RESUMO
Molecular information has been gathered from fossilized dental enamel, the best-preserved tissue of vertebrates. However, the association of morphological features with the possible mineral and organic information of this tissue is still poorly understood in the context of the emerging area of paleoproteomics. This study aims to compare the morphological features and chemical composition of dental enamel of extinct and extant terrestrial vertebrates of Crocodylia: Purussaurus sp. (extinct) and Melanosuchus niger (extant), and Rodentia: Neoepiblema sp. (extinct) and Hydrochoerus hydrochaeris (extant). To obtain structural and chemical data, superficial and internal enamel were analyzed by Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (SEM-EDS). Organic, mineral, and water content were obtained using polarizing microscopy and microradiography on ground sections of four teeth, resulting in a higher organic volume than previously expected (up to 49%). It is observed that both modern and fossil tooth enamel exhibit the same major constituents: 36.7% Ca, 17.2% P, and 41% O, characteristic of hydroxyapatite. Additionally, 27 other elements were measured from superficial enamel by inductively coupled mass spectrometry (ICP-MS). Zinc was the most abundant microelement detected, followed by Pb, Fe, Mg, and Al. Morphological features observed include enamel rods in the rodent teeth, while incremental lines and semiprismatic enamel were observed in the alligator species. The fossil enamel was in an excellent state for microscopic analyses. Results show that all major dental enamel's physical, chemical, and morphological features are present both in extant and extinct fossil tooth enamel (>8.5 Ma) in both taxa.
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We used two fossil teeth from South American Pleistocene mammals to obtain subsuperficial acid etching samples. We employed samples from the species Notiomastodon platensis and Myocastor cf. coypus for the enamel etchings. The controls included an extant rodent (rat). After the first etching was discarded, a second 20-s etching (i.e., subsuperficial) was directly collected with a ZipTip and injected into an LTQ Orbitrap Velos for MS analysis. The peptides were identified with different software programs that used Peptide Spectrum Match (PSM) and de novo sequencing including similarity search strategies. Most of the peptides that were recovered from the enamel of the fossils belonged to enamel-specific proteins. To our knowledge, this is the first study that has described the recovery of enamel peptide molecules from extinct South American taxa, indicating that enamel peptide data from late Pleistocene fossils can be employed as an additional parameter for phylogenetic analysis, and that the sample can be obtained by a very conservative acid etching, with almost no damage to the fossils. SIGNIFICANCE: This study shows that it is possible to obtain information based on plenty of ancient peptides recovered from subsuperficial enamel of fossil teeth from South American Pleistocene. The quality of the data suggests that peptides are likely the best preserved biomolecules under certain harsh environmental conditions. The recovery procedure only lasted 20 s and was minimally destructive to the fossils. This opens a myriad of new possibilities for the study of the past.
Assuntos
Fósseis , Peptídeos , Animais , Esmalte Dentário , Filogenia , RatosRESUMO
Eutherian dentition has been the focus of a great deal of studies in the areas of evolution, development, and genomics. The development of molar teeth is regulated by an antero-to-posterior cascade mechanism of activators and inhibitors molecules, where the relative sizes of the second (M2) and third (M3) molars are dependent of the inhibitory influence of the first molar (M1). Higher activator/inhibitor ratios will result in higher M2/M1 or M3/M1. Pax9 has been shown to play a key role in tooth development. We have previously shown that a G-quadruplex in the first intron of Pax9 can modulate the splicing efficiency. Using a sliding window approach with we analyzed the association of the folding energy (Mfe) of the Pax9 first intron with the relative molar sizes in 42 mammalian species, representing 9 orders. The Mfe of two regions located in the first intron of Pax9 were shown to be significantly associated with the M2/M1 and M3/M1 areas and mesiodistal lengths. The first region is located at the intron beginning and can fold into a stable G4 structure, whereas the second is downstream the G4 and 265 bp from intron start. Across species, the first intron of Pax9 varied in G-quadruplex structural stability. The correlations were further increased when the Mfe of the two sequences were added. Our results indicate that this region has a role in the evolution of the mammalian dental pattern by influencing the relative size of the molars.
Assuntos
Evolução Biológica , Eutérios/anatomia & histologia , Dente Molar/anatomia & histologia , Fator de Transcrição PAX9/metabolismo , Animais , Eutérios/metabolismo , Quadruplex G , ÍntronsRESUMO
Dental enamel is formed by rod-like structures, the enamel prisms. Groups of prisms are packed together in successive horizontal layers of alternating directions, known as Hunter-Schreger bands (HSBs). HSBs are the major microstructural characteristic of mammalian enamel. The pattern of HSBs can vary among mammalian species and this variability may provide relevant information regarding the species life history and taxon identification. In human HSBs can be used as a biometric-based parameter for personal identification in automated systems. The analysis of HSBs has been hampered by technical difficulties. The low contrast between light and dark bands and variations in light intensity may hinder the observation of HSBs in digital images. This article describes a simple and efficient computational procedure that greatly enhances the contrast and minimizes the differences in the intensity of illumination in HSBs images. Its use can significantly increase the quality and the number of HSBs that can be recorded in intact teeth.
Assuntos
Esmalte Dentário/ultraestrutura , Aumento da Imagem/métodos , Dente/ultraestrutura , Humanos , Processamento de Imagem Assistida por Computador/métodosRESUMO
BACKGROUND: Type 1 diabetes mellitus (T1DM) largely affects children, occurring therefore at the same period of deciduous and permanent teeth development. The aim of this work was to investigate birefringence and morphology of the secretory stage enamel organic extracellular matrix (EOECM), and structural and mechanical features of mature enamel from T1DM rats. METHODS: Adult Wistar rats were maintained alive for a period of 56 days after the induction of experimental T1DM with a single dose of streptozotocin (60 mg/kg). After proper euthanasia of the animals, fixed upper incisors were accurately processed, and secretory stage EOECM and mature enamel were analyzed by transmitted polarizing and bright field light microscopies (TPLM and BFLM), energy-dispersive x-ray (EDX) analysis, scanning electron microscopy (SEM), and microhardness testing. RESULTS: Bright field light microscopies and transmitted polarizing light microscopies showed slight morphological changes in the secretory stage EOECM from diabetic rats, which also did not exhibit statistically significant alterations in birefringence brightness when compared to control animals (P > .05). EDX analysis showed that T1DM induced statistically significant little increases in the amount of calcium and phosphorus in outer mature enamel (P < .01) with preservation of calcium/phosphorus ratio in that structure (P > .05). T1DM also caused important ultrastructural alterations in mature enamel as revealed by SEM and induced a statistically significant reduction of about 13.67% in its microhardness at 80 µm from dentin-enamel junction (P < .01). CONCLUSIONS: This study shows that T1DM may disturb enamel development, leading to alterations in mature enamel ultrastructure and in its mechanical features.
Assuntos
Esmalte Dentário/ultraestrutura , Animais , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Testes de Dureza , Microscopia Nuclear , Ratos Wistar , Espectrometria por Raios XRESUMO
OBJECTIVE: This study aimed to analyze the concentration and activity of carbonic anhydrase (CA) VI in the saliva of school children. We investigated the relationship among caries, CA VI concentration/activity, flow rate, pH, and buffering capacity. MATERIALS AND METHODS: Seventy-four school children were divided into a caries-free group and a caries group. Clinical examinations were conducted by one examiner according to World Health Organization criteria + early caries lesions. Salivary flow rate, pH, and buffering capacity were analyzed. Salivary CA VI concentration and activity were evaluated by ELISA and zymography, respectively. The data were analyzed using Student's t test and the Mann-Whitney test, and Pearson and Spearman correlation analyses were also done. In multivariate modeling, associations between variables were expressed as odds ratios. RESULTS: The results showed that salivary flow rate, salivary pH, and BC were significantly higher in the saliva of caries-free children. Also, the salivary CA VI concentration was significantly higher in the saliva of caries-free children. The salivary CA VI activity was higher in children with caries. We found a negative correlation between BC and dental caries. Also, in the caries group we found a positive correlation between the concentration and the activity of CA VI and a negative correlation between BC and CA VI activity. A negative correlation between salivary pH and CA VI concentration was observed in the caries-free group. A high activity of CA and a low salivary flow rate were associated with dental caries. CONCLUSION: These results support the conclusion that dental caries is highly affected by the activity of CA VI in saliva as well as by the salivary flow rate.
Assuntos
Anidrases Carbônicas/análise , Anidrases Carbônicas/fisiologia , Cárie Dentária/epidemiologia , Saliva/química , Saliva/enzimologia , Soluções Tampão , Criança , Estudos Transversais , Humanos , Concentração de Íons de Hidrogênio , SalivaçãoRESUMO
In most mammalian species enamel prisms are regularly arranged in layers of alternating directions forming an angle of approximately 90°. These successive layers of prisms are known as Hunter-Schreger bands (HSBs). The analysis of HSBs may provide valuable information regarding the species life history, taxon and personal identification, with evident applicability in physical anthropology and forensics. Obtaining good quality digital images of HSBs in intact specimens is not always a feasible task. The major problems are the low contrast of images; the reflection of incident light, which may create areas of intense shine in digital images; and the abrupt decrease in the degree of illumination that occurs after light crosses the vertical cracks, frequently present in enamel. We show here that the area of intense shine can be minimized by a polarizing filter coupled to the camera objective, and the filling of enamel cracks with corn oil can reduce refraction of light in enamel cracks. These procedures can significantly increase the quality and the area of HSBs that can be recorded in intact teeth.
Assuntos
Esmalte Dentário/anatomia & histologia , Imagem Óptica/métodos , Dente/anatomia & histologia , HumanosRESUMO
BACKGROUND: Chronic periodontitis represents a complex disease that is hard to control and is not completely understood. Evidence from past studies suggests that there is a key role for DNA methylation in the pathogenesis of periodontitis. However, all reports have applied technologies that investigate genes in a low throughput. In order to advance in the knowledge of the disease, we analyzed DNA methylation variations associated with gene transcription using a high-throughput assay. Infinium® HumanMethylation450 (Illumina) was performed on gingival samples from 12 periodontitis cases and 11 age-matched healthy individuals. Methylation data of 1,284 immune-related genes and 1,038 cell cycle-related genes from Gene Ontology (GO) and 575 genes from a dataset of stably expressed genes (genes with consistent expression in different physiological states and tissues) were extracted from a microarray dataset and analyzed using bioinformatics tools. DNA methylation variations ranging from -2,000 to +2,000 bp from the transcription start site (TSS) were analyzed, and the results were tested against a differential expression microarray dataset between healthy and periodontitis gingival tissues. Differences were evaluated using tests from the R Statistical Project. RESULTS: The comparison of probes between periodontitis and normal gingival tissues showed that the mean methylation scores and the frequency of methylated probes were significantly lower in genes related to the immune process. In the immune group, these parameters were negatively correlated with gene expression (Mann-Whitney test, p < 2.2e - 16). CONCLUSIONS: Our results show that variations in DNA methylation between healthy and periodontitis cases are higher in genes related to the immune-inflammatory process. Thus, DNA methylation must be modulating chromatin regions and, consequently, modulating the mRNA transcription of immune-inflammatory genes related with periodontitis, impacting the prognosis of disease.
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INTRODUCTION: The aim of this study was to compare the gelatinolytic activity of matrix metalloproteinase (MMP)-2 and MMP-9 and the expression of tissue inhibitor of matrix metalloproteinase (TIMP)-2 and myeloperoxidase protein (MPO) in clinically healthy human pulp and inflamed pulp tissue specimens. METHODS: Twenty dental pulps clinically diagnosed as inflammatory tissues and 20 healthy pulp tissues from enclosed third molars were harvested and evaluated. The gelatinolytic activity for MMP-2 and MMP-9 was assessed by using the zymography technique, TIMP-2 gene expression was evaluated using the enzyme-linked immunosorbent assay, and MPO was determined using the MPO assay. RESULTS: Data showed increased levels of MMP-9, active MMP-2, TIMP-2, and MPO in inflammatory pulp tissues compared with healthy tissues (P < .05). No statistical difference could be observed for pro-MMP-2 (P > .05). CONCLUSIONS: Although all samples were associated with MMP-2 expression, the active form of this MMP was observed only in inflamed pulps. Inflamed pulps showed an up-regulation of MMP-9, TIMP-2, and MPO.
Assuntos
Polpa Dentária/enzimologia , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Peroxidase/análise , Pulpite/enzimologia , Inibidor Tecidual de Metaloproteinase-2/análise , Exposição da Polpa Dentária/enzimologia , Precursores Enzimáticos/análise , Ensaio de Imunoadsorção Enzimática , Gelatinases/análise , Humanos , Inibidores de Proteases/análise , Regulação para CimaRESUMO
Parathyroid hormone participates in the metabolism of mineralized tissue. Its role in the formation of dentine is, as yet, incompletely understood. In the present study we analyzed the effect of transient (1 and 24-h/cycle) or continuous hPTH (1-34) treatment in odontoblast-like cells (MDPC-23) to the following parameters: mineral deposition detected by alizarin red, mRNA expression of the type I collagen (COL1), alkaline phosphatase (ALP), biglycan (BGN), matrix metalloproteinase 2 (MMP-2) and dentine sialophosphoprotein (DSPP) quantified by qRT-PCR. MMP-2 and ALP activities were quantified by zymography and colorimetric assay, respectively. The results showed that compared to Control group: intermittent PTH administration (1 and 24-h/cycle) decreased the mineral deposition and ALP activity. DSPP gene expression was not detected in both control and PTH treated cells. The PTH administration for 24-h/cycle increased the ALP, BGN and COL1 mRNA expression and continuous PTH treatment increased BGN and COL1 mRNA expression. Zymography assays showed that compared to Control group: PTH treatment for 1-h/cycle increased the total MMP-2 secretion and the continuous treatment decreased the secreted levels of MMP-2 active-form. Taken together, the results shown that PTH may regulate the odontoblast-like cells-induced secretion, and potentially this hormone can affect in vivo odontoblasts functions.
Assuntos
Odontoblastos/efeitos dos fármacos , Hormônio Paratireóideo/administração & dosagem , Fosfatase Alcalina/efeitos dos fármacos , Animais , Antraquinonas , Biglicano/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/efeitos dos fármacos , Colorimetria , Corantes , Esquema de Medicação , Proteínas da Matriz Extracelular/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Camundongos , Hormônio Paratireóideo/farmacologia , Fosfoproteínas/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/efeitos dos fármacos , Sais de Tetrazólio , TiazóisRESUMO
OBJECTIVES: Interleukin (IL)-8 is an important chemokine for regulation of the inflammatory response. A single nucleotide polymorphism (SNP) reference sequence (rs) 4073 in the IL8 gene has been shown to regulate IL-8 levels after stimulation with lipopolysaccharide. This study investigates the transmission pattern of the IL8 rs4073 risk allele A and its association with susceptibility to aggressive periodontitis (AgP) in families and in a case-control cohort of unrelated individuals from a Brazilian population. DESIGN: Genotyping was performed by standard polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) in 13 nuclear families and 184 unrelated subjects. Statistical analysis was performed using the transmission disequilibrium test (TDT) for the family dataset and Chi-square test and multivariate logistic regression modelling for the case-control dataset. RESULTS: TDT analyses did not detect evidence of over transmission of IL8 rs4073 alleles in affected and unaffected family members (allele T: 52%; allele A: 48%; p=0.2252). How expected, analyses of cases and unrelated controls showed a significant and inverse association of age with AgP; however, a lack of association between genotypes, ethnic groups and generalized AgP was observed. CONCLUSIONS: The SNP (rs4073) was not associated with AgP in unrelated individuals and there is no evidence of over transmission of the alleles in families with AgP, from Brazilian individuals.
Assuntos
Periodontite Agressiva/genética , Interleucina-8/genética , Polimorfismo de Nucleotídeo Único , Alelos , Brasil , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
INTRODUCTION: The aim of this study was to analyze the contribution of nonresident progenitor/stem cells and hematopoietic cells to reparative dentinogenesis. METHODS: Parabiosis was established between C57BL/6-TgN(ACTbEGFP)10sb/J transgenic mice (GFP+) and C57BL/6 wild-type mice (GFP-) to ensure blood cross-circulation between animals. Reparative dentinogenesis was stimulated by pulp exposures and capping on the first maxillary molar in the GFP- mice. Histologic sections of injured molars from GFP- mice were analyzed by epifluorescence microscopy to examine the contributions of GFP+ cells (nonresident progenitor cells and hematopoietic cells originating from GFP+ mice) to reparative dentinogenesis. RESULTS: GFP+ cells were detected in close association with reparative dentin formed at the site of pulp exposure in the maxillary first molars of the GFP- mice. CONCLUSIONS: The present study suggests the participation of the nonresident progenitor cells and hematopoietic cells in reparative dentinogenesis.
Assuntos
Dentinogênese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Parabiose/métodos , Células-Tronco/fisiologia , Fosfatase Ácida/análise , Compostos de Alumínio/uso terapêutico , Animais , Biomarcadores/análise , Compostos de Cálcio/uso terapêutico , Resinas Compostas/química , Circulação Cruzada/métodos , Materiais Dentários/química , Capeamento da Polpa Dentária/métodos , Exposição da Polpa Dentária/patologia , Exposição da Polpa Dentária/terapia , Dentina Secundária/fisiologia , Combinação de Medicamentos , Citometria de Fluxo , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Isoenzimas/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos Animais , Dente Molar/patologia , Dente Molar/fisiopatologia , Odontoblastos/patologia , Óxidos/uso terapêutico , Agentes de Capeamento da Polpa Dentária e Pulpectomia/uso terapêutico , Cimentos de Resina/química , Silicatos/uso terapêutico , Dióxido de Silício/química , Fosfatase Ácida Resistente a Tartarato , Zircônio/químicaRESUMO
OBJECTIVE: The purpose of this study is to investigate the effects of intermittent parathyroid hormone (PTH) administration on the apposition rate and structural features of dentine from mouse incisors. METHODS: Young male A/J Unib mice were treated daily for 6 and 10 days with 40 µg/kg of hPTH 1-34 or a vehicle. Dentine apposition rates measured by fluorescent labels (tetracycline and calcein) and alkaline phosphatase (ALP) plasma levels were evaluated after 6 days of treatment. Knoop microhardness testing and element content measurements in at.% of calcium (Ca), phosphorus (P), oxygen (O), and magnesium (Mg) in the peritubular and intertubular dentine were performed by Energy Dispersive X-ray (EDX) microanalysis via Scanning Electron Microscopy (SEM) after 10 days of treatment. RESULTS: Histometric analysis revealed an increase of 5% in the apposition rate of dentine and 25% in the ALP plasma levels in the PTH treated group. In addition, knoop microhardness testing revealed that the animals treated with PTH had a greater microhardness (11%). EDX microanalysis showed that PTH treatment led to increases in P (23%) and Ca (53%) at.% content, as well as the Ca/P ratio (24%) in peritubular dentine. The chemical composition of intertubular dentine did not vary between the groups. CONCLUSIONS: These findings indicate that intermittent administration of hPTH (1-34) increases apposition and mineralization of the dentine during young mice incisor formation.
Assuntos
Dentina/efeitos dos fármacos , Dentina/metabolismo , Hormônio Paratireóideo/farmacologia , Fosfatase Alcalina/sangue , Animais , Cálcio/análise , Dentina/ultraestrutura , Microanálise por Sonda Eletrônica , Fluoresceínas/administração & dosagem , Fluoresceínas/farmacologia , Dureza/efeitos dos fármacos , Magnésio/análise , Masculino , Camundongos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Hormônio Paratireóideo/administração & dosagem , Fósforo/análise , Distribuição AleatóriaRESUMO
This study evaluated the effect of zinc methacrylate (ZM) on the inhibition of matrix metalloproteinase 2 (MMP-2) and the ultimate tensile strength (UTS) of an experimental polymer. Enzymes secreted from mouse gingival tissues were analyzed by gelatin zymography in buffers containing 5 mM CaCl(2) (Tris-CaCl(2)) in 50 mM Tris-HCl buffer with various concentrations of ZM (0.5, 1, 2, 4, 8, and 16 mM). The matrix metalloproteinases present in the conditioned media were characterized by immunoprecipitation. The polymer UTS evaluation was performed in eight groups with various concentrations of ZM (0, 0.5, 1, 2.5, 5, 10, 20, and 30 wt.%), in a mechanical testing machine. MMP-2 (62 kDa) was detected in the zymographic assays and inhibited by ZM in all tested concentrations. UTS data were submitted to one-way ANOVA and Tukey's test (α = 0.05), and no significant differences were observed among groups, except in the polymer containing 30% ZM, presenting a significantly lower value when compared with the control group (p < 0.05). The results suggest that ZM inhibits MMP-2 expression in all concentrations tested, while small concentrations did not affect the ultimate tensile strength of the polymer. Zinc methacrylate is a metalloproteinase inhibitor that can be copolymerized with other methacrylate monomers. Yet, the addition of ZM did not affect the resin bond strength. Thus, in vivo tests should be performed to evaluate the performance of this material.
Assuntos
Materiais Dentários/química , Inibidores de Metaloproteinases de Matriz , Metacrilatos/química , Polietilenoglicóis/química , Ácidos Polimetacrílicos/química , Compostos de Zinco/química , Animais , Meios de Cultivo Condicionados , Inibidores Enzimáticos/farmacologia , Gengiva/enzimologia , Teste de Materiais , Metaloproteinase 2 da Matriz , Metacrilatos/farmacologia , Camundongos , Estresse Mecânico , Resistência à Tração , Técnicas de Cultura de Tecidos , Compostos de Zinco/farmacologiaRESUMO
INTRODUCTION: The aim of this study was to evaluate the effect of 10% ascorbic acid or 10% sodium ascorbate on organic matrix collagen of bovine dentin root canal walls after irrigation with 5.25% sodium hypochlorite (NaOCl), 17% ethylenediaminetetraacetic acid (EDTA), or 0.9% sodium chloride. METHODS: Eighty bovine incisors were randomly divided into 8 groups (n = 10): group 1, 0.9% sodium chloride (control); group 2, 5.25% NaOCl + 17% EDTA (NaOCl + EDTA); group 3, 5.25% NaOCl + 17% EDTA + 10% ascorbic acid (NaOCl + EDTA + AA); group 4, 5.25% NaOCl + 17% EDTA + 10% sodium ascorbate (NaOCl + EDTA + SA); group 5, 5.25% NaOCl (NaOCl); group 6, 17% EDTA; group 7, 10% ascorbic acid (AA); and group 8, 10% sodium ascorbate (SA). Teeth were chemomechanically prepared, submitted to histologic processing, and stained with Sirius Red dye to be analyzed under polarized light microscopy. Absorbance assay was also performed to confirm the loss of collagen. RESULTS: NaOCl + EDTA and NaOCl groups presented a significantly different birefringence pattern compared with the control group (P < .05). The measurement of the optical retardations of NaOCl + EDTA + SA indicated that this group was not statistically different from the control group. Although the measurement of the optical retardations of NaOCl + EDTA + AA was statistically different from the control group, the results were significantly higher than for NaOCl + EDTA. The birefringence of EDTA, AA, and SA groups was not statistically different from that of control group. The absorbance assay of NaOCl + EDTA and NaOCl groups confirmed the loss of collagen (P < .05). CONCLUSIONS: It is possible to conclude that 5.25% NaOCl, whether associated or not with 17% EDTA, causes birefringence alterations and loss of dentin collagen. These alterations reduced the ability of Sirius Red to bind with collagen fiber molecules. The reductions in the optical retardation values could be reversed by the application of either 10% ascorbic acid or 10% sodium ascorbate after 5.25% sodium hypochlorite and 17% EDTA irrigation.
Assuntos
Colágeno/efeitos dos fármacos , Dentina/efeitos dos fármacos , Substâncias Redutoras/farmacologia , Irrigantes do Canal Radicular/farmacologia , Animais , Ácido Ascórbico/farmacologia , Birrefringência , Bovinos , Colágeno/análise , Colágeno/química , Cavidade Pulpar/efeitos dos fármacos , Dentina/química , Ácido Edético/farmacologia , Matriz Extracelular/efeitos dos fármacos , Microscopia de Polarização , Distribuição Aleatória , Hipoclorito de Sódio/farmacologiaRESUMO
AIM: The objective of this study was to analyse the status of DNA methylation in the promoter region of the toll-like receptor (TLR)2 and TLR4 genes in gingival tissue samples from healthy subjects, smokers and non-smokers affected by chronic periodontitis. MATERIAL AND METHODS: Genomic DNA and total RNA were purified from gingival tissue using the TRIZOL reagent protocol. Genomic DNA was then digested by methylation-sensitive restriction enzymes, amplified by polymerase chain reaction (PCR), electrophoresed on a 10% polyacrylamide gel and stained using SYBR Gold. Real-time PCR was also performed to verify the transcript levels. RESULTS: The CpG dinucleotides analysed were observed to be unmethylated in the majority of DNA samples of the three groups and statistical differences were not found among groups (p>0.05). However, a trend towards methylation was observed in the TLR2 HhaI site in the samples of the periodontitis non-smoker groups. In fact, the analysis of all CpG sites together shows which complete methylation is observed in the shortest level in the samples of periodontitis non-smoker group. The analysis of transcript levels demonstrated no difference among groups (p>0.05). CONCLUSION: The results demonstrated major unmethylation of the TLR4 gene promoter in all groups. However, the results for the TLR2 gene promoter are inconclusive; this gene was found as a mosaic of methylated and unmethylated DNA in the majority of samples of the three groups and we also observed a trend towards the DNA methylation of CpG sites recognized by the HhaI enzyme.
Assuntos
Periodontite Crônica/genética , Gengiva/metabolismo , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Adulto , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Periodontite Crônica/imunologia , Periodontite Crônica/metabolismo , Ilhas de CpG/genética , Metilação de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fumar/genética , Estatísticas não Paramétricas , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
The aim of this study was to evaluate the effect of different concentrations of triethylene glycol dimethacrylate (TEGDMA) on the inhibition of matrix metalloproteinase 2 (MMP-2). Mouse gingival explants were cultured overnight in DMEM and the expression of secreted enzymes was analyzed by gelatin zymography in buffers containing 5 mM CaCl(2) (Tris-CaCl(2)) in 50 mM Tris-HCl buffer with the addition of TEGDMA at different concentrations (0.62%, 1.25%, 2.5%, or 5.0% (v/v)). The gelatinolytic proteinase present in the conditioned media was characterized as matrix metalloproteinase by means of specific chemical inhibition. The matrix metalloproteinases present in the conditioned media were characterized as MMP-2 by immunoprecipitation. The eletrophoretic bands were scanned and the transmittance values were analyzed. Data was plotted and submitted to linear regression to investigate MMP-2 inhibition as a function of TEGDMA concentration. Three major bands were detected in the zymographic assays. These bands were characterized as MMP-2. Zymogene (72 kDa), intermediate (66 kDa) and active forms of MMP-2 (62 kDa) were inhibited by TEGDMA in a dose-dependent way. These findings suggest that TEGDMA could inhibit MMP-2 expression even at small concentrations.
Assuntos
Resinas Compostas/farmacologia , Materiais Dentários/farmacologia , Gengiva/enzimologia , Inibidores de Metaloproteinases de Matriz , Polietilenoglicóis/farmacologia , Ácidos Polimetacrílicos/farmacologia , Animais , Resinas Compostas/administração & dosagem , Meios de Cultivo Condicionados , Relação Dose-Resposta a Droga , Ácido Edético/farmacologia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Imunoprecipitação , Indicadores e Reagentes , Teste de Materiais , Metaloproteinase 2 da Matriz/análise , Camundongos , Polietilenoglicóis/administração & dosagem , Ácidos Polimetacrílicos/administração & dosagem , Corantes de Rosanilina , Inibidores de Serina Proteinase/farmacologia , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
The formation of an ordered enamel organic extracellular matrix (EOECM) seems to be a crucial step for the proper formation of the enamel mineral phase. The ordered supramolecular structure of the EOECM in the secretory stage can be analyzed using polarizing microscopy, as it is strongly birefringent. Excessive fluoride (F) ingestion during tooth development can cause enamel fluorosis, leading to increased porosity in mature enamel. We analyzed the effects of F on the birefringence of the EOECM in the A/J, CBA, and DBA/2 strains of mice given 0, 11.25, and 45 ppm of fluoride in drinking water. In the CBA and DBA/2 strains, the 11.25 and 45 ppmF groups presented a significant decrease in optical retardation (OR) when compared with the respective 0 (CBA 11.25 ppmF p = 0.0056 and 45 ppmF p < 0.0001; DBA/2 11.25 and 45 ppmF p < 0.05). ORs in A/J 0 ppmF were significantly higher than in 45 (p < 0.0001). The enamel of the A/J strain was more severely affected by fluoride than it was in the other strains of mice and exhibited the lowest levels of fluoride in plasma, whereas its normal secretory enamel presented a significantly higher protein absorbance than it did in CBA and DBA mice (p = 0.0099 and p = 0.0025, respectively). The results showed that experimental fluorosis can alter the supramolecular organization of EOECM in the secretory stage of amelogenesis and that the susceptibility to dental fluorosis seems to be influenced by the inherent characteristics of the developing enamel.
Assuntos
Esmalte Dentário/efeitos dos fármacos , Esmalte Dentário/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fluoretos/farmacologia , Animais , Osso e Ossos/metabolismo , Dieta , Fluoretos/sangue , Fluorose Dentária/patologia , Camundongos , Microscopia de Polarização , Pigmentação/efeitos dos fármacosRESUMO
OBJECTIVES: PAX9 belongs to the Pax family of transcriptional factor genes. This gene is expressed in embryonic tissues such as somites, pharyngeal pouch endoderm, distal limb buds and neural crest-derived mesenchyme. Polymorphisms in the upstream promoter region of the human PAX9 have been associated with human non-syndromic tooth agenesis. In the present study, we verified the in vitro mRNA expression of this gene and the luciferase activity of two constructs containing promoter sequences of the PAX9 gene. MATERIAL AND METHODS: Embryonic tissues were obtained from digits, face, and midbrain/hindbrain regions. Fragments containing PAX9 promoter sequences were cloned into reporter plasmids and were transfected into the different cell cultures. mRNA were extracted from primary cell cultures. RESULTS: The semi-quantitative RT-PCR results showed that in vitro E13.5 limb bud and CNS cells express PAX9, but cells derived from the facial region do not. Moreover, the luciferase assay showed that protein activity of the constructed vector was weaker than pgl3 -basic alone. CONCLUSIONS: The present results suggest that the promoter sequences analyzed are not sufficient to drive PAX9 gene transcription.
Assuntos
Anodontia/genética , Perfilação da Expressão Gênica , Luciferases/análise , Fator de Transcrição PAX9/genética , Transcrição Gênica , Animais , Células Cultivadas , Humanos , Luciferases/genética , Fator de Transcrição PAX9/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas , RNA Mensageiro , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVES: PAX9 belongs to the Pax family of transcriptional factor genes. This gene is expressed in embryonic tissues such as somites, pharyngeal pouch endoderm, distal limb buds and neural crest-derived mesenchyme. Polymorphisms in the upstream promoter region of the human PAX9 have been associated with human non-syndromic tooth agenesis. In the present study, we verified the in vitro mRNA expression of this gene and the luciferase activity of two constructs containing promoter sequences of the PAX9 gene. MATERIAL AND METHODS: Embryonic tissues were obtained from digits, face, and midbrain/hindbrain regions. Fragments containing PAX9 promoter sequences were cloned into reporter plasmids and were transfected into the different cell cultures. mRNA were extracted from primary cell cultures. RESULTS: The semi-quantitative RT-PCR results showed that in vitro E13.5 limb bud and CNS cells express PAX9, but cells derived from the facial region do not. Moreover, the luciferase assay showed that protein activity of the constructed vector was weaker than pgl3 -basic alone. CONCLUSIONS: The present results suggest that the promoter sequences analyzed are not sufficient to drive PAX9 gene transcription.