Experimental Furcal Perforation Treated with MTA: Analysis of the Cytokine Expression.

The aim of this study was to evaluate mineral trioxide aggregate (MTA)-induced cytokine expression in mice after experimental furcal perforation. BALB/c mice (n=5) were subjected to induced furcal drilling of the maxillary first molar followed by MTA sealing in the left side (experimental group) and paraffin sealing in the right side (control group). Animals were euthanized at 7, 14 and 21 days after sealing the perforations. The expression levels of the IFN-γ, TNF-α, IL-10, IL-4, TGF-ß and RANKL genes were investigated by real time polymerase chain reaction (PCR) in the teeth and surrounding tissues. In the experimental groups, after the 7th day, there was a down-regulation of the mRNA levels of TNF-α and IL-4 compared to the 14th day (p<0.05). In these groups, the mRNA levels of RANKL, IFN-γ and TNF-α were statistically higher after 14 days compared to 21 days post-MTA sealing (p<0.05). The level of IL-10 mRNA was increased at the 21st day (p<0.05). The mRNA expression of TGF-ß did not exhibit any statistically relevant results. There was a statistical down-regulation of IL-4 gene expressions when control and experimental groups were compared at days 7 and 21. In conclusion, MTA sealing favored the expression of pro-inflammatory cytokines in the intermediate phase of the immuno-inflammatory response (14th day). The reduction of these cytokines in later phase of the response was probably due to immunoregulation by IL-10.

Effects of immediate and delayed intraradicular preparation on bond strength of fiber posts.

BACKGROUND: To assist the retention of restorations prepared in endodontically treated teeth, fiber posts are widely used in dental practice. The ideal time to prepare space for the post is still controversial. AIM: The purpose of this study was to evaluate the effects of immediate and delayed postspace preparation on the retention of the self-adhesive resin cement. MATERIALS AND METHODS: Twelve bovine teeth were used with sectioned roots standardized to 19 mm. The teeth were properly instrumented and filled with gutta-percha and eugenol-free cement AH Plus. Two experimental groups (n = 6) were created based on the different times of post preparation (immediate and delayed). Using cemented cylindrical fiber posts, the teeth were put in acrylic resin and polyvinyl chloride tubes, where the cuts were made. Two slices were obtained from the cervical third, two from the medium third and two from the apical third. Subsequently, the samples were subjected into push-out tests. Statistical analysis were performed using ANOVA and Tukey's test (P = 5%). RESULTS: The results indicated that, for all of the evaluated thirds, delayed preparation showed greater bond strength than immediate preparation. CONCLUSION: A delayed post preparation of the root space, following the root canal filling, is recommended.

Experimental Furcal Perforation Treated with MTA: Analysis of the Cytokine Expression

The aim of this study was to evaluate mineral trioxide aggregate (MTA)-induced cytokine expression in mice after experimental furcal perforation. BALB/c mice (n=5) were subjected to induced furcal drilling of the maxillary first molar followed by MTA sealing in the left side (experimental group) and paraffin sealing in the right side (control group). Animals were euthanized at 7, 14 and 21 days after sealing the perforations. The expression levels of the IFN-γ, TNF-α, IL-10, IL-4, TGF-β and RANKL genes were investigated by real time polymerase chain reaction (PCR) in the teeth and surrounding tissues. In the experimental groups, after the 7th day, there was a down-regulation of the mRNA levels of TNF-α and IL-4 compared to the 14th day (p<0.05). In these groups, the mRNA levels of RANKL, IFN-γ and TNF-α were statistically higher after 14 days compared to 21 days post-MTA sealing (p<0.05). The level of IL-10 mRNA was increased at the 21st day (p<0.05). The mRNA expression of TGF-β did not exhibit any statistically relevant results. There was a statistical down-regulation of IL-4 gene expressions when control and experimental groups were compared at days 7 and 21. In conclusion, MTA sealing favored the expression of pro-inflammatory cytokines in the intermediate phase of the immuno-inflammatory response (14th day). The reduction of these cytokines in later phase of the response was probably due to immunoregulation by IL-10.

O objetivo desse artigo foi avaliar a expressão das citocinas induzidas por MTA após a perfuração experimental de furca, em camundongos. Camundongos Balb/c (n=5) foram submetidos à perfuração induzida da furca do primeiro molar superior, seguido pelo selamento da mesma com MTA no lado esquerdo (grupo experimental) e com parafina no lado direito (grupo controle). Os animais foram sacrificados 7, 14 e 21 dias após o tratamento da perfuração. A expressão gênica dos níveis de IFN-γ, TNF- α, IL-10, IL-4, TGF- β e RANKL foram investigadas por PCR em tempo real nos dentes e tecidos adjacentes. No grupo experimental, após 7 dias, houve uma diminuição da expressão dos níveis de TNF- α e IL-4 comparados ao 14° dia (p<0,05). Nesses mesmos grupos, os níveis de mRNA de RANKL, IFN- γ e TNF- α foram estatisticamente maiores após 14 dias comparados a 21 dias após o tratamento com MTA (p<0,05). Os níveis de IL-10 estavam aumentados no 21^o dia (p<0,05). A expressão de mRNA do TGF- β não apresentou alteração estatisticamente relevante. Houve uma redução estatística da expressão gênica da IL-4 quando os grupos controle e experimental foram comparados nos dias 7 e 21. Em conclusão, o selamento com MTA favoreceu a expressão de citocinas pró-inflamatórias na fase intermediária da resposta imuno-inflamatória (14^o dia). A redução dessas citocinas, na fase tardia da resposta, ocorreu provavelmente devido à imunoregulação da expressão de IL-10.

Methylation pattern of the CD14 and TLR2 genes in human dental pulp.

INTRODUCTION: Pattern recognition receptors, such as toll-like receptor 2 (TLR-2) and TLR-4, participate in the activation of immune cells by microorganisms in dental pulp. However, the expression levels of pattern recognition receptors can be modulated by epigenetic factors, especially DNA methylation. In this study, the methylation status of the TLR-2 and CD14 (TLR4 co-receptor) genes in healthy and inflamed human dental pulp was examined. METHODS: The Methyl-Profiler DNA Methylation qPCR Assay was used to verify the DNA methylation patterns. RESULTS: No differences in the methylation patterns were observed between the 2 groups. Most DNA was unmethylated in both groups. CONCLUSIONS: The hypomethylation of TLR2 and CD14 genes is a usual feature in human dental pulp.

Avaliação do padrão de metilação em genes relacionados à resposta imune em polpa dental humana / Evaluation of pattern of mehylation in genes related to the immune response in human dental pulp

Receptores de reconhecimento padrão (PRRs), como o Toll-like receptor 2 (TLR-2) e o TLR4, participam da ativação do sistema imune por microrganismos quando a polpa dental está inflamada. Supões-se que seus níveis de expressão em tecido pulpar humano podem ser modulados por fatores epigenéticos, especialmente a metilação do DNA. O objetivo deste estudo foi avaliar o padrão de metilação dos genes TLR-2 e CD14 - um correceptor do TLR4 - em polpas dentais saudáveis e inflamadas. Adicionalmente, verificou-se a associação entre o padrão de metilação do gene interferon-gama (IFN-y), previamente por nós identificado, com sua expressão gênica em polpas dentárias...

Hypomethylation of tumor suppressor genes in odontogenic myxoma.

Odontogenic myxoma (OM) is an ectomesenchymal benign odontogenic tumor characterized by spindle or stellate-shaped cells embedded in an abundant myxoid or mucoid extracellular matrix. DNA methylation is characterized by the addition of methyl groups in cytosines within CpG islands in the promoter gene. DNA methylation can decrease the expression of tumor suppressor genes and contribute to the development of neoplastic lesions. The aim of study was to evaluate the methylation pattern of the tumor suppressor genes P16 (CDKN2A), P21 (CDKN1A), P27 (CDKN1B), P53 (TP53) and RB1 in OM and dental pulp. Methylation was evaluated using methylation-specific polymerase chain reaction (PCR). The transcription was studied in some cases by using reverse transcription quantitative PCR. A higher frequency of unmethylated P27, P53, and RB1 samples was observed in the OM when compared with the dental pulp. OM expressed mRNA of all the genes evaluated. Considering all the samples together, the expression of Rb was higher in the unmethylated samples compared with the partially methylated samples. This investigation revealed hypomethylation of the genes P27, P53, and RB1 in OM. In addition, methylation of tumor suppressor genes was found to be an usual event in normal dental pulp.

Methylation pattern of IFN-γ and IL-10 genes in periodontal tissues.

UNLABELLED: DNA methylation is characterized by the addition of methyl groups in cytosines within CpG islands. Unmethylated CpGs are related to transcriptionally active structure, whereas methylated CpG recruits methyl-binding proteins that promote chromatin compaction. DNA methylation can influence the expression of cytokines and affect the development of periodontal disease. OBJECTIVES: The purpose of the present study was to evaluate the methylation status of the interferon gamma (IFN-γ) and interleukin-10 (IL-10) genes in periodontal tissues. DESIGN: Methylation-specific polymerase chain reaction (MSP) and DNA sequencing analysis were used to verify the DNA methylation status of the IFN-γ and IL-10 genes, respectively, in samples from subjects without periodontitis and individuals with chronic periodontitis. Histological sections stained by hematoxylin-eosin were used for histopathological evaluation of samples. RESULTS: The methylation status of the IFN-γ and IL-10 genes was similar among the groups. Most of the samples were positive for IFN-γ methylation. Only 11% of the periodontitis group showed unmethylated DNA. Considering the IL-10 gene, no unmethylated sample was observed. The profile of total or partial methylation was detected in CpGs evaluated. CONCLUSIONS: The results showed evidence that methylation of IFN-γ and IL-10 genes is a usual feature on periodontal tissues. Further studies are needed to determine the functional relevance of these alterations.

Hypomethylation of tumor suppressor genes in odontogenic myxoma

Odontogenic myxoma (OM) is an ectomesenchymal benign odontogenic tumor characterized by spindle or stellate-shaped cells embedded in an abundant myxoid or mucoid extracellular matrix. DNA methylation is characterized by the addition of methyl groups in cytosines within CpG islands in the promoter gene. DNA methylation can decrease the expression of tumor suppressor genes and contribute to the development of neoplastic lesions. The aim of study was to evaluate the methylation pattern of the tumor suppressor genes P16 (CDKN2A), P21 (CDKN1A), P27 (CDKN1B), P53 (TP53) and RB1 in OM and dental pulp. Methylation was evaluated using methylation-specific polymerase chain reaction (PCR). The transcription was studied in some cases by using reverse transcription quantitative PCR. A higher frequency of unmethylated P27, P53, and RB1 samples was observed in the OM when compared with the dental pulp. OM expressed mRNA of all the genes evaluated. Considering all the samples together, the expression of Rb was higher in the unmethylated samples compared with the partially methylated samples. This investigation revealed hypomethylation of the genes P27, P53, and RB1 in OM. In addition, methylation of tumor suppressor genes was found to be an usual event in normal dental pulp.

O mixoma odontogênico (MO) é um tumor odontogênico benigno de origem mesenquimal caracterizado pela presença de células fusiformes ou estreladas dispostas em abundante matriz extracelular mucóide. A metilação do DNA é caracterizada pela adição de grupos metil em citosinas constituintes de ilhas CpG na região promotora do gene. A metilação pode diminuir a expressão de genes supressores de tumor e contribuir para o desenvolvimento de lesões neoplásicas. O objetivo deste trabalho foi avaliar o padrão de metilação nos genes P16 (CDKN2A), P21 (CDKN1A), P27 (CDKN1B), P53 (TP53), RB1 nos MO e na polpa dental. A metilação foi avaliada pela reação em cadeia da polimerase específica para a metilação. A transcrição dos genes foi estudada em alguns casos pela reação da transcriptase reversa (PCR quantitativa). Uma maior frequência de amostras não metiladas para os genes P27, P53 e RB1 foi observada nos MO quando comparados à polpa dental. Os MO expressaram RNAm de todos os genes avaliados. Considerando todas as amostras juntas, a expressão de Rb foi maior em amostras não metiladas comparadas as amostras parcialmente metiladas. Esta investigação mostrou a hipometilação dos genes P27, P53 e RB1 nos MO. Adicionalmente, a metilação nos genes supressores de tumor é um evento frequente em polpa dental normal.

Methylation pattern of the IFN-gamma gene in human dental pulp.

INTRODUCTION: DNA methylation is characterized by the addition of methyl groups in cytosines within cytosine-phosphate-guanine (CpG) islands. Unmethylated islands are related with transcriptionally active structure, whereas methylated DNA recruits methyl-binding proteins that promotes chromatin compaction. Although epigenetic events can influence the expression of cytokines, such events have not been investigated in dental pulp yet. The purpose of the present study was to evaluate the methylation status of the interferon gamma (IFN-gamma) gene in human dental pulp affected by inflammation compared with pulp tissue of impacted molar teeth and to verify the impact of methylation status in the expression pattern of the gene. METHODS: Methylation-specific polymerase chain reaction (MSP) was used to verify the DNA methylation status of the IFN-gamma gene in 16 human dental pulps affected by inflammation and in 16 pulp samples of impacted molar teeth. Histologic sections stained by hematoxylin-eosin were used for histopathological evaluation, and the expression of IFN-gamma was assessed by quantitative real-time PCR (qPCR). RESULTS: Although total methylation was observed in 43.75% of the samples of normal dental pulp tissues, partial methylation or unmethylation was found in 93.75% of the samples of inflamed pulp tissues. All the samples with total methylation in MSP showed no transcription of IFN-gamma. The qPCR results showed expression of IFN-gamma in 5 of 10 samples with partial methylation. CONCLUSION: The present study gives the first evidence of the possible participation of epigenetic events in the development of dental pulp inflammation.