Sphingosine-1-phosphate hinders the osteogenic differentiation of dental pulp stem cells in association with AKT signaling pathways / 国际口腔科学杂志·英文版

Sphingosine-1-phosphate (S1P) is an important lipid mediator that regulates a diverse range of intracellular cell signaling pathways that are relevant to tissue engineering and regenerative medicine. However, the precise function of S1P in dental pulp stem cells (DPSCs) and its osteogenic differentiation remains unclear. We here investigated the function of S1P/S1P receptor (S1PR)-mediated cellular signaling in the osteogenic differentiation of DPSCs and clarified the fundamental signaling pathway. Our results showed that S1P-treated DPSCs exhibited a low rate of differentiation toward the osteogenic phenotype in association with a marked reduction in osteogenesis-related gene expression and AKT activation. Of note, both S1PR1/S1PR3 and S1PR2 agonists significantly downregulated the expression of osteogenic genes and suppressed AKT activation, resulting in an attenuated osteogenic capacity of DPSCs. Most importantly, an AKT activator completely abrogated the S1P-mediated downregulation of osteoblastic markers and partially prevented S1P-mediated attenuation effects during osteogenesis. Intriguingly, the pro-inflammatory TNF-α cytokine promoted the infiltration of macrophages toward DPSCs and induced S1P production in both DPSCs and macrophages. Our findings indicate that the elevation of S1P under inflammatory conditions suppresses the osteogenic capacity of the DPSCs responsible for regenerative endodontics.

Blockade of PD-L1/PD-1 signaling promotes osteo-/odontogenic differentiation through Ras activation / 国际口腔科学杂志·英文版

The programmed cell death ligand 1 (PD-L1) and its receptor programmed cell death 1 (PD-1) deliver inhibitory signals to regulate immunological tolerance during immune-mediated diseases. However, the role of PD-1 signaling and its blockade effect on human dental pulp stem cells (hDPSCs) differentiation into the osteo-/odontogenic lineage remain unknown. We show here that PD-L1 expression, but not PD-1, is downregulated during osteo-/odontogenic differentiation of hDPSCs. Importantly, PD-L1/PD-1 signaling has been shown to negatively regulate the osteo-/odontogenic differentiation of hDPSCs. Mechanistically, depletion of either PD-L1 or PD-1 expression increased ERK and AKT phosphorylation levels through the upregulation of Ras enzyme activity, which plays a pivotal role during hDPSCs osteo-/odontogenic differentiation. Treatment with nivolumab (a human anti-PD-1 monoclonal antibody), which targets PD-1 to prevent PD-L1 binding, successfully enhanced osteo-/odontogenic differentiation of hDPSCs through enhanced Ras activity-mediated phosphorylation of ERK and AKT. Our findings underscore that downregulation of PD-L1 expression accompanies during osteo-/odontogenic differentiation, and hDPSCs-intrinsic PD-1 signaling inhibits osteo-/odontogenic differentiation. These findings provide a significant basis that PD-1 blockade could be effective immunotherapeutic strategies in hDPSCs-mediated dental pulp regeneration.

Experimentally induced diabetes mellitus influences expression of VEFGand CD68 in rat teeth pulp / La diabetes mellitus inducida experimentalmente influye en la expresión de VEGF y CD68 en la pulpa de dientes de rata

The purpose of this study was to examine the expression levels of the dental pulp to elucidate the role of Vascular Endothelial Growth Factor (VEGF) and CD68 on vascular angiogenesis, inflammation and odontoblast differentiation in the pulp tissue of diabetic rats depending on the effect of possible damage induced by diabetes. Wistar rats were used in the study, divided into two groups. Control group was fed with standard rat chow and drinking water ad libitum for 8 weeks. Single dose of streptozotocin (STZ) (55 mg/kg), was disolved in sodium citrate buffer and administered by intraperitoneal injection. Blood glucose concentration of rats exceeding 250 mg/dl were accepted as diabetic. Rats were sacrificed under anesthesia. Tissues were immediately dissected, fixed and embedded in paraffin and cut with a microtome then examined under light microscope. In the cross-sections of pulp tissue of diabetic group; the dilation of blood vessels besides hemorrhage and a significant increase in inflammatory cells were seen. The expression of VEGF in the blood vessel endothelial cells of the pulp was increased. VEGF showed positive reaction for degenerative odontoblast cells in the pulp. In this study, increase in VEGF and CD68 expressions in pulp tissue due to the effect of diabetes was thought to delay pulp treatment by inducing soft tissue damage and hypoxia.

El propósito de este estudio fue examinar los niveles de expresión en la pulpa dental para dilucidar el papel del Factor de Crecimiento Endotelial Vascular (VEGF) y el CD68 en la angiogénesis, la inflamación y la diferenciación de odontoblastos en el tejido pulpar de ratas diabéticas, dependiendo del efecto de daño inducido por la diabetes. Se utilizaron ratas Wistar divididas en dos grupos. El grupocontrol se alimentó con comida estándar para ratas y agua potable ad libitum durante 8 semanas. Se administró mediante inyección intraperitoneal dosis única de estreptozotocina (STZ) (55 mg / kg), se disolvió en tampón de citrato de sodio. La concentración de glucosa en sangre de ratas que excedían los 250 mg / dl se aceptó como diabética. Las ratas fueron sacrificadas bajo anestesia. Los tejidos se disecaron de inmediato, se fijaron en parafina y se cortaron para luego ser examinados con un microscopio óptico. En las secciones transversales del tejido pulpar del grupo diabético se observó la dilatación de los vasos sanguíneos además de hemorragia y un aumento significativo de células inflamatorias. La expresión de VEGF se incrementó en las células endoteliales de los vasos sanguíneos de la pulpa. VEGF mostró una reacción positiva para las células odontoblásticas degenerativas en la pulpa. El aumento en la expresión de VEGF y CD68 en el tejido de la pulpa debido al efecto de la diabetes puede retrasar el tratamiento de la pulpa al inducir hipoxia y daños en los tejidos blandos.

Effect of dental bleaching on pulp oxygen saturation in maxillary central incisors - a randomized clinical trial

Abstract Objective To assess pulp oxygen saturation levels (SaO2) in maxillary central incisors after dental bleaching. Materials and Methods 80 participants (160 teeth) were randomly allocated to four groups: G1 In-office bleaching with two applications of 35% hydrogen peroxide (HP) (20 minutes), followed by at-home bleaching with 10% carbamide peroxide (CP) (2 hours/day for 16 days); G2 - Same protocol as G1, plus desensitizing toothpaste; G3 - In-office bleaching with 35% HP and one application of placebo gel (20 minutes), followed by at-home bleaching with 10% CP (2 hours/day for 16 days); and G4 - Same protocol as G3, plus desensitizing toothpaste. Pulp SaO2 levels were measured before (T0) and immediately after (T1) in-office bleaching; on the 5th (T2), 8th (T3), 12th (T4), and 16th days of at-home bleaching (T5); and on the 7th (T6) and 30th (T7) days. Mean (SD) pulp SaO2 levels were compared within groups by generalized estimating equations (GEE) and Student's t-test (P<0.05). Results Mean pulp SaO2 at T0 was 84.29% in G1, 84.38% in G2, 84.79% in G3, and 85.83% in G4. At T1, these values decreased to 81.96%, 82.06%, 82.19%, and 81.15% in G1, G2, G3, and G4 respectively, with significant difference in G4 (P<0.05). During home bleaching, pulp SaO2 levels varied in all groups, with 86.55%, 86.60%, 85.71%, and 87.15% means at T7 for G1, G2, G3, and G4, respectively; G2 presented significant difference (P<0.05). Conclusions Pulp SaO2 level in maxillary central incisors was similar at baseline, reducing immediately after in-office bleaching, regardless of using desensitizing toothpaste and increasing at 30 days after dental bleaching.

Effect of at-home bleaching on oxygen saturation levels in the dental pulp of maxillary central incisors

Abstract The present study assessed oxygen saturation (SaO2) levels before, during, and after at-home bleaching treatment in the pulps of healthy maxillary central incisors. SaO2 levels were measured in 136 healthy maxillary central incisors using a pulse oximeter. The bleaching protocol consisted of 10% carbamide peroxide gel placed in individual trays and used for four hours daily for 14 days. SaO2 levels were assessed before bleaching (T0), immediately after the first session (T1), on the 7th day of treatment (T2), on the 15th day (the day following the last session) (T3), and 30 days after completion of the bleaching protocol (T4). Data were statistically analyzed using generalized estimating equations (GEE), Student's t test (p<0.05) and Pearson's correlation. Mean pulp SaO2 levels were 85.1% at T0, 84.9% at T1, 84.7% at T2, 84.3% at T3, and 85.0% at T4. Gradual reductions in SaO2 levels were observed, with significant differences (p<0.001) during the course of home bleaching treatment. However, 30 days after the end of the bleaching protocol, SaO2 levels returned to baseline levels. Home bleaching caused a reversible transient decrease in SaO2 levels in the pulps.

Resumo Este estudo verificou o grau de saturação de oxigênio (SaO2) pulpar antes, durante e após o clareamento dental caseiro em incisivos centrais superiores hígidos. O nível de SaO2 foi verificado em 136 incisivos centrais superiores hígidos usando oxímetro de pulso. A técnica de clareamento empregou peróxido de carbamida 10% em moldeira individual por quatro horas diárias durante 14 dias. Os níveis de SaO2 foram analisados antes do clareamento (T0), imediatamente após a primeira sessão (T1), no sétimo dia de tratamento (T2), no décimo quinto dia (um dia após a última sessão) (T3) e 30 dias após o término do clareamento dental (T4). A análise estatística utilizou o modelo de equações de estimações generalizadas (GEE), teste t de Student (p<0,05) e correlação de Pearson. Os níveis médios de SaO2 pulpar foram 85,1% em T0, 84,9% em T1, 84,7% em T2, 84,3% em T3 e 85,0% em T4. Foi observada uma redução gradual dos níveis de SaO2, com diferenças significantes (p<0,001) durante o clareamento dental caseiro. No entanto, 30 dias após o término do clareamento dental, houve retorno aos valores iniciais. O clareamento dental caseiro provocou uma diminuição transitória reversível no grau de SaO2 pulpar.

Cambios morfofuncionales en el periodoncio asociados al movimiento dentario por tratamiento ortodóncico / Morfofunctional changes in the periodontium associated to the teeth movement due to orthodontic treatment

Los cambios morfofuncionales que se producen en las estructuras de soporte dentario durante el movimiento ortodóncico involucran procesos bioquímicos, histológicos y fisiológicos. Desde hace más de un siglo, existen disímiles teorías que tratan de explicarlos; sin embargo, todavía se siguen realizando estudios a fin de comprenderlos más a fondo. En la presente comunicación se ofrece una actualización secuencial y resumida de dichos episodios, con el propósito de incrementar el nivel de conocimientos sobre el tema y mejorar la calidad en la atención ortodóncica.

Morfofunctional changes which take place in the supporting structures during the orthodontic movement involve biochemical, histological and physiologic processes. For more than one century, dissimilar theories exist that try to explain to them; however, studies are still being carried out in order to understand them thoroughly. In the present communication a sequential and summarized updating of these episodes, with the purpose of increasing the knowledge on the topic and improving the quality in the orthodontic care.

Oxygen Saturation in the Dental Pulp of Maxillary and Mandibular Molars - Part 2

Abstract This study determined the oxygen saturation (SaO2) in dental pulp of healthy maxillary and mandibular molars. Mean of SaO2 was evaluated in 112 maxillary and mandibular molars using pulse oximetry. Quantitative variables were described by mean and standard deviation. Variables with symmetric distribution were compared by Student t test and Mann-Whitney test. Pearson's correlation coefficient was used to correlate quantitative variables. Analysis of variance was used to assess differences in SaO2 levels between the molar groups, followed by post-hoc Tukey. The significance level established at p<0.05. Mean of oxygen saturation for the 112 molar dental pulps was 85.09%. There was no significant correlation (r=-0.007; p=0.977) between the mean of SaO2 of molar pulps with patient´s indicator finger (92.89%). There was a significant difference (p=0.037) between the mean of SaO2 of the first (85.76%) and second maxillary molars (81.87%), and it was not significant (p=0.1775) between the first and second mandibular molars. Maxillary molars had lower pulpal SaO2 (83.59%) than mandibular molars (86.89%) (p=0.018). The mean of the patient's response time to the cold stimulus was 1.12 s (maxillary molars 1.25 s and mandibular molars 0.99 s)(p=0.052). There was no significant correlation between the time response of the patient to the cold stimulus and the SaO2 for molars. The mean oxygen saturation level was 85.09%. The mandibular molars presented higher SaO2 level than maxillary molars.

Resumo Este estudo determinou o nível de saturação de oxigênio (SaO2) em polpas dentais hígidas de molares. O nível de SaO2 foi avaliado em 112 molares superiores e inferiores usando oxímetro de pulso. As variáveis quantitativas foram descritas pela média e desvio padrão. As variáveis com distribuição simétrica foram comparadas pelo teste t de Student e teste de Mann-Whitney. O coeficiente de correlação de Pearson foi utilizado para correlacionar variáveis quantitativas. A análise de variância foi utilizada para avaliar as diferenças nos níveis de SaO2 entre os grupos de molares, seguido de Tukey pós-hoc. A significância foi estabelecida em 0,05. O nível médio de SaO2 para as polpas de 112 molares foi de 85,09%, não havendo correlação com a média de SaO2 do dedo indicador do paciente (92,89%). Houve diferença significativa entre o nível médio de SaO2 dos primeiros molares superiores (85,76%) e os segundos molares superiores (81,87%) e não foi significativo entre os primeiros e os segundos molares inferiores. Os molares superiores apresentaram menor nível de SaO2 (83,59%) do que os molares inferiores (86,89%). A média do tempo de resposta do paciente ao estímulo com frio foi de 1,12 s (molares superiores 1,25 segundos e molares inferiores 0,99 segundos). Não houve correlação significativa entre o tempo de resposta do paciente ao estímulo com frio e o nível de saturação de oxigênio para os molares. Em resumo, o nível médio de saturação de oxigênio foi de 85,09%. Os molares inferiores apresentaram maior nível de SaO2 do que os molares superiors

Oxygen Saturation in the Dental Pulp of Maxillary Premolars in Different Age Groups - Part 1

Abstract The aim of this study was to determine oxygen saturation levels in the dental pulp of maxillary premolars in different age groups. A total of 120 human maxillary premolars with normal dental pulps were selected covering the following age groups: 20-24, 25-29, 30-34, 35-39 and 40-44 years (n=24 each group). Oxygen saturation was assessed using pulse oximetry. Analysis of variance was used to assess differences in oxygen saturation levels and Tukey's test was used to identify the age groups that differed from each other. Significance was set at 0.05. Mean oxygen saturation of 120 premolars was 86.20% considering all age groups. Significantly reduced levels were found in the oldest group compared to the other groups: 40 to 44 years - 80.00% vs. 89.71, 87.67, 88.71, and 84.80% for age groups 20-24, 25-29, 30-34, 35-39 years, respectively. The mean oxygen saturation levels were similar between 20 and 39 years of age (86.20%) in the whole sample, but reduced significantly in the 40-44-year age group, suggesting that older patients present lower oxygen saturation results even in the absence of pulp tissue injury.

Resumo Este estudo determinou os níveis de saturação de oxigênio (SaO2) em polpas dentárias de pré-molares superiores em diferentes faixas etárias. Foram selecionados 120 pré-molares superiores humanos com polpas dentárias normais, abrangendo os seguintes grupos etários: 20-24, 25-29, 30-34, 35-39 e 40-44 anos (n=24 para cada grupo). A saturação de oxigênio foi avaliada utilizando oximetria de pulso. A análise de variância foi utilizada para avaliar diferenças nos níveis de saturação de oxigênio, e o teste de Tukey foi utilizado para identificar os grupos etários que diferiam uns dos outros. A significância foi estabelecida em 0,05. A saturação média de oxigênio foi de 86,20% considerando todos os grupos etários. Níveis significativamente reduzidos foram encontrados no grupo de indivíduos de maior idade em comparação aos outros grupos: 40 a 44 anos - 80,00% vs. 89,71, 87,67, 88,71 e 84,80% para os grupos etários 20-24, 25-29, 30-34, 35-39 anos. Os níveis médios de saturação de oxigênio foram semelhantes entre os 20 e os 39 anos de idade (86,20%), mas reduziram-se significativamente na faixa etária de 40-44 anos, sugerindo que os pacientes mais idosos apresentam menor saturação de oxigênio mesmo na ausência de lesão do tecido pulpar.

Immunohistochemical expression of biglycan and decorin in the pulp tissue of human primary teeth during resorption

Primary teeth are interesting models that can be used to study physiological and pathological processes involving cells and extracellular matrices in hard and soft tissues. This study investigated the expression and distribution of biglycan and decorin-the non-collagenous components of the extracellular matrix-in primary teeth tissue, during physiological root resorption. Thirty healthy human primary teeth were grouped together according to root length: Group I - two-thirds root length, Group II - one-third root length, and Group III - teeth with no root. The streptavidin-biotin-peroxidase immunohistochemical method was used with antibodies against the previously named antigens. The proteoglycans studied were found in the pulp and dentin extracellular matrix in all groups without any differences in the proteins, among the groups. Biglycan was observed mainly in predentin and in pulp connective tissue in the resorption area. In addition, decorin was observed mainly in pulp connective tissue, but near the resorption area. Biglycan and decorin were distributed differentially in the dental tissues. The present immunohistocytochemical data, combined with previously reported data, suggest that these proteoglycans could be involved in regulating the physiological resorption process in healthy primary teeth.

CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

ABSTRACT Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 – 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation. .

Dental pulp stem cells express proteins involved in the local invasiveness of odontogenic myxoma

Little is known about the histogenesis of the odontogenic myxoma (OM). Dental pulp stem cells could be candidate precursors of OM because both OM and the dental pulp share the same embryological origin: the dental papilla. For the purpose of comparing OM and stem cells, this study analyzed the expression of two proteins related to OM invasiveness (MMP-2 and hyaluronic acid) in human immature dental pulp stem cells (hIDPSCs). Three lineages of hIDPSCs from deciduous and permanent teeth were used in this study. Immunofluorescence revealed positive reactions for MMP-2 and hyaluronic acid (HA) in all hIDPSCs. MMP-2 appeared as dots throughout the cytoplasm, whereas HA appeared either as diffuse and irregular dots or as short fibrils throughout the cytoplasm and outside the cell bodies. The gene expression profile of each cell lineage was evaluated using RT-PCR analysis, and HA was expressed more intensively than MMP-2. HA expression was similar among the three hIDPSCs lineages, whereas MMP-2 expression was higher in DL-1 than in the other cell lines. The expression of proteins related to OM invasiveness in hIDPSCs could indicate that OM originates from dental pulp stem cells.

Micrometric measurement of the density of stained odontoblast processes

The embryological, structural and functional unit of the dentine-pulp complex shares the odontoblast, located in the border of the dentine pulp, with basal nuclei and organelles. The odontoblast process emerges from its apical pole. It is formed by microtubules, microfilaments and vesicles covered by membranes penetrating the dentinal tubules, isolated from the inter-tubular matrix, along the extent of the dentine. The objective of this study was to evaluate the efficacy of three staining techniques: hematoxylin-eosin, periodic acid-Schiff and Schmorl, by staining the process, from beginning to end, and compare the results with the erosion technique. Thirty human teeth were employed in the trial; after their extraction the pulp was fixated, the pieces demineralized in nitric acid at 8%, the collagen filaments eliminated with Type II Collage-nase, the tissue was stained, and the measurements were made. The portions with no pulp were prepared with the erosion technique. Results: Comparing the best results obtained by staining with the values obtained with the erosion technique, the former showed lower values. Conclusion: Staining techniques show lower density of the staining processes compared with the dentinal tubules in the erosion technique.

Células-tronco mesenquimais derivadas da polpa de dente humano: caracterização e estudos funcionais em modelo experimental de epilepsia / Mesenchymal stem cells derived from human tooth pulp: characterization and functional studies in an experimental model of epilepsy

Terapia celular é o conjunto de métodos e abordagens tecnológicas com a utilização de células no tratamento de doenças. A terapia com células-tronco tem despertado grande interesse na comunidade científica devido à capacidade que essas células indiferenciadas têm de preservar sua própria população e de se diferenciar em células dos diversos tecidos. Encontrar a fonte de célula-tronco apropriada para uso terapêutico depende de diversos fatores, como a sua capacidade de proliferação e estabilidade citogenética, assim como suas características fenotípicas e seu potencial de diferenciação. A epilepsia é uma desordem neurológica que acomete de 1 a 3% da população mundial. É caracterizada pela presença de crises espontâneas recorrentes (CER) e possui seu tratamento clínico baseado no emprego de drogas anti-epilépticas (DAES). O uso de células tronco apresenta-se como uma alternativa ao tratamento desta patologia. Este trabalho tem como objetivos caracterizar as células-tronco mesenquimais de polpa de dente decíduo humano e avaliar seu papel terapêutico no modelo de epilepsia do lobo temporal induzido por lítio-pilocarpina em ratos Wistar. As células da polpa de dente humano utilizadas nesse estudo foram submetidas à avaliação fenotípica, do seu potencial de diferenciação, e da estabilidade cromossômica. Para verificar os efeitos terapêuticos das células-tronco de polpa de dente em modelo experimental de epilepsia os animais foram divididos nos seguintes grupos: SE-salina (n=10), que no transplante recebeu solução salina; SE-CDLH1 (n=13) que recebeu células-tronco de polpa de dente humano (107 células/animal), SE-CMO (n=8) que recebeu células mononucleares de medula óssea (107 células/animal) e o grupo controle (n=10) que não foi submetido ao status epilepticus (SE). As células foram transplantadas por via intraperitoneal. Foram realizadas análises, em diferentes espaços de tempo para avaliar migração celular por imunofluorescência e as crises espontâneas recorrentes. Para avaliar alterações de memória foi estudado o desempenho dos animais no labirinto aquático de Morris (LAM). As CDLH1 apresentaram estabilidade cromossômica (até a 8ª passagem); características imunofenotípicas de células mesenquimais, com alta expressão de CD105, CD73, CD 44, CD90, CD166, CD54, OCT-4, e STRO-1; capacidade de diferenciação nas linhagens adipogênicas, osteogênicas e condrogênicas. As CDLH1 migraram para o cérebro e baço dos ratos. Dos resultados terapêuticos observou-se a redução das crises espontâneas recorrentes nos animais tratados após 30 dias do SE e transplante, mas as células não foram capazes de bloquear essas crises (observado 60 dias pós-SE). Obtendo-se os mesmos resultados com o transplante de CMO. Já no estudo da memória não houve diferença estatística na latência de escape nos animais dos grupos epilépticos tratado e não-tratado). Os animais do grupo controle (não epiléptico), apresentaram retenção de memória em relação aos animais dos grupos SE-salina e SE-CDLH1. Novos estudos devem ser realizados para que se possa estabelecer a utilização ideal das células-tronco mesenquimais derivadas da polpa de dente humano em relação às necessidades terapêuticas da epilepsia.

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.

Lipopolysaccharide-induced dental pulp cell apoptosis and the expression of Bax and Bcl-2 in vitro

Lipopolysaccharide exerts many effects on many cell lines, including cytokine secretion, and cell apoptosis and necrosis. We investigated the in vitro effects of lipopolysaccharide on apoptosis of cultured human dental pulp cells and the expression of Bcl-2 and Bax. Dental pulp cells showed morphologies typical of apoptosis after exposure to lipopolysaccharide. Flow cytometry showed that the rate of apoptosis of human dental pulp cells increased with increasing lipopolysaccharide concentration. Compared with controls, lipopolysaccharide promoted pulp cell apoptosis (P < 0.05) from 0.1 to 100 μg/mL but not at 0.01 μg/mL. Cell apoptosis was statistically higher after exposure to lipopolysaccharide for 3 days compared with 1 day, but no difference was observed between 3 and 5 days. Immunohistochemistry showed that expression of Bax and Bcl-2 was enhanced by lipopolysaccharide at high concentrations, but no evident expression was observed at low concentrations (0.01 and 0.1 μg/mL) or in the control groups. In conclusion, lipopolysaccharide induced dental pulp cell apoptosis in a dose-dependent manner, but apoptosis did not increase with treatment duration. The expression of the apoptosis regulatory proteins Bax and Bcl-2 was also up-regulated in pulp cells after exposure to a high concentration of lipopolysaccharide.

Effects of sonicated Prevotella intermedia, Fusobacterium nucleatum and Lactobacillus casei extracts on interleukin-8 production by human dental pulp cells.

The objective of this study was to determine the effects of Prevotella intermedia, Fusobacterium nucleatum and Lactobacillus casei on the production of IL-8 by human dental pulp cells. Human dental pulp cells from teeth of young patients (aged 18-25 years) were cultured and tested with sonicated P. intermedia ATCC 25611, F. nucleatum ATCC 25586 and L. casei ATCC 4646 extracts. IL-8 secreted into the culture supernatants were measured at 6, 12 and 24 hours using a quantitative sandwich enzyme immunoassay technique. Cell viability was evaluated using trypan blue exclusion technique. IL-8 production by human dental pulp cells increased significantly at 12 and 24 hours after exposure to P. intermedia and F. nucleatum, whereas L. casei extract exhibited low IL-8 production. The sonicated bacterial extracts did not significantly affect viability or total number of dental pulp cells.

Efeito da isquemia sobre a atividade glicolítica dentinária de molares de ratos / Effect of ischemia on the dentin glycolytic activity of rats molar teeth

Uma avaliaçäo da atividade glicolítica dentinária foi feita em dentes molares de ratos em condiçöes de isquemia tendo em vista que a circulaçäo pulpar é importante na manutençäo da resistência dentinária à carie dentária. Coroas dentárias dos dentes molares de ratos controles e isquêmicos foram trituradas em um gral de porcelana contendo água ou KOH 1N, centrifugadas e nos sobrenadantes dosados ácido láctico, fosfato inorgânico, glicose e glicogênio, respectivamente. Os resultados demonstram que o estado isquêmico provoca um aumento da atividade glicolítica dentinária de forma semelhante ao dos tecidos moles, isto é, aumento do conteúdo de ácido e fosfato inorgânico e diminuiçäo de glicose e glicogênio