Effect of ethylenediaminetetraacetic acid and hyaluronic acid on the viability and cytokine expression of periodontal ligament fibroblasts.

BACKGROUND/AIMS: Dental root cell proliferation following tooth avulsion has not been well researched. Understanding the effects of dry time and dentin treatment influences on cell proliferation is essential to provide evidence-based guidelines for tooth replantation. The study evaluated the viability of periodontal ligament fibroblasts (PLF) in contact with roots, submitted to surface treatments with ethylenediaminetetraacetic acid (EDTA) and hyaluronic acid (HA) at different times, including to quantify inflammatory cytokines interleukin (IL)-6, IL-8, IL-1ß and TNF-α expressed by PLF. The adhesion of fibroblasts to treated root surfaces was also evaluated. MATERIAL AND METHODS: One hundred and eight cementum discs from bovine teeth were randomly divided into groups according to time periods of being dry (n = 12) as follows: (i) fresh discs that were not kept dry, (WD); (ii) dry for 1 hour (1 hd); and (iii) dry for 24 hours (24 hd). The discs were subdivided into 3 subgroups (n = 12) according to surface treatments: (iv) no treatment, (v) treatment with EDTA, (vi) treatment with HA. The discs were placed in 96-well plates, and PLF were seeded and kept in contact with the discs for 48 hours. Cell viability on the surface of the discs was assessed by XTT, and PLF adhesion was evaluated using scanning electron microscopy (SEM). Quantification of cytokines was performed using enzyme-linked immunosorbent assay (ELISA). Data were submitted to ANOVA and Tukey's test (α = .05). RESULTS: Surface treatment had a statistically significant effect on the cell viability in groups WD (P = .03), 1 hd (P = .01) and 24 hd (P = .048). PLF in contact with dried root surfaces expressed more cytokines without treatment with IL-6 decreasing the expression when treated with HA for 24 hours. SEM also showed adhesion of PLF to the surface of all discs at all time periods. CONCLUSION: EDTA + HA is an alternative treatment for cases of avulsed teeth as it promoted adhesion and increased viability of PLF.

Calcium Hydroxide improves epoxy sealer adhesion on root dentin / O hidróxido de cálcio melhora a adesão de selante resinoso à dentina radicular

Objetivo: O intuito deste estudo é avaliar a adesão de um cimento endodôntico (AH Plus) em canais radiculares após o uso de diferentes protocolos de EDTA 17% e o uso de medicação intracanal (ICM) à base de hidróxido de cálcio em veículo aquoso. Material e Métodos: Para isso, 72 dentes humanos unirradiculares foram instrumentadas até # 50 e divididos em seis grupos (n = 12). Grupo 1: EDTA por 3 min; Grupo 2: 3 mL de EDTA + 3 min de EDTA; Grupo 3: 3 ml de EDTA + 3 min de EDTA + 30 segundos de agitação ultra-sônica; Grupo 4: EDTA durante 3 min + ICM; Grupo 5: 3 ml de EDTA + 3 min de EDTA + ICM; Grupo 6: 3 ml de EDTA + 3 min de EDTA + 30 segundos de agitaçãosônica + ICM. Os canais radiculares foram preenchidos com cimento endodôntico após cada protocolo e, após 7 dias foram preparados para o teste de push-out. Os dados foram analisados utilizando ANOVA dois fatores (p < 0,05). Resultados: Não foi observado diferença estatística na resistência de união nos 3 diferentes protocolos de EDTA a 17%. No entanto, o uso de ICM aumentou significativamente a resistência de adesão. Conclusão: a medicação intracanal à base de hidróxido de cálcio melhorou a resistência de união do AH Plus às paredes dentinárias, independentemente do protocolo de EDTA. (AU)

Objective: The aim of this study is to evaluate the adhesion of an endodontic sealer (AHPlus) in root canals after the use of different protocols of 17% EDTA and the use of intracanal medication (ICM) based on calcium hydroxide in aqueous vehicle. Material and Methods: For this, 72 single-rooted human teeth were instrumented up to #50 and divided into six groups (n = 12). Group 1: EDTA for 3 min; Group 2: 3 mL of EDTA + 3 min of EDTA; Group 3: 3 mL of EDTA + 3 min of EDTA + 30 seconds of ultrasonic agitation; Group 4: EDTA for 3 min + ICM; Group 5: 3 mL of EDTA + 3 min of EDTA + ICM; Group 6: 3 mL of EDTA + 3 min of EDTA + 30 seconds of ultrasonic agitation + ICM. The root canals were filled with endodontic sealer after each protocol and after 7 days they were prepared to the push-out test. The data were analyzed using twoway ANOVA (p< 0.05). Results: It was observed no statistically difference in bond strength in the 3 different 17% EDTA protocols. However, the use of ICM increased significantly the resistance adhesion. Conclusion: Intracanal medication based on calcium hydroxide improved the bond strength of AHPlus to dentin walls, regardless of the EDTA protocol. (AU)

Efficacy of different techniques for removal of calcium hydroxide-chlorhexidine paste from root canals.

The purpose of this study was to evaluate the efficacy of different techniques for removal of combined calcium hydroxide [Ca(OH)2] and chlorhexidine paste from root canals. Fifty single-rooted human teeth were prepared by oscillatory and rotary systems and filled with a paste of Ca(OH)2 and 2% chlorhexidine gel. After incubation for 14 days, the specimens were divided into 5 groups (n = 10), and the medication was removed by 1 of 5 different procedures. In group 1 (control), removal procedures involved a master apical file, foraminal debridement, and 5 mL of saline solution applied with the NaviTip irrigation needle. Group 2 was treated the same as group 1, but in addition 0.5 mL of 17% ethylenediaminetetraacetic acid was used for 3 minutes. In group 3, ultrasonic agitation was performed for 1 minute. Group 4 was treated as group 2, but the NaviTip FX needle was used for irrigation. In group 5, a master apical file, foraminal debridement, and 3-minute application of 5 mL of citric acid were used. After the root-cleaning procedures, the crowns were removed at the cementoenamel junction, and the roots were split longitudinally into halves. The success of intracanal medicament removal was observed under stereoscopic microscope and scanning electron microscope. Remnants of Ca(OH)2 were found in all experimental groups, regardless of the removal technique used. There was no statistically significant difference in cleanliness in the apical third of the root canal among groups 1, 2, and 3. Group 4 showed the best and group 5 the worst results with statistically significant differences. Overall, the NaviTip FX irrigation needle technique was more efficient in removing a Ca(OH)2-chlorhexidine paste from the root canal.

Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC / LRP6 and Frizzled6 effects in the endothelial differentiation of DPSC

O objetivo deste estudo foi avaliar a função da via de sinalização Wnt/β-catenina através dos receptores LRP6 e Frizzled6 na diferenciação de células-tronco de polpa dental de dentes permanentes (DPSC) em células endoteliais. DPSC foram transduzidas com marcadores eGFP e vetores lentivirais shRNA (LRP6, Frizzled6 ou vetor vazio - controle) para os experimentos. Os testes in vitro avaliaram a expressão de GSK3-β e β-catenina por western blot na presença de rhWnt1 e rhVEGF165 e de VEGFe CXCL-8 (IL-8) por ELISA. A expressão de marcadores endoteliais (western blot e PCR) e formação de túbulos capilares foram analisados após a diferenciação endotelial das DPSCs. In vivo, fatias dentárias/matrizes condutivas semeadas com DPSCs-shRNA foram implantadas em subcutâneo de dorso de camundongos imunodeprimidos por 28 dias e o número de vasos sanguíneos foi determinado por imunohistoquímica para eGFP e coloração por HE. β-catenina ativa foimais expressa em shRNA-LRP6 e shRNA-Frizzled6 que nas células controle, e sua expressão aumentou com a suplementação com rhVEGF165 e rhWnt1. A expressão de GSK3-β fosforilado foi menor, porém também aumenta ou permanece estável com rhVEGF165 ou rhWnt1. Quanto à expressão de VEGF, em shRNA-Frizzled6 foi maior que nas células controle e em shRNA-LRP6 (p<0,05), enquanto que a expressão de IL8 foi menor em shRNA-LRP6, diferindo estatisticamente das outras células. A expressão dos marcadores endoteliais CD31 eVEGFR2 diminuiu nas células shRNA-LRP6, enquanto que em shRNA Frizzled6a expressão de VEGFR2 foi aumentada. A formação de túbulos capilares de shRNA-Frizzled6 e shRNA-LRP6 foi menor quando comparado ao controle, porém shRNA-Frizzled6 obteve uma tendência de aumento na proliferação de capilares em 144h. In vivo, DPSC-shRNALRP6 apresentou menor número de capilares formados quando comparados com as outras duas células (p<0,05). Coletivamente, os resultados deste estudo sugerem que a via de sinalização Wnt/β-catenina regula a diferenciação...

The aim of this study was to evaluate the function of Wnt/β-cateninsignaling through LRP-6 and Frizzled-6 receptors in the differentiation ofdental pulp stem cells from permanent teeth (DPSC) into endothelial cells.DPSC were transduced with EGFP-tagged lentiviral shRNA vectors(LRP6, Frizzled6 or empty vector - control) for experiments. In vitro assayevaluated GSK3-β and β-catenin expression by western blot in rhWnt1and rhVEGF165 presence, and VEGF and CXCL-8 (IL-8) expression byELISA. Endothelial markers expression (western blot and PCR) and tubeformation were analyzed after endothelial differentiation of DPSCs. In vivo,tooth slices/scaffolds seeded with transduced DPSCs were implantedsubcutaneously in back of immunodefficient mice and blood vessels werecounted per immunohistochemistry for eGFP and HE staining. Active β-catenin was more expressed in shRNA-LRP6 and shRNA-Frizzled6 thanin control cells, and increased with rhVEGF165 and rhWnt1supplementation. Phosphorylated GSK3-β expression was lower, howeveralso increased or maintained with rhVEGF165 or rhWnt1. VEGF expressionwas higher in shRNA-Frizzled6 than in control and shRNA-LRP6 (p<0,05),IL8 expression was lower in shRNA-LRP6, with statistically difference ofthe others cells. Endothelial markers CD31 and VEGFR2 expressiondecreased in shRNA-LRP6, but VEGFR2 expression increased in shRNAFrizzled6.shRNA-Frizzled6 and shRNA-LRP6 tube formation was lowerwhen compared to control, however shRNA-Frizzled6 had tendency toincrease proliferation in 144h. In vivo, shRNA-LRP6 showed fewer bloodvessels formed than other cells (p<0,05). Collectively, the results of thisstudy suggest that Wnt/β-catenin signaling regulates endothelialdifferentiation of DPSC through LRP6.

Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC / LRP6 and Frizzled6 effects in the endothelial differentiation of DPSC

O objetivo deste estudo foi avaliar a função da via de sinalização Wnt/β-catenina através dos receptores LRP6 e Frizzled6 na diferenciação de células-tronco de polpa dental de dentes permanentes (DPSC) em células endoteliais. DPSC foram transduzidas com marcadores eGFP e vetores lentivirais shRNA (LRP6, Frizzled6 ou vetor vazio - controle) para os experimentos. Os testes in vitro avaliaram a expressão de GSK3-β e β-catenina por western blot na presença de rhWnt1 e rhVEGF165 e de VEGFe CXCL-8 (IL-8) por ELISA. A expressão de marcadores endoteliais (western blot e PCR) e formação de túbulos capilares foram analisados após a diferenciação endotelial das DPSCs. In vivo, fatias dentárias/matrizes condutivas semeadas com DPSCs-shRNA foram implantadas em subcutâneo de dorso de camundongos imunodeprimidos por 28 dias e o número de vasos sanguíneos foi determinado por imunohistoquímica para eGFP e coloração por HE. β-catenina ativa foimais expressa em shRNA-LRP6 e shRNA-Frizzled6 que nas células controle, e sua expressão aumentou com a suplementação com rhVEGF165 e rhWnt1. A expressão de GSK3-β fosforilado foi menor, porém também aumenta ou permanece estável com rhVEGF165 ou rhWnt1. Quanto à expressão de VEGF, em shRNA-Frizzled6 foi maior que nas células controle e em shRNA-LRP6 (p<0,05), enquanto que a expressão de IL8 foi menor em shRNA-LRP6, diferindo estatisticamente das outras células. A expressão dos marcadores endoteliais CD31 eVEGFR2 diminuiu nas células shRNA-LRP6, enquanto que em shRNA Frizzled6a expressão de VEGFR2 foi aumentada. A formação de túbulos capilares de shRNA-Frizzled6 e shRNA-LRP6 foi menor quando comparado ao controle, porém shRNA-Frizzled6 obteve uma tendência de aumento na proliferação de capilares em 144h. In vivo, DPSC-shRNALRP6 apresentou menor número de capilares formados quando comparados com as outras duas células (p<0,05). Coletivamente, os resultados deste estudo sugerem que a via de sinalização Wnt/β-catenina regula a diferenciação ...

The aim of this study was to evaluate the function of Wnt/β-cateninsignaling through LRP-6 and Frizzled-6 receptors in the differentiation ofdental pulp stem cells from permanent teeth (DPSC) into endothelial cells.DPSC were transduced with EGFP-tagged lentiviral shRNA vectors(LRP6, Frizzled6 or empty vector - control) for experiments. In vitro assayevaluated GSK3-β and β-catenin expression by western blot in rhWnt1and rhVEGF165 presence, and VEGF and CXCL-8 (IL-8) expression byELISA. Endothelial markers expression (western blot and PCR) and tubeformation were analyzed after endothelial differentiation of DPSCs. In vivo,tooth slices/scaffolds seeded with transduced DPSCs were implantedsubcutaneously in back of immunodefficient mice and blood vessels werecounted per immunohistochemistry for eGFP and HE staining. Active β-catenin was more expressed in shRNA-LRP6 and shRNA-Frizzled6 thanin control cells, and increased with rhVEGF165 and rhWnt1supplementation. Phosphorylated GSK3-β expression was lower, howeveralso increased or maintained with rhVEGF165 or rhWnt1. VEGF expressionwas higher in shRNA-Frizzled6 than in control and shRNA-LRP6 (p<0,05),IL8 expression was lower in shRNA-LRP6, with statistically difference ofthe others cells. Endothelial markers CD31 and VEGFR2 expressiondecreased in shRNA-LRP6, but VEGFR2 expression increased in shRNAFrizzled6.shRNA-Frizzled6 and shRNA-LRP6 tube formation was lowerwhen compared to control, however shRNA-Frizzled6 had tendency toincrease proliferation in 144h. In vivo, shRNA-LRP6 showed fewer bloodvessels formed than other cells (p<0,05). Collectively, the results of thisstudy suggest that Wnt/β-catenin signaling regulates endothelialdifferentiation of DPSC through LRP6

Coronal bacterial leakage in root canals filled with single cone technique and different endodontic sealers / Infiltração coronária microbiana em canais radiculares obturados pela técnica do cone único com diferentes cimentos endodônticos

Objective: To evaluate coronal bacterial leakage comparing five endodontic sealers (AH Plus, Apexit Plus, Copaifera sp oil, EndoREZ and Polifil), and comparing root canals filled with EndoREZ sealer/ EndoREZ® Points and EndoREZ sealer/conventional gutta-percha points. Material & Methods: 84 human teeth were prepared and filled with gutta-percha points using the single cone technique. Roots were randomly divided into 6 groups: Apexit Plus, AH Plus, Copaifera sp oil, Polifil, EndoREZ, and EndoREZ/EndoREZ Points. After setting time, the roots were incorporated in a leakage model, which upper chamber contained a suspension of Streptococcus mutans, and lower chamber a broth. Leakage was assessed for turbidity in lower chamber for 60 days. Statistic analysis was performed using the nonparametric Kaplan-Meier method (p<0.05). Results: All experimental groups presented leakage during the study’s period. The medium time of leakage was: Apexit Plus and AH Plus 6.3 days, Polifil 5.1 days, Copaifera 1.2 days, and both EndoREZ groups infiltrated in the first day. Conclusions: There was no statistically significant difference between the sealers Apexit Plus, AH Plus and Polifil, but they prevented leakage better than Copaifera sp oil and both EndoREZ groups. However, none of the tested sealers was capable of resisting coronal bacterial leakage for more than 22 days.

Objetivo: Avaliar a infiltração coronária microbiana de cinco cimentos endodônticos (AH Plus, Apexit Plus, Copaiba, EndoREZ and Polifil), e comparar canais obturados com cimento EndoREZ/ cones EndoREZ e canais com cimento EndoREZ/ cones de guta-percha. Material e Métodos: 84 raízes de dentes humanos uniradiculados tiveram seus canais preparados e obturados pela técnica do cone único. As raízes foram divididas em 6 grupos: Apexit Plus, AH Plus, Copaiba, Polifil, EndoREZ e EndoREZ/ cones EndoREZ. Após endurecimento dos cimentos, as raízes foram adaptadas a um modelo de infiltração, cuja câmara superior continha uma suspensão de Streptococcus mutans, e a inferior um meio de cultura, deixando a porção apical da raiz imersa. A infiltração foi verificada diariamente pelo turvamento na câmara inferior, por um período de 60 dias. Os dados foram avaliados pela análise estatística não paramétrica Kaplan-Meier (p<0,05). Resultados: Todos os grupos experimentais apresentaram infiltração no período do experimento, contudo o tempo máximo foi de 22 dias. O tempo médio de infiltração foi: Apexit Plus 6,3 dias, AH Plus 6,3 dias, Polifil 5,1 dias, Copaiba 1,2 dias, e em ambos os grupos do cimento EndoREZ todos os espécimes infiltraram no primeiro dia. Conclusão: Não houve diferença estatisticamente significante entre os cimentos Apexit Plus, AH Plus e Polifil, mas estes apresentaram melhores resultados que Copaifera e ambos os grupos do EndoREZ. Porém, nenhum cimento foi capaz de impedir a infiltração coronária microbiana por mais de 22 dias.

Evaluation of apical leakage in root canals filled with different sealers / Avaliação do desempenho de diferentes cimentos obturadores no selamento apical de canais radiculares

Objective: To evaluate the apical leakage exhibited by root canals filled with gutta-percha points and three different endodontic sealers. Material and Methods: Thirty-five human molars were used and had their palatal (maxillary molars) and distal roots (mandibular molars) sectioned, standardized and instrumented with Mtwo rotary system. The roots were filled through active lateral condensation technique and divided into three groups (n=10), according to the endodontic sealer employed: G1- AH Plus, G2- Fill Canal, G3- MTA Fillapex. All groups were filled by gutta-percha points and endodontic sealer. Gutta-percha points were immersed into sodium hypochlorite for 24 h to achieve disinfection. After the filling procedure, the roots were immersed into Indian ink for posterior diaphanization and obtainment of the images through stereomicroscopy. By analyzing the images in Adobe Illustrator CS5 software, the level of apical leakage was determined. The data obtained were submitted to Kruskal Wallis and Dunn tests, with level of significance set at 5%. Results: Statistically significant differences were found between G1 and G3. G2 did not show statistically significant differences. G1 exhibited the smallest apical leakage mean (12.85), followed by G2 (109.84) and G3 (101.15). Conclusions: Root canal obturation with gutta-percha points and AH plus sealer through lateral condensation technique provided lower apical leakage rates than the other endodontic sealers evaluated

Objetivo: Avaliar a infiltração apical apresentada em canais radiculares obturados com guta-percha e três diferentes cimentos obturadores. Material e Métodos: Foram utilizados 35 molares humanos, os quais tiveram suas raízes linguais (molares superiores) e distais (molares inferiores) seccionadas, padronizadas e instrumentadas com o sistema rotatório Mtwo. As raízes foram obturadas através da técnica de condensação lateral ativa e divididas em 3 grupos (n=10), de acordo com o cimento obturador utilizado: G1- AH Plus, G2- Fill Canal, G3- MTA Fillapex. Todos os grupos foram obturados com o conjunto guta-percha e cimento obturador, sendo que a guta-percha utilizada permaneceu 24 h imersa em hipoclorito de sódio, para sua desinfecção. Após a obturação, as raízes foram imersas em corante tinta da Índia, para posterior diafanização e obtenção de imagens através de estereomicroscópio. Através da análise das imagens no programa Adobe Illustrator CS5 foi determinado o nível de infiltração apical. Os dados obtidos foram submetidos aos testes estatísticos de Kruskal Wallis e Dunn, com nível de significância 5%. Resultados: Verificou-se presença de diferença estatística significante entre o G1 e G3, sendo que G2 não diferiu estatisticamente dos demais grupos. O G1 apresentou a menor média de infiltração apical (12.85), seguida pelo G2 (109.84) e G3 (101.15). Conclusões: A obturação de canais radiculares com cones de guta-percha e cimento AH plus através da técnica de condensação lateral proporciona baixos índices de infiltração apical, quando comparada aos demais cimentos obturadores avaliados

Avaliação in vitro da citotoxicidade e genotoxicidade de diferentes cimentos endodônticos em fibroblastos V79 / In vitro evaluation of cytotoxicity and genotoxicity of different root canal sealers in V79 cells

Cimentos endodônticos podem liberar componentes tóxicos que interagem com os tecidos periapicais. A proposta deste trabalho foi avaliar a citotoxicidade e genotoxicidade de quatro cimentos endodônticos (EndoREZ, RoekoSeal, AHPlus e cimento experimental à base de óleo-resina da Copaifera multijuga) utilizando ensaios biológicos. Os cimentos foram mixados e incubados em estufa de umidade relativa a 37°C com 5% CO2 por 24 h. A exposição do meio de cultura aos cimentos ocorreu após 12 h do endurecimento dos cimentos. Células V79 foram expostas às diluições dos extratos por 24 h e a sobrevivência celular foi mensurada fotometricamente. A viabilidade celular foi mensurada pelo teste de MTT em espectrofotômetro e a genotoxicidade, indicada pela formação de micronúcleos, foi determinada após 24h do período de exposição. Os resultados da taxa de sobrevivência celular e a quantidade de danos ao DNA foram estatisticamente analisados pelo teste de Kruskal-Wallis (p<0,05). A citotoxicidade em relação ao grupo controle decresceu na seguinte ordem EndoREZ > Copaíba ≥ AH Plus > Roeko Seal. A formação de micronúcleos (MN) para indicar a genotoxicidade foi induzida somente pelos extratos do EndoREZ, sendo mais genotóxico que o EMS (controle positivo)

Endodontic sealers could release toxic components that may interact with periapical tissues. The purpose of this study was to evaluate the cytotoxicity and genotoxicity of four endodontic sealers (EndoREZ, RoekoSeal, AHPlus and experimental root canal sealer- based on Copaifera multijuga oil-resin) using routine cell biology techniques. The sealers were mixed and incubated in a humidified incubator at 37°C with 5% CO2 in air for 24 h. The exposure of the culture medium to the sealers occurred after 12 h of cure. V79 cells were exposed to dilutions of this extracts for 24 h and cell survival was measured photometrically. The cell viability was measured by MTT test in a spectrophotometer and the genotoxicity as indicated by the formation of micronuclei was determined after a 24 h exposure period The results of the cell survival rates and the amount of DNA damage was statistically analyzed for Kruskal-Wallis (p<0.05) test. The ranking of cell survival from the most to the least toxic material was: EndoREZ> Copaíba ≥ AH Plus > RoekoSeal. The formation of micronuclei (MN) to indicate genotoxicity was induced only by extracts of EndoREZ, being more genotoxic than EMS (positive control)

Longitudinal bond strength evaluation using the deproteinized dentin technique.

This study evaluated bond strength to dentin as a result of storage time for conventional adhesive systems (with or without collagen) that had been deproteinized with 10% sodium hypochlorite (NaOCl). For this study, 72 human molars were sectioned in a mesiodistal axial plane and embedded in acrylic resin; at that point, the vestibular and lingual surfaces were worn down with abrasive paper. Acid etching was performed for 15 seconds (using 37% phosphoric acid) and the specimens were divided into 12 groups (n = 6), depending on the adhesive system used, the dentin treatment performed, and the length of evaluation (24 hours or six months). A resin composite was inserted over the prepared area with the aid of a metal matrix. Following a mechanical shear test, fractured surfaces were analyzed by stereomicroscope and the data were submitted to ANOVA and Tukey's test. It was concluded that the dentin deproteinization treatment with 10% NaOCl improved the bond strength in five of the six groups. The bond strength after 24 hours was significantly higher than the bond strength measured after six months. Of the three adhesive systems tested in this study, DenTASTIC UNO demonstrated the lowest bond strength.