Exopolysaccharide dispelled by calcium hydroxide with volatile vehicles related to bactericidal effect for root canal medication

ABSTRACT Objective: Enterococcus faecalis is the dominant microbial species responsible for persistent apical periodontitis with ability to deeply penetrate into the dentin. Exopolysaccharides (EPS) contribute to the pathogenicity and antibiotic resistance of E. faecalis. Our aim was to investigate the antimicrobial activity of calcium hydroxide (CH), camphorated parachlorophenol (CMCP), and chlorhexidine (CHX) against E. faecalis in dentinal tubules. Material and Methods: Decoronated single-canal human teeth and semicylindrical dentin blocks were incubated with E. faecalis for 3 weeks. Samples were randomly assigned to six medication groups for 1 week (n=10 per group): CH + 40% glycerin-water solution (1:1, wt/vol); CMCP; 2% CHX; CH + CMCP (1:1, wt/vol); CH + CMCP (2:3, wt/vol); and saline. Bacterial samples were collected and assayed for colony-forming units. After dentin blocks were split longitudinally, confocal laser scanning microscopy was used to assess the proportion of viable bacteria and EPS production in dentin. Results: CMCP exhibited the best antimicrobial activity, while CH was the least sensitive against E. faecalis (p<0.05). CHX showed similar antimicrobial properties to CH + CMCP (1:1, wt/vol) (p>0.05). CH combined with CMCP inhibited EPS synthesis by E. faecalis, which sensitized biofilms to antibacterial substances. Moreover, increasing concentrations of CMCP decreased EPS matrix formation, which effectively sensitized biofilms to disinfection agents. Conclusion: The EPS matrix dispelled by CH paste with CMCP may be related to its bactericidal effect; the visualization and analysis of EPS formation and microbial colonization in dentin may be a useful approach to verify medicaments for antimicrobial therapy.

Ex-vivo Effect of Intracanal Medications Based on Ozone and Calcium Hydroxide in Root Canals Contaminated with Enterococcus faecalis

This ex vivo study evaluated the antibacterial effect of intracanal medications in root canals contaminated with Enterococcus faecalis. Fifty single-rooted human teeth were contaminated with E. faecalis (ATCC 29212) and incubated at 37°C for 21 days. The specimens were randomly divided into 5 groups according to the intracanal medication used: OZ-PG: ozonized propylene glycol; CH/CPMC: calcium hydroxide/camphorated paramonochlorophenol; OZ-PG/CH ozonized PG/CH; PC: positive control group (no medication); and NC: negative control group (no contamination). The samples were collected after 7 days (post-medication) and 14 days (final). Bacterial growth was checked by counting the colony-forming units (CFU). OZ-PG and CH/CPMC reduced significantly the CFU counts compared with PC in the post-medication and final samples, with no statistically significant differences among them. On the other hand, OZ-PG/CH did not reduce significantly the number of bacteria compared with PC. In conclusion, among the evaluated medications OZ-PG and CH/CPMC were the most effective against E. faecalis.

Resumo Este estudo ex vivo avaliou o efeito antibacteriano de medicações intracanal em canais radiculares contaminados com Enterococcus faecalis. Cinquenta dentes humanos unirradiculares foram contaminados com E. faecalis (ATCC 29212) e incubados a 37°C durante 21 dias. Os espécimes foram aleatoriamente divididos em diferentes grupos de acordo com a medicação intracanal utilizada: PG-OZ: propilenoglicol ozonizado; HC/PMCC: hidróxido de cálcio/paramonoclorofenol canforado; PG-OZ/CH; CP: controle positivo (sem medicação); e CN: controle negativo (sem contaminação). As amostras foram coletadas após 7 dias (pós-medição) e 14 dias (final). O crescimento bacteriano foi verificado através da contagem das unidades formadoras de colônias (UFC). PG-OZ e HC/PMCC reduziram estatisticamente o número de bactérias quando comparados com o CP nas amostras pós-medição e final, sem diferenças estatísticas entre si. Por outro lado, PG-OZ/HC não reduziu significativamente o número de bactérias em comparação com o CP. Em conclusão, entre as medicações avaliadas, PG-OZ e HC/PMCC foram as mais eficazes contra E. faecalis. .

Camphor modulates TRPV3 cation channels activity by interacting with critical pore-region cysteine residues

TRPV3 ion channels mediate thermo-transduction, nociception, inflammation and dermatitis in mammals. TRPV1-4 proteins have been shown to have conserved cysteine-residues in the pore-forming regions. These residues participate in channel activation via S-nitrosylation of channel proteins. Camphor is a commonly used ligand for TRPV3 channels. Thus the knowledge about the potential binding/interacting site[s] for camphor will help to design effective and potent analgesic compounds. In an overlap-extension PCR method, following primer-pairs were used to mutate conserved cysteine-residues in the pore-region of TRPV3 channels; GATTGAGAATcCTCCAAGGACAAAAAGGAC, TRPV3-C612S-Fw and GTCCTTGGAGgACTTCTCAATCAGTCAGTGAGG, TRPV3-C612S-Rv primers pair. And for TRPV3-C619S: GGACTCcAGTTCCTATGGCCAGC, TRPV3-C619S-Fw and GCTGGCCATAgGAACTGGAGTCC, TRPV3-C619S-Rv respectively. All cDNA constructs were confirmed by DNA-sequencing and used to make cRNAs. Oocytes expressing mTRPV3-C619S and mTRPV3-C612S mutant channels were challenged with 2-APB [1 mM], camphor [10 mM] and dihydrocarveol [10 mM] either at -40 mV or +40 mV holding potentials in voltage-clamp experiments. Responses of both mutants to 2-APB were similar to wild-type mTRPV3. Interestingly, responses to camphor were totally lost in mTRPV3-C619S mutant, while responses to dihydrocarveol remained intact. In contrast mTRPV3-C612S displayed slightly altered [16 +/- 2% reduction] phenotype with respect to camphor sensitivity. It is concluded that pore-region cysteines play critical role in camphor sensitivity of TRPV3 ion channels

Influence of serum and necrotic soft tissue on the antimicrobial effects of intracanal medicaments

The purpose of this study was to investigate the influence of serum and necrotic soft tissue on the antimicrobial activity of intracanal medicaments. The medicaments tested were: calcium hydroxyde/glycerin paste, calcium hydroxide/chlorhexidine paste, calcium hydroxide/camphorated paramonochlorophenol/glycerin paste, and chlorhexidine/zinc oxide paste. Survival of Enterococcus faecalis and Candida albicans exposed to the medicaments tested in the presence or absence of serum or necrotic tissue was monitored in three in vitro experiments where samples for culturing were taken at different time periods. The overall results demonstrated that the antimicrobial activity of all intracanal medicaments tested was slowed down in the presence of necrotic tissue. Calcium hydroxide pastes in glycerin or chlorhexidine were significantly affected by serum. Of the medicaments tested in this study, the least affected was the calcium hydroxide/camphorated paramonochlorophenol/glycerin paste.

O objetivo deste estudo foi avaliar a influência do soro e de tecido mole necrosado na atividade antimicrobiana de medicamentos intra-canais. Os medicamentos testados foram pastas de hidróxido de cálcio/glicerina, hidróxido de cálcio/clorexidina, hidróxido de cálcio/paramonoclorofenol canforado/glicerina e clorexidina/óxido de zinco. A sobrevivência de Enterococcus faecalis e Candida albicans expostos aos medicamentos na presença ou ausência de soro ou tecido necrosado foi monitorada em três experimentos in vitro nos quais amostras para cultura foram avaliadas em diferentes períodos de tempo. No geral, os resultados demonstraram que a atividade antimicrobiana de todos os medicamentos testados foi retardada na presença de soro ou de tecido necrosado. As pastas de hidróxido de cálcio em glicerina ou clorexidina foram significativamente afetadas pelo soro. Dos medicamentos testados, o menos afetado foi a pasta de hidróxido de cálcio/paramonoclorofenol canforado/glicerina.

Antibacterial and antioxidant activities of the essential oil of Artemisia echegarayi Hieron. (Asteraceae) / Actividad antibacteriana y antioxidante del aceite esencial extraído de Artemisia echegarayi Hieron. (Asteraceae)

Artemisia echegarayi Hieron. (Asteraceae) is commonly known in Argentina as “ajenjo”. Many studies report high efficacy of essential oils against food-borne pathogenic bacteria. The antimicrobial activity and minimal inhibitory concentration of A. echegarayi essential oil were evaluated against seven bacterial species of significant importance in food hygiene, by using the disc diffusion assay and the micro-well dilution method, respectively. Volatile components of the extract were analyzed by gas chromatography-mass spectrometry and major components were determined. Furthermore, the essential oil was tested for its antioxidant activity. The essential oil inhibited the growth of gram-positive and gram-negative tested bacteria, with the exception of Proteus mirabilis. A. echegarayi essential oil presented the lowest minimal inhibitory concentration against Listeria monocytogenes and Bacillus cereus. Two terpenes, thujone and camphor, were identified from this essential oil as the principal constituents responsible for antibacterial activity. The oil showed a free radical scavenging activity equivalent to 50% of the reference compound. These preliminary studies showed promising results since this essential oil may provide an alternative to promote its use as a natural food additive.

Artemisia echegarayi Hieron. (Asteraceae), conocida como “ajenjo”, es una planta típica de la región de Cuyo (Argentina). En este trabajo se evaluó la actividad antimicrobiana in vitro y la concentración inhibitoria mínima del aceite esencial extraído de sus partes aéreas frente a especies bacterianas que con frecuencia contaminan los alimentos. Se utilizaron las técnicas de difusión con discos en agar y microdilución en placa respectivamente. Además, se determinó la actividad antioxidante de este aceite esencial in vitro por espectrofotometría. En general, tanto las bacterias gram-positivas como las gram-negativas fueron inhibidas por este aceite, con excepción de Proteus mirabilis. Listeria monocytogenes y Bacillus cereus resultaron ser las bacterias más sensibles. El análisis por croma-tografía en fase gaseosa y espectrometría de masa permitió la identificación cualitativa y cuantitativa de los componentes mayoritarios del aceite esencial del ajenjo. Entre ellos, la tuyona y el alcanfor se destacaron como los principales responsables de la actividad antibacteriana observada. Los datos preliminares obtenidos en el presente estudio sugieren que el aceite esencial de Artemisia echegarayi representa una alternativa para promover su empleo como aditivo natural en alimentos.

Concentration of hydrogen ions in several calcium hydroxide pastes over different periods of time

The purpose of this in vitro study was to measure the concentration of hydrogen ions (pH) of calcium hydroxide [(Ca(OH)2] pastes combined with different vehicles over 7 periods of time. The Ca(OH)2 was manipulated with the following vehicles: i: sterile water; ii: iodoform plus sterile water; iii: local anesthetics (Lydocaine 2 percent with 1: 100,000 epinephrine); iv: polyethyleneglycol; v: glycerin; vi: 2.0 percent chlorhexidine gel; vii: camphorated paramonochlorophenol (CMCP); viii: (CMCP) + glycerin; and ix: polyethyleneglycol plus CMCP. The pastes were made on a glass plate to toothpaste consistency and the pH was measured at the following times: 5 min, 1, 24, 48 h; 7, 14 and 28 days. The data were statistically analyzed (Kruskal-Wallis at p<0.05). At 5 min, 1 and 24 h, the pH of all tested pastes ranged from 13.05 to 11.16. At 48 h and 7 days the pH of all tested pastes ranged from 11.66 to 8.92. At 14 and 28 days almost all pastes had pH means lower than 10. In conclusion, the mean pH of all tested calcium hydroxide pastes decreased with the time. Pastes made with aqueous vehicles (especially with sterile water), followed by oily vehicles (especially with CMCP + glycerin), held the highest pH means over the periods of time tested.

O objetivo deste estudo in vitro foi mensurar a concentração de ions hidroxila (pH) de pastas de hidróxido de cálcio manipuladas com diversos veículos em 7 intervalos de tempo. As pastas foram manipuladas com os seguintes veículos: (i) água destilada estéril; (ii) idodofórmio + água destilada estéril; (iii) anestésico local (Lidocaína a 2 por cento com 1:100.000 epinefrina); (iv) polietilenoglicol (Calen); (v) glicerina; (vi) clorexidina gel 2 por cento; (vii) paramonoclorofenol canforado (PMCC); (viii) PMCC + glicerina; e (ix) PMCC + polietilenoglicol (Calen PMCC). As pastas foram manipuladas em consistência de pasta de dente e os pH mensurados 5 min; 1, 24, 48 h; 7, 14 e 28 dias após manipulação. Os resultados foram analisados estatisticamente através do teste Kruskal-Wallis (p<0,05). Aos 5 min, 1 e 2 h após manipulação o pH de todas as pastas ficou entre 13.05 e 11.16. Aos 48 h e 7 dias após a manipulação, o pH de todas as pastas testadas variou de 11.66 a 8.92. Aos 14 e 28 dias, quase todas as pastas mostraram pH menor que 10. Concluiu-se que o pH de todas as pastas hidróxido de cálcio decresceram de acordo com o tempo. Pastas feitas com veículos aquosos (especialmente com água destilada), seguida de veículos oleosos (especialmente com CMCP + glicerina) mantiveram as maiores médias de pH durante os períodos testados.

Residual antibacterial activity of chlorhexidine digluconate and camphorated p-monochlorophenol in calcium hydroxide-based root canal dressings

The purpose of this study was to evaluate the residual antibacterial activity of several calcium hydroxide [Ca(OH)2]-based pastes, placed in root canals of dogs' teeth with induced chronic periapical lesions. Root canals were instrumented with the ProFile rotary system and filled with 4 pastes: G1 (n=16): Ca(OH)2 paste + anesthetic solution; G2 (n=20): Calen® paste + camphorated p-monochlorophenol (CMCP); G3 (n=18): Calen®; and G4 (n=18): Ca(OH)2 paste + 2 percent chlorhexidine digluconate. After 21 days, the pastes were removed with size 60 K-files and placed on Petri plates with agar inoculated with Micrococcus luteus ATCC 9341. Pastes that were not placed into root canals served as control. After pre-diffusion, incubation and optimization, the inhibition zones of bacterial growth were measured and analyzed by Mann-Whitney U test at 5 percent significance level. All pastes showed residual antibacterial activity. The control samples had larger halos (p<0.05). The mean residual antibacterial activity halos in G1, G2, G3 and G4 were 7.6; 10.4; 17.7 and 21.4 mm, respectively. The zones of bacterial growth of G4 were significantly larger than those of G1 and G2 (p<0.05). In conclusion, regardless of the vehicle and antiseptic, all Ca(OH)2-based pastes showed different degrees of measurable residual antibacterial activity. Furthermore, unlike CMCP, chlorhexidine increased significantly the antibacterial activity of Ca(OH)2.

Este estudo objetivou mensurar a atividade anti-bacteriana residual de várias pastas à base de hidróxido de cálcio [Ca(OH)2], após permanecerem em canais radiculares de cães com lesões periapicais crônicas induzidas. Os canais radiculares foram instrumentados com o sistema Profile e preenchidos com quatro diferentes pastas: G1-Ca(OH)2 p.a./solução anestésica (n=16), G2-Calen®/PMCC (n=20), G3-Calen® (n=18) e G4-Ca(OH)2 p.a./solução de digluconato de clorexidina a 2 por cento (n=18). Transcorridos 21 dias, amostras foram recuperadas dos canais radiculares com limas Kerr #60 e colocadas em placas de Petri com ágar semeado com Micrococcus luteus ATCC 9341. Pastas não aplicadas nos canais radiculares serviram como controle. Após pré-difusão, incubação e otimização, as zonas de inibição do crescimento bacteriano foram mensuradas e analisadas pelo teste de Mann-Whitney (a<0.05). Verificou-se que todas as pastas apresentaram ação antibacteriana residual, com maiores halos para as amostras-controles. As médias dos halos de inibição para G1, G2, G3 e G4 foram 7,6; 10,4; 17,7 e 21,4 mm, respectivamente. Os halos de inibição de G4 foram significativamente superiores as de G1 e G2 (p<0.05). Portanto, independentemente do veículo, todas as pastas à base de Ca(OH)2 determinaram, em diferentes magnitudes, atividade anti-bacteriana residual mensurável. Ademais, diferentemente do PMCC, a solução de digluconato de clorexidina a 2 por cento ampliou significativamente, a atividade anti-bacteriana residual do hidróxido de cálcio.

Susceptibility of some oral microorganisms to chlorhexidine and paramonochlorophenol

Tendo em vista a necessidade de se utilizarem agentes antimicrobianos durante a terapia endodôntica, o presente estudo tem por objetivo determinar as concentrações inibitórias mínimas (CIMs) de digluconato de clorexidina e de paramonoclorofenol (PMC) frente a cepas de microrganismos freqüentemente isolados dos canais radiculares infectados. Ambos os agentes foram testados por meio de testes de diluição em meio sólido contra Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Candida albicans, Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella denticola e Prevotella melaninogenica. As CIMs de clorexidina variaram de 2,67 a 80,00 µg/ml, e as CIMs de PMC variaram de 46,67 a 213,33 µg/ml. A maior CIM do paramonoclorofenol foi frente a E. faecalis, entretanto E. coli foi o microrganismo mais susceptível. A maior CIM do digluconato de clorexidina foi frente a P. aeruginosa, entretanto E. coli e P. denticola foram os microrganismos mais susceptíveis. Como os valores de CIM observados são bem menores do que as concentrações usadas normalmente na terapia endodôntica, sugere-se que ambos os agentes são efetivos na redução microbiana no canal radicular.

Activity of endodontic antibacterial agents against selected anaerobic bacteria

The antimicrobial activity of substances used as antibacterial agents (solutions of 10 calcium hydroxide, camphorated paramonochlorophenol - PMCC, 2 chlorhexidine digluconate and 10 castor oil plant detergent) on anaerobic bacteria (Fusobacterium nucleatum ATCC 25586, Prevotella nigrescens ATCC 33563, Clostridium perfringens ATCC 13124 and Bacteroidesfragilis ATCC 25285), using a broth dilution technique, was evaluated in vitro. For determination of minimum inhibitory and minimum bactericide concentrations (MIC and MBC), two culture broths, Reinforced Clostridial Medium (RCM) and supplemented Brucella, standardized inoculum and serially diluted solutions were used. All antibacterial agents presented antimicrobial activity that varied for different bacteria. There were no differences in the performance of the two broths. Chlorhexidine digluconate was the most effective, with the lowest MICs, followed by castor oil detergent, PMCC and calcium hydroxide. C. perfringens and B. fragilis were the most resistant bacteria to all agents.

Activity of endodontic antibacterial agents against selected anaerobic bacteria%Atividade antimicrobiana de agentes antibacterianos utilizados em Endodontia sobre bactérias anaeróbias estritas

Foi avaliada in vitro a capacidade antimicrobiana de substâncias utilizadas como agentes antimicrobianos (solução de hidróxido de cálcio 10 por cento, paramonoclorofenol canforado - PMCC, digluconato de clorexidina 2 por cento e detergente de mamona 10 por cento) sobre bactérias anaeróbias (Fusobacterium nucleatum ATCC 25586, Prevotella nigrescens ATCC 33563, Clostridium perfrigens ATCC 13124 e Bacteroides fragilis ATCC 25285), utilizando-se a técnica da diluição em caldo. Para a determinação das concentrações inibitória e bactericida mínimas (CIM e CBM), dois caldos, Reinforced Clostridial Medium (RCM) e Brucella suplementado, inóculo padronizado e diluições seriadas foram utilizados. Todos os agentes antimicrobianos apresentaram atividade antimicrobiana, variando para as diferentes espécies bacterianas, não havendo diferença entre os caldos utilizados. O digluconato de clorexidina demonstrou a melhor eficiência, com as menores CIMs, seguido pelo detergente de mamona, PMCC e hidróxido de cálcio. C. perfringens e B. fragilis foram as espécies mais resistentes aos agentes

Evaluation of antimicrobial efficacy of various root canal filling materials for primary teeth.

Four materials viz. zinc oxide-eugenol, iodoform paste, Kri paste, Maisto's paste and Vitapex (Calcium hydroxide + iodoform) were tested for their antibacterial effect against the aerobic and anerobic bacteria, viz. Staphylococcus aureus, Streptococcus viridans, Streptococcus faecalis, Bacteroides melaninogenicus and mixed bacterial culture; obtained from infected non-vital deciduous anterior teeth. The antimicrobial sensitivity was checked on BHI-agar plates using well method. The results showed that all the 4 materials were distinctly different from each other in their antimicrobial activity. Maisto's paste was invariably the superior most in its antibacterial efficacy (in comparison to all the 5 micro-organism strains). Iodoformized zincoxide eugenol appeared to be the second best followed by Kri paste. Vitapex showed the least antibacterial activity.

Atividade antibacteriana da pasta de hidróxido de cálcio/paramonoclorofenol canforado/glicerina contendo diferentes proporçöes de iodofórmio sobre bactérias anaeróbias estritas e facultativas / The antibacterial activity of calcium hydroxide camphorated paramonochlorophenol glycerin past containing differet amounts of iodorform against obligate and facultative anaerobic bacteria

A atividade antibacteriana de pasta à base de hidróxido de cálcio/paramonoclorofenol cnforado/glicerina (H/P/G) contendo diferentes proporções de iodofórmio foi testada contra 3 bactérias anaeróbias estritas (Porphyromonas endodontalis, Prophyromonas gingivalis e Prevotella intermedia) e 3 anaeróbias facultativas (enterococcus faecallis, Staphylococcus aureuse e Streptococcus sanguis). Para fins comparativos, testou-se também os efeitos antibacterianos de pastas à base de iodofórmio ou hidróxido de cálcio em glicerina. Os resultados demonstraram que a adição de iodofórmio à pasta H/P/G näo interferiu em sua propriedades antibacterianas. A pasta de hidróxido de cálcio e glicerina não paresentou qualquer efeito inibitório sobre as espécies bacterianas testadas.