrafollicular, and peripheral types. Ameloblastomas are common, have an aggressive behavior and recurrent course, and are rarely metastatic, while AOTs are hamartomatous benign lesions derived from the complex system of the dental lamina or its remnants. With advances in the elucidation of molecular signaling mechanisms in cells, the cytodifferentiation of epithelial tumor cells in ameloblastomas and AOTs can be identified using different biomarkers. Therefore, it is suggested that comprehensive pathological observation including molecular genetic information can provide a more reliable differential diagnosis for the propagation and prognosis of ameloblastomas and AOTs. This study aimed to review the current concepts of ameloblastomas and AOTs and to discuss their clinico-pathological features relevant to tumorigenesis and prognosis.

The Epithelial-Mesenchymal Transition During Tooth Root Development

Hertwig's epithelial root sheath (HERS) consists of bilayered cells derived from the inner and outer dental epithelia and plays important roles in tooth root formation as well as in the maintenance and regeneration of periodontal tissues. With regards to the fate of HERS, and although previous reports have suggested that this entails the formation of epithelial rests of Malassez, apoptosis or an epithelialmesenchymal transformation (EMT), it is unclear what changes occur in the epithelial cells in this structure. This study examined whether HERS cells undergo EMT using a keratin-14 (K14) cre:ROSA 26 transgenic reporter mouse. The K14 transgene is expressed by many epithelial tissues, including the oral epithelium and the enamel organ. A distinct K14 expression pattern was found in the continuous HERS bi-layer and the epithelial diaphragm were visualized by detecting the beta-galactosidase (lacZ) activity in 1 week postnatal mice. The 2 and 4 week old mice showed a fragmented HERS with cell aggregation along the root surface. However, some of the lacZ-positive dissociated cells along the root surface were not positive for pan-cytokeratin. These results suggest that the K14 transgene is a valuable marker of HERS. In addition, the current data suggest that some of the HERS cells may lose their epithelial properties after fragmentation and subsequently undergo EMT.

Surgical and Orthodontic Treatment of Unicystic Ameloblastoma Related to an Impacted Molar Tooth in the Mandible: Case Report

Morphological analysis of the enamel organ in rats treated with fluoxetine

Purpose: Previous studies have evaluated the presence of serotonin in the dental epithelia and mesenchyme during odontogenesis, suggesting its participation in tooth development. Materials and methods: Here, we used fluoxetine, a selective serotonin re-uptake inhibitor, at a dose of 10 mg/kg, administered for 20 days during pregnancy in 12 Wistar rats to examine the influence of this drug on the development of the enamel organ of the upper first molars of rat fetuses at 17 days of intra-uterine life (i.u.l.), and at one, five and ten days postpartum. The pregnant rats were anesthetized with xylazine at 10 mg/kg and ketamine at 25 mg/kg. The fetuses were removed and beheaded; their jaws were removed, and the upper jaws were exposed. The tissues were fixed in Bouin's fixative, decalcified in 5 percent nitric acid for 4 - 12 h, conventionally processed for microscopy, and embedded in paraffin. Serial sections of approximately 5 mm were obtained and stained with hematoxylin and eosin, as well as periodic acid-Schiff. Results and conclusion: Morphological analysis showed no structural changes in the experimental group compared to the controls, suggesting that, at the dose used, fluoxetine does not interfere with serotonin-mediated development of the enamel organ or the process of amelogenesis.

Odontoameloblastoma: Report of two cases.

Odontoameloblastoma (OA) is a very rare mixed odontogenic neoplasm, characterized by the simultaneous occurrence of an ameloblastoma and a compound or complex odontoma in the same tumor mass. To date, less than 50 cases of OA and/or ameloblastic odontoma have been reported in the English dental literature. This neoplasm was called ameloblastic odontoma. The term OA was included in the 1971 WHO classification. In this study, we present two cases of OA, which we hope will contribute to the awareness and knowledge of surgeons regarding the existence of this odontogenic tumor so that patients having it may be treated and followed-up properly.

Utilização da matriz derivada do órgão do esmalte (endogain) em defeitos periodontais / The use of the enamel matrix derived organ (Emdogain) in periodontal defects

As doenças periodontais têm sua etiologia explicada pela presença do biofilme dental. Sabe-se que os produtos derivados do metabolismo bacteriano provocam alterações no padrão estrutural do periodonto, gerando alterações em cemento, ligamento periodontal e osso alveolar. A tentativa em se conseguir regeneração desses tecidos tem sido bastante estudada. O objetivo deste estudo foi realizar uma revisão da literatura sobre a utilização da matriz derivada do órgão do esmalte (Emdogain) em defeitos periodontais, em comparação com outras técnicas utilizadas. Concluímos que sua utilização promove formação de novo cemento e nova inserção, porém não se apresentou melhor que a regeneração tecidual guiada.

Evaluation of osteogenic activity and mineralization of cultured human dental papilla-derived cells

Experimental study of the dental follicle's function in tooth root development / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study role of dental follicle in tooth root development.</p><p><b>METHODS</b>Sixteen mandibular first molar dental germs from eight five-day postnatal Balb/c mice were divided into two groups randomly. Dental follicle of germs in one group was undetached and that of another group was removed. Subsequently, each of the germs was separately transplanted to back-muscles of adult nude mice. At seventh and fourteenth day after transplanting, the germs were collected, fixed, demineralized, dehydrated, and embedded in wax in sequence. Serial sections of 5 microm thick were made following the routine methods, stained with haematoxylin-eosin dying solution, and observed under a light microscope.</p><p><b>RESULTS</b>All implantations were located in the back-muscles with abundant capillary vasculature. Under microscope, although all tooth germs could further develop after grafting, tooth germs without dental follicle developed slowly with small size and low calcification compared to those with dental follicle. Although position of Hertwig's epithelial root sheath of all germs seemed no changing, roots of the group with dental follicle could further develop and the roots develop toward the apical direction; this tendency couldn't be seen in the germs of another group. Inflammatory cells could be seen in and out of the pulp cavity of the two groups at 7th day after grafting, while no obvious inflammatory cell was observed at 14th day after grafting.</p><p><b>CONCLUSION</b>Dental follicle play an important role in tooth root development. It probably can lead tooth root to develop in normal direction.</p>

Marsupialization in resolving dentigerous cysts: Case report

The effect of lead exposure in utero on the teeth eruption and enamel development of rat offspring / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effects of lead exposure at different levels in utero on the teeth eruption and enamel development of rat offsprings.</p><p><b>METHODS</b>27 pregnant SD rats were divided into three groups randomly: high level lead group (HLG), low level lead group (LLG) and control group with nine rats in each group. The three groups from the gestation day to the end of the gestation were given either deionized water in control group or deionized water containing 200 mg/L Pb2+ as lead acetate through drinking method in high level lead experimental group and 50 mg/L Pb2+ as lead acetate through drinking method in low level lead experimental group. The incisors of newborn rats were marked at the level of the gingival papilla on the 26th day after birth. On the 36th day, the incisors of newborn rats were marked again at the same level. Then the rat offsprings were killed and their blood was collected for lead analysis. The mandible incisors of rat offspring were separated and the content of Pb in incisors was determined by using a graphite furnace atomic absorption spectrometric method. The teeth of rat offspring were observed and the distance between two marks were measured by means of stereomicroscope. The ratio of calcium to phosphate of enamel of rat offspring was compared by electron probe microanalyses.</p><p><b>RESULTS</b>The level of blood lead in 200 mg/L, 50 mg/L treated rat offspring groups was higher than that in control group. The tooth lead of 200 mg/L, 50 mg/L treated rat offspring groups [(77.3 +/- 6.3), (27.8 +/- 4.5) microg/g] were higher than the control [(6.6 +/- 0.8) microg/g, P < 0.01]. Compared with the control group, the teeth of lead exposure experimental groups were smaller and severity of attrition was obvious and pulpal perforations were often observed. These appearances was more distinct in rats of high level lead experimental group. The incisors of lead-treated rat offspring erupted [(0.25 +/- 0.08), (0.30 +/- 0.09) mm/d] more slowly than control ones [(0.39 +/- 0.09) mm/d, P < 0.01]. The ratio of calcium to phosphate (Ca/P) decreased with the increase of lead exposure. It was found that Ca/P in lead exposure experimental groups (1.68 +/- 0.54), (1.37 +/- 0.47) was significantly lower than that in the control group (2.14 +/- 0.33).</p><p><b>CONCLUSION</b>Lead exposure in utero affects the normal eruption of teeth and enamel formation and the degree was related with the lead exposure level.</p>

Aspecto clinicopatológico e inmunohistoquímico en tres casos de ameloblastoma / Clinico-pathologic and immunohistochemical aspects in three ameloblastoma cases

El ameloblastoma es una de las neoplasias odontogénicas más comunes que se presentan en el maxilar inferior con mayor frecuencia, sector posterior, con localización intraósea y periférica (esta última, más rara). Los subtipos histológicos no influencian directamente en el tratamiento ni en el pronóstico porque generalmente se utilizan actos quirúrgicos radicales por la frecuencia de recidiva de los ameloblastomas. Se utilizó en biopsias las técnicas inmunohistoquímicas básicas y Mib-1 (Ki 67 en parafina) para determinar el índice de proliferación celular que indicaría la posible transformación maligna del ameloblastoma o su capacidad de recidiva. En un germen dentario, en estadio de campana, el órgano del esmalte mostró idéntica afinidad inmunohistoquímica con un índice de proliferación celular Mib 1 (Ki 67) de un 1 por ciento

Are there structural alterations in the enamel organ of offspring of rats with alloxan-induced diabetes mellitus?

A hipoplasia de esmalte é a mais comum dentre as alterações de desenvolvimento do dente humano, e ocorre com freqüência em dentes decíduos de filhos de mães diabéticas. O presente estudo experimental analisou, por meio de microscopia óptica e morfometria, o órgão do esmalte de incisivos inferiores de filhotes de ratas com diabetes aloxânico, induzido previamente à gestação. Os resultados mostraram que não foram observadas pela microscopia óptica alterações significantes nos germes dentais dos animais descendentes de ratas diabéticas, com exceção de um caso. A análise morfométrica dos órgãos do esmalte de ratos nascidos de mães diabéticas tratadas e não tratadas evidenciou as seguintes diferenças estatisticamente significantes: menor espessura da matriz de esmalte, menor altura dos ameloblastos e área de seus núcleos. Nos animais nascidos de ratas diabéticas tratadas, observou-se núcleos dos ameloblastos mais elípticos e aumento da área correspondente ao interstício do retículo estrelado. Estes resultados indicam que há alterações estruturais no órgão do esmalte de descendentes de ratas com diabetes aloxânico as quais poderiam induzir a hipoplasia do esmalte dental visto por microscopia eletrônica de varredura, possivelmente refletindo as alterações metabólicas observadas nesta condição. Estudos futuros devem ser realizados a fim de determinar se estas alterações são transitórias ou permanentes.

Expression and activity of matrix metalloproteinase-2 (MMP-2) in the development of rat first molar tooth germ

Tooth germ development is associated with morphological and biochemical changes of the dental papilla and enamel organ. Enzymes with gelatinolytic activities were studied by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzymography in tooth germ of newborn to 15-day-old rats. Three major bands with gelatinolytic activity were detected at all periods and characterized as the latent and active forms of MMP-2 using their molecular weight and activity dependent on Zn++ and Ca++ ions as criteria. Expression and activity of MMP-2 increased progressively from 0 to 15 days after birth. Mechanical separation of the tooth germ from 10-day-old rats showed that the gelatinolytic activity was localized mainly in the dental papilla and not the dental organ. These data indicate that the expression and activity of MMP-2 varies during the development and maturation of rat first molar tooth germ.

Apoptose na involução do retículo estrelado de germes dentários de molares de rato / Apoptosis in the involuting stellate reticulum of rat molar tooth germs

A síntese de esmalte inicia-se na regiao correspondente ao ápice da futura cúspide do dente em formaçao, e prossegue pelas vertentes do mesmo, em direçao a alça cervical. Assim que ocorrer a deposiçao da primeira camada de esmalte, o retículo estrelado (RE) adjacente inicia um processo de involuçao, o qual, envolve uma série de eventos, tais como: diminuiçao dos espaços intercelulares, invasao vascular, reduçao do número de células e aparecimento de células do tipo macrófago. Sabe-se que, o desaparecimento de várias células embrionárias durante o desenvolvimento de tecidos e órgaos, ocorre via apoptose, e que, macrófagos sao fagócitos relacionados com a remoçao de corpos apoptóticos. Assim sendo, decidimos examinar regioes do RE em involuçao, com a finalidade de verificar se apoptose está envolvida com este processo. Para isso, utilizamos germes dentários de primeiros molares de ratos Wistar com I, 3, e 5 dias de idade, os quais, foram fixados em formaldeído para o método TUNEL; forrnol-cálcio-sacarose, para detecçao de fosfatase ácida (método da pararosanilina hexazotizada); e em glutaraldeído-formaldeído para microscopia de luz e microscopia eletrônica. Através da microscopia de luz, observamos a presença de corpos basofílicos, tanto disperses pela matriz intercelular,, como, aparentemente, no interior de células macrófagos-like do RE. A presença dê estruturas TUNEL positivas, coradas em marrom,, confirma os dados observados através da microscopia de luz indicando fragmentaçao nuclear (corpos apoptóticos). Através da técnica da pararosanilina hexazotizada,, células arredondadas apresentaram granulaçoes intensamente positivas para fosfatase ácida, indicando atividade lisosômica. Nos cortes ultrafinos, foram observadas imagens de células do RE com núcleo contendo uma massa de cromatina periférica, eletron-opaca e com forma de meia-lua (típica de apoptose). Também observamos a presença de corpos apoptóticos, de tamanho e forma variados, no citoplasma de células do retículo, o que indica que estariam sendo englobados por estas células residentes. Células do tipo macrófago do retículo estrelado apresentam no citoplasma vacúolos contendo material de eletron-opacidade variável o que sugere tanto remoçao da matriz intercelular do retículo, como também a internalizaçao e digestao de corpos apoptóticos. Estas imagens nos levam a crer que, o fenômeno de morte celular programada (apoptose) ocorre nas células do RE e está relacionada com a involuçao ...(au)