Pulpectomia: publicações, evidências científicas e influência da pandemia de Covid-19 / Pulpectomy: publications, scientific evidence and the influence of the COVID-19 pandemic

Objetivou-se analisar o panorama das publicações sobre pulpectomia, de forma geral, além de verificar as evidências acerca do potencial de redução de endotoxinas pelo hidróxido de cálcio e investigar a influência da pandemia de COVID-19 em relação à realização de procedimentos endodônticos em Odontopediatria. Para tanto, foram realizados três estudos. O primeiro visou mapear e discutir, por meio de uma revisão bibliométrica, o panorama das publicações científicas acerca da pulpectomia em dentes decíduos utilizando os termos "pulpectomy", "root canal", "child" AND "preschoolar". O segundo estudo delineou-se como uma revisão sistemática, seguida de metanálise objetivando verificar se existem evidências de redução de endotoxinas antes e depois do uso de hidróxido de cálcio como medicação intracanal em canais radiculares infectados. Riscos de avaliação de viés e metanálises foram realizados. A certeza de evidência foi determinada pelo GRADE. O terceiro estudo pretendeu verificar as possíveis influências da pandemia de COVID-19 na realização de procedimentos endodônticos em Odontopediatria, por meio da aplicação de um questionário eletrônico composto por perguntas divididas em 3 domínios: (1) informações profissionais; (2) gerenciamento de biossegurança e (3) protocolo endodôntico de atendimento antes e durante a pandemia. Após a realização dos três estudos, foi identificada uma tendência na pesquisa científica para a utilização de instrumentação mecanizada e pastas obturadoras mais biocompatíveis e eficazes. Foi possível observar ausência de estudos clínicos multicêntricos sobre procedimentos de pulpectomia em dentes decíduos. A maioria dos estudos foi publicada em periódicos de Odontopediatria, apresentaram ampla variedade de protocolos de tratamento e foram realizados principalmente em países em desenvolvimento, denotando claro viés nas opções de tratamento oferecidas para crianças em todo o mundo, uma vez que o acesso aos serviços de anestesia geral em países desenvolvidos favorece a exodontia dos dentes com comprometimento pulpar. Além disso, concluiu-se que o hidróxido de cálcio reduz os níveis de endotoxinas quando usado como medicação intracanal, mas é incapaz de eliminá-las completamente em dentes permanentes, independentemente de sua associação com alguma solução irrigante. Não foram recuperados artigos em dentes decíduos. No terceiro estudo, a amostra final foi composta por 313 participantes de todos os estados brasileiros. A maioria dos participantes era do sexo feminino (90,35%), e mais da metade cursou Odontopediatria em instituição privada (50,25%) há mais de dez anos (53,63%) e trabalha em metrópole (54,75%). No geral, a maioria dos respondentes atualmente prática Odontopediatria exclusivamente em clínicas privadas (61,63%). Durante a pandemia de COVID-19 foi possível ver um aumento na complexidade dos casos planejados previamente e da quantidade de urgências odontológicas. Levando isso em consideração, muitos odontopediatras sentiram a necessidade de se reinventarem, priorizando técnicas minimamente invasivas e com um menor tempo de consulta. Com isso, pôde-se notar uma tendência pela busca do tratamento endodôntico não instrumental (p<0,01), justamente por ser uma técnica mais rápida e barata. Apesar de já existirem muitos trabalhos publicados acerca da endodontia em dentes decíduos, conforme constatado nos dois primeiros estudos, observa-se que ainda são necessárias novas investigações nessa área. (AU)

The objective was to analyze the overview of publications on pulpectomy, in general, in addition to verifying the evidence about the potential for endotoxin reduction by calcium hydroxide and to investigate the influence of the COVID-19 pandemic in relation to the performance of endodontic procedures in Pediatric Dentistry. Therefore, three studies were carried out. The first aimed to map and discuss, through a bibliometric review, the panorama of scientific publications about pulpectomy in primary teeth using the terms "pulpectomy", "root canal", "child" AND "preschooler". The second study was designed as a systematic review, followed by a meta-analysis aiming to verify if there is evidence of endotoxin reduction before and after the use of calcium hydroxide as an intracanal medication in infected root canals. Bias assessment risks and meta-analyses were performed. The certainty of evidence was determined by GRADE. The third study aimed to verify the possible influences of the COVID-19 pandemic on the performance of endodontic procedures in Pediatric Dentistry, through the application of an electronic questionnaire composed of questions divided into 3 domains: (1) professional information; (2) biosafety management and (3) endodontic care protocol before and during the pandemic. After carrying out the three studies, a trend in scientific research towards the use of mechanized instrumentation and more biocompatible and effective filling pastes was identified. It was possible to observe the absence of multicenter clinical studies on pulpectomy procedures in primary teeth. Most studies were published in pediatric dentistry journals, presented a wide variety of treatment protocols, and were carried out mainly in developing countries, denoting a clear bias in the treatment options offered to children around the world, since access to care services general anesthesia in developed countries favors extraction of teeth with pulp involvement. Furthermore, it was concluded that calcium hydroxide reduces the levels of endotoxins when used as an intracanal medication but is unable to completely eliminate them in permanent teeth, regardless of its association with some irrigating solution. No articles in primary teeth were retrieved. In the third study, the final sample consisted of 313 participants from all Brazilian states. Most of the participants were female (90.35%), and more than half had studied Pediatric Dentistry at a private institution (50.25%) for over ten years (53.63%) and worked in a metropolis (54.75% ). Overall, the majority of respondents currently practice Pediatric Dentistry exclusively in private clinics (61.63%). During the COVID-19 pandemic, it was possible to see an increase in the complexity of previously planned cases and the number of dental emergencies. Taking this into account, many pediatric dentists felt the need to reinvent themselves, changing case plans, prioritizing minimally invasive techniques and with less chair time. Thus, a trend towards the search for non-instrumental endodontic treatment (p<0.001) could be noted, precisely because it is a faster and cheaper technique. Although there are many published works about endodontics in deciduous teeth, as seen in the first two studies, it was observed that further investigations are still needed. (AU)

The impact of Distinctive Root Canal Instrumentation Systems on Endotoxin Lessening from the Root Canal: A Systematic Review and Meta-Analysis

ABSTRACT Objective: To determine the impact of distinctive instrumentation systems of the root canals on the endotoxin lessening through the root canals. Material and Methods: From the electronic databases, MEDLINE, PubMed, Cochrane Library, Embase, ISI, Google Scholar have been used to perform a systematic literature review between 2015 and 2020. Therefore, a software program (Endnote X9) has been utilized for managing electronic titles. Searches were performed with keywords, "root canal," "instrumentation," "endotoxin," "root canal preparation," "biofilm" "endodontics," and "lipopolysaccharide." This systematic review has been conducted on the basis of the key consideration of the PRISMA Statement-Preferred Reporting Items for the Systematic Review and Meta-analysis. Results: Hence, 163 potentially important abstracts and research topics have been discovered by electronic searches and three studies (3 RCTs) have been included. According to the outputs, any statistically significant differences have been not found between the rotary files and reciprocation (SMD 0.51, 95% CI [0.11, 0.90], p=0.011) (I2 = 49.5%; p=0.138). Conclusion: Analyses indicated that instrumentation methods decreased the content of endotoxin from the root canals.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

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.

Root canal contamination or exposure to lipopolysaccharide differentially modulate prostaglandin E 2 and leukotriene B 4 signaling in apical periodontitis

Abstract Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.

Lipopolysaccharides' Cytotoxicity on Human Umbilical Cord Mesenchymal Stem Cells

Abstract Objective: To show the cytotoxicity of Porphyromonas gingivalis lipopolysaccharide (LPS) on human umbilical cord mesenchymal stem cells (HUCMSCs) to better understand the characteristics for its application in regenerative procedures under periodontopathogen LPS influence. Material and Methods: Ultrapure Porphyromonas gingivalis LPS was used in this study. This research used a frozen stock HUCMSCs, previously confirmed by flow cytometry. The biological characteristics, such as cell morphology, proliferation, and protein expression, were screened. To check the cytotoxicity, HUCMSCs were cultured and divided into two groups, the control group and LPS group with various concentrations from 25 to 0.39 µg/mL. MTT assay was done and the cells were observed and counted. The significance level was set at 5%. Results: The percentage of living HUCMSCs on LPS group were not significantly different among concentrations (p>0.05) from 25 to 0.39 µg/mL, even though there were slight mean decrease between groups, but they were not significant. The duration of 24 hours of exposure of LPS does not significantly lower HUCMSCs viability. Conclusion: LPS does not affect the viability of HUCMSCs. The lower the concentration of LPS, the higher the viability of HUCMSCs.

A Protocol to Perform Systemic Lipopolysacharide (LPS) Challenge in Rats / Protocolo para realizar reto sistémico con lipopolisacárido (LPS) en ratas

Abstract 19. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. In animals, intraperitoneal administration of LPS, stimulates innate immunity and the production of proinflammatory cytokines. LPS provides an inflammatory stimulus that activates the neuroimmune and neuroendocrine systems resulting in a set of responses termed sickness behavior. The purpose of this protocol is to describe step-by-step the preparation and procedure of application of intraperitoneal injection of LPS in rats, as a guide for those researchers that want to use this assay to mount an inflammatory response. LPS intraperitoneal challenge in rats has been widely used to evaluate antiinflammatory reagents and to address basic scientific questions.

Resumen 23. El lipopolisacárido (LPS) es un componente de la membrana externa de las bacterias Gram negativas. En animales, la administración intraperitoneal de LPS estimula la inmunidad innata y la producción de citoquinas proinflamatorias. El LPS proporciona un estímulo inflamatorio que activa el sistema neuroinmunológico y el sistema neuroendocrino, lo que da como resultado un conjunto de respuestas denominadas conductas de enfermedad. El propósito de este protocolo es describir paso a paso la preparación y el procedimiento de aplicación de la inyección intraperitoneal de LPS en ratas, como una guía para aquellos investigadores que desean utilizar este método para estimular una respuesta inflamatoria en el animal. La estimulación con LPS en ratas, aplicada intraperitonealmente, se ha utilizado ampliamente para evaluar reactivos antiinflamatorios y para abordar preguntas básicas de investigación científica.

Periapical bone response to bacterial lipopolysaccharide is shifted upon cyclooxygenase blockage

Abstract Objectives: Infection, inflammation and bone resorption are closely related events in apical periodontitis development. Therefore, we sought to investigate the role of cyclooxygenase (COX) in osteoclastogenesis and bone metabolism signaling in periapical bone tissue after bacterial lipopolysaccharide (LPS) inoculation into root canals. Methodology: Seventy two C57BL/6 mice had the root canals of the first molars inoculated with a solution containing LPS from E. coli (1.0 mg/mL) and received selective (celecoxib) or non-selective (indomethacin) COX-2 inhibitor. After 7, 14, 21 and 28 days the animals were euthanized and the tissues removed for total RNA extraction. Evaluation of gene expression was performed by qRT-PCR. Statistical analysis was performed using analysis of variance (ANOVA) followed by post-tests (α=0.05). Results: LPS induced expression of mRNA for COX-2 (Ptgs2) and PGE2 receptors (Ptger1, Ptger3 and Ptger4), indicating that cyclooxygenase is involved in periapical response to LPS. A signaling that favours bone resorption was observed because Tnfsf11 (RANKL), Vegfa, Ctsk, Mmp9, Cd36, Icam, Vcam1, Nfkb1 and Sox9 were upregulated in response to LPS. Indomethacin and celecoxib differentially modulated expression of osteoclastogenic and other bone metabolism genes: celecoxib downregulated Igf1r, Ctsk, Mmp9, Cd36, Icam1, Nfkb1, Smad3, Sox9, Csf3, Vcam1 and Itga3 whereas indomethacin inhibited Tgfbr1, Igf1r, Ctsk, Mmp9, Sox9, Cd36 and Icam1. Conclusions: We demonstrated that gene expression for COX-2 and PGE2 receptors was upregulated after LPS inoculation into the root canals. Additionally, early administration of indomethacin and celecoxib (NSAIDs) inhibited osteoclastogenic signaling. The relevance of the cyclooxygenase pathway in apical periodontitis was shown by a wide modulation in the expression of genes involved in both bone catabolism and anabolism.

Cytotoxicity of intracanal dressings on apical papilla cells differ upon activation with E. faecalis LTA

Abstract Objective The aim of this study was to investigate the cytotoxic effects of modified triple antibiotic paste and an experimental composition using calcium hydroxide on lipoteichoic acid (LTA)-primed apical papilla cells (APC). Material and Methods Human APC were tested for in vitro cytotoxicity of modified Triple Antibiotic Paste (mTAP - Ciprofloxacin, Metronidazole and Cefaclor at 1:1:1) and of a paste of Ciprofloxacin, Metronidazole and Calcium hydroxide (CMC - 1:1:2) and modified CMC (mCMC - 2:2:1) by using MTT assay. The substances were reconstituted in DMEM at 1,000 µg/mL and » serially diluted before being kept in contact with cells for 1, 3, 5 and 7 days. Further, cells were primed with 1 µg/mL of Enterococcus faecalis LTA for 7 days prior to the viability test with 1,000 µg/mL of each substance. Statistical analysis was performed using one-way analysis of variance (ANOVA) and two-way ANOVA respectively followed by Tukey's post-test. Significance levels were set at p<0.05. Results In the first assay, the higher cytotoxic rates were reached by mTAP for all experimental periods. CMC was found toxic for APC at 5 and 7 days, whereas mCMC did not affect the cell viability. Only CMC and mCMC were able to induce some cellular proliferation. In the second assay, when considering the condition with medium only, LTA-primed cells significantly proliferated in comparison to LTA-untreated ones. At this context, mTAP and CMC showed similar cytotoxicity than the observed for LTA-untreated cells, while mCMC was shown cytotoxic at 7 days only for LTA-primed APC. Comparing the medications, mTAP was more cytotoxic than CMC and mCMC. Conclusion mTAP showed higher cytotoxicity than CMC and mCMC and the effect of topic antimicrobials might differ when tested against apical papilla cells under physiological or activated conditions.

Análise in vitro da N-Acetilcisteína em fibroblastos do ligamento periodontal estimulados por LPS bacteriano / In vitro analysis of N-Acetylcysteine in periodontal ligament fibroblasts stimulated by bacterial LPS

O objetivo deste estudo foi avaliar in vitro o comportamento biológico celular da N-Acetilcisteína (NAC), diante da estimulação ou não pelo LPS bacteriano. Foram utilizados fibroblastos do ligamento periodontal, que ficaram em contato por 48 horas com as substâncias testadas: NAC, Hidróxido de cálcio p.a. (HC), Lipopolissacarídeo de Escheria Coli (LPS), NAC + LPS e HC + LPS. Para os grupos NAC + LPS, HC + LPS e LPS, as células foram estimuladas com 2µg/mL de LPS por 24 horas, previamente aos tratamentos descritos. Foram realizados os testes biológicos de viabilidade celular (XTT), Espécies Reativas de Oxigênio (ROS), Elisa (para as citocinas IL-6, IL-8, IL-10, IL-1ß e TNF-α) e Micronúcleo (MNT). Os dados foram analisados estatisticamente pela análise descritiva e pelos testes ANOVA e Kruskall Wallis, seguido do teste Dunn (p˂0.05) para os testes de XTT, ROS e MNT, e para o teste Elisa foi realizado cálculo das médias e desvio padrão. Os resultados obtidos mostraram que NAC teve um bom comportamento, frente à agressão provocada pelo LPS, quanto à produção de ROS. HC apresentou maior viabilidade celular que NAC, embora NAC não tenha apresentado citotoxicidade. Em relação à expressão das citocinas, NAC foi capaz de reduzir o potencial inflamatório do LPS quando da análise do TNF- α e IL-1ß. NAC foi capaz de reduzir a genotoxicidade do LPS, como mostrado pelo teste MNT. Concluiu-se que NAC apresentou um comportamento biológico satisfatório, pela viabilidade celular dos fibroblastos, pela redução da geração de ROS e do potencial inflamatório provocado pelo LPS, e por reduzir a sua genotoxicidade (AU)

The purpose of this study was to evaluate in vitro the action of N-Acetylcysteine (NAC) and calcium hydroxide (CH) on biological activity, either without or with LPS stimulation in periodontal ligament fibroblasts (PDLF) cells. PDLF were placed in contact with NAC, CH, NAC + LPS, LPS and CH + LPS for 48 hours. PDLF were stimulated by bacterial LPS for 24 hours in NAC + LPS, LPS and CH + LPS groups, before the substances described above are applied. The LPS and NAC effect on cell viability was measured using a XTT test. Reactive oxygen species (ROS) production was evaluated using ROS/superoxide detection kit. Inflammatory cytokines (IL-6, IL-8, IL-10, IL-1ß and TNF-α) were evaluated by enzyme-linked immunosorbent (ELISA) assay. Genotoxicity was measured using micronucleus test (MNT). The means and standard deviation for all tests were calculated. Data were analyzed statistically by descriptive analysis, ANOVA and Kruskall Wallis tests, followed by Dunn test (p˂.05). CH was superior to NAC on cellular viability, although NAC was not cytotoxic. The results showed that NAC was able to reduce ROS production of LPS. NAC was able to reduce the inflammatory potential of LPS by decreasing the TNF-α and IL-1ß release. NAC was able to reduce the genotoxicity of LPS. It was concluded that NAC showed a satisfactory biological activity, presenting a minimal effect on cell viability of PDLF cells and reducing ROS production and inflammatory potential provoked by LPS, and to decrease its genotoxicity(AU)

Bacterial endotoxin adhesion to different types of orthodontic adhesives

Abstract Bacterial endotoxin (LPS) adhesion to orthodontic brackets is a known contributing factor to inflammation of the adjacent gingival tissues. Objective The aim of this study was to assess whether LPS adheres to orthodontic adhesive systems, comparing two commercial brands. Material and Methods Forty specimens were fabricated from Transbond XT and Light Bond composite and bonding agent components (n=10/component), then contaminated by immersion in a bacterial endotoxin solution. Contaminated and non-contaminated acrylic resin samples were used as positive and negative control groups, respectively. LPS quantification was performed by the Limulus Amebocyte Lysate QCL-1000™ test. Data obtained were scored and subjected to the Chi-square test using a significance level of 5%. Results There was endotoxin adhesion to all materials (p<0.05). No statistically significant difference was found between composites/bonding agents and acrylic resin (p>0.05). There was no significant difference (p>0.05) among commercial brands. Affinity of endotoxin was significantly greater for the bonding agents (p=0.0025). Conclusions LPS adhered to both orthodontic adhesive systems. Regardless of the brand, the endotoxin had higher affinity for the bonding agents than for the composites. There is no previous study assessing the affinity of LPS for orthodontic adhesive systems. This study revealed that LPS adheres to orthodontic adhesive systems. Therefore, additional care is recommended to orthodontic applications of these materials.

Toll-like receptor agonists Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells

Abstract IL-10 expressing regulatory B cells (B10) play a key role in immune system balance by limiting excessive inflammatory responses. Effects of toll-like receptor signaling and co-stimulatory molecules on B10 activity during innate and adaptive immune responses are not fully understood. Objective This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and IL-10 competency in vitro. Material and Methods Spleen B cells were isolated from C57BL/6J mice with or without formalin-fixed P. gingivalis immunization. B cells were cultured for 48 hours under the following conditions: CD40L, CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of fixed P. gingivalis. Percentages of CD1dhiCD5+ B cells were measured by flow cytometry. IL-10 mRNA expression and secreted IL-10 were measured by real-time quantitative PCR and by ELISA respectively. Results P. gingivalis LPS plus CD40L significantly increased CD1dhiCD5+ B cell percentages and secreted IL-10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels were significantly increased in CD40L+LPS treated group compared with CD40L treatment group in the absence of P. gingivalis. CpG plus CD40L significantly decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL-10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group. Conclusions P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses.

Influência da galectina-3 na resposta de neutrófilos a patógenos periodontais / Influence of galectin-3 on neutrophil response to periodontal pathogens

Galectina-3, uma proteína que se liga a -galactosídeos, é expressa por neutrófilos e inúmeras evidências indicam que esta molécula atua como uma possível reguladora da resposta imune. Sabe-se que galectina-3 ao ligar com LPS pode levar a formação de oligômeros, que podem alterar o limiar de ativação de células da resposta imune inata. Apesar de existirem diversos estudos que mostram a influência de galectina-3 na resposta de neutrófilos frente a componentes bacterianos, os resultados são em sua maioria contraditórios e inconclusivos. Para elucidar a influência da galectina-3 na reposta imune inata a patógenos periodontais, o presente trabalho avaliou a atividade antimicrobiana in vitro de neutrófilos, isolados de camundongos selvagens (WT) ou geneticamente deficientes de galectina-3 (Gal-3KO), previamente estimulados com LPS de Aa e Pg. Os resultados não evidenciaram diferenças significativas no número de unidades formadoras de colônia (UFC) recuperadas das culturas de neutrófilos provenientes de animais deficientes de galectina-3 e do grupo controle (WT). Contudo, a estimulação de neutrófilos com LPS por 18 horas levou a redução no número de UFC recuperadas das culturas, quando comparado com as culturas estimuladas com LPS por apenas 3 horas.

Galectin-3, a protein that binds -galactosides, is expressed by neutrophils and numerous evidences indicate that this molecule acts as a possible regulator of the immune response. It is known that galectin-3 binding to LPS can lead to the formation of oligomers and thus changing the activation threshold of cells of the innate immune response. Although there are several studies that show the influence of galectin-3 in neutrophil response against bacterial components, the results are conflicting and inconclusive in their majority. To elucidate the influence of galectin-3 in the innate immune response to periodontal pathogens, the present study evaluated the in vitro antimicrobial activity of neutrophils, isolated from wild-type or galectin-3 deficient mice, previously stimulated with LPS of Aa and Pg. The results showed no significant differences in the number of colony forming units (CFU) recovered from cultured galectin-3 deficient neutrophils or control group. However, in a 18 hours time course of LPS stimulation, we observed reduction in the number of CFU, when compared to 3 hours of LPS stimulation.

QMix® irrigant reduces lipopolysacharide (LPS) levels in an in vitro model

AbstractThe presence of endotoxin inside the root canal has been associated with periapical inflammation, bone resorption and symptomatic conditions.Objectives To determine, in vitro, the effect of QMix® and other three root canal irrigants in reducing the endotoxin content in root canals.Material and Methods Root canals of single-rooted teeth were prepared. Samples were detoxified with Co-60 irradiation and inoculated with E. coli LPS (24 h, at 37°C). After that period, samples were divided into 4 groups, according to the irrigation solution tested: QMix®, 17% EDTA, 2% chlorhexidine solution (CHX), and 3% sodium hypochlorite (NaOCl). LPS quantification was determined by Limulus Amebocyte Lysate (LAL) assay. The initial counting of endotoxins for all samples, and the determination of LPS levels in non-contaminated teeth and in contaminated teeth exposed only to non-pyrogenic water, were used as controls.Results QMix® reduced LPS levels, with a median value of 1.11 endotoxins units (EU)/mL (p<0.001). NaOCl (25.50 EU/mL), chlorhexidine (44.10 EU/mL) and positive control group (26.80 EU/mL) samples had similar results. Higher levels were found with EDTA (176.00 EU/mL) when compared to positive control (p<0.001). There was no significant difference among EDTA, NaOCl and CHX groups. Negative control group (0.005 EU/mL) had statistically significant lower levels of endotoxins when compared to all test groups (p<0.001).Conclusion QMix® decreased LPS levels when compared to the other groups (p<0.001). 3% NaOCl, 2% CHX and 17% EDTA were not able to significantly reduce the root canal endotoxins load.

Monitoring the effectiveness of root canal procedures on endotoxin levels found in teeth with chronic apical periodontitis

Objective: The aim of this study was to monitor the effectiveness of root canal procedures by using different irrigants and intracanal medication on endotoxin levels found in root canals of teeth with chronic apical periodontitis. Material and Methods: Thirty root canals of teeth with pulpal necrosis associated with periapical lesions were selected and randomly divided into groups according to the irrigants used: GI - 2.5% NaOCl, GII - 2% chlorhexidine (CHX) gel, and GIII - saline solution (SS) (all, n=10). Samples were collected with sterile/apyrogenic paper points before (S1) and after root canal instrumentation (S2), after use of 17% ethylenediaminetetraacetic acid (EDTA) (S3), and after 30 days of intracanal medication (Ca(OH)2+SS) (S4). A turbidimetric kinetic Limulus Amebocyte Lysate assay was used for endotoxin measurement. Results: Endotoxins were detected in 100% of the root canals investigated (30/30), with a median value of 18.70 EU/mL. After S2, significant median percentage reduction was observed in all groups, irrespective of the irrigant tested: 2.5% NaOCl (99.65%) (GI), 2% CHX (94.27%) (GII), and SS (96.79%) (GIII) (all p<0.05). Root canal rinse with 17% EDTA (S3) for a 3-minute period failed to decrease endotoxin levels in GI and a slight decrease was observed in GII (59%) and GIII (61.1%) (all p>0.05). Intracanal medication for 30 days was able to significantly reduce residual endotoxins: 2.5% NaOCl (90%) (GI), 2% CHX (88.8%) (GII), and SS (85.7%) (GIII, p<0.05). No differences were found in the endotoxin reduction when comparing s2 and s4 treatment groups. Conclusion: Our findings demonstrated the effectiveness of the mechanical action of the instruments along with the flow and backflow of irrigant enduring root canal instrumentation for the endotoxin removal from root canals of teeth with chronic apical periodontitis. Moreover, the use of intracanal medication for 30 days contributed for an improvement ...

Efeitos de diferentes concentrações de dextrose e lipopolissacarídeo no comportamento corrosivo da liga Ti-6Al-4V com superfície usinada e tratada com duplo ataque ácido / Effects of different concentrations of dextrose and lipopolysaccharide on the corrosion behavior of cpTi with machined and double acid-etched surfaces

Objetivos: Avaliar o comportamento corrosivo da liga Ti-6Al-4V em função de sua superfície polida ou condicionada por meio de duplo ataque ácido, em simulador de fluidos corporais (SFC) com diferentes concentrações de dextrose (0; 5mM; 7,5 mM e 15 mM) e lipopopolissacarídeo (LPS) (0; 0,15 μg/mL; 15 μg/mL; 150 μg/mL), usados isolados ou associados. Materiais e Métodos: Discos de liga Ti-6Al-4V (2 mm de espessura e 15 mm de diâmetro) foram confeccionados com diferentes padrões de superfície. Para o ensaio eletroquímico (n=3), testes padrões como potencial de circuito aberto, espectroscopia de impedância eletroquímica (EIE) e teste potenciodinâmico foram conduzidos em solução de fluido corpóreo (SBF) com diferentes concentrações de dextrose e LPS. As superfícies dos discos foram caracterizadas pela microscopia eletrônica de varredura (MEV), microscopia de força atômica (MFA), e por meio da rugosidade de superfície e da microdureza. Os dados quantitativos foram analisados pelo teste de correlação de Pearson e T-teste independente, com nível de significância de 5%. Resultados: Nos parâmetros de corrosão, houve forte correlação do LPS com os valores de Ipass (densidade de corrente de passivação), Cdl (capacitância) e Rp (resistência de polarização) (p<0,05) para o titânio (Ti) com tratamento de superfície por duplo ataque ácido. A associação de dextrose e LPS foi correlacionada com Icorr (densidade de corrente de corrosão) e Ipass (p<0,05). Em relação à cinética de corrosão, os grupos tratados com ácido apresentaram aumento significante nos valores de Cdl e redução nos valores de Rp (P<0,05, T-teste). Quanto à topografia, houve aumento da rugosidade de superfície para ambas as superfícies (R2=0,726, p=0,0001 para Ti polido; R2=0,405, p=0,036 para Ti ácido). A microdureza do Ti polido diminuiu (p<0,05) e do Ti ácido aumentou (p=0,0001). A MFA mostrou alteração na microestrutura do Ti por meio do aumento da espessura superficial principalmente no grupo com associação...

Objectives: To analyse the corrosion behavior of Ti-6Al-4V with different surfaces (machined and modified by treatment with double acid-etching), on simulator body fluids (SBF) with different concentrations of dextrose (0; 5 mM, 7.5 mM and 15 mM) and lipopolysaccharide (LPS) (0; 0.15 μg/mL; 15 μg/mL; 150 μg/mL), used isolated or in combination. Materials and Methods: Ti-6Al-4V disks (2 mm in thickness and 15 mm in diameter) were fabricated with different surfaces. For electrochemical assay (n = 3), open circuit potential, electrochemical impedance spectroscopy (EIS) and potentiodynamic tests were conducted in SBF with different concentrations of dextrose and LPS. The surfaces of the disks were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and by surface roughness and microhardness. Data were analyzed by Pearson correlation and independent t-test at a significant level of 5%. Results: For the corrosion parameters, a strong correlation of LPS with Ipass (passivation current density), Cdl (capacitance) and Rp (polarization resistance) values (P <.05) for Ti surface treated by double acid- etching was noted. The combination of dextrose and LPS was correlated with Ipass and Icorr (corrosion current density) (P <.05). Regarding the corrosion kinetics the acid-treated groups showed significant increase in Cdl values and reduced values of Rp (P <.05, T-test). In terms of topography, there was an increase in surface roughness for both surfaces (R2 = 0.726, p = 0.0001 for machined Ti, R2 = 0.405, p = 0.036 for Ti acid). The machined Ti exhibited reduction in microhardness (P <.05), while Ti acid showed increased microhardness (p = 0.0001) vs baseline. The AFM showed changes in the microstructure of Ti by increasing the thickness of surface mainly in the association of dextrose and LPS. Conclusions: The combination of dextrose and LPS affected the corrosion behavior of Ti-6Al-4V surface treated with double acid-etching. These results...

Efeitos de diferentes concentrações de dextrose e lipopolissacarídeo no comportamento corrosivo da liga Ti-6Al-4V com superfície usinada e tratada com duplo ataque ácido / Effects of different concentrations of dextrose and lipopolysaccharide on the corrosion behavior of cpTi with machined and double acid-etched surfaces

Objetivos: Avaliar o comportamento corrosivo da liga Ti-6Al-4V em função de sua superfície polida ou condicionada por meio de duplo ataque ácido, em simulador de fluidos corporais (SFC) com diferentes concentrações de dextrose (0; 5mM; 7,5 mM e 15 mM) e lipopopolissacarídeo (LPS) (0; 0,15 μg/mL; 15 μg/mL; 150 μg/mL), usados isolados ou associados. Materiais e Métodos: Discos de liga Ti-6Al-4V (2 mm de espessura e 15 mm de diâmetro) foram confeccionados com diferentes padrões de superfície. Para o ensaio eletroquímico (n=3), testes padrões como potencial de circuito aberto, espectroscopia de impedância eletroquímica (EIE) e teste potenciodinâmico foram conduzidos em solução de fluido corpóreo (SBF) com diferentes concentrações de dextrose e LPS. As superfícies dos discos foram caracterizadas pela microscopia eletrônica de varredura (MEV), microscopia de força atômica (MFA), e por meio da rugosidade de superfície e da microdureza. Os dados quantitativos foram analisados pelo teste de correlação de Pearson e T-teste independente, com nível de significância de 5%. Resultados: Nos parâmetros de corrosão, houve forte correlação do LPS com os valores de Ipass (densidade de corrente de passivação), Cdl (capacitância) e Rp (resistência de polarização) (p<0,05) para o titânio (Ti) com tratamento de superfície por duplo ataque ácido. A associação de dextrose e LPS foi correlacionada com Icorr (densidade de corrente de corrosão) e Ipass (p<0,05). Em relação à cinética de corrosão, os grupos tratados com ácido apresentaram aumento significante nos valores de Cdl e redução nos valores de Rp (P<0,05, T-teste). Quanto à topografia, houve aumento da rugosidade de superfície para ambas as superfícies (R2=0,726, p=0,0001 para Ti polido; R2=0,405, p=0,036 para Ti ácido). A microdureza do Ti polido diminuiu (p<0,05) e do Ti ácido aumentou (p=0,0001). A MFA mostrou alteração na microestrutura do Ti por meio do aumento da espessura superficial principalmente no grupo com associação...

Objectives: To analyse the corrosion behavior of Ti-6Al-4V with different surfaces (machined and modified by treatment with double acid-etching), on simulator body fluids (SBF) with different concentrations of dextrose (0; 5 mM, 7.5 mM and 15 mM) and lipopolysaccharide (LPS) (0; 0.15 μg/mL; 15 μg/mL; 150 μg/mL), used isolated or in combination. Materials and Methods: Ti-6Al-4V disks (2 mm in thickness and 15 mm in diameter) were fabricated with different surfaces. For electrochemical assay (n = 3), open circuit potential, electrochemical impedance spectroscopy (EIS) and potentiodynamic tests were conducted in SBF with different concentrations of dextrose and LPS. The surfaces of the disks were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and by surface roughness and microhardness. Data were analyzed by Pearson correlation and independent t-test at a significant level of 5%. Results: For the corrosion parameters, a strong correlation of LPS with Ipass (passivation current density), Cdl (capacitance) and Rp (polarization resistance) values (P <.05) for Ti surface treated by double acid- etching was noted. The combination of dextrose and LPS was correlated with Ipass and Icorr (corrosion current density) (P <.05). Regarding the corrosion kinetics the acid-treated groups showed significant increase in Cdl values and reduced values of Rp (P <.05, T-test). In terms of topography, there was an increase in surface roughness for both surfaces (R2 = 0.726, p = 0.0001 for machined Ti, R2 = 0.405, p = 0.036 for Ti acid). The machined Ti exhibited reduction in microhardness (P <.05), while Ti acid showed increased microhardness (p = 0.0001) vs baseline. The AFM showed changes in the microstructure of Ti by increasing the thickness of surface mainly in the association of dextrose and LPS. Conclusions: The combination of dextrose and LPS affected the corrosion behavior of Ti-6Al-4V surface treated with double acid-etching. These results...

Caracterização da ação modulatória de citocinas inflamatórias pelo óleo de Melaleuca alternifolia e seus componentes (terpinen-4ol e alfa-terpineol) em macrófagos humanos ativados por lipopolissacarídeos de Porphyromonas gingivalis e Escherichia coli / Characterization of modulatory action of inflammatory cytokines by Melaleuca alternifolia oil and its components (terpinnen-4ol and alpha-terpineol) in human macrophages activated lipopolysaccharide of Porphyromonas gingivalis and Escherichia coli

O óleo de Melaleuca alternifolia (TTO) tem propriedades antimicrobianas e somado a isso, sugere-se que apresente características anti-inflamatórias. Este estudo investigou o potencial do TTO e seus componentes (terpinen-4-ol e alfa-terpineol) em modular citocinas e os mecanismos intracelulares participantes desta ação. Células monocíticas (U937) foram diferenciados em macrófagos com 40ng/mL de 12-miristato 13-acetato de forbol. A citotoxicidade dos óleos foi determinada com o ensaio de redução de Metil-tetrazolium (MTT). A habilidade em modular a produção das citocinas após o estímulo com LPS de Porphyromonas gingivalis (agonista de TLR2) e Escherichia coli (agonista de TLR4), foi estabelecida por meio de ensaios ELISA. Os efeitos dos compostos testados, na ativação de vias de sinalização intracelular, foram avaliados por western blot. As concentrações selecionadas pelo MTT foram: 0,015% e 0,004% para o TTO, 0,059% e 0,0073% para o terpinen-4-ol e 0,0064% e 0,0007% para alfa-terpineol. A influência do TTO na produção de citocinas foi mais marcante após estímulo de TLR4, com diminuição de IL-1ß, IL-6 e IL-10. O terpinen-4-ol reduziu a produção de INF-γ, IL-1ß, IL-6, IL-4, IL-10 e IL-17 após ativação tanto TLR4 quanto TLR2. O alfa-terpienol atua após ativação de TLR2 e 4 inibindo a produção de IL-1ß, IL-6 e IL-10. Nenhuma das vias analisadas (NF-kB, ERK, p38 MAP-Kinase) sofreram interferência dos óleos. O TTO tem ação modulatória inibindo produção de citocinas inflamatórias e o terpinen-4-ol é o seu maior componente ativo, entretanto este mecanismo não ocorre via inibição de NF-kB, ERK e p38 MAP-Kinase

The oil of Melaleuca alternifolia (TTO) has antimicrobial properties, coupled with this, it is suggested that it also has antiinflammatory characteristics. This study investigated the potential of TTO and its components (terpinen-4-ol and alphaterpineol) and cytokines in modulating the intracellular mechanisms that participate in this action. Monocytic cells (U937) were differentiated into macrophages with 40ng/mL 12-myristate 13-acetate phorbol. The olil cytotoxicity was determined with the reduction assay Methyl-tetrazolium (MTT). The ability to modulate cytokine production after stimulation with LPS of porphyromonasgingivalis (TLR2 agonist) and Escherichia coli (TLR4 agonist), was established by ELISA assays. The effects of the tested compounds, the activation of intracellular signaling pathways were evaluated by western blot. The concentrations selected by the MTT were 0.015% and 0.004% for the TTO, 0.059% and 0.0073% to terpinen-4-ol and 0.0064% and 0.0007% for alpha-terpineol. The influence of TTO in cytokine production was more marked after TLR4 stimulation, with decreased IL-1ß, IL-6 and IL-10. The terpinen-4-ol reduced the production of INF-γ, IL-1ß, IL-6, IL-4, IL-10 and IL-17 activation after both TLR4 as TLR2. The alpha-terpienol acts upon activation of TLR2 and four inhibiting the production of IL-1ß, IL-6 and IL-10. None of the analyzed pathways (NF-kB, ERK, p38 MAP-Kinase) suffered interference oils. The TTO has modulatory action by inhibiting the production of inflammatory cytokines and terpinen-4-ol is your greatest asset component however, this mechanism does not occur via inhibition of NF-kB, ERK and p38 MAP-kinase

Ca(OH)2 action on TNF-alpha and NO release in macrophage culture stimulated by Pseudomonas aeruginosa LPS / Ação do Ca(OH)2 sobre a produção de TNF-alfa e NO de cultura de macrófagos estimulada por LPS de Pseudomonas aeruginosa

Purpose: Calcium hydroxide [Ca(OH)2] interaction with the immune system to destroy or neutralize bacteria and their by-products is not completely understood. This study evaluated the calcium hydroxide ability to neutralize Pseudomonas aeruginosa lipopolysaccharides (LPS) using two different methods: nitric oxide (NO) stimulation and Tumoral Necrosis Factor-alpha (TNF-alpha) release in mice macrophage culture. Methods: For the NO assay, peritoneal exudate cells (PECs) were placed in contact with 25ìg/mL and 50ìg/mL LPS and LPS/Ca(OH)2 suspensions (50ìg/25mg and 25ìg/25mg). After incubation for 8h, Griess reagent was used, and the NO release was quantified. In the TNF-alpha assay the LPS solution was tested in 25ìg/mL, and the LPS/Ca(OH)2 suspension was tested in 25ìg/25mg concentration. After incubation for 24 hours, cells were fixed and stained with crystal violet. Absorbance values were obtained. Results were expressed in micromols. All tests were performed in triplicate. Results: The presence of Ca(OH)2 in both tested concentrations significantly reduced NO and TNF-alpha release. Conclusion: It can be concluded that bacteria LPS is a strong stimulus for NO and TNF-alpha release, but calcium hydroxide can neutralize it.

Objetivo: A ação do hidróxido de cálcio [Ca(OH)2] com o sistema imune e o mecanismo de neutralização das bactérias e seus subprodutos ainda não foi completamente esclarecida. Neste estudo foi avaliada a capacidade do Ca(OH)2 em neutralizar o lipopolissacarídeo (LPS) de Pseudomonas aeruginosa, utilizando-se duas metodologias: liberação de Óxido Nítrico (NO) e Fator de Necrose Tumoral Alfa (TNF-alfa) em cultura de macrófagos peritoneais de camundongos. Metodologia: No ensaio do NO, as células peritoneais foram expostas a uma solução de LPS (25mg/mL e 50mg/mL); e à suspensão de LPS/Ca(OH)2 em duas concentrações (50mg/ 25mg e 25mg/25mg). Após 8 horas de incubação, foi utilizado reagente de Griess, e a liberação de NO foi quantificada. No ensaio do TNF-alfa, a solução de LPS foi usada na concentração de 25mg/mL e o LPS/Ca(OH)2 a 25mg/25mg. Após 24 horas, as células foram fixadas e coradas com cristal violeta, e os valores de absorbância foram obtidos. Os resultados foram expressos em micromols. Todos os testes foram realizados em triplicata. Resultados: A presença de Ca(OH)2 nas duas concentrações avaliadas reduziu significativamente a liberação de NO e TNF-alfa. Conclusão: Pode-se concluir que o LPS bacteriano representa um forte estímulo para liberação destas citocinas, mas o hidróxido de cálcio foi capaz de neutralizar este efeito.

Efeito do processo inflamatório sobre as propriedades regenerativas das células-tronco do ligamento periodontal / Effect of inflammation on the regenerative properties of stem cells from periodontal ligament

Estudos recentes mostraram que o ligamento periodontal abriga populações celulares com características de células-tronco adultas, as quais são denominadas como células progenitoras ou indiferenciadas de origem mesenquimal. O sucesso da regeneração dos tecidos periodontais depende de uma série de eventos biológicos, os quais envolvem a migração e a proliferação das células indiferenciadas para o interior do defeito periodontal, e sua subsequente diferenciação em osteoblastos, cementoblastos e fibroblastos, acompanhada pela síntese de componentes das matrizes teciduais. Alguns estudos na área médica mostraram que as propriedades biológicas das células-tronco podem ser alteradas após a exposição tecidual a um processo inflamatório crônico, comprometendo o seu potencial regenerativo. Desta maneira, o presente estudo teve como objetivo realizar uma revisão da literatura das evidências disponíveis a respeito do impacto da doença periodontal inflamatória crônica sobre as propriedades regenerativas das células mesenquimais indiferenciadas, localizadas nas estruturas periodontais remanescentes.