Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.
Abstract Periodontal regeneration is still a challenge in terms of predictability and magnitude of effect. In this study we assess the biological effects of combining chemical root conditioning and biological mediators on three relevant cell types for periodontal regeneration. Material and Methods: Bovine dentin slices were conditioned with 25% citric acid followed by topical application of basic fibroblast growth factor (bFGF, 10 and 50 ng). We used ELISA to assess the dynamics of bFGF release from the dentin surface and RT-qPCR to study the expression of Runx2, Col1a1, Bglap and fibronectin by periodontal ligament (PDL) fibroblasts, cementoblasts and bone marrow stromal cells (BMSC) grown onto these dentin slices. We also assessed the effects of topical application of bFGF on cell proliferation by quantification of genomic DNA. Results: Acid conditioning significantly increased the release of bFGF from dentin slices. Overall, bFGF application significantly (p<0.05) increased cell proliferation, except for BMSC grown on non-conditioned dentin slices. Dentin substrate discretely increased expression of Col1a1 in all cell types. Expression of Runx2, Col1a1 and Fn was either unaffected or inhibited by bFGF application in all cell types. We could not detect expression of the target genes on BMSC grown onto conditioned dentin. Conclusion: Acid conditioning of dentin improves the release of topically-applied bFGF. Topical application of bFGF had a stimulatory effect on proliferation of PDL fibroblasts, cementoblasts and BMSC, but did not affect expression of Runx2, Col1a1, Bglap and fibronectin by these cells.
ABSTRACT The Mate Tea (Ilex paraguariensis A. St.-Hil.) is a climax species adapted to low light, and that is why is more cultivated in consortium with other species of woods and more intensively that their traditional exploration. To maintain adequate levels of productivity, it is necessary the use of a fertilizer that gives nutritional intake to plants. The levels of substances of plant secondary metabolism as methylxanthines and phenolic compounds may be affected by this type of intensive management since the shading and fertilization influence the physiology of the plant. Aiming the determination of the levels of totals methylxanthines and phenolic compounds, produced under relative brightness of 30, 45 and 60%, and conventional fertilization (N, P2O5 and K2O 15-05-30) and slow release (N, P2O5 and K2O 15-08-12), this study was proposed for two growing seasons 2011 e 2012. The fertilization of mate tea associated with the apparent brightness levels did not promote increased levels of totals methylxanthines and phenolic compounds. The apparent brightness of 30% established the best indicator to obtain methylxanthines, and the conventional fertilization had the highest fertilization production of methylxanthine, when subjected to the apparent brightness of 30 and 45 %. There was an increase of the content of phenolic compounds of the growing season 2011 to 2012 promoted by the fertilization treatment with FLL and FC under the apparent brightness of 30% and 60%.
RESUMO A erva-mate (Ilex paraguariensis A. St.-Hil.) é uma espécie climácica, bastante adaptada ao sombreamento, por isso, cada vez mais cultivada em consórcio com outras espécies madeiráveis, e de forma mais intensiva de que a sua exploração tradicional. Para manter níveis de produtividade adequados é necessário o uso de fertilizantes que deem aporte nutricional às plantas. Os teores de substâncias do metabolismo secundário da planta, como as metilxantinas e os compostos fenólicos, podem ser afetados por esse tipo de manejo mais intensivo, já que o sombreamento e a adubação influenciam a fisiologia da planta. Objetivando a determinação de teores de metilxantinas e compostos fenólicos totais em folhas de erva-mate, produzidas sob luminosidade aparente de 30, 45 e 60%, e fertilização convencional (FC) (N, P2O5 e K2O 15-05-30) e de liberação lenta (N, P2O5 e K2O 15-08-12), foi proposto esse estudo para duas estações de crescimento nos anos de 2011 e 2012. A fertilização da erva-mate associada aos níveis de luminosidade aparente não promoveram o aumento de teores de metilxantinas totais e compostos fenólicos totais. A luminosidade aparente de 30% estabeleceu o melhor indicador para obtenção de metilxantinas, sendo que a fertilização FC apresentou a maior produção de metilxantinas, quando submetida à luminosidade aparente de 30 e 45%. Houve incremento do teor dos compostos fenólicos da estação de crescimento 2011 para 2012 promovido pelos tratamentos fertilizados com FLL e FC sob a luminosidade aparente de 30% e 60%.
Abstract This study aimed to characterize the dynamics of suppressor of cytokine signaling (SOCS1) expression in a rat model of lipopolysaccharide-induced periodontitis. Wistar rats in the experimental groups were injected three times/week with LPS from Escherichia coli on the palatal aspect of the first molars, and control animals were injected with vehicle (phosphate-buffered saline). Animals were sacrificed 7, 15, and 30 days after the first injection to analyze inflammation (stereometric analysis), bone loss (macroscopic analysis), gene expression (qRT-PCR), and protein expression/activation (Western blotting). The severity of inflammation and bone loss associated with LPS-induced periodontitis increased from day 7 to day 15, and it was sustained through day 30. Significant (p < 0.05) increases in SOCS1, RANKL, OPG, and IFN-γ gene expression were observed in the experimental group versus the control group at day 15. SOCS1 protein expression and STAT1 and NF-κB activation were increased throughout the 30-day experimental period. Gingival tissues affected by experimental periodontitis express SOCS1, indicating that this protein may potentially downregulate signaling events involved in inflammatory reactions and bone loss and thus may play a relevant role in the development and progression of periodontal disease.
Abstract Several calcium silicate-based biomaterials have been developed in recent years, in addition to Mineral Trioxide Aggregate (MTA). The aim of this study was to evaluate the cytotoxicity, genotoxicity and apoptosis/necrosis in human osteoblast cells (SAOS-2) of pure calcium silicate-based cements (CSC) and modified formulations: modified calcium silicate-based cements (CSCM) and three resin-based calcium silicate cements (CSCR1) (CSCR 2) (CSCR3). The following tests were performed after 24 hours of cement extract exposure: methyl-thiazolyl tetrazolium (MTT), apoptosis/necrosis assay and comet assay. The negative control (CT-) was performed with untreated cells, and the positive control (CT+) used hydrogen peroxide. The data for MTT and apoptosis were submitted to analysis of variance and Bonferroni’s posttest (p < 0.05), and the data for the comet assay analysis, to the Kruskal-Wallis and Dunn tests (p < 0.05). The MTT test showed no significant difference among the materials in 2 mg/mL and 10 mg/mL concentrations. CSCR3 showed lower cell viability at 10 mg/mL. Only CSC showed lower cell viability at 50 mg/mL. CSCR1, CSCR2 and CSCR3 showed a higher percentage of initial apoptosis than the control in the apoptosis test, after 24 hours exposure. The same cements showed no genotoxicity in the concentration of 2 mg/mL, with the comet assay. CSC and CSCR2 were also not genotoxic at 10 mg/mL. All experimental materials showed viability with MTT. CSC and CSCR2 presented a better response to apoptosis and genotoxicity evaluation in the 10 mg/mL concentration, and demonstrated a considerable potential for use as reparative materials.
Mechanical instrumentation of the root surface causes the formation of a smear layer, which is a physical barrier that can affect periodontal regeneration. Although different procedures have been proposed to remove the smear layer, there is no information concerning how long the smear layer persists on root surfaces after instrumentation in vivo. This study assessed the presence of the smear layer on root surfaces over a 28-day period after subgingival instrumentation with hand instruments. Fifty human teeth that were referred for extraction because of advanced periodontal disease were scaled and root planed (SRP) by a single experienced operator. Ten teeth were randomly assigned to be extracted 7, 14, 21, and 28 days after SRP. Another 10 teeth were extracted immediately after instrumentation (Day 0, control group). The subgingival area of the instrumented roots was evaluated with scanning electron microscopy. Representative photomicrographs were assessed by a blinded and calibrated examiner according to a scoring system. A rapid and significant (p < 0.05, Z test) initial reduction in the amount of smear layer was observed at 7 days, and a further significant (p < 0.05) decrease was observed 28 days after SRP. Interestingly, even 28 days after SRP, the smear layer was still present on root surfaces. This study showed that the physiological elimination of the smear layer occurred in a biphasic manner: a rapid initial reduction was observed 7 days after instrumentation, which was followed by a slow process leading to a significant decrease 28 days after instrumentation.
Objective: The aim of this study was to evaluate a possible synergism between AGE-RAGE and TLR4 signaling and the role of p38 MAPK and NF-kB signaling pathways on the modulation of the expression of inflammatory cytokines and proliferation of cells from the innate and adaptive immune response. Material and Methods: T lymphocyte (JM) and monocyte (U937) cell lines were stimulated with LPS and AGE-BSA independently and associated, both in the presence and absence of p38 MAPK and NF-kB inhibitors. Proliferation was assessed by direct counting and viability was assessed by a biochemical assay of mitochondrial function. Cytokine gene expression for RAGe, CCL3, CCR5, IL-6 and TNF-α was studied by RT-PCR and RT-qPCR. Results: RAGE mRNA expression was detected in both cell lines. LPS and AGE-BSA did not influence cell proliferation and viability of either cell line up to 72 hours. LPS and LPS associated with AGE induced expression of IL-6 and TNF-α in monocytes and T cells, respectively. Conclusions: There is no synergistic effect between RAGE and TLR signaling on the expression of IL-6, TNF-α , RAGE, CCR5 and CCL3 by monocytes and lymphocytes. Activation of RAGE associated or not with TLR signaling also had no effect on cell proliferation and survival of these cell types.
Recently, new treatment approaches have been developed to target the host component of periodontal disease. This review aims at providing updated information on host-modulating therapies, focusing on treatment strategies for inhibiting signal transduction pathways involved in inflammation. Pharmacological inhibitors of MAPK, NFκB and JAK/STAT pathways are being developed to manage rheumatoid arthritis, periodontal disease and other inflammatory diseases. Through these agents, inflammatory mediators can be inhibited at cell signaling level, interfering on transcription factors activation and inflammatory gene expression. Although these drugs offer great potential to modulate host response, their main limitations are lack of specificity and developments of side effects. After overcoming these limitations, adjunctive host modulating drugs will provide new therapeutic strategies for periodontal treatment.
PURPOSE: This in vitro study aimed to evaluate the effect of different fruit juice drinks available in the Brazilian market on smear layer removal and dentinal tubules opening, as well as to verify the effect of toothbrushing subsequently to the juices exposure. METHODS: Dentin specimens were prepared and randomly distributed into the control group (distilled water) and twelve types of fruit juice drinks (cashew, orange, mandarin, apple, passion fruit, guava, strawberry, grape, mango, pear, peach, pineapple). The following treatments were applied: immersion or immersion + brushing. After preparation for SEM, photomicrographs were assessed using an index of smear layer removal. RESULTS: No significant differences regarding smear layer removal and dentinal tubules exposure could be observed between the groups after both treatments (Kruskal-Wallis, post-hoc paired comparisons, P>0.05). The control solution and the fruit juice drinks were not able to remove smear layer and to open dentinal tubules. Significant difference between the applied treatments was detected only for the mango juice group (Mann-Whitney, P<0.05). CONCLUSION: Under the experimental conditions, the different fruit juice drinks did not promote significant alterations on human radicular dentin morphology regardless of the subsequent application of brushing procedures.
OBJETIVO: O objetivo deste estudo in vitro foi avaliar o efeito de diferentes sucos de fruta disponíveis no mercado brasileiro sobre o grau de remoção de smear layer e abertura dos túbulos dentinários, assim como verificar o efeito da escovação realizada imediatamente após a exposição aos sucos. METODOLOGIA: Amostras de dentina foram preparadas e distribuídas aleatoriamente entre o grupo controle (água destilada) e doze tipos de suco de fruta (caju, laranja, tangerina, maçã, maracujá, goiaba, morango, uva, manga, pêra, pêssego, abacaxi). Os tratamentos aplicados foram: imersão ou imersão + escovação. Após preparo para MEV, fotomicrografias foram avaliadas utilizando-se um índice de remoção de smear layer. RESULTADOS: Não foram encontradas diferenças significativas entre os grupos em relação à remoção de smear layer e abertura dos túbulos após ambos os tratamentos (Kruskal-Wallis, comparações pareadas, P>0.05). A solução controle e os sucos de fruta não promoveram remoção de smear layer e abertura dos túbulos dentinários. Apenas no grupo do suco de manga foi verificada uma diferença significativa entre os tratamentos aplicados (Mann-Whitney, P<0.05). CONCLUSÃO: Com base na metodologia utilizada, pode-se concluir que os diferentes sucos de fruta não promoveram alterações significativas na morfologia da dentina radicular apesar da realização subsequente de procedimentos de escovação.
Studies involving bioacoustics analysis turned possible the identification of complex species groups, as well as the description and differentiation of cryptic species. In order to increase the knowledge on the species composition of amphibians in Araucaria Forests, we sampled eleven areas in the states of Paraná and Santa Catarina, which significantly increased the geographic distribution range of eight amphibian species: Ischnocnema henselii, Dendropsophus anceps, a currently classified as critically endangered in Paraná State, D. nahdereri, Scinax granulatus, Trachycephalus dibernardoi, Pseudis cardosoi, Leptodactylus araucaria and L. nanus. Some of the new records represent corrections of previous identifications for example: Ischnocnema henselii is a cryptic species with some populations in Paraná that have been previously mistaken to I. guentheri; Trachycephalus dibernardoi was mistaken for T. imitatrix and Leptodactylus nanus to L. marmoratus. We also characterize and describe the calls of all these species.
Estudos envolvendo análises bioacústicas têm possibilitado a identificação de espécies pertencentes a grupos complexos, bem como a descrição e diferenciação de espécies crípticas. Recorrendo a esta ferramenta e com o objetivo de aumentar o conhecimento sobre a composição de espécies de anfíbios em áreas de Floresta com Araucária, foram amostradas 11 áreas nos estados do Paraná e Santa Catarina. Os resultados ampliaram significativamente o conhecimento da distribuição geográfica de oito espécies de anfíbios anuros: Ischnocnema henselii, Dendropsophus anceps, atualmente classificada como criticamente ameaçada no Estado do Paraná, D. nahdereri, Scinax granulatus, Trachycephalus dibernardoi, Pseudis cardosoi, Leptodactylus araucaria e L. nanus. Alguns destes registros representam correções em identificações anteriores: Ischnocnema henselii é uma espécie críptica e algumas populações até o momento eram confundidas no Paraná com I. guentheri; Trachycephalus dibernardoi era identificada como T. imitatrix e Leptodactylus nanus como L. marmoratus. A caracterização e descrição dos cantos destas espécies também são apresentadas.
The patient's diet has been considered an important etiological factor of dentin hypersensitivity. The frequent ingestion of acidic substances can promote the loss of dental structure or remove the smear layer. The purpose of this study was to evaluate the degree of smear layer removal and dentinal tubules exposure by different natural orange juices. Extracted human teeth were submitted to manual scaling in order to develop the smear layer. Seventy dentin samples were obtained and distributed into the following groups: Control, lime orange, lime, valência orange, navel orange, mandarin, and tangerine. Each group included 2 methods of application: Topical and topical + friction. After preparation for SEM analysis, photomicrographs were assessed by a blind calibrated examiner using an index system. The Kruskal-Wallis test indicated a significant influence of the orange juices on smear layer removal. Significant difference was observed between navel orange, valência orange, mandarin and the control group (p < 0.05). These orange juices resulted in greater removal of the smear layer and greater opening of dentinal tubules. The comparison between the application methods for each group using the Mann-Whitney test showed that friction increased smear layer removal significantly only for lime orange and lime. The data suggest that certain natural orange juices are more effective in terms of smear layer removal and dentinal tubules exposure than others.
The purpose of this clinical study was to investigate if periodontal disease and rheumatoid arthritis (RA) are associated. The study included 39 RA patients (test group) and 22 age- and gender-matched healthy individuals (control group). Questionnaires on general and oral health were applied and a complete periodontal exam, including visible plaque, marginal bleeding, attachment loss (AL) and number of teeth present, was also performed by a single calibrated examiner. Diabetes mellitus patients and smokers were excluded. RA patients had fewer teeth, higher prevalence of sites presenting dental plaque and a higher frequency of sites with advanced attachment loss. Although the prevalence of dental plaque was higher in the test group (Chi-square test, p = 0.0006), the percentage of sites showing gingival bleeding was not different (Fisher’s exact test, p > 0.05). Based on our results, we suggest that there is an association between periodontal disease and RA.
Species richness, spatial distribution and the seasonal occurrence of anurans (adults and tadpoles) were studied throughout 15 months in an Araucaria forest remnant at Fazenda Rio Grande, Paraná State, Brazil. Thirty-two species were registered, which corresponds to the third largest anuran richness in Paraná State. The spatial distribution of the species was not uniform between habitats sampled: 46% of the species were associates with opened areas, occurring exclusively in habitats of open areas and/or in forest edges, 34% were associated with forest habitats, occurring, in habitats forest and/or forest edge, while that 13% were classified as generalists as they occurred in open formations, at the edge and interior of the forest fragments. The reproductive period of the species was correlated with rainfall, since 80% of the species were found in breeding activity the rainy season. About 40% of the registered anuran species were associated on the habitat features provided by the forest cover which reinforces the importance of conservation of Araucaria Forest remnants.
A riqueza de espécies, a distribuição espacial e a ocorrência sazonal de anuros (adultos e girinos) foram determinadas ao longo de 15 meses, em um remanescente da floresta de araucária no município de Fazenda Rio Grande, Paraná, Brasil. Foram registradas 32 espécies, sendo essa a área com a terceira maior riqueza registrada no Estado. A distribuição espacial das espécies não foi uniforme entre os hábitats amostrados: 46% das espécies foram associadas à área aberta, ocorrendo exclusivamente em hábitats de área aberta e/ou em borda florestal, 34% foram associadas à hábitats florestais, ocorrendo em hábitats florestais ou de borda florestal, enquanto que 13% foram classificadas como generalistas, pois ocorreram em área aberta, borda e interior de floresta. O período reprodutivo dos anuros foi fortemente relacionado com a chuvosa, quando cerca de 80% das espécies foram registradas em atividade de vocalização. Cerca de 40% das espécies registradas são associadas à hábitats florestais, o que reforça a urgência da preservação desses remanescentes de Floresta de Araucária.
Several antiseptic substances have been used as adjuncts to routine mechanical procedures of oral hygiene, based on their antimicrobial effects. The objective of this study was to assess in vitro the antimicrobial efficiency of a mouthwash containing Triclosan/Gantrez and sodium bicarbonate in comparison to both positive and negative controls. Standard strain samples of Escherichia coli, Pseudomonas aeruginosa, Actinomyces viscosus and Bacillus subtilis were used. Samples of Streptococcus mutans and Gram-negative bacilli were collected from 20 volunteers (10 with a clinically healthy periodontium and 10 presenting biofilm-associated gingivitis). Evaluation of the antimicrobial activity was performed by determining the minimal inhibitory concentration (MIC). The results indicated that the test solution inhibited the growth of both Gram-negative and Gram-positive microorganisms from the volunteers’ saliva as well as that of the standard strains at the MIC dilution of 1:20, whereas the MIC dilution of 0.12% chlorhexidine against the same bacteria was 1:80. Thus, even though the tested mouthrinse solution presented an in-vitro antimicrobial activity superior to that of a placebo, it was inferior to that of chlorhexidine.
Diversas substâncias antisépticas têm sido utilizadas como adjuntos aos procedimentos mecânicos rotineiros de higiene oral, com base em seus efeitos antimicrobianos. O objetivo deste estudo foi avaliar, in vitro, a eficiência antimicrobiana de um enxaguatório bucal contendo Triclosan/Gantrez e bicarbonato de sódio, em comparação a controles positivos e negativos. Linhagens padrão de Escherichia coli, Pseudomonas aeruginosa, Actinomyces viscosus e Bacillus subtilis foram utilizadas. Amostras de Streptococcus mutans e Bacilos Gram-negativos foram coletadas de 20 voluntários (10 com um periodonto clinicamente saudável e 10 com gengivite associada à presença de biofilme). A avaliação da atividade antimicrobiana foi realizada pela determinação da Concentração Inibitória Mínima (CIM). Os resultados mostraram que a solução teste inibiu o crescimento de microrganismos Gram-negativos e Gram-positivos da saliva dos voluntários, bem como das linhagens padrão na CIM de 1:20, enquanto que a CIM da diluição de clorexidina 0.12% contra as mesmas bactérias foi de 1:80. Desta forma, apesar de o enxaguatório bucal testado apresentar atividade antimicrobiana in vitro superior à do placebo, esta foi inferior à da Clorexidina.